Your body is always under attack by numerous foreign microscopic entities that want to infiltrate you and cause harm. Since there are billions of these foreign pathogens that exist in different shapes and sizes, it is difficult for your body to keep up. Your body's main challenge is to identify which is a harmful microorganism and which is not, and then executing a repeatable, seamless protocol that aims to eliminate all traces of the harmful pathogen before it exerts severe damage to your body's intracellular and extracellular structure, as well as vital organs. Every surface you contact, every medium you stroll in such as air and water, and every person you touch contains a multitude of microscopic organisms or infectious agents (i.e. bacteria, parasites, viruses) that are too small for your naked eyes to perceive. Fortunately, there is an army of cells patrolling inside and throughout your body via your lymphatic system, circulatory system and the nervous system that can detect and respond swiftly to any invading agent.
Most of you who never studied microbiology or cellular biology in school may not be aware your body has an immune system that is active 24/7, nor understand how it actually functions. In this blog post, I'll be discussing how your immune system works, the step-by-step guide of the typical immune response from start to finish, and how your immune system evolved over the generations to be complex and helped the human race survive for quite a long time.
How does foreign agents invade your body?
Did you know that:
- All of your body surfaces, including the gut, mucous membranes and the skin, are occupied by microbes. A majority of these microbes thrive in complex colonies within and on our bodies, which fosters a synergistic relationship between them and our bodies without harming us.
- Your body contains more bacterial cells than human cells, approximately 10:1 ratio.
The 5 mains categories of infectious agents include:
- Bacteria = Single-celled organisms that carry a single circular molecule of DNA, which encodes the genes for reproduction or other essential functions. They can appear spherical (coccus), rodlike (bacillus), or curved (vibrio, spirillum, or spirochete).
- Virus = They are strings of nucleic acid, either DNA or RNA, encased in a nuclear envelope such as a protein shell or a lipid shell. It is dormant outside of a living cell because it lacks the capacity to reproduce, hence it needs to invade a cell and hijack its metabolic machinery in order to reproduce.
- Fungi = These eukaryotic organisms produce spores, e.g. bread moulds, ringworm.
- Protozoa = These single-celled eukaryotes feed on organic matter such as other microorganisms or organic tissues and debris. e.g. Malaria.
- Helminth = Also known as parasitic worms, they are intestinal worms that transmit through soil and and infect the gastrointestinal tract e.g. hookworm, tapeworm
A newly identified infectious agent called:
- Prions = A type of protein that lead to abnormal folding of normal proteins in the brain, resulting in diseases such as Creutzfeldt-Jakob disease (CJD).
Foreign agents often enter our body through the common orifices such as the mouth, eyes, nose, or urogenital openings, as well as cuts, wounds, or bites that damage the skin barrier.
Microbes can also spread through other methods:
- Direct contact with infected skin, mucous membranes, or body fluids. e.g. Cold sores, sexually transmitted diseases.
- Indirect contact after an infected person touches a surface such as door handle / knob, table, fancet handle, where microbes remain before another person touches the same surfaces. Then that person touches their eye, mouth, or nose.
- Droplets containing infectious agents can spread through coughing, sneezing, or talking if they interact with mucous membranes of the eye, mouth, or nose of another person.
- Common vehicles e.g. Contaminated food, water, blood, or other vehicles, e.g. Salmonella and E. Coli
- Vectors, such as dogs, fleas, mites, rats, snails and ticks, that can carry and transmit diseases
- Airborne transmission: Pathogens spreading in the air as evaporated droplets or dust particles containing microorganisms for long periods of time.
What are antigens?
a. Antigen
- Every foreign invader such as a particulate matter or a pollen grain contains a molecule or molecular structure that binds to a specific antibody or T-cell receptor, known as an antigen.
- They can exist in the form of proteins, peptides, polysaccharides, lipids, nucleic acids, or other biomolecules or a solid particulate matter or a pollen grain.
- It may originate from within the body ("self-protein") or from the external environment ("non-self"). This presents a challenge for the body's immune system to identify and attack only "non-self" external antigens, as well as ignore the self-proteins due to negative selection of T cells in the thymus and B cells in the bone marrow.
- In 1899, Ladislas Deutsch (László Detre) first described the hypothetical substances halfway between bacterial constituents and antibodies as antigenic or immunogenic substances. He thought those molecules were precursors of antibodies. However, by 1903, he learnt that an antigen triggered the production of immune bodies (antibodies) and stated that the term "antigen" is a contraction of antisomatogen.
What are the different types of antigens?
i. Superantigen (SAg)
- Superantigens (SAgs) are a type of antigens that trigger excess activation of the immune system by causing non-specific activation of T-cells, which led to polyclonal T cell activation and significant cytokine release.
- They are created by a number of pathogenic viruses and bacteria likely as a defence mechanism against the immune system.
- Li et al. (1999) found SAgs are capable of activating up to 20% of the body's T-cells, which is about 20,000 - 200,000 times more compared a normal antigen-induced T-cell response.
- Examples of potent superantigens include Anti-CD3 and Anti-CD28 antibodies (CD28-SuperMAB).
Describe the superantigen structure
- They are produced intracellularly by bacteria and are released upon infection as extracellular mature toxins.
- Peterson et al. (2004) described the crystal structures of the enterotoxins as compact, ellipsoidal proteins characterised by a two-domain folding pattern consisting of an NH2-terminal β barrel globular domain known as the oligosaccharide / oligonucleotide fold. This fold is a long α-helix that diagonally spans the centre of the molecule, and a COOH terminal globular domain.
- Papageorgiou et al. (1999) reported the domains contains binding regions for the major histocompatibility complex class II (MHC class II) and the T-cell receptor (TCR), respectively.
How does superantigen interact with immune cells?
-- MHC Class II
- Alouf & Müller-Alouf (2003) found SAgs preferred the HLA-DQ form of the molecule because binding to the α-chain orients it in the best position to coordinate with the TCR.
- Mehindate et al. (1995) stated SAgs may bind to the polymorphic MHC class II β-chain (albeit less frequently), which is regulated by a zinc ion coordination complex between 3 SAg residues and a highly conserved region of the HLA-DR β chain.
- Petersson et al. (2004) found the zinc ion increases the affinity of the binding, as well as staphylococcal SAgs cross-linking MHC molecules via the α and β chains interactions. This induces cytokine expression and release in APCs, as well as co-stimulatory molecule production that increases the effectiveness of the cell's interaction with and activation of T-Cells.
-- T-Cell receptor
- The SAg binds to the variable region of the β-chain of the T-cell receptor (TCR) via its T-cell binding region. Since the human T-cell repertoire consists of only 50 types of Vβ elements and a number of SAgs can bind to multiple types of Vβ regions, one SAg can activate a significant proportion of the T-cell population.
- Studies by Brouillard et al. (2007) and Buonpane et al. (2005) noted some people respond more strongly to particularly SAgs due to variability in the types of T-cell regions.
-- Group II SAgs interact with the Vβ region using conformation-dependent processes, independent of specific Vβ amino acid side-chains.
-- Group IV SAgs interacts with all 3 CDR loops of certain Vβ forms.
- It occurs in a cleft between the small and large domains of the SAg, where SAg crams between the TCR and MHC. Li et al. (1998) detailed SAg displacing the antigenic peptide away from the TCR and evading the normal mechanism for T-cell activation.
- Arcus et al. (2000) posited the SAg's ability to stimulate T-cells is determined by its affinity for the TCR, with SPMEZ-2 being the most potent SAg discovered to date.
-- T-cell signalling
- The SAg cross-links the MHC and the TCR, which induces a signalling pathway that leads to cell proliferation and cytokine production.
- This is due to a cognate antigen activating a T-cell because of its high affinity interaction with the TCR for an extended period of time, and the SAg mimicking the temporal bonding.
- Watson & Lee (2006) discovered low levels of Zap-70 in T-cells activated by SAgs, which suggested impairment of the normal signaling pathway of T-cell activation
- Choi S & Schwartz (2007) postulated that Fyn rather than Lck is activated by a tyrosine kinase, resulting in the adaptive induction of anergy.
- Stiles & Krakauer (2005) stated that activation of both the protein kinase C pathway and the protein tyrosine kinase pathways lead to upregulated production of proinflammatory cytokines. Choi S & Schwartz (2007) highlighted that this signalling pathway impairs the calcium/calcineurin and Ras/MAPkinase pathways to a lesser extent.
Describe the effects of superantigen
-- Direct
- When superantigens stimulate APCs and T-Cells, it triggers an inflammatory response, focused on the action of Th1 T-helper cells. Stiles & Krakauer (2005) listed the major inflammatory molecules include gamma interferon (IFN-γ), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, monocyte chemoattractant protein 1 (MCP-1), IL-1, IL-2, IL-6 and TNF-α.
- Buonpane et al. (2005) found the uncoordinated excess release of cytokines overwhelms the body, leading to symptoms such as rashes, fever or worse, multi-organ failure, coma and death.
- Prolonged exposure to superantigen increases the generation of IL-4 and IL-10, which deletes activated T-Cells. Furthermore, these aforementioned cytokines downregulate production of IFN-gamma, MHC Class II, and costimulatory molecules on the surface of APCs, which leads to the formation of memory cells unresponsive to antigen stimulation.
- Yamaguchi et al. (1999) found MHC crosslinking activates a signalling pathway that suppresses haematopoiesis and upregulates Fas-mediated apoptosis.
- Studies found IFNα levels increase after prolonged exposure to superantigen, which triggers an autoimmune response, resulting in the autoimmune disease Kawasaki disease.
- Jebara & Geha (1996) reported superantigen activation in T-Cells produces CD40 ligand that activates isotype switching in B cells to IgG and IgM and IgE.
-- Indirect
- Vomiting = In cases of food poisoning, bacteria that produces SAg release a toxin that is highly resistant to heat. Llewelyn & Cohen (2002) noted an active region of the SAg molecule associated with triggering gastrointestinal toxicity.
- Induces recruitment of neutrophils to the site of infection independent of T-cell stimulation. This is caused by SAgs activating monocytic cells, which triggers the release of TNF-α. This increases expression of adhesion molecules that recruit leukocytes to infected regions. This results in inflamed lungs, intestinal tissue, and any organ with colonised bacteria.
- Reinforces the effects of endotoxins in the body by decreasing the threshold for endotoxicity. Schlievert (1982) discovered a synergistic relationship between endotoxin and SAg, which may correspond with a weakened immune system caused by SAg infection. This may result in more detrimental effects compared to typical bacterial infections.
- Jehara & Geha (1996) hypothesised SAgs play a role in the progression of sepsis in patients with bacterial infections.
ii. Allergen
- It is a type of antigen that stimulates an abnormally vigorous immune response in which the immune system combats a perceived threat that would otherwise be harmless to the body. Sensitivity to each allergen varies from person to person.
- Goldsby et al. defined allergen as an antigen that stimulates a type I-hypersensitivity reaction in atopic individuals through immunoglobulin E (IgE) responses.
What are the different types of allergens?
What is a seasonal allergy?
- I am one of many people who suffers from seasonal allergy every spring and summer, with common symptoms such as runny and irritated nose, nasal congestion, red eyes and sneezing.
- Around 1/3 of the world experience seasonal allergy during certain seasons, particularly during spring, summer, or autumn (fall) when trees or grasses pollinate. e.g. Elm, oak, and maple trees pollinate in the spring, whereas Bermuda, timothy and orchard grasses pollinate in the summer.
- If both of your parents experienced seasonal allergies in the past, there is a 66.7% chance you will experience seasonal allergies too. The risk decreases to 60% if only one of your parents experienced seasonal allergies.
- When an allergen enters your body predisposed to allergies, it induces an immune reaction and the generation of antibodies. These antibodies migrate to mast cells that live in your nose, eyes and lungs.
- When an allergen enters the nose more than once, mast cells release a variety of chemicals or histamines that irritate and inflame the membranes lining the nose and yield the symptoms of an allergic reaction: itching, sneezing, scratchy throat, and watery eyes.
Differences in symptoms between seasonal allergies and the common cold include:
-- Absence of fever
-- Runny and clear mucous secretions
-- Rapid and sequential sneezes
-- Itchy throat, ears and nose
-- Symptoms typically last longer than 7-10 days
- Researchers found a number of allergens fuse together to form a new type of allergy, a process known as cross-reaction. e.g. Grass pollen allergens can cross-react with food allergy proteins in vegetables such as carrots, celery, corn, lettuce and onion.
- A 2010 study discovered another type seasonal grass allergy in a number of rural areas that contained airborne particles of pollen intermingled with mould.
- Dr. Ruslan Medzhitov (2010) found protease allergens cleave the same sensor proteins that evolved to detect proteases produced by the parasitic worms, which indicated the human immune system evolved the allergic reaction as a defence mechanism to fight off parasites.
- In 2010, Weinmann published a report on seasonal allergies called “Extreme allergies and Global Warming”, which concluded climate change played a major role in worsening allergy triggers. 16 USA states were named as “Allergen Hotspots” for significant increases in allergenic tree pollen if global warming pollution continue to accumulate.
- Thus, researchers raised their concerns that global warming would be severely harmful to millions of asthmatics across USA whose asthma attacks are triggered by seasonal allergies.
- In 1952, Gregory & Hirst first suggested a fungal allergen called basidiospore as a possible airborne allergen that associated with asthma. The basidospore family include mushrooms, rusts, smuts, brackets, and puffballs. Examples of basidiospore species include Ganoderma, Pleurotus ostreatus, cladosporium, and Calvatia cyathiformis.
iii. Antigenic variation
- Sometimes known as antigenic alteration, antigenic variation is defined as the process of an infectious agent changing the structure of its proteins or carbohydrates on its surface in order to evade the host's immune response, as well as re-infection of previously infected hosts.
- They can be caused by gene conversion, site-specific DNA inversions, hypermutation, or recombination of sequence cassettes, which leads to expression of a heterogeneous phenotype of a clonal population of pathogen.
i. Bacteria
- Genus Neisseria: Neisseria meningitidis and Neisseria gonorrhoeae (the gonococcus) = Neisseria species vary their pili (protein polymers made up of subunits involved in bacterial adhesion and host immune response).
- Genus Streptococcus = The Steptococci vary their M-protein
- Mycoplasma
- Borrelia burgdorferi (linked to Lyme disease) = Surface lipoprotein VlsE varies due to recombination, leading to antigenic diversity. Wisniewski-Dyé (2008) found plasmid sections of the silent vls cassettes recombine with the vlsE gene to produce variants of the surface lipoprotein antigen.
ii. Protozoa
-- Trypanosoma brucei (Linked to sleeping sickness)
- It replicates extracellularly in the bloodstream of infected mammals and is targeted by several host immune systems such as the complement system, and the innate and adaptive immune systems.
- To shield itself from the immune response, it envelops itself with a dense, homogeneous coat (~10^7 molecules) of the variant surface glycoprotein (VSG).
