6.3 Innate & Adaptive Immunity Mechanisms

6.3 Innate & Adaptive Immunity Mechanisms

Independent study guide by OpenExamPrep.

Core Examination Pearl: Board examiners routinely evaluate the molecular steps of leukocyte extravasation and its genetic defect (LAD-1), the enzymes of the respiratory burst and their deficiency state (Chronic Granulomatous Disease), the convergence and biological products of the complement cascade (MAC deficiency and Neisseria risk), the fundamental comparison between MHC Class I and Class II antigen presentation, and the cytokine profiles driving CD4+ T-cell subsets (Th1, Th2, Th17, Treg).

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1. Innate Immunity: Barriers & Pattern Recognition Receptors

The innate immune system provides immediate, non-specific host defense against microbial invasion without requiring prior antigenic exposure or generating immunological memory.

Epithelial & Chemical Defenses of the Lower Extremity

• Stratum Corneum Keratin: Provides a physical, hydrophobic, desquamating mechanical barrier against fungal and bacterial adherence.
• Acid Mantle: Sebum-derived free fatty acids maintain a physiological skin surface pH of 4.5 to 5.5, inhibiting non-commensal pathogenic bacterial colonization.
• Antimicrobial Peptides (AMPs): Defensins and Cathelicidins (LL-37) produced by keratinocytes and neutrophils insert into microbial membranes, disrupting electrochemical gradients and forming lethal pores.
• Lysozyme: Cleaves $\beta$-(1,4) glycosidic bonds between NAG and NAM in bacterial peptidoglycan.

Pattern Recognition Receptors: Toll-Like Receptors (TLRs)

Innate cells recognize conserved microbial structures known as Pathogen-Associated Molecular Patterns (PAMPs) via germline-encoded Pattern Recognition Receptors (PRRs):

• Toll-Like Receptors (TLRs): Transmembrane leucine-rich repeat glycoproteins located on the cell surface or endosomal membranes:
  • TLR4: Recognizes Lipopolysaccharide (LPS / Endotoxin) of Gram-negative bacteria (assisted by accessory proteins CD14 and MD-2).
  • TLR2: Recognizes Peptidoglycan, Lipoteichoic acid of Gram-positive bacteria, and zymosan of fungi (heterodimerizes with TLR1 or TLR6).
  • TLR3: Located in endosomes; recognizes viral double-stranded RNA (dsRNA).
  • TLR5: Recognizes bacterial Flagellin.
  • TLR7 & TLR8: Located in endosomes; recognize viral single-stranded RNA (ssRNA).
  • TLR9: Located in endosomes; recognizes unmethylated CpG DNA motifs common to bacteria and DNA viruses.
• Signal Transduction Pathway: Surface TLRs engage the cytoplasmic adaptor protein MyD88 (except TLR3, which uses TRIF), activating interleukin-1 receptor-associated kinases (IRAKs) and the I$\kappa$B kinase (IKK) complex. IKK phosphorylates I$\kappa$B, targeting it for degradation and freeing the master transcription factor Nuclear Factor Kappa B (NF-$\kappa$B) to translocate to the nucleus. NF-$\kappa$B drives transcription of key pro-inflammatory cytokines: TNF-$\alpha$, IL-1$\beta$, IL-6, and chemokine IL-8.

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2. Neutrophil Extravasation & Leukocyte Adhesion Deficiency

Neutrophils are the primary cellular first responders to acute bacterial infection in podiatric soft tissues. Their recruitment from the postcapillary venule bloodstream into infected tissues follows a precise, sequential multistep adhesion cascade:

