Overview

• Type I hypersensitivity reactions, which are characterized by immediate allergic responses and mediated by IgE and Mast cell activation.
• “Atopy” is the susceptibility to develop immediate hypersensitivity reactions, and is characterized by elevated levels of IgE antibodies.
  — Atopy is influenced by genetic and environmental factors.

Primary humoral response to antigens

• When Helper T cells recognize the antigen-MHC complex, intercellular interactions activate the B cell and induce production of large quantities of antigen-specific IgE antibodies.
• Mast cell (aka, mastocyte) display high affinity IgE Fc receptors on their surfaces; basophils (which we haven’t shown) also have high-affinity IgE receptors.
• Circulating IgE antibodies readily bind these receptors, “sensitizing” the mast cell.
• The next time the antigen is present tissues, it binds with the IgE antibodies and cross-links the mast cell receptors.
  This triggers release of:
  — Cytoplasmic granules, which include histamines, proteases, and heparin
  — Membrane phospholipids, which comprise platelet activating factor and arachidonic acid: Prostaglandin D2 and Leukotrines B4, C4, and D4. Recall that prostaglandin causes vasodilation and increases vascular permeability; leukotrienes recruit pro-inflammatory leukocytes and promote bronchoconstriction.
  — Pro-inflammatory cytokines, including Tumor Necrosis Factor (TNF), IL-1, IL-4, and chemokines.
  These degranulation products mediate early and late phases of the hypersensitivity reaction.

Early Phase

• Vasodilation, vascular leakage, and increased glandular secretion; notice that are the early events of the acute inflammatory response.
• Anti-histamines, which prevent histamine binding, are often effective at suppressing the early phase.

Late Phase

• Eosinophil activity, particularly their degranulation and release of Major Basic Protein and Eosinophil Cationic Protein, among other proteins, and IL-4 and other cytokines.
• Broad-spectrum anti-inflammatory drugs, such as corticosteroids, can reduce eosinophilia; thus, they are more effective than anti-histamines at the late phase of type I reactions.

Skin Prick Test

• Clinically, allergy diagnosis can be achieved with skin prick tests, in which small quantities of common allergens are introduced via small pricks or scratches to the skin.
  — In the image, we can see that this patient had several positive reactions, which are characterized by wheal-like swelling and flare-like erythema, in response to antigens from spiders, moths, caterpillars, scorpion, tick, and, to a lesser extent, worker honey bees. In contrast, there were negative responses to spider web, caterpillar web, and house dust.

Common clinical manifestations of hypersensitivity type 1 reactions

• Asthma is characterized by bronchioles with thickened smooth muscle, inflamed mucosal linings with hyperactive goblet cells – in our sample, we can see that the lumen is filled with mucus. Upon antigen exposure, the bronchi constrict and the goblet cells are activated to secrete mucus, all of which reduces airflow.
  — Treatments include corticosteroids, leukotriene antagonists, and phosphodiesterase inhibitors, which suppress the inflammatory actions of the immune system.
• Allergic hypersensitivity can produce various skin reactions; eczema and hives are common.
  — Eczema (aka, atopic dermatitis) is characterized by red, scaly, flaky areas that may become “weepy;”
  flares can be treated with corticosteroids or topical calcineurin inhibitors, which block cytokine expression; thick emollients that maintain the skin barrier may prevent flares.
  — Hives are characterized by red areas with raised, swollen plaques; anti-histamines or corticosteroids are used to suppress edema and erythema.
• Allergic rhinitis involves inflammation and stimulation of the mucosa of the nasal passages and is often associated with allergic conjunctivitis.
  — Anti-histamines, corticosteroids, or immunotherapy are used to suppress inflammation, and avoidance of the responsible allergens is advised.
• Gastrointestinal allergic reactions are characterized by increased smooth muscle contraction and, therefore, peristalsis and increased gastric secretions; thus, vomiting and diarrhea occur.
  — Aside from allergen avoidance, antihistamines to suppress smooth muscle contraction may be helpful. If the allergy is severe, anaphylaxis, which we address next, can be treated with epinephrine.
• Systemic anaphylaxis, which can be fatal, has multifold effects:
  Bronchoconstriction, which limits airflow
  Edema, which, if in the larynx, further obstructs airflow
  Vasodilation, which can lead to hypotension and shock
  Cardiac arrhythmia or even cardiac arrest
  — Immediate treatment with epinephrine, which reverses the cardiopulmonary effects, is necessary to prevent serious organ damage or death.

Common Allergens

• Common environmental allergens include insect venom (i.e., bee stings), pollen, dust (specifically, the feces of dust mites), animal dander, and air pollution.
• Certain foods, specifically the proteins in some foods, can trigger allergic responses; nuts, fish, milk, eggs, soy, wheat, and gluten are the most common culprits.
• Some medications are responsible for type I reactions; specifically, penicillin, aspirin, and other non-steroidal anti-inflammatory drugs. Paradoxically, topical corticosteroids are also known to cause allergic reactions.

Long-term desensitization

Can be achieved by regular administration of increasing quantities of the antigen, which suppresses IgE activity.

• Hypersensitivity reactions are mediated by antibodies and T cells (if you are unfamiliar with antibodies and T cells, we recommend you review our adaptive immunity tutorials).
  — Types I, II, and II are antibody-mediated.
  — Type IV is T cell-mediated.

Type I Hypersensitivity

• Type I immediate hypersensitivity is typified by allergy.
  — Mediated by IgE antibodies and mast cell and basophil activation.
  — This reaction occurs within minutes of antigen presentation.