- Initially, the VSG coat protects the parasite from immune detection. Eventually, the host identifies VSG as a foreign antigen and subsequently launches an immune attack against the microbe.
- Since the protozoa's genome has over 1,000 genes that code for different VSG variants, located on the subtelomeric portion of large chromosomes, or on intermediate chromosomes.
- Stockdale et al. (2008) found they activate by gene conversion in a hierarchical order: (1) telomeric VSGs, (2) array VSGs, and (3) pseudogene VSGs.
- Hartley & McCulloch (2008) noted each new gene is swapped in turn into a VSG expression site (ES), because only 1 VSG can be expressed at any given time. This mechanism is partially dependent on homologous recombination of DNA, which is partially regulated by the interaction of the T. brucei BRCA2 gene with RAD51.
- Since T. brucei contains several possible expression sites, transcriptional regulation plays a role in antigen switching. Stockdale et al. (2008) explained a new VSG is either selected by transcriptional activation of a previously silent ES, or by recombination of a VSG sequence into the active ES.
- Despite the biological triggers resulting in VSG switching not fully understood, mathematical modelling conducted by Mideo et al. (2013) indicated that the ordered appearance of different VSG variants is determined by at least 2 key parasite-derived factors: differential activation rates of parasite VSG and density-dependent parasite differentiation.
-- Plasmodium falciparum
- This virus is the major aetiologic agent of human malaria with a complex life cycle that situates in both humans and mosquitoes. In the human host, the parasite spends a majority of its life cycle within hepatic cells and erythrocytes.
- Therefore, parasitised host cells expressing parasite proteins require constant alterations to avoid destruction by the host immune system. That protein is the dual purpose Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is encoded by the diverse var family of genes (~60 in total).
- The var family increasingly diversifies via several different mechanisms including exchange of genetic information at telomeric loci, as well as meiotic recombination.
- PfEMP1 acts to sequester infected erythrocytes from destruction in the spleen via adhesion to the endothelium.
- Kyes et al. (2007) found the parasite avoids the host immune system by altering the var allele encoding the PfEMP1 protein.
- Although it expresses multiple copies of only 1 identical protein, Scherf et al. (1998) hypothesised this var switching in P. falciparum is typically transcriptional. Kyes et al. (2007) demonstrated var swtiching occurs immediately after the parasite invades an erythrocyte.
- Ralph et al. (2005) used fluorescent in situ hybridisation analysis to demonstrate an association between the activation of var alleles and modified positioning of the genetic material to distinct “transcriptionally permissive” areas.
iii. Viruses
- Viral genomes generally mutate faster than bacterial genomes, with a few exceptions such as paramyxoviruses. In addition, viruses with shorter genomes have faster rates of mutation than longer genomes because they have a faster rate of replication.
- Dufy (2008) demonstrated a number of DNA viruses have the same high rates of antigenic variation as their RNA counterparts.
There are 6 different types of antigenic variation within viruses:
- Antigenic drift = Point mutations caused by imperfect replication of the viral genome.
- Antigenic shift = Viral genome is reassorted when a single host cell is infected with 2 viral cells. The genes of the 2 species intermingle and produce 256 new variations of the virus. e.g. Influenza every few years.
- Antigenic rift = Recombination of viral gene, also caused by 2 viral cells infecting the same host cell. Segments of each viral gene undergo recombination to generate new genes. e.g. H5N1 virus
- Antigenic sift = Direct transmission with a zoonotic strain of a virus, particularly during a spillover event.
- Antigenic lift = Viral transmission of host-derived gene. It involves viruses stealing host genes and then integrating them into their own viral genome, leading to encoded genes with increased virulence. e.g. pox virus vaccinia
- Antigenic gift: Intentional modification of a virus's genome by humans either in a lab setting or for the purpose of designing a bioweapon.
-- Influenza virus
- The antigenic characteristics of influenza viruses are determined by both hemagglutinin (H) and neuraminidase (N). A number of host proteases cleaves the peptide HA into 2 subunits: HA1 and HA2. If the amino acids at the cleave sites are lipophilic, the virus increases in virulence.
- Selection pressure in the environment leads to antigenic changes in the antigen determinants of HA, including locations undergoing both adaptive evolution and substitutions, which results in changes in the virus's antigenicity. Note that glycosylation of HA has no effect on either the antigenicity or the selection pressure.
- Antigenic variation of influenza virus occurs in two ways: (1) antigenic drift due to a change in a number of amino acids, and (2) antigenic shift due to the acquisition of new structural proteins from other animal hosts.
- Recombination between segments encoding for hemagglutinin and neuraminidase of avian and human influenza virus segments have historically lead to influenza pandemics. For example, the 1957 Asian flu was caused by the acquisition of 3 genes from Eurasian avian viruses and subsequent reassortment with 5 gene segements of the circulating human influenza strains.
-- HIV-1
- HIV-1 is notable for evading the immune response, and co-evolving amino acid mutations. For instance, a substitution in the specific site leads to a secondary mutation in another site.
- The extent and frequency of certain HLA allele targeting an epitope varies from person-to-person. Therefore, an individual's CTL response is restricted to a few epitopes of a specific HLA allele. Nevertheless, the epitopic repertoire increases with time because of viral escape.
- In individuals expressing a protective HLA B*27 allele, the first mutation situating in the Gag epitope KK10 occurs on position 6 from a Leucine (L) to a Methionine (M), followed by another mutation on position 2 from an Arginine (R) to a Lysine (K).
- Carlson & Brumme (2008) stated that application of selective pressure can accurately predict the pattern of HIV-1 evolution, as well as its escape pathways, which can aid in designing immunogens.
- The gp120 region of HIV-1 Env contacts its primary receptor called CD4, which is functionally conserved and susceptible to neutralising antibodies such as monoclonal antibody b12.
- Li et al. (2009) discovered substitutions in the region proximal to CD4 contact surface developed HIV-1's resistance to neutralisation by b12.
-- Flaviviruses
- This family of viruses includes a number of well-known viruses such as West Nile virus and Dengue virus. It has a prototypical envelope protein (E-protein) on its surface that gets targeted by virus neutralizing antibodies.
- This E protein is involved in the interaction with the receptor and avoiding the host immune system. It contains 3 major antigenic domains A, B and C that correspond to the 3 structural domains II, III and I.
- Structural domain III is a putative receptor binding domain and a target for antibodies for the infectious nature of flaviviruses to be neutralised.
- Amino acid substitutions of different positions in the domain III gene can result in varying antigenic differences, which lead to varying levels of neutralisation by antibodies.
- In other flaviviruses that result in dengue, louping ill and yellow fever, its mutations in the domain III of the E protein allow it to evade antibody neutralisation.
iv. Hapten
- A hapten is a small molecule that attach to a large carrier such as a protein, which trigger an immune response. Once the body has produced antibodies to a hapten-carrier adduct, the hapten may also bind to the antibody, but it won't trigger an immune response.
Examples of hapten include:
-- Aniline and its carboxyl derivatives (o-, m-, and p-aminobenzoic acid)
-- Urushiol, a toxin found in poison ivy
-- Hydralazine, a blood pressure-lowering drug that occasionally results in drug-induced lupus erythematosus.
-- Fluorescein, biotin, digoxigenin, and dinitrophenol.
v. Others
-- Tolerogen
= A substance that induces a specific immune non-responsiveness due to its molecular form. If its molecular form alters, a tolerogen transforms into an immunogen.
-- Immunoglobulin-binding protein
= Examples include protein A, protein G, and protein L. They attack antibodies by binding at positions outside of the antigen-binding site.
-- T-dependent antigen
= Antigens that require T cells' assistance to induce the formation of specific antibodies.
-- T-independent antigen
= Antigens that directly stimulate B cells.
-- Immunodominant antigens
= Antigens that dominate in their ability to produce an immune response.
b. Epitope
- Known as an antigenic determinant, an epitope is a segment of an antigen recognised by the immune system, particularly by antibodies, B-cells, or T-cells. It is a specific section of the antigen that an antibody binds to.
- The segment of an antibody that binds to the epitope is known as the paratope.
- Mahmoudi Gomari et al. (2020) found epitopes are also sequences derived from the host that are recognised by the immune system (i.e. autoimmune diseases), despite being non-self proteins.
- Huang & Honda (2006) classified epitopes into conformational epitopes and linear epitopes according to their structure and interaction with the paratope. Ferdous et al. (2019) estimated around 90% of epitopes are conformational.
Describe the functions of epitopes
-- T-Cell epitopes
- Steers et al. (2014) found T-cell epitopes are expressed on the surface of an APC, where they bind to major histocompatibility complex (MHC) molecules.
- Alberts et al. (2002) discovered the T-cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer epitopes, 13–17 amino acids in length. In addition, non-classical MHC molecules present non-peptidic epitopes such as glycolipids.
-- B-Cell epitopes
- It refers to the segment of an antigen bound by the immunoglobulin or antibodies.
- Regenmortel (2009) stated that a majority of B-cell epitopes are conformational, thus a minority are linear.
- Masked epitopes due to protein subunit aggregation are known as cryptotopes.
- When epitopes are only recognised in a specific quaternary structure and its residues spans multiple protein subjects, they are known as neotopes. However, they become unrecognisable when its subunits dissociate.
-- Cross-reactivity
- Originally postulated by Nobel laureate Niels Kaj Jerne, it refers to the paratope becoming the epitope for another antibody that will subsequently bind to it when an antibody binds to an antigen's epitope.
-- Linear
- A linear / sequential epitope is a linear sequence of amino acids (primary sequence) that is recognised by antibodies.
- When antigens are too large to bind entirely to a receptor, only a particular section of the antigen binds with a specific antibody, known as an epitope.
- The linear sequence of an epitopes is a segment of amino acids that doesn't usually present as a simple line of sequential proteins. When the antigen disintegrates in a lysosome, it generates small peptides that are recognised through the amino acids organised continuously in a line, thus are labelled linear epitopes.
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| This diagram illustrates recognition of epitopes in a linear fashion. Note: the same (coloured) segment of protein can be a part of more than 1 epitope. |
-- Conformational
- A conformational epitope is a sequence of sub-units (usually amino acids) composing an antigen that interact with a receptor presented by an immune cell.
- Whenever a receptor interacts with an undigested antigen, the interacting surface amino acids may be discontinuous with each other if the protein is unfolded. Conformational epitopes refer to discontinuous amino acids that combine in 3D conformation and interact with the receptor's paratope.
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| This diagram illustrates B-cells recognising conformational epitopes. Note how the segments widely separated in the primary structure have interacted with the three-dimensional tertiary structure forming part of the same epitope. |
c. Mimotope
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| Diagram of the increased safety of mimotopes compared with native allergens. a. Crosslinking of IgE receptors on mast cells requires polyclonal IgE binding to two different epitopes on the same allergen. b. A mimotope displayed on a carrier protein (e.g., a filamentous phage coat protein) would only bind to IgE from a single colony and would thus fail to crosslink the IgE receptors on mast cells |
- It is a macromolecule that mimics the structure of an epitope. This triggers an antibody response similar to one induced by a typical epitope.
- The term mimotope was first coined by Mario Geysen in 1986, which described peptides mimicking epitopes. Since then, the concept has extended to peptide mimic of all types of binding sites.
Where do antigens originate?
i. Exogenous antigens
- These antigens enter the body from the outside environment via inhalation, ingestion, or injection.
- The immune system's response to exogenous antigens is usually endocytosis or phagocytosis, followed by antigen-presenting cells (APCs) processing them into fragments before presenting them to T helper cells (CD4+) by class II histocompatibility molecules on their surface.
ii. Endogenous antigens
- They are produced within healthy cells as a result of normal cell metabolism, or of viral or intracellular bacterial infection. They include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens.
- Fragments of these antigens are presented on the APC's surface in the complex with MHC class I molecules. If activated cytotoxic CD8+ T cells recognise them, they release various toxins that lyse or cause apoptosis of the infected cell.
- To prevent cytotoxic cells from destroying cells just for presenting self-proteins, they are deleted due to tolerance (negative selection).
iii. Autoantigens
- It is a self-protein or protein complex recognised by the immune system.
- Under normal conditions, they usually aren't targeted by the immune system. In the case of autoimmune diseases, their associated T-cells still linger (rather than get deleted) and actually attack these autoantigens.
iv. Neoantigens
- They don't exist in the normal human genome. Compared to original self-proteins, they are involved in tumour control. This means the quality of the T-cell pool available for these antigens aren't impacted by central T-cell tolerance.
- Technologies such as MANA-SRM can directly detect and quantify neoantigens in order to systematically analyse T-cell reactivity against such neoantigens.
-- Viral antigens
- Epitopes derived viral open reading frames produce neoantigens associated with virus-associated tumours such as cervical cancer and a subset of head and neck cancers.
-- Tumour antigens
- Also known as tumour-specific antigens (TSAs), they are found only on the surface tumour cells presented by MHC class I or class II molecules.
- Tumour-associated antigens (TAAs) are commonly presented by both tumour cells and normal cells, which are recognised by cytotoxic T lymphocytes.
- Tumour antigens presented on a mutated receptor by a tumour cell are recognised by B-cells.
- In the case of human tumours lacking a viral aetiology, neo-epitopes are produced by tumour-specific DNA alterations.
v. Native Antigens
- They are antigens yet to be processed by an APC to smaller fragments. T-cells cannot bind to these antigens, whereas B-cells can bind to them.
What is antigen specificity?
- It refers to the host cells' ability to recognise an antigen specifically as a unique molecular entity and distinguish it from another with precision. It is the result of the side-chain conformations of the antigen.
What is immunity?
- When multicellular organisms resist the effects of harmful microorganisms, they are considered immune to them.
- Researchers appreciated immunity is a complex biological system comprised of both specific and nonspecific components that is capable of identifying and responding to foreign agents (non-self), and recognising and tolerating familiar agents (self).
Describe the history of immunity research
- Humans have been fascinated by the causes of disease and the concept of immunity for millennia. The earliest view of the cause of diseases was due to supernatural forces, and that illness was a form of theurgic punishment for "bad deeds" or "evil thoughts" afflicted upon the soul by the gods or by one's enemies.
- Classical Greek philosopher Hippocrates (Father of Medicine) hypothesised diseases were caused by changes or imbalance in 1 of the 4 humours (blood, phlegm, yellow bile or black bile).
- The first written descriptions of the concept of immunity may have been published by Athenian Thucydides around 430 BC. When the plague struck Athens, he wrote "the sick and the dying were tended by the pitying care of those who had recovered, because they knew the course of the disease and were themselves free from apprehensions. For no one was ever attacked a second time, or not with a fatal result."
- The practice of active immunotherapy may have started with Mithridates VI of Pontus (120-63 BC), who administered the blood of animals that fed upon snakes to develop immunity against snake venom.
- For about 2 millennia, poisons were thought to be the proximate cause of disease, therefore a concoction of ingredients was administered to cure poisoning during the Renaissance, labelled Mithridate (named after Mithridates VI of Pontus).