+-----------------------------------------------------------------------------------------+
|                           THE NEUTROPHIL EXTRAVASATION CASCADE                          |
+-----------+-----------------------+-----------------------+-----------------------------+
| Step      | Cellular Process      | Leukocyte Receptors   | Endothelial Ligands         |
+-----------+-----------------------+-----------------------+-----------------------------+
| Step 1    | Margination & Rolling | Sialyl Lewis X (CD15s)| P-Selectin & E-Selectin     |
| Step 2    | Tight Adhesion        | Beta-2 Integrin LFA-1 | ICAM-1 (CD54) &             |
|           |                       | (CD11a/CD18), Mac-1   | VCAM-1 (CD106)              |
| Step 3    | Diapedesis            | PECAM-1 (CD31)        | PECAM-1 (CD31)              |
| Step 4    | Chemotaxis (Homing)   | Chemokine Receptors   | C5a, LTB4, IL-8, fMLP       |
+-----------+-----------------------+-----------------------+-----------------------------+

Sequential Extravasation Steps

1. Margination & Rolling: Vasodilation driven by histamine and prostaglandins slows microvascular blood flow, allowing neutrophils to marginate along the vessel wall. Endothelial P-selectin (rapidly released from Weibel-Palade bodies by histamine or thrombin) and E-selectin (induced within hours by TNF-$\alpha$ and IL-1) bind to carbohydrate ligands on the neutrophil surface, primarily Sialyl Lewis X (CD15s). These low-affinity, transient bonds break under hemodynamic shear stress, causing the neutrophil to "roll" along the endothelium.
2. Tight (Firm) Adhesion: Locally produced chemokines (IL-8, C5a) bind neutrophil GPCRs, triggering "inside-out" signaling that alters the conformational state of leukocyte $\beta_2$-integrins from a low-affinity to a high-affinity extended conformation. Neutrophil integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) bind firmly to endothelial ligands ICAM-1 (CD54) and VCAM-1, arresting rolling.
3. Diapedesis / Transmigration: Neutrophils crawl toward endothelial intercellular junctions and squeeze between adjacent endothelial cells via homophilic interactions mediated by PECAM-1 (CD31) (Platelet Endothelial Cell Adhesion Molecule-1).
4. Chemotaxis: Once in the extravascular stroma, neutrophils migrate directionally along a chemical concentration gradient toward the site of infection. The four classic potent neutrophil chemoattractants are:
   • C5a (complement cleavage fragment)
   • Leukotriene B4 (LTB4) (arachidonic acid lipoxygenase metabolite)
   • Interleukin-8 (IL-8 / CXCL8) (macrophage-derived chemokine)
   • fMLP (bacterial N-formyl-methionyl peptides)

Leukocyte Adhesion Deficiency Type 1 (LAD-1)

• Genetic Etiology: Autosomal recessive mutation in the ITGB2 gene on chromosome 21, which encodes the common CD18 $\beta_2$-subunit of integrin heterodimers (LFA-1 [CD11a/CD18] and Mac-1 [CD11b/CD18]).
• Pathophysiology: Neutrophils can perform selectin-mediated rolling, but cannot undergo firm adhesion to ICAM-1. Neutrophils are incapable of transmigrating across the vascular wall into infected peripheral tissues.
• Classic Clinical Triad:
  1. Delayed Separation of the Umbilical Cord: Umbilical cord separation normally requires neutrophil-mediated tissue necrosis; in LAD-1, separation is delayed beyond 30 days of life.
  2. Recurrent Severe Bacterial Infections WITHOUT Pus Formation: Tissues develop necrotic ulcerations and cold abscesses devoid of neutrophils ("infections without pus").
  3. Profound Peripheral Leukocytosis / Neutrophilia: White blood cell counts are persistently elevated (often 30,000 to 100,000/$\mu$L) even between infections, as neutrophils cannot exit the circulation into peripheral tissues.

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3. Oxygen-Dependent Killing: The Respiratory Burst & CGD

Phagocytosis culminates in internalizing the microbe into a phagosome, which fuses with azurophilic and specific granules to form a phagolysosome. Microbicidal destruction proceeds via oxygen-dependent and oxygen-independent pathways.