Mechanism

• Initial exposure to antigen
  — IgE antibodies are released from mature B cells (plasma cells).
  — These antibodies bind to mast cells (and basophils, not shown); the mast cells are now “sensitized.”
• Subsequent exposure to the same antigen
  — Antigen binding cross-links the antibodies bound to mast cells, leading to degranulation.
  — As a result, cytokines, membrane phospholipids, and granules are released form the mast cell.
• Some key mediators of type I hypersensitivity that are released upon degranulation include histamine, leukotrienes, prostaglandins, and platelet activating factor.
  — Recall that these are the mediators of acute inflammation, which, when excessive, cause damage to the tissues.

Manifestations

• Early edema and erythema are often characterized by “wheal and flare” – the “wheal” is caused by vascular leakage and swelling; the “flare” is caused by vasodilation and reddening of the skin.
• The later stage of type I hypersensitivity is characterized by eosinophilia in the effected tissues; chemokines released during degranulation and leukocyte activation attracts eosinophils, which release proteins that cause more tissue damage.
• Anaphylaxis is a systemic and potentially fatal form of type I hypersensitivity; epinephrine is administered to reverse respiratory and cardiovascular effects.

Type II Hypersensitivity

• Type II antibody-mediated hypersensitivity is characterized by cytotoxic IgG or IgM complement activation.
• Reactions occur within 1-3 hours after antigen exposure.

Mechanisms

• Example: transfusion reaction
  — Donor red blood cell with surface antigens;
  — Host IgG antibodies bind those antigens, which initiates the complement cascade.
  — As a result, a Membrane Attack Complex (MAC) forms and allows water influx into the red blood cell, causing lysis.

Manifestations

• Blood cell destruction
• Goodpasture’s syndrome: damage to renal and lung tissue by anti-basement membrane antibodies.
• Cellular dysfunction: antibodies that bind to cellular receptors, altering their activity.
  — For example, IgG antibodies can bind TSH receptors, with inhibitory OR stimulatory consequences, depending on their configuration.

Type III Hypersensitivity

• Characterized by deposition of IgG or IgM antibody-antigen complexes (also called “immune complexes”) in vessels walls and/or tissues.
• Type III reactions occur 1-3 hours after antigen exposure.

Mechanism

• When IgG-bound antigen deposits in tissues, complement and neutrophils are activated, with tissue destruction as a result.

Manifestations

• Hypersensitivity pneumonitis, aka allergic alveolitis, is a localtype III response to inhaled antigens; historically common in farmers who inhaled mold, fungi, and other environmental pathogens, it is increasingly common in office workers exposed to microorganisms in humidifiers and air conditioning systems.
  — Immune complexes activate complement in the alveoli, the site of gas exchange in the lungs. Then, local Arthus reactions thicken the lung interstitium, making gas exchange more difficult.
• Serum sickness is a systemic example of a type III reaction;
  — It is induced by antibodies from other species. It is characterized by widespread effects, including rash, edema, joint pain, and fever.

Type IV Hypersensitivity

• Also called delayed hypersensitivity, it’s mediated by helper and cytotoxic T cells.
• These responses appear 1-3 days after exposure, because it takes additional time to recruit and activate T cells and their products.

Mechanisms

• In response to antigens, helper T cells release cytokines that recruit macrophages and neutrophils, which damage host tissues.
• Cytotoxic cells directly damage host tissues via granzymes.

Manifestations

• An example of helper T cell-mediated hypersensitivity is the tuberculin reaction test: a positive result, characterized by induration and erythema, indicates that an individual has been previously infected by M. tuberculosis.
• An example of cytotoxic T cell-mediated hypersensitivity is contact dermatitis; in response to certain organic or metallic substances, such as poison ivy, cytotoxic T cells destroy host tissues and cause an itchy, red, vesicular rash.

SELF-TOLERANCE

• Failure of self-tolerance results in autoimmune disorders, in which abnormal immune cells provoke a damaging immune response to otherwise benign molecules.
• These disorders have genetic components, often related to HLA alleles, and are often triggered by environmental or infectious stimuli. Furthermore, women are more often affected than are males, though the reasons for this are undetermined.
• Tolerance is achieved by selective removal of T and B cells that respond to self-antigen via central and peripheral mechanisms

Central Tolerance & Peripheral Tolerance

• Hematopoietic bone marrow houses the B and T cell precursors.
• B cells
  — During central tolerance, show that B cells remain in the bone marrow: here, they are exposed to self-antigen.
  — B cells that respond to self-antigen can undergo receptor editing, in which they develop new receptors that are do not recognize self-antigen, or, they can undergo apoptosis.
  — When self-reactive B cells in the periphery are exposed to self-antigen, they enter an unresponsive state of anergy. It is thought the absence of co-stimulation by T cells renders B cells anergic.
• T cells
• T cells undergo central tolerance in the thymus, where they are exposed to self-antigen by antigen-presenting epithelial cells.
• In response, they can differentiate to become T regulatory cells, or, undergo apoptosis.
• Peripherally, T cells reactive to self-antigens may become anergic and unresponsive (particularly in the absence of co-stimulation); or, they may be suppressed by T regulatory cells, which inactivates them; or, they may undergo apoptosis.

Unfortunately, some self-reactive B and T cells end up in the circulation, where they act as autoantibodies that cause chronic, often progressive, damage.

SYSTEMIC AUTOIMMUNE DISORDERS

Bear in mind that autoimmunity can also be organ-specific, as in multiple sclerosis or Type I diabetes mellitus.

Rheumatoid arthritis

• Tends to affect the synovial joints of the hands and feet early in the disorder.