- The term "immunes" was mentioned in a 60 BC poem called "Pharsalia" written by poet Marcus Annaeus Lucanus to describe a North African tribe's resistance to snake venom.
- The first clinical description of immunity may have been written by the Islamic physician Al-Razi in the 9th century in his work A Treatise on Smallpox and Measles ("Kitab fi al-jadari wa-al-hasbah'', translated 1848). He mentioned that exposure to these specific agents developed lasting immunity (although he didn't specifically use the term) in regards to smallpox and measles.
- Until the 19th century, the miasma theory hypothesised diseases such as cholera or the Black Plague were caused by a noxiously "bad air". This lead to the trend of plague doctors wearing a special costume comprising of an ankle-length overcoat and a bird-like beak mask, filled with sweet or strong-smelling substances (e.g. dried flowers such as roses and carnations, herbs such as lavender and peppermint, camphor, or a vinegar sponge) combined with boots, gloves, a wide-brimmed hat, and an outer over-clothing garment.
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| This is a copper engraving of Doctor Schnabel (i.e., Dr. Beak), a plague doctor in 17th-century Rome, circa 1656. |
- The modern word "immunity" originated from the Latin immunis, meaning exemption from military service, tax payments or other public services.
- In 1882, Ilya Mechnikov was the first scientist to publish a full theory of immunity, who identified the phenomenon of phagocytosis.
- Louis Pasteur's germ theory of disease began to explain the mechanisms of disease manifested by bacteria, and mechanisms of body's resistance against infection.
- In 1888, Emile Roux and Alexandre Yersin successfully isolated the diphtheria toxin.
- After the 1890 discovery of antitoxin based immunity to diphtheria and tetanus by Behring and Kitasato, the antitoxin was the first major success of modern therapeutic immunology.
- It's unknown when the first immunisations were officially administered, but Gherardi (2007) believed the Chinese established the practice of immunisation around 1000 AD by drying and inhaling powders derived from the crusts of smallpox lesions.
- There were records of inoculation in India, the Ottoman Empire, and east Africa around the 15th century, which involved poking the skin with powdered material derived from smallpox crusts. This same practice was introduced into the west by Lady Mary Wortley Montagu in 1721.
- In 1798, Edward Jenner introduced a safer method of the deliberate infection with cowpox virus to develop immunity against smallpox, which was labelled vaccination 2 years later. This is not to be confused with variolation, which referred to smallpox inoculation.
The Lymphatic System
Which organs are involved in the immune response?
- The lymphatic system is a network of lymph, lymphatic vessels, lymph nodes, lymphatic or lymphoid organs, and lymphoid tissues that forms part of your circulatory system and immune system.
- The vessels transport a clear fluid called lymph towards the heart. (Lymph is derived from the Latin word lympha meaning the deity of fresh water, "Lympha").
1. Primary Lymphoid Organs
These organs produce lymphocytes from immature progenitor cells and early clonal selection of lymphocyte tissues.
a. Bone Marrow
- This region is the site of T-Cell precursor generation and B-cell production and maturation. In adult humans, bone marrow is mostly located in the ribs, vertebrae, sternum, and bones of the pelvis.
- After maturation, B-Cells enter the circulatory system and travel to secondary lymphoid organs scavenging for any foreign pathogens.
- T-Cells travel from the bone marrow to the thymus, where they undergo further development and maturation. This occurs before they join B-cells in searching for pathogens.
b. Thymus
- Located in the upper front part of the chest, in the anterior superior mediastinum, behind the sternum, and in front of the heart, this organ is the site of T-Cell maturation.
- It is composed of immature T-Cells called thymocytes, as well as lining cells called epithelial cells that assist the development of thymocytes.
- T-cells undergo position selection or negative selection by interacting with immune receptors of the body and whether they react appropriately to them or not respectively.
2. Secondary Lymphoid Organs
These organs are responsible for maintenance of mature naive lymphocytes and triggering an adaptive immune response. They are the sites of lymphocyte activation by antigens, which lead to clonal expansion and affinity maturation.
a. Spleen
Its main functions include:
-- Immune cell production
-- Removal of particulate matter and aged blood cells, mainly red blood cells
-- Blood cell production during foetal life
- It generates antibodies in its white pulp and removes antibody-coated bacteria and antibody-coated blood cells via blood and lymph node circulation.
- The red pulp consists of mostly monocytes, which targets injured tissue, transforms into dendritic cells and macrophages as it augments tissue healing.
- It is regarded as the centre of activity of the mononuclear phagocyte system, which consists of the phagocytic cells located in reticular connective tissue.
- Until the 5th month of prenatal development, the spleen produces red blood cells. Then, after birth, the bone marrow takes over the responsibility of haematopoiesis.
- As a major lymphoid organ and a central player in the reticuloendothelial system, the spleen continues to generate lymphocytes, as well as store red blood cells and lymphocytes.
b. Lymph nodes
Structure of lymph nodes
- This kidney-shaped organ are present throughout the body by the lymphatic vessels, which act as major sites of lymphocytes including B-Cells and T-Cells. They act as filters for foreign particles including cancer cells, one of the important immune functions.
- Each lymph node is encased in a fibrous capsule, which extends inside a lymph node to form trabeculae. It is divided into outer cortex and the inner medulla, which contain numerous cells.
- The indent on the concave surface of the lymph node where lymphatic vessels exit and blood vessels enter and leave is called hilum.
- Lymph enters the convex side of a lymph node through several afferent lymphatic vessels and subsequently flows into a series of sinuses, as well as a space underneath the capsule called the subcapsular sinus, before entering the cortical sinuses.
- After permeating the cortex, lymph then accumulates in medullary sinuses, which drains into the efferent lymph vessels to exit the node at the hilum on the concave side.
- There are about 450 lymph nodes in an adult human body, and some of them can be felt when enlarged e.g. the axillary lymph nodes under the arm, the cervical lymph nodes of the head and neck and the inguinal lymph nodes near the groin crease.
Subdivisons:
- A lymph node is subdivided into compartments called nodules (or lobules). Each each nodule contains a region of cortex as well as a collection of follicle B cells, a paracortex of T cells, and a part of the medulla.
- The anatomy of a lymph node is segregated into the outer cortex and the inner medulla. The cortex region forms part of the outer portion of the node, under the capsule and the subcapsular sinus, which contains an outer part and a deeper part known as the paracortex.
- The outer cortex comprises of groups of primarily inactivated B cells called follicles. When these follicles are activated, they may develop into geminal centres.
- The deeper paracortex mainly consists of the T cells, which primarily communicate with dendritic cells.
- The medulla contains large blood vessels, sinuses and medullary cords that consists of antibody-secreting plasma cells.
Cell composition:
- Lymph nodes consist of a type of white blood cell called lymphocytes, which comprise of mainly B-Cells and T-Cells.
- B-Cells cluster in the outer cortex as follicular B cells in lymphoid follicles, whilst T cells and dendritic cells are located in the paracortex.
- The medulla consists of plasma cells, as well as macrophages which are also present within the medullary sinuses.
- As part of the reticular network, there are follicular dendritic cells in the B-cell follicle and fibroblastic reticular cells in the T-cell cortex.
Lymph flow:
- Lymph enters the convex side of a lymph node through multiple afferent lymphatic vessels as part of the network of lymphatic vessels.
- Subsequently, it flows into a space beneath the capsule called the subcapsular sinus.
- Then lymph flows into sinuses within the cortex via the trabecular sinus.
- It accumulates in medullary sinuses after traversing the cortex.
- It then drains into the efferent lymphatic vessels to exit the node at the hilum on the concave side.
- In order for smooth lymph flow, the channels within the node are lined by endothelial cells along with fibroblastic reticular cells.
- The endothelium of the subcapsular sinus is continuous with that of the afferent lymph vessel, as well as similar sinuses situated along the trabeculae and within the cortex.
Capsule:
- The node's interior is supported by a meshwork made of thin reticular fibres (reticulin) made of reticular connective tissue.
- The lymph node capsule is composed of dense irregular connective tissue and plain collagenous fibres, with several membranous processes or trabeculae extending from its internal surface.
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| This is an illustration of trabeculae inside lymph node tissue. |
Functions of lymph nodes
- Filter lymph for identification and combating infection using lymphocytes i.e. T-Cells and B-Cells.
- Lymph drain into and from lymph nodes via afferent and efferent vessels respectively.
- B-Cells enter the lymph node and into a lymphoid follicle, where they multiply and divide, each generating a different antibody. If a particular B-Cell is activated, that one will proceed to generate more antibodies or act as a memory cell to help the body fight future infection. Other unactivated B-Cells undergo apoptosis and die.
- Antigen-presenting cells enter the lymph system and then lymph nodes to present the antigen to T cells. APC activates once a T cell with the appropriate T cell receptor binds to it.
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| Diagram of locations of regional lymph nodes |
c. Tonsils
- Located in the back of your mouth, your tonsils are a pair of lymphoid organs that face into the aerodigestive tract, known as Waldeyer's tonsillar ring.
- You have the following tonsils: 1 Adenoid tonsil, 2 tubal tonsils, 2 palatine tonsils, and the lingual tonsils.
- Tonsils act as the immune system's first line of defence against ingested or inhaled foreign pathogens, which often combine with blood to aid in immune responses to common illnesses such as the common cold.
- The tonsil's surface have specialised antigen capture cells called Microfold cell (M cells), which is responsible for uptake of antigens produced by pathogens.
- The M-cells subsequently signal B cells and T cells in the tonsil that a pathogen is present, which triggers an immune response.
d. Mucosa-Associated Lymphatic Tissue (MALT)
- MALT is a system of small pockets of lymphoid tissue located in various submucosal membrane sites of the body, such as the eye, breast, gastrointestinal tract, lung, nasopharynx, thyroid, salivary glands, and skin.
- MALT comprises of lymphocytes such as T cells and B cells, as well as plasma cells and macrophages. Each of these immune cells interact with antigens that traverse the mucosal epithelium.
- It constitutes about 50% of the lymphoid tissue in human body.
-- BALT = Bronchus
-- CALT = Conjunctival
-- GALT = Gut
-- LALT = Larynx
-- NALT = Nasal
-- SALT = Skin
-- TALT = Testis
-- VALT = Vulvo-vaginal
Types of MALT:
-- O-MALT = Organised e.g. Tonsils of Waldeyer's tonsillar ring
-- D-MALT = Diffuse i.e. Not organised as a separately, macroscopically anatomically identifiable mass, tissue or organ.
3. Tertiary Lymphoid Organs (TLOs)
https://www.researchgate.net/figure/Plasticity-in-secondary-and-tertiary-lymphoid-organ-development-Lymph-node-development_fig2_7229162
- Yin et al. (20170 described TLOs as abnormal lymph node-like structures located in peripheral tissues at sites of chronic inflammation e.g. chronic infection, transplanted organs undergoing graft rejection, some cancers, autoimmune and autoimmune-related disease.
- They contain less lymphocytes, which means they only participate in the immune response when they detect antigens resulting in inflammation.
- Hiraoka et al. (2016) found TLOs have an active germinal centre, which is surrounded by a network of follicular dendritic cells (FDCs).
- Ruddle (2014) observed TLOs undergo different regulation processes compared to lymphoid tissues, which develop during ontogeny. However, they still drain interstitial fluid and transport lymphocytes in response to the same chemical messengers and gradients.
- Researchers found TLOs with an active germinal centre play a critical role in the immune response against a number of cancer types such as melanoma, non-small cell lung cancer, colorectal cancer and glioma, which increases the patient's longevity.
a. Other lymphoid tissue
- Lymphatic tissue associated with the lymphatic system is mainly connective tissue made of reticular fibres that contains various types of leukocytes (white blood cells), as well as lymphocytes.
b. Lymphatic vessels
- Your lymphatic vessels are thin-walled vessels, similar to blood vessels structurally, which carries lymph.
- They are lined by endothelial cells, contain a thin layer of smooth muscle, and an outer layer of connective tissue called adventitia to bind the lymph vessels to the surrounding tissue.
- The endothelial cells function to mechanically transport lymph, but the basement membrane on which it rests is discontinuous, which leads to leakages.
- The smooth muscle layer are arranged in a circular fashion around the endothelium, contracting or relaxing to adjust the diameter of the lumen.
- Smaller lymphatic vessels lack both the muscular layer and the outer adventitia. It links with other capillaries, enlarge, and takes up layers of adventitia and then smooth muscles.
- Shayas et al. (2006) stated lymph moves through vessels due to peristalsis, valves, and compression during contraction of adjacent skeletal muscle and arterial pulsation.
-- Lymph capillaries
- Lymph flow initiates with blind ending (closed at one end) highly permeable superficial lymph capillaries, which are made of endothelial cells with button-like junctions between them. This structure allows fluid to permeate when the interstitial pressure is sufficiently high.
- These junctions shaped like junctions are made of protein filaments such as platelet endothelial cell adhesion molecule-1 (PECAM-1).
- A valve system prevents the absorbed lymph from leaking back into the ISF. It features collagen fibres linked to lymphatic endothelial cells. When interstitial fluid pressure increases, the valve system separates the endothelial cells and permits the flow of lymph into the capillary for circulation.
- There is a system of semilunar valves that prevents back-flow of lymph along the lumen of the lymphatic vessel.
- Lymph capillaries have numerous anastomoses between them to form an interconnected network.
-- Lymph vessels
- Lymph capillaries drain into larger collecting (contractile) lymphatics, which transport lymph through peristalsis of smooth muscle walls, as well as valves to prevent the backward flow of lymph.
- When a collecting lymph vessel accumulates lymph from more capillaries, it enlarges until it becomes an afferent lymph vessel as it travels to the lymph node.
- Then the lymph percolates through the lymph node tissue and exits via an efferent lymph vessel, which drains directly into one of the (right or thoracic) lymph ducts, or another lymph node as its afferent lymph vessel.
- Lymph vessels consist of functional units divided by semilunar valves called lymphangions, which control lymph flow by contraction or relaxation of the encircling smooth muscle.
Describe the development of the lymphatic system
- Lymphatic tissues begin developing by the end of the 5th week of embryonic development.
- Lymphatic vessels develop from lymph sacs that extend from developing veins, which originate from mesoderm.
- The paired jugular lymph sacs at the junction of the internal jugular and subclavian veins are the first to appear.
- From the jugular lymph sacs, lymphatic capillary plexuses travel to the thorax, upper limbs, neck, and head.
- The unpaired retroperitoneal lymph sac located at the root of the mesentery of the intestine is the second lymph sac to appear, which develops from the primitive vena cava and mesonephric veins. From the retroperitoneal lymph sac, capillary plexuses and lymphatic vessels extend to the abdominal viscera and diaphragm. It links with the cisterna chyli, but disconnects with adjacent veins.