Key mediators and their effects:

• Helper T cells release interferon gamma and IL-17.
  — Interferon gamma activates macrophages
  — IL-17 recruits inflammatory neutrophils and monocytes.
• Macrophages release tumor necrosis factor and IL-1.
  — Trigger the release of degradative proteases.
• Activated T cells
  — Express RANKL, which promotes bone resorption.

Characteristic damage of rheumatoid arthritis in a zoomed in view:

• Inflammation of the synovium
  — Results in pannus formation, which comprises thickened synovial fluid with infiltrating inflammatory cells, fibroblasts, and synovial cells that erode the cartilage.
  — Over time, the pannus on opposing bones forms a fibrous ankylosis, which may then become bony ankylosis.
• These changes cause stiffness, swelling, and joint deformities.
  — In a photograph and x-ray, we can see swan neck deformity, a common consequence of rheumatoid arthritis in which the proximal interphalangeal joint is hyperextended and the distal interphalangeal joint is flexed (thus, it looks like the neck of a swan).

Serum Markers:

• Antibodies against citrullinated protein antigens (ACPAs), such as citrullinated fibrinogen, which is produced during inflammation.
• Rheumatoid factor is also found in patients with rheumatoid arthritis; however, despite its name, rheumatoid factor is non-specific to rheumatoid arthritis and is present in other autoimmune disorders.

Systemic Sclerosis

• Aka, scleroderma

Key mediators and their effects:

• Caused by a combination of autoimmunity, vascular damage, and fibrosis.
  — Tumor growth factor Beta is a key mediator of this disorder.

Serum Markers:

• Anti-centromere antibody (ACA)
• Anti-Scl-70

Limited Systemic Sclerosis

• Affects the skin of the fingers, forearms, and face; visceral involvement, if any, occurs late.
• Limited systemic sclerosis is characterized by the presence of anti-centromere antibodies.
  CREST
  Calcinosis, in which calcified nodules form under the skin; they may progress to lesions.
  Raynaud’s phenomena, in which the small vessels of the fingers and toes severely constrict in response to stress and/or cold.
  — Initially, reduced blood flow turns the fingers white; as oxygen levels fall, the fingers appear bluish; and, finally, after blood flow is re-established, they become red.
  Esophageal dysmotility is caused by collagen replacement of the muscularis layer of the esophagus; dysphagia and/or heart burn can result.
  Sclerodactyly occurs when hands take on a claw-like appearance because fibrosis of the dermis pulls the skin taught; dermal tightening can also occur in the face.
  Telangiectasia is characterized by dilation of superficial micro-vessels; it commonly looks like a rash on the face or chest.

Diffuse Systemic Sclerosis

• Manifests as widespread cutaneous fibrosis with quick progression to the viscera.
• It is associated with Scl-70.
• Examples of Organ Effects:
  — Pulmonary fibrosis, in which the interstitial tissue becomes thick and fibrotic, can occur. Pulmonary fibrosis and hypertension are the leading causes of death from systemic sclerosis.
  — Renal failure, due to systemic vascular damage and hypertension.
  — Pericarditis and pericardial effusion.
  — Damage to the villous structures in the small intestine, which leads to malabsorption.

Sjögren’s syndrome

• Characterized by damage to the lacrimal and salivary glands, and is largely mediated by interferons (types 1 & 2).
• Key autoantibodies are anti-SSA/Ro, anti-SSB/La, which are anti-nuclear autoantibodies (which are also implicated in the pathogenesis of systemic lupus erythematosus).

Xerostsomia

• Aka, dry mouth
• Can lead to ulcers and increased dental caries
• In a histological sample of the minor salivary glands of the lip, we indicate the leukocyte infiltration that has damaged the tissues.

Keratoconjuctivitis sicca

• Aka, dry eye.
• Occurs when the lacrimal gland, which is located laterally above the eye, does not produce tears.
• Thus, the eyes become dry, irritated, and more susceptible damage; vision may be impaired.
• Furthermore, Sjörgen’s syndrome may have non-glandular effects, particularly in the joints, lungs, kidneys, and cardiovascular system.

Be aware that systemic lupus erythematosus is another autoimmune disorder with multi-organ effects; we discuss it in more detail, elsewhere.

Chronic inflammation occurs when tissue injury and repair attempts overlap.

• It is characterized by infiltration of mononuclear cells, particularly macrophages and lymphocytes, which interact dynamically over the course of inflammation (recall that acute inflammation is characterized by neutrophil infiltration).
• As a result of infectious agents and/or prolonged inflammatory response, tissue damage occurs.
• Tissue repair attempts comprise angiogenesis (formation of blood vessels, which ultimately regress) and fibrosis, aka, scarring.
• Because macrophages constitute the primary leukocyte in chronic inflammation, we’ll focus on their origins and activation; however, be aware that other leukocytes, including neutrophils, can be found at sites of chronic inflammation.

MACROPHAGE ORIGINS & ACTIVATION

• Macrophages arise from hematopoietic cells that reside in the bone marrow of adults (in the fetus, hematopoietic cells reside in the yolk sac and liver).
  — These cells give rise to various blood cell lines, including the monocytes, which circulate in the vasculature.
• Outside of the circulation, monocytes differentiate to become macrophages, and reside scattered throughout the connective tissues of the body.
  – Additionally, tissue resident macrophages reside in the liver (Kupffer cells), spleen and lymph nodes (sinus histocytes), central nervous system (microglial cells) and in the alveoli of the lungs.
  – Collectively, we can refer to these macrophages as the mononuclear phagocyte system (formerly called the “reticuloendothelial system”; some authors even reject the notion of a “mononuclear phagocyte system”).
• Upon activation, macrophages engage in various activities:
  – Presentation of antigens to T lymphocytes.
  – Production of cytokines, which mediate inflammatory responses.
  – Production of growth factors and enzymes that promote tissue repair and inhibit inflammation.
• To accomplish these goals, macrophages have dynamic phenotypes reflective of their microenvironments.