- The paired posterior lymph sacs are the final lymph sacs to appear, which develop from the iliac veins. They generate capillary plexuses and lymphatic vessels of the abdominal wall, pelvic region, and lower limbs. It also links with the cisterna chyli, but disconnects from adjacent veins.
- Besides the anterior part of the sac from which the cisterna chyli develops, mesenchymal cells infiltrate all lymph sacs to transform them into lymph nodes.
Describe the function of the lymphatic system
The functions of the lymphatic system include:
-- Removal of interstitial fluid from tissues
-- Absorption and transport of fatty acids and fats as chyle from the digestive system
-- Transport white blood cells to and from the lymph nodes into the bones
-- The lymph transports antigen-presenting cells, such as dendritic cells, to the lymph nodes where an immune response is triggered.
i. Fat absorption
- Lymph vessels situated at the start of the gastrointestinal tract (usually the small intestine) are called lacteals.
- During digestion, lipids are transferred to the lymphatic system for transport to the blood circulation via the thoracic duct. An exception includes medium-chain triglycerides passively diffusing from the GI tract to the portal system. Nutrients diffused into the circulatory system are processed by the liver after passing through systemic circulation.
- The lymph originating in the lymphatics of the small intestine is called chyle.
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| Nutrients in food are absorbed via intestinal vili into the blood and lymph. Long-chain fatty acids (and other lipids with similar fat solubility such as medicines) are absorbed into the lymph, where it is enveloped inside chylomicrons. Then they move via the thoracic duct of the lymphatic system and finally enter the bloodstream via the left subclavian vein, therefore bypassing the liver's first-pass metabolism. |
ii. Immune function
- The lymphatic system is the major site for immune cells to develop and mature for an immune response. Immune cells require presentation or detection of antigens by other immune or dendritic cells to be activated.
- Upon the recognition of an antigen, an immunological cascade is triggered to activate and recruit more immune cells to produce more antibodies and cytokines, as well as recruit other immune cells such as macrophages.
When was the lymphatic system discovered?
- It's thought the first person to mention the lymphatic system was Hippocrates in his 5th century BC in his work On Joints.
- Between the 1st and 2nd century AD, a Roman physician named Rufus of Ephesus identified the axillary, inguinal and mesenteric lymph nodes as well as the thymus.
- In 3rd century BC, a Greek anatomist named Herophilos was the first mention lymphatic vessels when he described lacteals draining into the hepatic portal veins, and thus into the liver.
- Greek physician Galen supported the findings of Ruphus and Herophilos, who described the lacteals and mesenteric lymph nodes in his dissection of apes and pigs in the 2nd century AD.
- In the mid 16th century, Gabriele Falloppio explained the lacteals as "coursing over the intestines full of yellow matter."
- In around 1563, an anatomy professor Bartolomeo Eustachi described the thoracic duct in horses as vena alba thoracis.
- In 1622, physician Gaspare Aselli identified lymphatic vessels of the intestines in dogs and labelled them venae albae et lacteae, which are now known as the lacteals.
- In 1651, Jean Pecquet identified a link between the thoracic duct and the lacteals after he discovered a white fluid coalescing with blood in a dog's heart. He suspected that fluid to be chyle as its flow increased when abdominal pressure was increased. He traced the fluid from the thoracic duct to a chyle-filled sac that he called the chyli receptaculum, which is now known as the cisternae chyli.
- Further studies found the lacteal link to the venous system via the thoracic duct, meaning it didn't terminate at the liver.
- In 1652, a Swede named Olaus Rudbeck (1630–1702) discovered certain transparent vessels in the liver that contained a non-white clear fluid, therefore he named them hepatico-aqueous vessels.
- Thomas Bartholin learned such hepatico-aqueous vessels exist not just in the liver, but throughout the body, and named them "lymphatic vessels".
- This lead to a fierce disagreement between one of Bartholin's pupils, Martin Bogdan, and Rudbeck, whom he accused of plagiarism.
What is lymph?
- Lymph is a fluid that flows through the lymphatic system via lymph vessels to lymph nodes.
- The term is derived from Latin, lympha meaning "water", or the name of the ancient Roman deity of fresh water, Lympha.
- Its composition is similar but not identical to that of blood plasma. Lymph that exits a lymph node contains mainly lymphocytes, whereas lymph that exists in the human digestive system called chyle contains primarily triglycerides (fat), hence the milky appearance.
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| This diagram illustrates the formation of interstitial fluid from blood. According to the starling forces labelled, the hydrostatic pressure is higher proximally, which drives fluid out. On the other hand, oncotic forces are higher distally, which drags fluid in. |
What is the immune system?
Kurzegesagt:
Ted-Ed:
Crash Course:
- Inside your body is a complex network of biological processes that protects you from diseases and harmful pathogens.
- Your body's first line of defence are the physical barriers, i.e. skin, that blocks pathogens such as bacteria and viruses from entering the organism.
- If a pathogen infiltrates these barriers, your second line of defence against the pathogens is an immediate, but non-specific response called the innate immune response.
- If pathogens successfully evade the innate response, there is a third line of defence signalled by the innate immune system that is specific to the antigen called the adaptive immune system.
- It is labelled "adaptive" because it adapts its response during an infection to augment its recognition of the pathogen. This helps the adaptive immune system to unleash faster and potent attacks every time the same pathogen is detected.
How does your immune system protect you?
1. Surface barriers
- Surface barriers against infection include mechanical, chemical, and biological barriers. Examples include the leaves' cuticle, insects' exoskeleton, externally deposited eggs' shells and membranes, and skins.
- Since surface barriers alone doesn't fully protect organisms from the environment, there are backup systems in place to protect your vital organs such as the lungs, intestines, and the genitourinary tract. Examples include the coughing and sneezing to propel pathogens and other irritants out of the respiratory tract.
-- Antimicrobial peptides such as the β-defensins released by the skin and respiratory tract
-- Enzymes such as lysozyme and phospholipase A2 in saliva, tears, and breast milk
-- Acidic vaginal secretions following menarche
-- Defensins and zinc in semen to eliminate pathogens.
-- Gastric acid in the stomach
- Examples of biological barriers include commensal flora in the genitourinary and gastrointestinal tracts to compete with pathogenic bacteria for food and space, as well as alter the environmental conditions such as pH and iron levels.
2. Innate Immune System
- The innate immune response is an older evolutionary defence mechanism that is commonly found in plants, fungi, insects, and primitive multicellular organisms.
The main functions of the innate immune response include:
-- Recruit immune cells to infection sites by secreting chemical factors, including chemical mediators called cytokines
-- Activate the complement cascade to identify bacteria, activate cells, and trigger removal of antibody complexes or dead cells
-- Identify and remove foreign substances present in organs, tissues, blood and lymph, by specialised white blood cells
-- Activate the adaptive immune system through antigen presentation
-- Act as a physical and chemical barrier to infectious agents. This occurs via physical measures, such as skin, and chemical measures, such as clotting factors in blood, which are released following a contusion or other injury that severs the first-line physical barrier. This is not to be confused with a second-line physical or chemical barrier, such as the blood-brain barrier, which protects the nervous system from pathogens that have already gained access to the host.
What are the anatomical barriers?
i. Inflammation
- Inflammation is a complex biological process that features immune cells, blood vessels, and molecular mediators responding to harmful stimuli, such as pathogens, damaged cells, or irritants.
- Note that inflammation is not synonymous with infection since infection refers to the interaction between the action of pathogen invasion and the reaction of the body's inflammatory response.
- On the other hand, inflammation refers to body's immunovascular response to a pathogen. Words ending in the suffix -itis relate to inflammation, which clinical health care providers may refer to infection. e.g. urethritis
What are the causes of inflammation?
-- Physical: Burns, frostbite, physical injury (blunt or penetrating), foreign bodies (e.g. splinters, dirt and debris), trauma, ionising radiation
-- Biological: Infection by pathogens, immune reactions due to hypersensitivity, stress
-- Chemical: Chemical irritants, toxins, alcohol
-- Psychological: Excitement
What are the differences between acute and chronic inflammation?
What are the cardinal signs of inflammation?
1. Pain (Dolour): Secretion of chemicals such as bradykinin and histamine that stimulate nerve endings
2. Heat (Calour): Increased blood flow at body core temperature to the inflamed site
3. Redness (Rubour): Increased blood flow at body core temperature to the inflamed site
4. Swelling (Tumour): Accumulation of fluid
5. Loss of function (Functio laesa): Variety of causes
- The first 4 classical signs were described by Celsus (ca. 30 BC–38 AD). There is a debate whether the loss of function was added by Galen, or by Thomas Sydenham and Virchow.
How does acute inflammation work?
- Acute inflammation is triggered by resident immune cells already existing in the affected tissue, such as macrophages, dendritic cells, histiocytes, Kupffer cells and mast cells.
- The cells have receptors on its surface known as pattern recognition receptors (PRRs), which recognise (i.e. bind) 2 subclasses of molecules: pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).
- PAMPs are molecules that are associated with numerous pathogens, which are distinct from host molecules. Whereas, DAMPs are molecules that are associated with host-related injury and cell damage.
- At the onset of an infection, burn, or other injuries, one of the PRRs recognises a PAMP or DAMP, which activates these cells. This results in the release of inflammatory mediators that manifest in the clinical signs of inflammation.
- This triggers vasodilation, which increases blood flow throughout the body to result in redness (rubour) and increased heat (calour).
- Increased permeability of the blood vessels leads to an exudation (leakage) of plasma proteins and fluid into the tissue (oedema), which results in swelling (tumour).
- A number of the secreted mediators such as bradykinin increase the sensitivity to pain (hyperalgesia or dolour). Mediators also stimulate the blood vessels to allow the migration of leukocytes, mainly neutrophils and macrophages, to travel out of the blood vessels (extravasation) and into the tissue.
- The neutrophils migrate along a chemotactic gradient generated by the local cells to reach the site of injury.
- The loss of function (functio laesa) may be due to a neurological reflex in response to pain.
- A number of acellular biochemical cascade systems that involves preformed plasma proteins act in parallel to trigger and propagate the inflammatory response. They include the complement system activated by bacteria and the coagulation and fibrinolysis systems activated by necrosis.
Describe the vascular component
- Vascular events associated with acute inflammation include increased flow of plasma fluid, which carries crucial proteins such as fibrin and immunoglobulins (antibodies) towards the inflamed tissue.
- When tissue macrophages and mastocytes bind to PAMPs, they release vasoactive amines such as histamine and serotonin, as well as eicosanoids such as prostaglandin E2 and leukotriene B4 in order to remodel the local vasculature.
- Released mediators such as nitric oxide vasodilate blood vessels and increase its permeability to immune cells, which increases net blood plasma flow from the blood vessel to the tissue space. This results in tissue swelling called oedema.
- This leaked fluid contains various antimicrobial mediators from the plasma such as complement, lysozyme and antibodies, which damage and opsonise the microbes prior to the cellular phase of inflammation.
- In the case of a lacerating wound, platelets, coagulants, plasmin and kinins aim to clot the wounded area and stimulate haemostasis. These clotting mediators provide a structural staging framework at the inflammatory tissue site in the form of a fibrin lattice in order to set the foundation for wound repair and phagocytic debridement.
List of plasma cascade systems
- Complement system
- Kinin-kallikrein system
- Coagulation system
- Fibrinolysis system
List of plasma-derived mediators
Describe the cellular component
This component features leukocytes, which situate in the bloodstream and move into the inflamed tissue via extravasation to aid in inflammation.
(a) Leukocyte Extraversion
1. Chemoattraction
- The presence of pathogens become recognised and then activates macrophages in the affected tissue to release cytokines such as IL-1, TNFα and chemokines.
- L-1, TNFα and C5a stimulate the endothelial cells of blood vessels near the site of infection to express cellular adhesion molecules, including selectins.
- This leads to the creation of chemokines that bind to proteoglycans, which form a gradient in the inflamed tissue and along the endothelial wall.
-- Cytokines:
- IL-1 activates resident lymphocytes and vascular endothelia.
- TNFα increases vascular permeability and activates vascular endothelia.
- CXCL8 (IL-8) creates a chemotactic gradient that directs leukocytes towards the site of tissue injury/infection, as well as activates leukocyte integrins.
2. Rolling adhesion
- Carbohydrate ligands on the circulating leukocytes bind to selectin molecules on the inner wall of the vessel, with marginal affinity. This decelerates the leukocytes and drives them to roll along the inner surface of the vessel wall.
- This leads to the formation and disintegration of transitory bonds and disintegration between selectins and their ligands.
- Different types of leukocytes express P-selectin glycoprotein ligand-1 (PSGL-1) that binds to P-selectin on the endothelial cell, which causes the leukocyte to roll along the endothelial surface.
- The glycosylation pattern of PSGL-1 attenuates this interaction, meaning a number of glycovariants of PSGL-1 have unique affinities for different selectins, which allows endothelial cells to migrate to specific sites within the body.
- Cytokines also result in the expression of integrin ligands such as ICAM-1 and VCAM-1 on endothelial cells, which regulate the adhesion and decelerate the speed of leukocytes.
-- Selectins:
- P-Selectins = Expressed on activated endothelial cells and platelets, which bind PSGL-1 as a ligand. Its synthesis can be stimulated by cytokines such as thrombin, leukotriene B4, complement fragment C5a, histamine, TNFα or LPS. They also trigger the externalisation of Weibel-Palade bodies in endothelial cells, which lead to the presentation of pre-formed P-selectins on the endothelial cell surface.
- E-Selectins = Expressed on activated endothelial cells, it binds PSGL-1 and ESL-1. It is synthesised shortly after P-selectin synthesis, induced by cytokines such as IL-1 and TNFα.
- L-Selectins = They are constitutively expressed on some leukocytes, and are known to bind GlyCAM-1, MadCAM-1 and CD34 as ligands.
If L-selectin isn't present, the immune response would be significantly slower, P-selectins tend to bind to each other with high affinity. However, this rarely occurs since the receptor-site density is lower than with the smaller E-selectin molecules. Therefore, this increases the initial leukocyte rolling speed, which extends the slow rolling phase.
3. Tight adhesion
- Macrophages release chemokines to activate the rolling leukocytes to trigger the switching of surface integrin molecules from the default low-affinity state to a high-affinity state.
- Endothelial cells release chemokines and soluble factors to aid in juxtacrine activation of integrins.
-- Integrins:
β2 integrins on rolling leukocytes bind endothelial cellular adhesion molecules, which restricts cell movement.
- Anderson & Anderson (1976) used SEM images to discover homing receptors on microvilli-like tips on leukocytes that allow them to escape the blood vessel and enter the tissue.
- Wiese et al. (2009) discovered selectins (E-selectin, L-selectin, and P-selectin) played a role in strengthening bonds between the leukocytes and the vessel walls under higher force.
- Despite the interest into this phenomenon, more research is required to understand why increasing shear that elevates the force applied to adhesive bonds would decelerate the cell's rolling motion until an optimal shear is reached where rolling velocity is minimal.