Macrophage Activation

Be aware that some texts describe two distinct types of macrophage activation, classical and alternative, and the resulting macrophages as either Type 1 (aka, classical type) or Type 2 (aka, alternative type); however, research now suggests that these should not be thought of as dichotomous types, but, rather, as potential phenotypes that macrophages express in response to environmental stimuli.

Pro-inflammatory effects:

• Stimulation by specific cytokines, such as interferon gamma (aka, type II interferon) and/or by microbes activate macrophages to produce inducible nitric oxide, reactive oxygen species, and lysosomal enzymes, which are antimicrobial.
• These activated macrophages also produce inflammatory cytokines and chemokines:
  – Tumor necrosis factor and chemokines activate lymphocytes.
• Activated T lymphocytes produce interferon gamma, which, as we’ve indicated, triggers pro-inflammatory macrophage activation. Thus, T lymphocytes can act as part of a positive feedback loop with pro-inflammatory activated macrophages.

Anti-inflammatory effects:

• Anti-inflammatory stimuli include interleukins 13 and 4 (IL-13, IL-4).
  – In response to these stimuli, activated macrophages produce cytokines, such as interleukin-10 (IL-10), that inhibit inflammatory activity of T lymphocytes, natural killer cells, and macrophages; thus, interleukin 10 prevents excessive inflammation and damage to host cells.
  – These “alternatively activated” macrophages also produce growth factors that promote tissue repair, including vascular endothelial growth factor (VEGF), which facilitates angiogenesis, and transforming growth factor beta (TGF-ß), which facilitates deposition of extracellular matrix proteins for fibrosis.
• Interleukins 13 and 4 (IL-13 and IL-4) are produced by T lymphocytes; thus, T lymphocytes can also promote anti-inflammatory macrophage activation.

Fibrosis

Unfortunately, the pro-inflammatory feedback loop can be maladaptive when prolonged macrophage-lymphocyte interactions promote chronic inflammation, as seen in fibrosis of the lung.
– In the example, chronic inflammation led to the deposition of extracellular proteins at the cost of healthy lung alveolar tissues.

• Fibrosis is characterized by excessive collagen deposition in response to persistent stimulation; it results in tissue loss and organ failure.
  – Some common pathological conditions caused by organ fibrosis include liver cirrhosis, constrictive pericarditis, scleroderma, and lung fibrosis disorders.

• The acute inflammatory response is activated in the presence of infectious agents and/or damaged tissues.
• Acute inflammation triggers vascular and cellular responses that deliver cells and proteins to the site of cell injury.
  Key steps of this process include:
  – Recognition of inflammatory agents.
  – Leukocyte and plasma protein recruitment from the blood to the tissues.
  – Leukocyte activation.
  – Control and termination of inflammatory reactions, which are otherwise harmful to healthy cells.

DETAILS

Recognition of offending agents

• Cellular receptors for microbes exist in the plasma membranes, endosomes, and cytosol of host cells.
  – For example: Toll-like receptors (TLR) enable dendritic and other “sentinel cells” to recognize invading microbes.

Other sensors are specialized to recognize signs of host cell damage:

• Cytosolic sensors recognize various molecules, such as uric acid, ATP, DNA, and reduction of intracellular potassium concentrations, that indicate cellular damage.
  – For example: Multi-protein cytosolic complexes called inflammasomes respond to the cytosolic sensors and trigger the release of cytokines, which, as we’ll see, are key mediators of the inflammatory response.

• Circulating proteins act as pattern recognition molecules that recognize invaders by their display of abnormal, non-self patterns.
  – For example: mannose-binding lectin protein binds to mannose, which is a characteristic microbial sugar; after binding, MBL facilitates microbe ingestion and activates the immune system.

Recruitment of plasma proteins and leukocytes from the blood

1. Vasodilation and increased permeability of the vessel wall:

• Occur in response to inflammatory mediators, importantly: histamine, prostaglandins, platelet activating factor (PAF), thromboxane A2 (generated from prostaglandins), bradykinin, and leukotrienes.

2. Plasma proteins and fluid exit the vessel (aka, exudation); this process can lead to excess fluids in the interstitial tissues, a condition called “edema.”
   – Clinical correlation: fibrinous pericarditis is a form of acute inflammation; as a result of the inflammatory response, fibrin and leukocytes infiltrate the pericardium (specifically the visceral pericardium, aka, the epicardium) and can cause “friction rub.”
3. Neutrophil recruitment from the blood involves: Capture, Rolling, Adhesion, Diapedesis, and Chemotaxic Migration.

• Capture:
  – Capture occurs via E-selectins, which are a type of cell adhesion molecule.
  – Cytokines, specifically Tumor Necrosis Factor (TNF) and Interleukin-1, upregulate the expression of E-selectins on the endothelial lining of the vessel.
  – Correspondingly, neutrophils express PSGL-1 (P-selectin glycoprotein ligand -1), which binds with selectins.
• Rolling:
  – Achieved via binding with P-selectins; their expression is upregulated by cytokines, thrombin, and histamine.
• Adhesion:
  – Firmer adhesion occurs when endothelial ICAM-1 (Intracellular Adhesion Molecule) binds with neutrophil LFA-1 ligands (Lymphocyte Function-Associated).
• Diapedesis:
  – The process of movement across the vessel wall typically occurs via the paracellular route, and is assisted by PECAM-1 (Platelet Endothelial Cell Adhesion Molecule).
  – Once outside of the vessel, neutrophils generate more cytokines, which further promotes the inflammatory response.
• Chemotaxic Migration:
  – Chemokines guide neutrophils to the site of inflammation along chemotactic gradients.

Phagocytosis and destruction of inflammatory agents

We’ll use neutrophil destruction of microbes as an example.

1. Neutrophil recognition of the microbe via sensors.
2. The neutrophil engulfs the microbe and moves it into a phagosome.
3. Lysosomes merge with the phagosome, which exposes the microbe to lysosomal degradative enzymes in a phagolysosome.
4. Lysosomal enzymes, reactive oxygen species (ROS, aka, reactive oxygen intermediates), and inducible nitric oxide (iNO) destroy the microbe.
   – Inflammatory cytokines, such as interferon gamma, trigger the production of ROS and iNO within the lysosomes and phagolysosomes.

• NETs
  In addition to phagocytosis, neutrophils can produce extracellular traps (aka, NETs) to destroy infective pathogens.
  – In this process, the neutrophil exudes its nuclear materials to envelop the microbes in chromatin and concentrated antimicrobial peptides and enzymes.

• Macrophages
  Although neutrophils are the primary leukocytes active in acute inflammation, other cell types, particularly macrophages, have important roles.
  – Macrophages release both pro- and anti-inflammatory cytokines that mediate the inflammatory response; they also release growth factors and enzymes that promote tissue repair. We learn more about the complex actions of macrophages, elsewhere.

Control and Termination

Control and, ultimately, termination of the acute inflammatory response is necessary to avoid destruction of healthy host cells. Thus, it is not surprising that the mechanisms for control are built into the process:

• Neutrophils have short half-lives outside of the blood stream, so their destructive capabilities in the tissues are limited.
• Lipoxins, which are secreted by neutrophils and macrophages, prohibit continued recruitment of new neutrophils.
• Also, as we mentioned earlier, macrophages release various anti-inflammatory molecules.

Possible outcomes of acute inflammation:

• In many cases, full resolution occurs, in which return to normal tissue functioning is possible.
• In others, scarring or fibrosis occurs, in which the damaged tissues are replaced by connective tissues.
• Chronic inflammation results when inflammatory agents persist; we’ll learn more about chronic inflammation, elsewhere.

Pharmacological correlation

• NSAIDS – non-steroid anti-inflammatory drugs – inhibit cyclooxygenase (COX), which is the enzyme responsible for prostaglandin synthesis.
• By inhibiting COX production, these medications, which include aspirin and ibuprofen, limit inflammation and pain. Elsewhere, we’ll learn their affects on blood coagulation.

• T cells are lymphocytes that directly or indirectly eradicate pathogens.
• They respond to intracellular targets, as opposed to the B cells of the humoral arm, which respond to extracellular microbes.
• Key events in the life cycle of the T cell:
  — They arise from stem cells in the bone marrow.
  — T cells mature in the thymus (T is for Thymus): maturation involves positive and negative selection, and gives rise to naïve (non-activated) cells defined by the presence of either CD4 or CD8 proteins on their surfaces.
  — In the secondary lymphoid organs, such as the lymph nodes and spleen, naïve T cells are activated by antigens; the naïve T cells become functional effector cells.
  — After the pathogen is eradicated, most of the effector cells undergo apoptosis; otherwise, they pose a potential danger to the host cells.
  — Some of the T cells differentiate to become memory cells, which will respond if/when the host is exposed to the same antigen – thus, the secondary response to subsequent exposure can occur much faster.

MHC

Major Histocompatibility Complex (MHC) molecules present peptide antigens that activate T-cells.

Class I MHC molecules

• Present fragments of antigens that are synthesized endogenously – i.e., peptides derived from viral antigens produced within the cells.
• Class I MHC molecules are only recognized by naïve CD8+ T cells and their Cytotoxic T cell descendants.

Class II MHC molecules

• Present fragments from extracellular microbes and pathogens – i.e., peptides derived from extracellular microbes.
• Class II MHC molecules are only recognized by naïve CD4+ T cells and their Helper T cell descendants.

T CELL MATURATION

• In the histological sample, we indicate the cortex of a lobule, which is where positive selection occurs, and, the medulla, which is where negative selection occurs.
• “Thymic education” entails two selective mechanisms that eliminate T cells that would otherwise harm the host:
  — In positive selection, immature T cells are exposed to cortical epithelial cells displaying self-MHC complexes:
  The T cells that recognize the MHC complexes survive
  Those that fail to recognize the MHC complexes undergo apoptosis.
  — Then, in negative selection, T cells are exposed to MHC complexes with self-antigen:
  Those T cells that do NOT respond to the self-antigen survive
  The T cells that DO respond undergo apoptosis.
  — Thus, positive selection ensures that the surviving T cells can recognize the MHC complex, which is necessary for their activation, while negative selection ensures that self-destructive T cells are eliminated.
  — Ultimately, thymic maturation produces three main types of T cells, which we designate based on their unique cell surface proteins: CD8+, CD4+, and CD4+/CD25+.
  CD4+/CD25+ T cells are Regulatory T cells; they can suppress the activity of the other T cell types via expression of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4).
  — In addition to the CD proteins, naïve T cells also express receptors (T-Cell Receptors, TCRs) for specific antigens; binding with their specific antigen induces their activation.