- Thomas et al. (2004) proposed the catch bond hypothesis that suggests the increased force on the cell reduces off-rates and extends the bond lifetimes and stabilising the rolling step of leukocyte extravasation.
- Experiments by Yago et al. (2004) uncovered evidence of a force-dependent decrease in off-rates governed flow-enhanced rolling of L-selectin–bearing microspheres or neutrophils on PSGL-1.
- The theory suggests catch bonds allow increasing force to convert short bond lifetimes into long bond lifetimes in order to reduce rolling velocities and increase the regularity of rolling steps as shear increases from the threshold to an optimal value. As shear increases, the transitions to slip bonds shorten their bond lifetimes and increase rolling velocities and decrease rolling regularity. It is posited that force-dependent alterations of bond lifetimes direct L-selectin–dependent cell adhesion above and below the shear optimum. Yago et al. (2010) stated catch bonds may be a possible biological mechanism for flow-enhanced cell adhesion.
4. (Endothelial) Transmigration
- This step involves reorganisation of the leukocytes' cytoskeleton and elongation of pseudopodia through the gaps between endothelial cells. The gaps form from the interactions of the leukocytes with the endothelium, as well as autonomously through endothelial mechanics. This passage of cells through the intact vessel wall is called diapedesis.
- PECAM proteins situated on the leukocyte and endothelial cell surfaces interact and effectively drag the cell through the endothelium, known as leukocyte transmigration.
- After passing through the endothelium, leukocytes subsequently pass through the basement membrane into the interstitial fluid, however the method employed is poorly understood.
- Finally, leukocytes migrate along a chemotactic gradient towards the injury or infection.
(b) Phagocytosis
- First described by Canadian physician William Osler in 1876, and later studied and named by Élie Metchnikoff in the early 1880s, phagocytosis (derived from the Ancient Greek φαγεῖν (phagein) 'to eat', and κύτος, (kytos) 'cell') is a process that involves a cell (phagocyte) engulfing a large particle (≥ 0.5 μm) with its plasma membrane. It results in an internal compartment called the phagosome.
- The main phagocytes include dendritic cells, eosinophils, osteoclasts, macrophages, monocytes, and neutrophils, with the last three being the most active in the immune response.
- Phagocytosis occurs when a foreign pathogen, e.g. a bacterial cell, binds to molecules or receptors on the surface of the phagocyte. The phagocyte subsequently enlarges itself to encapsulate the bacterium to engulf it.
- The bacterium is trapped inside a phagocytic compartment called a phagosome. Within a minute, the phagosome merges with either a lysosome or a granule to form a phagolysosome.
- The bacterium would be killed by a number of mechanisms within several minutes. However, dendritic cells and macrophages take more time to kill the bacterium, which may be several hours.
- Macrophages are known to engulf copious amounts of material and release undigested material out into the tissue. This debris serves as a molecular signal to recruit more phagocytes from the blood.
What are the initiaing receptors?
- A phagocytes has a number of receptors on its surface to bind to foreign material, which include opsonin receptors, scavenger receptors, and Toll-like receptors.
- Scavenger receptors bind to a large range of molecules on the surface of bacterial cells, whereas toll-like receptors bind to more specific molecules.
What are the different methods of phagocytosing bacteria?
a. Oxygen-dependent intracellular
Phagocytosis of bacteria increases oxygen consumption (known as a respiratory burst), which produces antimicrobial reactive oxygen molecules. Since these oxygen compounds are toxic to both the invader and the cell itself, they are contained in compartments inside the cell.
- Superoxide is an oxygen-rich bacteria-killing substance that converts to hydrogen peroxide and singlet oxygen by an enzyme called superoxide dismutase. They also react with hydrogen peroxide to produce hydroxyl radicals, which are also toxic to invading microbes.
- Myeloperoxidase is released from neutrophil granules into the phagolysosome after granules fuse with the phagolysosome. Then it uses hydrogen peroxide and chlorine to create hypochlorite, which is a toxic antibacterial substance. Since myeloperoxidase contains a heme pigment, it gives secretions in neutrophils, such as pus and infected sputum, its green appearance.
b. Oxygen-independent intracellular
-- Electrically charged proteins, such as defensins, that damage the bacterium's membrane
-- Lysozymes that perforate the bacterial cell wall
-- Lactoferrins remove essential iron from bacteria, and are present in neutrophil granules.
-- Proteases and hydrolytic enzymes, such as hyaluronidase, lipase, collagenase, elastase, ribonuclease, deoxyribonuclease, that digest the proteins of destroyed bacteria.
c. Extracellular
-- Interferon-γ stimulates macrophages to produce nitric oxide, which kills microbes adjacent to the phagocyte. Subsequently, activated macrophages produce and secrete a cytokine called tumour necrosis factor (TNF), which kills cancer cells and virus-infected cells, as well as activate other cells of the immune system.
What are the names of cell-derived mediators?
ii. Complement System
- Known as the complement cascade, the complement system serves to augment (complement) the ability of antibodies and phagocytic cells to remove microbes and damaged cells from an organism, promote inflammation, and attack the pathogen's cell membrane.
- In 1888, American-British bacteriologist George Nuttall discovered sheep blood serum killed the bacterium that causes anthrax. When the serum was heated, its killing function disappeared.
- In 1891, German bacteriologist Hans Ernst August Buchner observed the same property of blood in his experiments, and named the killing function "alexin", meaning "to ward off" in Greek.
- By 1894, researchers demonstrated serum extracted from guinea pigs that had recovered from cholera killed the cholera bacterium in vitro. Even after the serum was inactivated by heat, it still maintained its ability to protect the guinea pigs from illness due to cholera bacteria.
- Belgian scientist Jules Bordet, of the Pasteur Institute in Paris, proposed this phenomenon had 2 components, one that maintained a "sensitising" effect after being heated and one (alexin) whose toxic effect was lost after being heated.
- Moreover, the heat-stable component is thought to associate with immunity against specific microorganisms, whereas the heat-sensitive component is thought to associate with the non-specific antimicrobial activity conferred by all normal sera.
- It wasn't until 1899, when Paul Ehrlich renamed the heat-sensitive component "complement" as part of his theory of the immune system. He theorised the immune system involved a group of cells that have specific receptors on their surface to recognise antigens.
- When the body is immunised against the antigen, the levels of these special receptors increase, which subsequently shed from the cells to circulate in the blood. Ehrlich labelled these receptors "amboceptors" to emphasise their bifunctional binding capacity, which includes recognition and binding to a specific antigen, as well as recognition and binding to the heat-labile antimicrobial component of fresh serum.
- Thus, Ehrlich named this heat-labile component "complement", as it "complements" the cells of the immune system.
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| Physician Paul Ehrlich won Nobel Prize in Physiology of Medicine in 1908. |
There are 3 different complement pathways:
a. Classical Complement Pathway = Membrane attack - by rupturing the cell wall of bacteria
- It is initiated by the binding of antigen-antibody complexes to the C1q protein. The globular regions of C1q recognise and bind to the Fc region of antibody isotypes IgG or IgM.
- In addition, those regions of C1q bind to bacterial and viral surface proteins, apoptotic cells, and acute phase proteins. C1q is part of the inactive C1 complex comprising of six molecules of C1q, two molecules of C1r, and two molecules of C1s.
- The binding of C1q triggers conformational changes and the activation of the serine protease C1r, which subsequently cleaves and activates the serine protease C1s.
- The activated C1s then cleaves C4 into C4a and C4b, and C2 into C2a and C2b.
- The larger fragments C4b and C2b combine to become C4b2b, a C3 convertase. C3 convertase then cleaves C3 into C3a and C3b.
- While the anaphylatoxin C3a interacts with its C3a receptor (C3aR) to recruit leukocytes, C3b plays a role in downstream complement activation.
- C3b combines with C3 convertase (C4b2b) to create C5 convertase (C4b2b3b), which then cleaves C5 into C5a and C5b.
- C5a is also an anaphylatoxin that interacts with its cognate C5a receptor (C5aR) to recruit leukocytes.
- Subsequent interactions between C5b and other terminal components C6, C7, C8, and C9 create the membrane attack complex (MAC) or the C5b-9 complex that opens pores on the target cell membranes to lysing.
b. Alternative Complement Pathway = Phagocytosis - by opsonising antigens via C3b
- This pathway is initiated by the C3b protein directly binding a microbe, or by foreign materials and damaged tissues. It is continuously activated at a mild level, caused by spontaneous C3 hydrolysis due to the breakdown of the internal thioester bond. One notable difference is the pathway doesn't require pathogen-binding antibodies to be activated.
- The change in C3's shape allows the binding of plasma protein Factor B, which allows Factor D to cleave Factor B into Ba and Bb.
- Bb remains bound to C3(H2O) to form C3(H2O)Bb, a complex known as a fluid-phase C3-convertase. This alternative convertase cleaves C3 proteins into C3a and C3b.
- Since this convertase is unstable, it binds to a serum protein called properdin to create the stable complex C3bBbP, which subsequently binds to an additional C3b to form alternative pathway C5-convertase [(C3b)2 BbP]
- Then the complement system follows the same path as the other pathways regardless of the means of activation (alternative, classical, or lectin). C5-convertase cleaves C5 into C5a and C5b. C5b binds sequentially to C6, C7, C8 and subsequently to several molecules of C9 to form membrane attack complex (MAC).
Since C3b is freely moving and abundant in the plasma, it can bind to either a host cell or a pathogen surface. To prevent complement activation from proceeding on the host cell, there are a number of regulatory proteins that disrupt the complement activation process:
- Complement Receptor 1 (CR1 or CD35) and DAF (decay accelerating factor also known as CD55) competes with Factor B in binding with C3b on the cell surface and may displace Bb from an already formed C3bBb complex.
- A plasma protease called complement factor I cleaves C3b into its inactive form, iC3b, to prevent the formation of a C3 convertase. However, it requires a C3b-binding protein cofactor such as complement factor H, CR1, or Membrane Cofactor of Proteolysis (MCP or CD46) to activate.
- Complement Factor H inhibits the formation of the C3 convertase by competing with factor B for binding to C3b, which triggers the breakdown of C3 convertase. Moreover, it acts as a cofactor for Factor I-mediated cleavage of C3b.
- CFHR5 (Complement Factor H-Related protein 5) binds to act as a cofactor for factor I, has decay accelerating activity and preferentially binds to C3b at host surfaces.
c. Lectin Pathway = Inflammation - by attracting macrophages and neutrophils
Known as the lectin complement pathway, it involves MBL creating oligomers of subunits, which are trimers (i.e. 6- and 18-subunit oligomers correspond to a dimer and a hexamer, respectively). Note that this pathway doesn't recognise an antibody bound to its target.
- This pathway is initiated by mannose-binding lectin (MBL) or ficolin binding to certain sugars. MBL binds to mannose, glucose, or other sugars with 3- and 4-OH groups situated in the equatorial plane, in terminal positions on carbohydrate or glycoprotein components of microorganisms including bacteria e.g. Salmonella, Listeria, and Neisseria strains.
- Multimers of MBL form a complex with protease zymogens called MASP1 (Mannose-binding lectin-Associated Serine Protease), MASP2 and MASP3.
- When the carbohydrate-recognising heads of MBL bind to specifically arranged mannose residues on the pathogen's surface, MASP-1 and MASP-2 are activated to cleave complement components C4 and C2 into C4a, C4b, C2a, and C2b.
- Situated in MBL complex are two smaller MBL-associated proteins (MAps) called MBL-associated protein of 19 kDa (MAp19) and MBL-associated protein of 44 kDa (Map44). MASP-1, MASP-3 and MAp44 are alternative splice products of the MASP1 gene, whereas MASP-2 and MAp19 are alternative splice products of the MASP-2 gene. Degn et al. (2009) suggested the function of MAp44 is a competitive inhibitor of lectin pathway activation, by displacing MASP-2 from MBL, hence preventing cleavage of C4 and C.
- If C4b doesn't bind to bacterial cell membranes, it deactivates and then combines with C2a to form the classical C3 convertase (C4bC2a) on the surface of the pathogen, rather than the alternative C3 convertase (C3bBb) involved in the alternative pathway.
- Jacqueline Stanley (2002) found C4a and C2b act as potent cytokines, with C4a triggering degranulation of mast cells and basophils and C2b increasing vascular permeability.
iii. White Blood Cells (Leukocytes)
- Also known as leukocytes, white blood cells (WBCs) are nucleated, which distinguishes them from the anucleated red blood cells and platelets.
- They are produced and derived from multipotent cells in the bone marrow known as haematopoietic stem cells (HSCs), which are then classified by cell lineage depending on the progenitor they evolve into.
- Myeloid cells (myelocytes) include neutrophils, eosinophils, mast cells, basophils, and monocytes. Monocytes are further subdivided into dendritic cells and macrophages.
- Lymphoid cells (lymphocytes) include T cells (subdivided into helper T cells, memory T cells, cytotoxic T cells), B cells (subdivided into plasma cells and memory B cells), and natural killer (NK) cells.
- WBCs are named after the physical appearance of a blood sample after centrifugation. Leukocyte is derived from the Greek roots leuk- meaning "white" and cyt- meaning "cell". WBCs are found in the buffy coat, a thin, typically white layer of nucleated cells between the sedimented red blood cells and the blood plasma.
a. Mast Cell
- Known as a mastocyte or labrocyte, mast cells are resident cells of connective tissue filled with granules rich in histamine and heparin.
- They were first described by Paul Ehrlich in his 1878 doctoral thesis on the basis of their unique staining characteristics and large granules.
Describe the structure of mast cells
- A granulated cell that contain histamine and an anticoagulant called heparin.
- It releases histamine and other inflammatory mediators when the Fc region of immunoglobulin E (IgE) binds to them and when IgE's paratopes bind to an antigen.
- They circulate in an immature form, only maturing once in a tissue site, which determines its characteristics.
- Mast cell differentiation and growth is stimulated by T cell-derived interleukin 3.
- They exist in most tissues characteristically surrounding blood vessels and nerves, particularly near the boundaries between the outside world and the internal milieu, such as the skin, mucosa of the lungs, and digestive tract, as well as the mouth, conjunctiva, and nose.
What are the functions of mast cells?
- Activated mast cells either selectively release (piecemeal degranulation) or rapidly release (anaphylactic degranulation) mediators that stimulate inflammation, from storage granules into the local microenvironment.
They are activated to undergo degranulation by allergens through:
-- Cross-linking with immunoglobulin E receptors (e.g., FcεRI)
-- Physical injury via pattern recognition receptors for damage-associated molecular patterns (DAMPs)
-- Microbial pathogens via pattern recognition receptors for pathogen-associated molecular patterns (PAMPs)
-- Various compounds via their associated G-protein coupled receptors (e.g., morphine through opioid receptors) or ligand-gated ion channels.
- They express a high-affinity receptor (FcεRI) for the Fc region of IgE, the least abundant type of antibody.