T CELL ACTIVATION

• Activating Cells:
  — MHC class I molecules are displayed by all nucleated cells (in other words, most body cells except red blood cells).
  — MHC class II molecules are displayed by dendritic cells, macrophages, and B cells – because of this unique ability, these are referred to as “antigen-presenting cells.”
  However, be aware that B cells do not activate naïve T cells; they stimulate mature Helper T cells as part of their own activation (discussed in detail, elsewhere).
  — As we learned earlier, CD8+ and CD4+ T cells recognize different MHC classes; this means that they can only be activated by cells displaying the appropriate MHC molecules.
• A general example:

1. An antigen-presenting cell, such as a dendritic cell, recognizes and engulfs a microbe.
2. It digests the microbe and re-packages a peptide fragment with an MHC class II molecule on its surface.
3. The MHC- antigen complex is recognized by a naïve CD4+ cell, which is subsequently activated. Notice that if the antigen-presenting cell displayed only antigens complexed with MHC class II molecules, the CD4+ cell would not have recognized it.

Activating mechanisms in more detail:

• 2-signal activation of a CD8+ T cell, which differentiates to become a cytotoxic cell.
  — The cell surface of the CD8+ has the T-cell Receptor Complex (TCR complex), which consists of the following components:
  The T-cell receptor, which is specific to the peptide antigen displayed by the MHC molecule; CD3 proteins; and, the CD8 protein, which recognizes and interacts with the MHC class I molecule of the nucleated cell.
  — The representative nucleated cell displays the class I MHC – antigen complex.
  — The interaction between the TCR complex and nucleated cell allows for the second signal, which involves co-stimulationbetween CD28 and B7-2.
  — Activation triggers proliferation, aka, cloning, of the T cell and differentiation into the effector type – which, for CD8+ cells, is the Cytotoxic T cell.
  These processes are guided by cytokines, which are released by T cells and antigen-presenting cells.
• 2-signal activation of a CD4+ T cell, which differentiates to become a Helper T cell.
  — The dendritic cell surface displays the MHC II-antigen complex.
  — The CD4+ cell has the TCR complex: the T-cell receptor, which is specific to the antigen; the CD3 molecules; and, the CD4 protein that interacts with the MHC II molecule on the antigen-presenting cell.
  — The second signal comprises co-stimulation: interaction between CD28 on the surface of the T cell and B7-2 on the dendritic cell.
  — Activation results in proliferation and differentiation to effector cells.

Effector Cell Functions:

• Cytotoxic T cells directly kill pathogen-bearing cells via the following steps:

1. The T cell recognizes the MHC I – antigen complex.
2. Docking brings the two cell membranes in close association.
3. The T cell releases perforins, which form a pore in the infected cell’s membrane.
4. The cytotoxic cell releases granzymes, which move through the pore and trigger apoptosis of the infected cell.

• Helper T cells, the products of activated CD4+ cells, have multiple roles in both innate and adaptive responses:
  — They amplify the innate response via cytokine release and recruitment of neutrophils and macrophages.
  — They activate B cells, which mediate the humoral arm of the adaptive immune response.
  — They activate cytotoxic T cells, in part by upregulating the expression of co-stimulatory molecules on dendritic cells.
  — Superantigens, such as Staphylococcus bacteria, are super potent activators of CD4+ cells.

• 4 subsets of Helper T cells:
  — Under the influence of interferon-gamma and IL-12, cells of subset Th1 develop.
  They engage in anti-viral activity, macrophage activation, and induce cytotoxic T cell differentiation; when unregulated, they are associated with autoimmune diseases.
  This subset produces IL-2 and interferon-gamma.
  — Under the influence of IL-4, Th-2 develop.
  This subset is particularly important in defense against worms and in mobilization of eosinophils; they are associated with allergies and asthma.
  They produce IL-4, IL-5, and Il-13.
  — Tissue growth factor-beta, IL-6, IL-1, and IL-23 induce differentiation of subset Th17, which recruit neutrophils and monocytes.
  They are also associated with autoimmune disease.
  They produce IL-17 and IL-22.
  — Follicular helper T cells differentiation is thought to require interaction with B cells.
  Follicular helper T cells promote the humoral immune response and produce IL-21.

• The humoral response is mediated by B cells, which produce antibodies that act combat infectious agents.
• Class switching and somatic hypermutation provide diversity in antibody specificity and response, which protects the host from a wide variety of infections.
• Two types of antibody-mediated immunity:
  — Passive immunity occurs when an individual is given antibodies to infectious agents.
  — Active immunity occurs when an individual’s immune system produces antibodies in response to infection.

B CELL MATURATION

• B-cell maturation is antigen-independent (in other words, B cell maturation does not require antigen interactions).
  — Occurs within the bone marrow.
• Pre-B cells are characterized by μ-chains.
  — Upon stimulation by Burton’s tyrosine kinase (BTK), the pre-B cell transitions to an immature B cell; it expresses the antibody IgM (immunoglobulin M) as its B cell receptor (aka, BCR).
• Immature B cells undergo negative selection, also called clonal deletion, which removes B cells that bind with self-antigen.
• The surviving B cells become naïve mature cells, characterized by both IgM and IgD.
• These cells exit the bone marrow and travel via the circulation to the secondary lymphoid organs for activation.
  In our histology sample, we highlight the splenic nodules and lymphoid germinal centers where B cells encounter activating antigens.

B CELL ACTIVATION

• Most antigens require input from Helper T cells to activate naïve B cells; this is referred to as Thymus-Dependent activation.

1. Naïve B cells bind and internalize antigen.
2. Within the B cell, the antigen is complexed with MHC II and displayed on the B cell surface.
3. When a Helper T cell recognizes and interacts with the antigen-MHC II complex, the B cell is activated.
4. The B cell proliferates and differentiates, giving rise to memory cells, which participate in subsequent immune responses, and, plasma cells, which secrete large quantities of antibodies that circulate in the blood to fight infection.

• Thymus-independent B cell activation occurs when antigens, such as polysaccharide bacterial products, directly activate B cells. Write that thymus-independent B cell activation results in minimal class switching.

Details of B and T cell interactions:

Two key signals are required

• The first signal involves MHC recognition by the T cell, and is necessary for the second signal.
  — The B cell displays the antigen-MHC II complex, and that it is recognized by the T-cell receptor (TCR) and CD4 proteins of the Helper T cell.
• The second signal involves co-stimulation by B and T cell proteins:
  — Binding of B7-2 with CD-28 triggers cytokine activation.
  — Binding of CD40 with CD40L (L is for ligand) triggers class switching and affinity maturation.
• Cytokines released by the Helper T cell influence class switching from IgM/IgD to the other isotypes (IgA, IgG, and IgE).

ANTIBODIES

Key Functions

Antibodies do not directly kill pathogens.

• Neutralize microbes by blocking their extracellular receptors and inhibiting their attachment to host cells; antibody binding can also inhibit viral replication, stopping the spread of infection.
• Antibodies can opsonize microbes, which means they bind to them and make it easier for phagocytes to recognize and engulf them.
• Activate the complement cascade, leading to formation of membrane attack complexes (MAC) and, consequently, microbe lysis.
• Agglutination occurs when antibodies bind multiple cell-bound antigens simultaneously, causing them to clump; precipitation, which we haven’t shown, occurs when antibodies simultaneously bind multiple soluble antigens. In both cases, binding makes antigen capture and phagocytosis easier.
• Finally, show that antibody-dependent cellular cytotoxicityoccurs when antibody-coated cells are targeted by natural killer cells.

Primary and secondary humoral immune responses:

• At the time of first exposure, antibody concentration is low; exposure provokes the primary response, which is characterized by a rise in low-affinity antibody concentration.
  — When infection is cleared, antibody concertation dips back down (but notice that it is not as low as pre-exposure levels).
• Upon subsequent exposure to the same antigen, the secondary response rapidly produces a spike in high-affinity antibodies; thus, the secondary response is “primed” by the first.

Antibody Structure:

Because IgG is a the most abundant antibody, we’ll use it as an example of representative antibody structure; however, be aware that IgM and IgA look quite different.

• Antibodies, also called immunoglobulins, are glycoproteins that comprise heavy and light chains.
• They can be separated into fragments and regions:
  — Fab: F = fragment, ab = antigen-binding; notice that IgG has two Fab’s.
  Paratope binds the eptiope of a specific antigen.
  — Fc end: c = crystallizable region, but it can also be remembered because the C fragment interacts with Fc Cell surface receptors.
• Variable and constant regions; the variable regions differ across antibodies, while the constant region is constant.

Isotypes:

• IgM is the first antibody formed during B cell development; it opsonizes antigens and fixes complement.
• IgD, the second antibody type formed, can bind bacteria and activate B cells.
• IgG, which we drew above, is the predominant antibody type during secondary responses; it opsonizes bacteria, fixes complement, and neutralizes toxins.
  — Importantly, it passes from maternal to fetal blood via the placenta, and is an example of naturally acquired passive immunity.
• IgA is the predominant antibody in secretions, including breast milk (another example of passive immunity); it neutralizes antigens and blocks their adhesion to mucosal surfaces.
• IgE provides defense against parasites; it is associated with hypersensitivity and allergic reactions.

• The first line of defense comprises physical and chemical barriers that prevent pathogen entry into the body.
• The second line of defense comprises the internal cells, complement system and other circulating proteins, and pathogen recognition receptors.
• Some participants of the innate immune system also activate the adaptive arm:
  – Some pathogen receptors trigger B and T cell responses of the adaptive system; the toll-like receptors are especially important.
  – Dendritic cells and macrophages present antigens to T cells, which then participate in the cellular response to pathogens; thus, dendritic cells and macrophages are called “Antigen presenting cells.”

1ST LINE OF DEFENSE

Physical Barriers
– Keratinized squamous epithelia of the skin physically prohibits entry into the body.
Chemical Barriers
– Low surface pH of the skin, vagina, and stomach; this barrier is referred to as the acid mantle.
– Mucus creates another type of chemical barrier. It is secreted by goblet cells, and comprises antimicrobial lysozymes and sticky mucin that traps microbes and prevents binding to host cells.

• When physical and chemical barriers are breached, internal defenses activate.

INTERNAL INNATE DEFENSES

Cellular Defenses

Leukocytes

• Neutrophils and macrophages are phagocytic cells that engulf and destroy microbes; recall that they are early responders in the acute inflammatory response.
• Eosinophils and mast cells, release pro-inflammatory molecules, such as histamine.

Natural killer cells

• Often considered a specialized lymphocyte; these cytotoxic cells are regulated via inhibitory and activating signals.
  – Healthy cells display MHC I, which inhibits the natural killer cell; also, the natural killer cell’s activator receptor is unbound.
  – Virus-infected cells have diminished expression of MHC I molecules; also, NK activation receptor is stimulated, which results in destruction of the infected cell.

Dendritic cells

• Bind with antigen and trigger cytokine release; recall that cytokines mediate the inflammatory response.

Complement System

The complement system, specifically through the actions of proteins C3a and C3b, destroys microbes.
– Comprises inactive proteins that circulate in the blood; of these proteins, the products of C3 cleavage (C3a and C3b) have multifold functions.