- Plasma cells produce IgE antibodies to coat the mast cells with high affinity, which are typically specific to one particular antigen.
- In allergic reactions, mast cells are still unactivated until an allergen binds to IgE already coated upon the cell. Pulendran & Ono (2008) stated other membrane activation events can either prime mast cells for subsequent degranulation or act in synergy with FcεRI signal transduction.
- Allergens bind to the antigen-binding sites, situated on the variable regions of the IgE molecules bound to the mast cell surface.
- When the allergen binds to 2 or more IgE molecules (cross-linking), it stimulates the mast cell to release a unique, stimulus-specific set of mediators through degranulation known as chemotaxis.
List of mediators released into the extracellular environment during mast cell degranulation include:
- Serine proteases, such as tryptase and chymase
- Histamine (2–5 picograms per mast cell)
- Serotonin
- Proteoglycans, mainly heparin (active as anticoagulant) and some chondroitin sulfate proteoglycans
- Adenosine triphosphate (ATP)
- Lysosomal enzymes: β-hexosaminidase, β-glucuronidase, arylsulfatases
- Newly formed lipid mediators (eicosanoids): Thromboxane, prostaglandin D2, leukotriene C4, platelet-activating factor
- Cytokines: TNF-α, basic fibroblast growth factor, interleukin-4, stem cell factor, chemokines (e.g. eosinophil chemotactic factor)
- Reactive oxygen species
6. These chemical mediators trigger characteristic symptoms of an allergy. For example, histamine dilates post-capillary venules, activates the endothelium, and increases blood vessel permeability, which results in oedema (swelling), warmth, redness, and attraction of other inflammatory cells to the site of release, as well as depolarised nerve endings (hence itching and pain).
- In the nervous system
They typically situate in a number of structures that mediate visceral sensory (e.g. pain) or neuroendocrine functions, or along the blood–cerebrospinal fluid barrier, including:
-- Pituitary stalk
-- Pineal gland
-- Thalamus
-- Hypothalamus
-- Area postrema
-- Choroid plexus
-- Dural layer of the meninges near meningeal nociceptors
- Ren et al. (2020) found mast cells demonstrate the same general functions in the central nervous system as in the body, which includes effecting or regulating allergic responses, autoimmunity, innate and adaptive immunity, and inflammation.
- Carobotti et al. (2015) stated mast cells serve as the main effector cell in response to pathogens that can affect the gut–brain axis across all bodily systems.
- In the gut
- Studies conducted in 2014 and 2015 found mucosal mast cells in the GIT situate in close proximity to sensory nerve fibres, which communicate bidirectionally.
- Wouters, Vicario & Santos (2015) found degranulated mast cells release mediators (e.g., histamine, tryptase, and serotonin) that activate, sensitise, and up-regulate membrane expression of nociceptors (i.e., TRPV1) on visceral afferent neurons via their receptors (respectively, HRH1, HRH2, HRH3, PAR2, 5-HT3). This, in turn, leads to neurogenic inflammation, visceral hypersensitivity, and intestinal dysmotility (i.e., impaired peristalsis).
- Moon, Befus & Kulka (2014) discovered neuronal activation triggers neuropeptide (e.g. substance P and calcitonin gene-related peptide) signalling to mast cells where they bind to their associated receptors and induce degranulation of a set of mediators (β-Hexosaminidase, cytokines, chemokines, PGD2, leukotrienes, and eoxins).
Describe the physiology of mast cells
-- High-affinity IgE receptor, FcεR1
Expressed on the mast cell's surface is the high affinity IgE-receptor, FcεR1, whose structure is a tetramer made of one alpha (α) chain, one beta (β) chain, and two identical, disulfide-linked gamma (γ) chains.
- The binding site for the IgE molecule consists of an extracellular portion of the α chain made of two domains that are similar to Ig.
- One transmembrane domain contains of an aspartic acid residue, whereas the other one contains a short cytoplasmic tail.
- The β chain contains a single immunoreceptor tyrosine-based activation motif ITAM in the cytoplasmic region, while each γ chain has one ITAM on the cytoplasmic region.
- When the ITAMs of the β and γ chains are phosphorylated by tyrosine, it triggers the signalling cascade from the receptor, which activates the mast cell.
- FcεR1 on Type 2 helper T cells,(Th2) and many other cell types lack the β chain, thus signalling is mediated only by the γ chain. The reason for this is the α chain containing endoplasmic reticulum retention signals that causes the α-chains to remain degraded in the ER.
- In rats, the assembly of the α chain containing the co-transfected β and γ chains masks the ER retention and allows the α-β-γ complex to be exported to the golgi apparatus to the plasma membrane.
- Kinet (1999) found only the γ complex is needed to counterbalance the α chain ER retention in humans.
-- Allergen process
- The Lyn tyrosine kinase situates on the cytoplasmic end of the FcεR1 β chain. The antigen cross-links the FcεR1 molecules, and Lyn tyrosine kinase phosphorylates the ITAMs in the FcεR1 β and γ chain in the cytoplasm. Subsequently, the Syk tyrosine kinase gets recruited to the ITAMs located on the γ chains. This activates the Syk tyrosine kinase, triggering phosphorylation.
- Rivera et al. (2002) stated Syk functions as a signal amplifying kinase activity due to the fact that it targets multiple proteins, triggering its activation.
- Li et al. (1992) reported the antigen stimulated phosphorylation causes the activation of other proteins in the FcεR1-mediated signalling cascade.
-- Degranulation and fusion
- The linker for activation of T cells (LAT) is a crucial adaptor protein activated by Syk phosphorylation, which can be moderated by phosphorylation to produce new binding sites.
- When Phospholipase C gamma (PLCγ) binds to LAT, it becomes phosphorylated, and subsequently plays a role in catalysing phosphatidylinositol bisphosphate breakdown to yield inositol trisphosphate (IP3) and diacyglycerol (DAG). Furthermore, IP3 increases calcium levels, and DAG activates protein kinase C (PKC).
- The tyrosine kinase FYN phosphorylates Grb2-associated-binding protein 2 (Gab2), which binds to phosphoinositide 3-kinase, which also activates PKC.
- Abbas et al. (2011) found PKC causes the activation of myosin light-chain phosphorylation granule movements, which disassembles the actin–myosin complexes to allow granules to interact with the plasma membrane. This results in the fusion of the mast cell granule with the plasma membrane.
- Soluble N-ethylmaleimide sensitive fusion attachment protein receptor (SNARE) complex mediates the fusion process, with Rab3 guanosine triphosphatases and Rab-associated kinases and phosphatases regulating granule membrane fusion in resting mast cells.
-- MRGPRX2 mast cell receptor
- Human mast-cell-specific G-protein-coupled receptor MRGPRX2 is involved in recognising pathogen associated molecular patterns (PAMPs) and initiating an antibacterial response.
- Pundir et al. (2019) found MRGPRX2 binds to competence stimulating peptide (CSP) 1 - a quorum sensing molecule (QSM) produced by Gram-positive bacteria.
- This induces signal transduction to a G protein and activates mast cell, which triggers the release of antibacterial mediators including ROS, TNF-α and PRGD2. These mediators recruit other immune cells to inhibit bacterial growth and biofilm formation.
-- List of enzymes
b. Natural Killer Cell (NK Cells)
- Also known as NK Cells, or large granular lymphocytes (LGL), natural killer cells are cytotoxic lymphocytes in the rapidly expanding family of innate lymphoid cells (ILC), representing 5–20% of all circulating lymphocytes in humans.
- In 1966, Dr. Henry Smith at the University of Leeds School of Medicine published the first study to state that untreated lymphoid cells confer a natural immunity to tumours. He concluded the "phenomenon appear[ed] to be an expression of defence mechanisms to tumour growth present in normal mice."
- In the 1970s, doctoral student Rolf Kiessling and postdoctoral fellow Hugh Pross discovered a unique type of lymphocyte was responsible for "natural" or spontaneous cytotoxicity in the mouse, and Hugh Pross and doctoral student Mikael Jondal made the same discovery in the human.
-- NK subsets
- CD56(bright) NK Cells are similar to T helper cells because they release cytokines. They are primarily found in bone marrow, secondary lymphoid tissue, liver, and skin.
- CD56(dim) NK Cells are mainly found in the peripheral blood, and are known to kill cells. They are positive with CD16, the key mediator of antibody-dependent cellular cytotoxicity (ADCC).
- COVID19 patients had decreased levels of only CD56(bright) NK cells. Patients with severe forms of COVID19 had decreased levels of CD56(dim) NK cells.
-- NK Cell Receptors
i. Activating receptors
- Ly49 (homodimers) = They are part of the C-type lectin family receptors that are multigenic in mice, but are pseudogenic in humans.
- NCR (natural cytotoxicity receptors) = Type 1 transmembrane proteins of the immunoglobulin superfamily, When activated, they mediate NK killing and release of IFNγ. They bind viral ligands such as haemagglutinins and haemagglutinin neuraminidases, some bacterial ligands and cellular ligands related to tumour growth such as PCNA.
- CD16 (FcγIIIA) = Involved in antibody-dependent cell-mediated cytotoxicity, especially binding to IgG.
ii. Inhibiting receptors
- Killer-cell immunoglobulin-like receptors (KIRs) = Part of a multigene family of recently evolved Ig-like extracellular domain receptor. Main receptors for both classical MHC I (HLA-A, HLA-B, HLA-C) and nonclassical Mamu-G (HLA-G) in primates and non-primates. Iannello et al. (2008) found regular cells express MHC class 1, thus are recognised by KIR receptors and NK cell killing is inhibited.
- CD94/NKG2 (heterodimers) = A C-type lectin family receptor conserved in both rodents and primates and identifies nonclassical (also nonpolymorphic) MHC I molecules such as HLA-E.
- ILT or LIR (immunoglobulin-like receptor) = Recently discovered members of the Ig receptor family.
- Ly49 (homodimers) = Functional homologues of KIRs in mice, including the expression pattern. They are receptors for classical (polymorphic) MHC I molecules.
Describe the function of NK Cells
- Cytolytic granule mediated cell apoptosis
- NK Cells have small cytotoxic granules in their cytoplasm consisting of proteins such as perforin and proteases known as granzymes.
- Upon release them adjacent to cells slated for killing, perforin creates pores in the cell membrane of the target cell, forming an aqueous channel through which the granzymes and associated molecules can enter, triggering either apoptosis or osmotic cell lysis. α-defensins and antimicrobial molecules are also released to disrupt the bacterial cell walls, ultimately killing the bacteria.
- Antibody-dependent cell-mediated cytotoxicity (ADCC)
- Antibodies opsonise infected cells, so immune cells are able to detect them. FcγRIII (CD16) receptors expressed on NK cells recognise the antibodies that bind to antigens, which activates NK cells, results in the release of cytolytic granules.
- Cytokine-induced NK and Cytotoxic T lymphocyte (CTL) activation
- Cytokines released by immune cells upon viral infection alert NK cells the presence of viral pathogens in the infected area. Examples of cytokines involved in NK cell activation include IL-12, IL-15, IL-18, IL-2, and CCL5.
- Interferons or macrophage-derived cytokines also activate NK cells, which play a role in containing viral infections while the adaptive immune response generates antigen-specific cytotoxic T cells that can eliminate the infection.
- In order to eliminate viral infections, NK cells release the molecules IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, while TNFα acts to promote direct NK tumour cell killing.
- Missing 'self' hypothesis
- The mechanisms NK cells use to protect the body against pathogens is not well understood, but researchers suggest the recognition of an "altered self" plays a role.
- NK cells contain 2 types of surface receptors to regulate their cytotoxic activity: activating receptors and inhibitory, including NK cell immunoglobulin-like receptors.
- Inhibitory receptors recognise MHC class I alleles, which allows NK cells to preferentially target cells that possess low cells of MHC class I molecules. This type of NK cell target interaction is called "missing-self recognition", coined by Klas Kärre and co-workers in the late 90s.
- MHC Class I molecules are the main mechanism cells use to present viral or tumour antigens to cytotoxic T-Cells. Both intracellular microbes and tumours employ an evolutionary adaptation in the form of chronic down-regulation of MHC I molecules. This makes infected cells undetectable to T-cells, which augments its evasion from T cell-mediated immunity.
- Lodoen & Lanier (2005) thought NK cells evolved as an evolutionary response to the loss of MHC, eliminating CD4/CD8 action, which forces another immune cell to serve the same function.
- Tumour cell surveillance
- In both mice and humans, NKs play a fundamental role in tumour immunosurveillance by directly triggering the death of tumour cells, even if surface adhesion molecules and antigenic peptides are absent.
- Vivier et al. (2011) pointed out that the NK cells' critical role in eliminating pathogens during innate immune response because T-Cells are incapable of recognising pathogens without surface antigens. For example, when NK cells detect the presence of tumour cells, they activate and subsequently produce and release cytokines.
- If a tumour cell is identified as "self" by the immune system, it doesn't trigger inflammation, hence a T-Cell response.
- Instead, NKs release a number of cytokines, such as IFNγ, tumour necrosis factor α (TNFα), and interleukin (IL-10), with the latter two functioning as proinflammatory and immunosuppressors, respectively.
- Activated NK cells and subsequently generated cytolytic effector cells influences the effectiveness of macrophages, dendritic cells, and neutrophils in the immune response, which results in antigen-specific T and B cell responses.
- Terunuma et al. (2008) found NK cells lyse tumour cells not via antigen-specific receptors, but with alternative receptors such as NKG2D, NKp44, NKp46, NKp30, and DNAM. NKG2D is a disulfide-linked homodimer that recognises a number of ligands usually expressed on tumour cells, including ULBP and MICA.
- NK cells express the FcR (FC-gamma-RIII = CD16) that binds the Fc portion of IgG class antibodies. This allows NK cells to target certain cells that experienced a humoral response through antibody-dependant cytotoxicity (ADCC). However, it is dependent on the affinity of the Fc receptor, which can be high, intermediate or low. FcR affinity is determined by the amino acid in position 158 of the protein, which can be phenylalanine (F allele) or valine (V allele).
- Clearance of senescent cells
- Antonangeli et al. (2019) stated NK cells directly kill senescent cells and release cytokines, which subsequently activate macrophages that also kills senescent cells.
- Prata et al. (2018) found NK cells, as well as CD8+ cytotoxic T-lymphocytes, use NKG2D receptors to detect senescent cell, before using perforin pore-forming cytolytic protein to kill those cells.
- Adaptive NK cells
- Rölle et al. (2013) discovered evidence of NK cells exhibiting adaptive immune functions such as dynamic expansion and contraction of subsets, increased longevity and a form of immunological memory.
- Sun et al. (2009) discovered the protective memory functions of NK cells induced by murine cytomegalovirus (MCMV) and the role of receptor Ly49 directly recognising the MCMV-ligand m157 in generating the adaptive NK cell response.