• Three pathways lead to C3 cleavage:
  – In the classical pathway, C1 is “fixed” to antibody-antigen complexes, which initiates a cascade of events that lead to C3 cleavage.
  – The alternative pathway is triggered by spontaneously activated C3b.
  – The lectin pathway is triggered when lectins, such as, mannose-binding lectin, binds microbial sugars and marks them for phagocytosis.
• Effects of cleaved C3:
  – C3a has pro-inflammatory effects; it recruits neutrophils and macrophages.
  – C3b opsonizes microbes, which involves binding to pathogens and marking them for phagocytosis.
  – Membrane Attack Complexes (MAC): C3b combines with other complement proteins (C5b, C6, C7, C8, and C9) to form a pore in the membrane of the microbe; massive water influx through the MAC lyses the microbe.

Circulating proteins with antimicrobial effects

• Defensins are positively charged peptides that insert pores into microbe membranes and trigger lysis. Defensins are particularly active in the GI and respiratory tracts (be aware that some authors include defensins as part of the first line immune defenses, too).
• Interferons are antiviral proteins that inhibit virus replication and activate natural killer cells to enhance destruction of infected cells.
• Acute-phase proteins promote opsonization and/or activate the complement system.
  The liver is a major source of these proteins, which include:
  – C-reactive protein, serum amyloid A, and the collectins.
  – Some important examples of collectins are pulmonary surfactant proteins that fight pathogens in the lungs, and mannose-binding lectin, which, as we learned, can activate the complement system.

Pathogen recognition receptors (PRRs)

We address these individually, but be aware that they often coordinate to effectively eradicate pathogens.

• Toll-like receptors sense a wide variety of pathogens, including bacteria, myocbacteria, viruses, and fungi.
  – They are present in both cellular and endosomal membranes.
  – Upon stimulation, they trigger the release of pro-inflammatory cytokines and interferons.
• NOD-like receptors (Nucleotide-binding Oligomerization Domain-like) are cytosolic sensors that also respond to diverse stimuli:
  – Some NOD-like receptors recognize bacterial wall peptidoglycans and trigger pro-inflammatory cytokine release.
  – Some NOD-like receptors respond to microbial and non-microbial materials via inflammasomes, which are protein complexes that can trigger cell death and recruit pro-inflammatory cells.
• RIG-like receptors trigger interferon release in response to viral RNA.
• Cytosolic DNA sensors induce interferon release in response to DNA from damaged cells.

Pathogens

Disease-causing or harmful microorganisms

Antigens

Material that can evoke an immune response

TWO BRANCHES OF THE IMMUNE SYSTEM

1) Innate Branch – non-specific, fast

• Physical barriers such as skin or chemical barriers
• Chemokines are a chemical signal produced by damaged cell to alert the body to danger and act as a homing signal for immune cells
• Neutrophils are the first type of phagocytic cell to arrive
• Monocytes arrive and mature into macrophages which engulf and destroy pathogens
• Inflammation (response to tissue damage) has four clinical signs: redness, heat, swelling and pain

2) Adaptive Branch – specific, slow, systemic, memory

• Humoral immunity – B cells (matured into plasma cells) producing antibodies (Y-shaped proteins)

• Cell-mediated immunity – Cytotoxic T cells recognize infected cells and kill them while helper T cells act as the general of the immune army and release chemical signals that activate various immune cell types

GROSS ANATOMY

• The splenic artery and vein enter at the hilum.
• Blood vessels travel within the lieno-renal ligament (aka, splenorenal ligament), which connects the intraperitoneal spleen to the posterior abdominal wall.

HISTOLOGY

Capsule

• Invaginates into the parenchyma as trabeculae, which divide the tissues into lobules; although omitted here for simplicity, blood vessels pass through the trabeculae.
• Comprises collagen, elastic, and smooth muscle fibers.
• The mesothelium, which is the outermost layer, forms the trabeculae.

Red pulp

• Is highly vascularized; this tissue is responsible for filtering of damaged red blood cells and other particles.
• Stroma comprises reticular cells and fibers, plasma cells, and macrophages.
• These cells and fibers constitute the splenic cords (of Billroth), which are interspersed between the sinusoids.
• Sinusoids comprise elongated endothelial cells on a discontinuous basement membrane; though not visible here, reticular fibers also encircle the sinusoids.
  • Blood flow through the spleen is complicated, and authors disagree on whether circulation is open and/or closed. In closed circulation, the blood flows from arterial vessels directly to the sinusoids; in open circulation, arterial vessels open into the red pulp, through which the blood percolates towards the sinusoids.

White pulp, which comprises nodules of lymphoid aggregations; this is the immune component of the spleen.

• A nodule comprises lymphocytes, primarily B cells, and antigen-presenting cells; recall that B cells participate in the immune response against blood-borne antigens.
• Perilymphoid red pulp surrounds the nodule.
• Germinal center is at center of the nodule; its size diminishes with age, and that inter-individual variation exists.
• Mantle and marginal zones form rings of darker-staining areas.
• A central artery passes through each nodule; it is a branch of the trabecular artery, and, in humans, is located peripherally in the nodule.
  • As it passes through the white pulp, T-cells surround the vessel, forming the peri-arteriolar lymphoid sheath (PALS).
  • Though not shown here, the central artery exits the white pulp and terminates in macrophage-sheathed capillaries, of the red pulp. These capillaries may either drain directly into the splenic sinusoids or, instead, into the spaces of the red pulp.

Notice that the spleen does NOT have a cortex and medulla.

Be aware that we’ve simplified some aspects of splenic histology; there is a great deal of intertextual variation regarding the details of splenic tissue structure and function.

Furthermore, although rodent models are often applied to studies of human anatomy and physiology, they are not viable in the case of the spleen because there are significant differences in the rodent and human spleens.