- Gumá et al. (2004) found an NK cell subset carrying the activating receptor NKG2C (KLRC2) expanded in response to human cytomegalovirus, as well as other infections such as Chikungunya virus, Hantavirus, HIV, or viral hepatitis.
- Nevertheless, more research is required to understand whether these viral infections stimulate the adaptive NKG2C+ NK cells or whether other infections lead to re-activation of latent HCMV.
- Hammer et al. (2018) hypothesised adaptive NK cells utilise the activating receptor NKG2C (KLRC2) to directly bind to human CMW-derived peptide antigens and respond to peptide recognition with activation, expansion, and differentiation.
- Pregnancy
- Successful pregnancies requires the suppression of the mother's immune system because a majority of pregnancies involve 2 parents who don't have matching tissues.
- The differences between "uterine NK cells" (uNK cells) and peripheral NK cells include lesser cytotoxic ability and slightly different receptor profile, while similarities include being in the CD56bright NK cell subset and potent cytokine secretion.
- Bulmer et al. (2010) found uNK cells are highly abundant in utero in early pregnancy, comprising about 70% of leukocytes situated there.
- Kopcow et al. (2005) found uNK cells induce cell cytotoxicity in vitro, but with lesser activity than peripheral NK cells.
- Trophoblast cells downregulate HLA-A and HLA-B to defend against cytotoxic T cell-mediated death, which usually stimulate NK cells by missing self recognition.
- Lash et al. (2010) suggested the selective retention of HLA-E (a ligand for NK cell inhibitory receptor NKG2A) and HLA-G (a ligand for NK cell inhibitory receptor KIR2DL4) by the trophoblast prevented NK cell-mediated death.
- Seshadri & Sunkara (2013) didn't find any significant difference with uNK cells in women with recurrent miscarriage compared with controls. Nevertheless, a greater proportion of peripheral NK cells situate in women with recurrent miscarriages compared to control groups.
- Once NK cells interact with HLA-C, the cytokines they secrete to regulate their function and stimulate trophoblastic proliferation include TNF-α, IL-10, IFN-γ, GM-CSF and TGF-β. For example, IFN-γ dilates and narrows the walls of maternal spiral arteries to increase blood flow to the implantation site.
- Evasion by tumour cells
- Tumour cells may evade the immune response by shedding decoy NKG2D soluble ligands. Vivier et al. (2011) found these ligands bind NK cell NKG2D receptors, which triggers a false NK response and consequently generates competition for the receptor site.
- Prostate cancer tumour cells use this method of evasion to avoid recognition from CD8 cells due to their ability to downregulate expression of MHC class 1 molecules.
c. Basophils
- Basophils are the least common type of granulocyte, representing about 0.5% to 1% of circulating white blood cells. Its name is derived from a combination of terms meaning "base-loving", since they are susceptible to staining by basic dyes. They were discovered by German physician Paul Ehrlich in 1879.
- Its structure includes large cytoplasmic granules obscuring the cell nucleus under the microscope when stained. If the basophil is unstained, the nucleus is visible and it usually has two lobes.
- They play fundamental roles in inducing inflammatory reactions during immune response, as well as in the formation of acute and chronic allergic diseases, e.g. anaphylaxis, asthma, atopic dermatitis and hay fever.
Describe the function of basophils
- Anticoagulant heparin prevents rapid blood clotting.
- The vasodilator histamine increases blood flow to the tissues at sites of ectoparasite infection, e.g., ticks.
- They have protein receptors on their cell surface that bind IgE, an immunoglobulin involved in macroparasite defence and allergy.
- They play important roles in both parasitic infections and allergies.
- It is inhibited by a glycoprotein called CD200, which is produced by human genome as well as herpesvirus-6, herpesvirus-7, and herpesvirus-8.
- Activated basophils degranulate to release histamine, proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes (e.g. elastase and lysophospholipase), as well as lipid mediators like leukotrienes (LTD-4), and several cytokines such as IL-4.
- IL-4 plays an important role in the development of allergies and the production of IgE antibody by the immune system.
d. Eosinophils
- This type of white blood cell is a granulocyte that develop during hematopoiesis in the bone marrow before migrating into the blood circulation, where they are terminally differentiated and do not multiply.
- The term eosinophil means "acid-loving", which refers to their large acidophilic cytoplasmic granules that have high affinity for acids.
- Using the Romanowsky method, basophil granules appear brick-red after staining with a red dye called eosin.
- Granules within the cellular cytoplasm include a range of mediators such as deoxyribonucleases (DNase), eosinophil peroxidase, lipase, plasminogen, ribonuclease (RNase), and major basic protein.
- In humans, 1–3% of white blood cells are eosinophils, and they are about 12–17 micrometres in size with bilobed nuclei. They situate in the tissue as they are released into the bloodstream as neutrophils.
- They are usually located in the medulla and the junction between the cortex and medulla of the thymus, as well as, in the lower gastrointestinal tract, lymph nodes, ovaries, spleen and uterus.
How do eosinophils develop?
- Lambrecht & Hamma (2015) found both TH2 and ILC2 cells express the transcription factor GATA-3, which promotes the production of TH2 cytokines, including the interleukins (ILs).
- Studies reported findings of IL-5 controlling the development of eosinophils in the bone marrow, as they differentiate from myeloid precursor cells. Uhm et al. (2012) stated transcription factors such as GATA and C/EBP determines their lineage fate.
- Before they leave the bone marrow, eosinophils produce and store numerous secondary granule proteins.
- After maturation, eosinophils circulate in blood and migrate to inflammatory sites in tissues, or to sites of helminth infection in response to chemokines such as CCL11 (eotaxin-1), CCL24 (eotaxin-2), CCL5 (RANTES), 5-hydroxyicosatetraenoic acid and 5-oxo-eicosatetraenoic acid, and certain leukotrienes such as leukotriene B4 (LTB4) and MCP1/4.
- Another TH2 cytokine called interleukin-13 primes eosinophilic exit from the bone marrow by lining vessel walls with adhesion molecules such as VCAM-1 and ICAM-1.
- Activated eosinophils undergo cytolysis, where the disintegration of the cell releases eosinophilic granules found in extracellular DNA traps.
- High concentrations of these DNA traps leads to cellular damage, as the granules they contain results in ligand-induced secretion of eosinophilic toxins, which lead to structural damage.
- Wagner et al. (2007) suggested the non-coding RNA EGOT regulates eosinophil granule protein expression.
Describe the functions of eosinophils
- Upon activation, eosinophils effector functions include producing the following molecules:
-- Cationic granule proteins and their release by degranulation
-- Reactive oxygen species e.g. hypobromite, superoxide, and peroxide (hypobromous acid, which is preferentially produced by eosinophil peroxidase)[17]
-- Lipid mediators like the eicosanoids from the leukotriene (e.g., LTC4, LTD4, LTE4) and prostaglandin (e.g., PGE2) families[18]
-- Enzymes, such as elastase
-- Growth factors such as TGF beta, VEGF, and PDGF[19][20]
-- Cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-13, and TNF alpha
- Combats viral infections, which is indicated by the abundance of RNases they contain within their granules, and in fibrin removal during inflammation.
- Mediators of allergic responses and asthma pathogenesis
- Combats helminth (worm) colonisation, which is elevated in the presence of certain parasites.
- Postpubertal mammary gland development
- Oestrus cycling
- Allograft rejection
- Neoplasia
- Antigen presentation to T-cells
- Tissue damage and inflammation in many diseases, including asthma. Colin Sanderson (1992) found increased levels of interleukin-5 upregulates the expression of adhesion molecules. This subsequently facilitates the adhesion of eosinophils to endothelial cells, thus triggering inflammation and tissue damage.
When eosinophils are activated by an immune stimulus, they degranulate to release a set of cytotoxic granule cationic proteins that induce tissue damage and dysfunction. They include:
-- Major basic protein (MBP)
-- Eosinophil cationic protein (ECP)
-- Eosinophil peroxidase (EPX)
-- Eosinophil-derived neurotoxin (EDN)
-- Eosinophil cationic protein (ECP)
-- Eosinophil peroxidase (EPX)
-- Eosinophil-derived neurotoxin (EDN)
- ECP and EDN are ribonucleases with antiviral activity.
- MBP triggers mast cell and basophil degranulation, and is suggested to play a role in peripheral nerve remodelling.
- ECP produces toxic pores in the membranes of target cells to facilitate entry of other cytotoxic molecules to the cell. Their functions include inhibiting proliferation of T cells, suppressing antibody production by B cells, inducing degranulation by mast cells, and stimulating fibroblast cells to secrete mucus and glycosaminoglycan.
- EPX produces reactive oxygen species and reactive nitrogen intermediates that induce oxidative stress in the target, resulting in cell death by apoptosis and necrosis.
e. Professional Phagocytes
i. Monocytes
- Monocytes are the largest type of leukocyte (white blood cell) that differentiate macrophages and conventional dendritic cells. They have a non-granulated cytoplasm, which classifies them as granulocytes. They contain unilobar nuclei, which shelter azurophil granules. The archetyle geometry of the monocyte nucleus is mainly ellipsoidal (or bean-shaped or kidney-shaped).
- In an adult human, Swirski et al. (2009) estimated around 50% of the monocytes are stored in the spleen.
- They compose 2-10% of all leukocytes in the human body and elicit a number of functions in the immune response.
-- Replenishing resident macrophages under normal conditions.
-- Migration within approximately 8-12 hours in response to inflammation signals from sites of infection in the tissues.
-- Differentiation into macrophages or dendritic cells to effect an immune response.
Ziegler-Heitbrock et al. (2010) identified at least three types of monocytes in human blood:
-- (1) Classical Monocyte = High expression of CD14 cell surface receptor [CD14++ CD16− monocyte]
-- (2) Non-Classical Monocyte = Low expression of CD14 and co-expression of the CD16 receptor [CD14+CD16++ monocyte]
-- (3) Intermediate Monocyte = High expression of CD14 and low expression of CD16 (CD14++CD16+ monocytes)
- Ghattas et al. (2013) suggested "intermediate" monocytes are more of a subpopulation, rather than a developmental step, because they comparatively express high levels of surface receptors involved in reparative processes (e.g. vascular endothelial growth factor receptors type 1 and 2, CXCR4, and Tie-2). Moreover, they tend to be enriched in the bone marrow.
- Said et al. (2010) demonstrated activated monocytes express high levels of PD-1, particularly in CD14+CD16++ monocytes as compared to CD14++CD16− monocytes. When the ligand PD-L1 binds to PD-1 receptor, it triggers the production of IL-10, which subsequently activates CD4 Th2 cells and inhibits CD4 Th1 cell function.
How do they develop?
- The precursors of monocytes are generated at the bone marrow, known as monoblasts, which are bipotent cells that differentiate from haematopoietic stem cells. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body where they differentiate into macrophages and dendritic cells. They constitute 3 - 8% of the leukocytes in the bloodstream.
- Swirski et al. (2009) estimated about 50% of the body's monocytes are stored in clusters in the red pulp's Cords of Billrot of the spleen.
What are the functions of monocytes?
- Phagocytosis = Process of consuming microbes and particles before digesting and destroying this material. It involves opsonising proteins such as antibodies or complement coating the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognise pathogens. Another method of destroying infected host cells is antibody-dependent cell-mediated cytotoxicity.
- Antigen presentation = Microbial fragments that linger after phagocytosis may serve as antigens, which are incorporated into MHC molecules and subsequently trafficked to the cell surface of monocytes, as well as macrophages and dendritic cells. This results in activation of T lymphocytes, which then triggers a specific immune response against the antigen.
- Cytokine production = Example cytokines generated by monocytes include TNF, IL-1 and IL-12 that elicit either pro-inflammatory or delayed anti-inflammatory effects.
- Cells produce a number of mediators that regulate the chemotaxis and other functions of monocytes. They include chemokines such as monocyte chemotactic protein-1 (CCL2) and monocyte chemotactic protein-3 (CCL7), as well as several arachidonic acid metabolites such as Leukotriene B4 and members of the 5-Hydroxyicosatetraenoic acid and 5-oxo-eicosatetraenoic acid family of OXE1 receptor agonists (e.g., 5-HETE and 5-oxo-ETE). Furthermore, Sozzani et al. (1996) found N-Formylmethionine leucyl-phenylalanine and other N-formylated oligopeptides are generated by bacteria to activate the formyl peptide receptor.
ii. Macrophage
- Abbreviated as Mφ, MΦ or MP, derived from Greek μακρός (makrós) = large, φαγεῖν (phagein) = to eat, macrophages are white blood cells of the immune system that serve to engulf and digest anything that does not have surface proteins specific to healthy body cells, including cancer cells, cellular debris, microbes, foreign substances, etc.
What are the different types of macrophages?
- They situate in essentially all tissues, where they patrol for potential pathogens by amoeboid movement. A collection of macrophages station at strategic points where microbes or foreign particles are likely to invade or accumulate. This is known as the mononuclear phagocyte system.
What are the functions of macrophages?
(1) Phagocytosis
- Removes dying or dead cells and cellular debris
- Ingests aged neutrophils during the early stages of chronic inflammation
- Verma & Saraf (2017) found macrophages remain at strategic locations such as vital organs (lungs, liver, spleen), bone, neural tissue and connective tissue in case any foreign materials still linger undigested, and additional macrophages need to be recruited.
- Usually macrophage performs phagocytosis of a pathogen by initially trapping it in a phagosome, which subsequently fuses with a lysosome to create a phagolysosome. Then, enzymes and toxic peroxides digest the pathogen to become loose waste material that gets expelled.
- Some bacteria, such as Mycobacterium tuberculosis, have demonstrated resistance to phagocytosis. YashRoy (2000) stated typhoidal Salmonellae stimulates their own phagocytosis by host macrophages in vivo, and uses lysosomes to inhibit digestion. Hence, they use macrophages for their own replication, which leads to macrophage apoptosis.
(2) Adaptive Immunity
- After they digest a pathogen, they present its antigen to the corresponding helper T-Cell. This is executed by integrating the antigen into the T-Cell's membrane and expressing it attached to an MHC Class II molecule. This results in the production of antibodies that bind to the same antigens of those specific pathogens, which aids in phagocytosis performed by macrophages.
- Regulates immune responses and activate inflammation by releasing a variety of monokines such as enzymes, complement proteins, and regulatory factors such as IL-1.
- Carries receptors for lymphokines to be stimulated into pursuing microbes and tumour cells.
- Defends against tumour cells and somatic cells infected with fungus or parasites.
-- Macrophage Subtypes
(3) Muscle Regeneration
- Tidball et al. (1999) elucidated the first wave of macrophages arrive during periods of increased muscle use that corresponds with muscle membrane lysis and membrane inflammation, which can enter and degrade the contents of injured muscle fibres.
- Schiaffino & Partridge (2008) suggested their levels peak roughly 24 hours after the onset of some form of muscle cell injury or reloading, before plummeting after 48 hours.
- The second wave involves the non-phagocytic macrophages dispersing near regenerative fibres, which peak between 2 and 4 days and maintains those levels for several days during muscle tissue rebuild.
- Schiaffino & Partridge (2008) hypothesised macrophages secrete soluble substances that impact the proliferation, differentiation, growth, repair, and regeneration of muscle, however it is unknown what factor regulates these effects.
- Bréchot et al. (2008) pointed out the macrophages' role in augmenting tissue repair isn't specific to a particular muscle. Moreover, they found macrophages accumulate in numerous tissues during the healing process phase following injury.
(4) Wound Healing
- A 2009 review stated the macrophages' role in wound healing involves initially replacing polymorphonuclear neutrophils as the predominant cells in the wound by day two after injury.
- Lorenz & Longaker (2003) found growth factors released by platelets and other cells attract monocytes from the bloodstream through blood vessel walls to the wound. It is estimated the levels of monocytes migrating to the wound peaks 1 - 1.5 days after the injury occurs. When monocytes are situated in the wound, they mature into macrophages.
- Macrophages play the important roles of phagocytosing bacteria and damaged tissue, and eliminating damaged tissue by releasing proteases.
- During the 3rd and 4th post-wound days, macrophages release a number of factors such as growth factors and other cytokines to attract cells involved in the proliferation stage of dealing to the infected area.
- Greenhalgh (1998) found macrophages are activated by the low oxygen levels of their surroundings to secrete factors that trigger and accelerate angiogenesis, as well as activate cells that repair the epithelial layers, generate granulation tissue and organise a new extracellular matrix.
(5) Limb Generation
- Souppouris (2013) asserted macrophages play a role in the typical limb regeneration based on its observations in salamanders.
- Godwin et al. (2013) discovered that the lack of macrophages lead to failure of limb regeneration and a scarring response in the salamander.
(6) Iron Homeostasis
- Macrophages typically destroy expired erythrocytes in the spleen and liver. The iron released from the haemoglobin is either stored internally in ferritin or is released into the circulation via ferroportin. When systemic iron levels increase, or hepcidin levels increase in inflammation, this acts on macrophage ferroportin channels, which result in iron lingering within the macrophages.
(7) Pigment Retainment
- Melanophages are a subset of macrophages situated in the tissues that serve to absorb pigment, either native to the organism or exogenous (such as tattoos), from extracellular space.
- Mishima (1967) found melanophages only accumulate phagocytosed melanin in lysosome-like phagosome, which is different to dendritic junctional melanocytes.
- Baranska et al. (2018) discovered this process occurs repeatedly until the pigment from the dead dermal macrophages is phagocytosed by newer macrophages, which preserves the tattoo in the same place.
(8) Tissue Homeostasis
- Gosselin et al. (2014) stated every tissue possesses its own specialised population of resident macrophages, which have reciprocal interconnections with the stroma and functional tissue.
- Muller et al. (2014) found these resident macrophages are non-migratory or sessile, which provide essential growth factors to support the physiological function of the tissue (e.g. macrophage-neuronal crosstalk in the guts), as well as actively defend the tissue from inflammatory damage.
iii. Neutrophil
- Also known as neutrocytes or heterophils, neutrophils are granulocytes that compose 40% to 70% of all white blood cells in humans. The standard normal range for neutrophil count is 2.5 - 7.5 x 109/L.
- By itself, human neutrophils have an average diameter of 8.85 µm. When they bind to a surface, its average diameter increases to 12–15 micrometers (µm) in peripheral blood smears.
- Its nucleus contains multiple lobes joined by chromatin, classifying them as polymorphonuclear cells. Zucker-Franklin (1988) found the nucleolus disappears as the neutrophil matures.
- Karni et al. (2001) found around 17% of female neutrophil nuclei that contains the inactivated X chromosome resemble the appearance of a drumstick.
- In the cytoplasm, the Golgi apparatus is relatively small, mitochondria and ribosomes are considered scarce, and the rough endoplasmic reticulum is absent. Roughly a third of the 200 granules within the cytoplasm are azurophilic.
- Neutrophils undergo segmentation as they mature, dividing into 3 - 5 segments.
- Edwards (1994) found neutrophils in the bloodstream change shape when activated from spherical to amorphous or amoeba-like that extends pseudopods in search of antigens.
- Sanchez et al. (1973) discovering consumption of basic sugars such as glucose, fructose, or sucrose found in honey and orange juice decreased the capacity of neutrophils to engulf bacteria, whereas starches had no negative effect on neutrophils. Moreover, fasting was found to bolster the neutrophils capacity to phagocytise bacteria.
- A 2007 study by the Whitehead Institute of Biomedical Research stated neutrophils preferentially reacted to certain types of sugars. The researchers found neutrophils preferentially digested beta-1,6-glucan targets compared to beta-1,3-glucan targets.
How long is the lifespan of neutrophils?
- Tak et al. (2013) estimated the average lifespan of inactivated human neutrophils in the circulation to be between 5 and 135 hours.
- Wheater & Stevens (2002) found that activated neutrophils place themselves adjacent to the blood vessel endothelium and are subject to selectin-dependent capture, followed by integrin-dependent adhesion.
- Akbar et al. (2022) discovered neutrophils are secreted from the spleen into the bloodstream after myocardial infarction.
- Since neutrophils are more numerous than the long-living monocyte/macrophage phagocytes, a pathogen is likely to initially encounter a neutrophil upon entry into the body.
- Experts hypothesised neutrophils have a shorter lifespan in order to minimise propagation of pathogens parasitising phagocytes, which allows surrounding immune cells to capture and destroy those pathogens. In addition, another advantage of the neutrophil's shorter lifespan is limiting the damage of antimicrobial products to the host tissues during inflammation.
Neutrophil antigens
Chu et al. (2013) identified 5 sets of neutrophil antigens: HNA1 - 5.
-- HNA-1a,b,c are located on the low affinity Fc-γ receptor IIIb (FCGR3B :CD16b)
-- HNA-2a is located on CD177.
-- HNA-3a,b are both located on the seventh exon of the CLT2 gene (SLC44A2).
-- HNA-4a,b and HNA-5a,b are located in the β2 integrin. Furthermore, HNA-4 is located on the αM chain (CD11b), whereas HNA-5 is located on the αL integrin unit (CD11a).
Neutrophil subpopulations
- Researchers discovered two functionally unequal subpopulations of neutrophils according to their varying levels of their reactive oxygen metabolite generation, membrane permeability, enzyme system activity, and ability to be inactivated.
- One subpopulation of neutrophils demonstrated high membrane permeability (neutrophil-killers), high production of reactive oxygen metabolites, and inactivation after interacting with the substrate.
- The other subpopulation of neutrophils (neutrophil-cagers) generate reactive oxygen species less intensively, which don't bind to substrate and preserve their activity.
What are the functions of neutrophils?
(1) Chemotaxis
- The process of chemotaxis occurs via amoeboid movement, which allows neutrophils to migrate toward sites of infection or inflammation. Neutrophils uses its surface receptors to detect chemical gradients of molecules such as interleukin-8 (IL-8), interferon gamma (IFN-γ), C3a, C5a, and Leukotriene B4, in order to direct the path of their migration.
- Serhan et al. (2010) found neutrophils express a range of specific receptors that detect complement, cytokines such as interleukins and IFN-γ, chemokines, lectins, and other proteins, as well as endothelium and opsonin (via Fc receptors).
- Pantarelli & Welch (2018) stated neutrophils uses the lipid products of PI3Ks to regulate activation of Rac1, hematopoietic Rac2, and RhoG GTPases of the Rho family, which are required for cell motility. In addition, they asserted Rac-GTPases modulate cytoskeletal dynamics and facilitate neutrophils adhesion, migration, and spreading. These Rac-GTPases accumulate asymmetrically to the plasma membrane at the leading edge of polarised cells.
- Since PI3Ks and their lipid products spatially modulate Rho GTPases and organise the neutrophil's leading edge, it may play a fundamental in establishing leukocyte polarity.
- Lämmermann et al. (26) demonstrated neutrophil swarming in mice, which suggests they migrate with coordination towards sites of inflammation.
(2) Anti-microbial function
- Ear & McDonald (2008) found neutrophils release cytokines upon arrival at the point of infection, which escalates inflammatory reactions by other types of immune cells.
- Hickey & Kubes (2009) identified 3 methods neutrophils use to direct attack micro-organisms: phagocytosis, degranulation, and generation of neutrophil extracellular traps.
(3) Phagocytosis
- Edwards (1994) stated neutrophils recognise their targets coated in opsonins before they approach and engulf microorganisms or viral particles. As phagocytes, they engulf and kill microbes to form a phagosome into which reactive oxygen species and hydrolytic enzymes are secreted.
- When the enzyme NADPH oxidase activates, it generates a respiratory burst of the reactive oxygen species, superoxide. Superoxide then decays spontaneously via enzymes known as superoxide dismutases (Cu/ZnSOD and MnSOD), to hydrogen peroxide, which is then converted to hypochlorous acid (HClO), by the green heme enzyme myeloperoxidase.
- Segal (2005) hypothesised the bactericidal properties of HClO are responsible for the destruction of bacteria phagocytosed by the neutrophil, or the activation of proteases.
- The interaction between neutrophils and microbes and its products tend to impact neutrophil turnover. However, the effects are such interactions often vary, specific to certain microbes, which range from prolonging the neutrophil lifespan to rapid neutrophil lysis after phagocytosis.
- Studies discovered microbes such as Chlamydia pneumoniae and Neisseria gonorrhoeae delay the apoptosis of neutrophils and/or PICD (phagocytosis-induced cell death), which extends the neutrophil lifespan.
- Kobayashi et al. (2017) discovered microbes such as Streptococcus pyogenes changes the neutrophil fate after phagocytosis by inducing rapid cell lysis and/or accelerating apoptosis to the point of secondary necrosis.
(4) Degranulation
Degranulation is the process of releasing various proteins in 3 types of granules, each of which have antimicrobial functions.
(5) Extracellular Traps
- Brinkmann et al. (2004) discovered activated neutrophils released web-like structures of DNA as part of the 3rd mechanism for destruction of bacteria. Known as neutrophil extracellular traps (NETs), they consist of a web of fibres composed of chromatin and serine proteases that serve to trap and destroy extracellular microbes.
- Researchers hypothesised NETs produced abundant levels of antimicrobial components that bind, disintegrate, and destroy microbes independent of phagocytic uptake. Furthermore, NETs may act as a physical barrier to minimise the spread of pathogens throughout the body. Clark et al. (2007) suggested NETs plays a crucial role in sepsis with its ability to trap bacteria.
- Monteith et al. (2021) discovered the formation of NETs promote macrophage bactericidal activity during infection.
- Gupta et al. (2007) observed activated NETs are involved in inflammatory diseases such as preeclampsia, a pregnancy-related inflammatory disorder.
- Hoyer & Nahrendorf (2017) suggested NETs play a role in cardiovascular disease because they affect the formation of thrombi in coronary arteries. Studies reported NETs promote the formation of thrombus in vitro, as well as in vivo.
- Zuo et al. (2020) hypothesised NETs play a role in the creation of blood clots in cases of severe COVID-19.
iv. Dendritic Cell (DCs)
- They are antigen-presenting cells (also known as accessory cells) of the mammalian immune system.
- Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They also serve as messengers between the cells of innate and the adaptive immune systems.
- DCs were first observed by Paul Langerhans (i.e. Langerhans cells) in the late 19th century. However, they were labeled 'dendritic cells' by Ralph M. Steinman and Zanvil A. Cohn in 1973. Their discovery of dendritic cells playing a principal role in the adaptive immune response helped them earn the Albert Lasker Award for Basic Medical Research in 2007 and the Nobel Prize in Physiology or Medicine in 2011.
- Liu et al. (2007) estimated mice DCs are replenished from the blood at a rate of 4000 cells per hour, and experience a limited number of divisions in the spleen across 10 to 14 days.
What are the different types of DCs?
i. Primate - In vivo
ii. In blood
This nomenclature was proposed by the nomenclature committee of the International Union of Immunological Societies.
- CD1c+ myeloid DCs = Produces chemokines
- CD141+ myeloid DCs = Cross-presentation
- CD303+ plasmacytoid DCs = Produces IFN-α
iii. In vitro
Ohjimoto et al. (2007) proposed the following dendritic cells cultured in vitro:
-- Mo-DC / MDDC = Cells matured from monocytes
-- HP-DC = Cells derived from hematopoietic progenitor cells.
Describe the life of DCs
- Hematopoietic bone marrow progenitor cells initially differentiate into immature dendritic cells, which are characterised by high endocytic activity and low T-cell activation potential.
- Immature DCs use pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) to scavenge the surrounding environment for pathogens such as viruses and bacteria. The function of TLRs is recognising particular chemical signatures located on subsets of pathogens.
- Other immature DCs may phagocytose minute traces of membrance from live own cells, known as nibbling.
- Once immature DCs interact with a presentable antigen, they activate into mature DCs, then migrate to a lymph node.
- Immature DCs phagocytose pathogens and disintegrate their proteins into tiny debris. Once they mature, those fragments are presented at their cell surface via MHC molecules. Simultaneously, DCs upregulate cell-surface receptors that serve as co-receptors in T-Cell activation such as CD80 (B7.1), CD86 (B7.2), and CD40, which significantly augments their ability to activate T-Cells.
- DCs upregulate a chemotactic receptor called CCR7, which stimulates the DC to travel through the bloodstream to the spleen or through the lymphatic system to a lymph node.
- Once DCs reach the lymph node or spleen, they function as antigen-presenting cells. Their role involves presenting antigens derived from the pathogen, as well as non-antigen specific co-stimulatory signals to helper T-cells and killer T-cells as well as B-cells, which activates them. In addition, DCs stimulates T-cell tolerance to test the responsiveness of T-Cells.
- Maverakis et al. (2015) found a number of C-type Lectin receptors (CLRs) on the surface of DCs that signal DCs the appropriate time trigger immune tolerance instead of lymphocyte activation.
- Professional antigen-presenting cells such as macrophages, B-lymphocytes and dendritic cells activate a resting helper T-cell upon presentation of a matching antigen.
- In non-lymphoid organs, memory T-Cells are activated by macrophages and B-Cells, whereas both memory and naive T-cells are activated by dendritic cells.
- In the lymph node and secondary lymphoid organs, all 3 aforementioned APCs activate naive T-Cells.
- Smith et al. (2004) stated mature DCs activate antigen-specific naive CD8+ T cells, however DCs need to communicate with CD4+ helper T Cells in order to produce CD8+ memory T cells.
- Hoyer et al. (2014) reported the assistance from CD4+ T Cells stimulates the activation and empowerment of matured dendritic cells to efficiently stimulate CD8+ memory T cells. Furthermore, CD8+ activation requires concurrent communication of CD4+ T helper cells, CD8+ T cells and dendritic cells.
I'll delve into the details of lymphocytes (T-Cells and B-Cells) and the adaptive immune response in the next blog post.
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