The Respiratory Burst Pathway

The respiratory burst is a rapid metabolic surge characterized by a 20- to 100-fold increase in non-mitochondrial oxygen consumption, catalyzed by three sequential enzymes:

                    NADPH Oxidase
1.  2 O₂ + NADPH ──────────────────> 2 O₂•⁻ (Superoxide) + NADP⁺ + H⁺

                    Superoxide Dismutase (SOD)
2.  2 O₂•⁻ + 2 H⁺ ─────────────────────────> H₂O₂ (Hydrogen Peroxide) + O₂

                    Myeloperoxidase (MPO) + Cl⁻
3.  H₂O₂ + Cl⁻ + H⁺ ───────────────────────────> HOCl (Hypochlorous Acid / Bleach) + H₂O

1. NADPH Oxidase (Phox Complex): A multi-subunit enzyme complex assembled on the phagolysosomal membrane (comprising membrane subunits gp91phox and p22phox, and cytosolic subunits p47phox, p67phox, and Rac). Catalyzes the transfer of electrons from NADPH to molecular oxygen, generating superoxide anion ($O_2^{\bullet-}$).
2. Superoxide Dismutase (SOD): Spontaneously or enzymatically converts superoxide into hydrogen peroxide ($H_2O_2$).
3. Myeloperoxidase (MPO): A heme-containing enzyme stored within neutrophil azurophilic granules (which imparts the green color to pus and sputum). In the presence of chloride ($Cl^-$), MPO catalyzes the conversion of $H_2O_2$ into hypochlorous acid (HOCl / the active germicidal agent in bleach). HOCl is the most potent, rapid bactericidal oxidant generated by human leukocytes.

Chronic Granulomatous Disease (CGD)

• Genetic Etiology: Defect in any subunit of the NADPH oxidase complex. The most common form (~65–70%) is X-linked recessive, caused by a mutation in the CYBB gene encoding the gp91phox membrane subunit. Autosomal recessive forms involve p47phox or p22phox.
• Pathophysiology: Phagocytes cannot produce superoxide ($O_2^{\bullet-}$) or downstream endogenous $H_2O_2$. When phagocytosing non-catalase-producing bacteria (such as Streptococcus pneumoniae), the microbes generate metabolic $H_2O_2$ that the host cell's intact MPO can utilize to kill the bacteria. However, when infected by catalase-positive organisms, the microbial catalase rapidly breaks down the organism's own $H_2O_2$ into $H_2O$ and $O_2$. Deprived of both endogenous and exogenous $H_2O_2$, the neutrophil is completely powerless to kill the microbe.
• Pathogen Vulnerability (Catalase-Positive Organisms): Recurrent, life-threatening granulomatous infections and abscesses of the skin, lymph nodes, lungs, and liver. The classic board-tested pathogens are:
  • Staphylococcus aureus (most common bacterial pathogen)
  • Serratia marcescens (Gram-negative bacillus)
  • Burkholderia cepacia (highly refractory)
  • Nocardia species (branching filamentous bacterium)
  • Aspergillus species (most common fungal killer in CGD)
• Diagnostic Testing:
  • Dihydrorhodamine (DHR) 123 Flow Cytometry (Preferred Test): Neutrophils stimulated in vitro oxidize non-fluorescent DHR to fluorescent rhodamine 123. Normal cells show intense fluorescence; CGD neutrophils exhibit complete absence of fluorescence.
  • Nitroblue Tetrazolium (NBT) Slide Test (Historical): Yellow, soluble NBT dye is taken up by neutrophils. Intact NADPH oxidase reduces NBT to insoluble, dark blue formazan crystals. In CGD, cells remain colorless / fail to turn blue.

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4. The Complement System: Pathways, Convertases & Deficiencies

The complement system is a biochemical cascade comprising over 30 circulating serum proteins and cell-surface receptors that operate in a tightly regulated proteolysis cascade.

Complement Activation Pathways

+-----------------------------------------------------------------------------------------+
|                              THE THREE COMPLEMENT PATHWAYS                              |
+-----------------------+-----------------------------+-----------------------------------+
| Pathway               | Primary Initiator / Trigger | Initial Protease Complex          |
+-----------------------+-----------------------------+-----------------------------------+
| 1. Classical Pathway  | Antigen-Antibody Complexes  | C1q binds Fc region of            |
|                       | (IgM or IgG1, IgG3)         | C1r/C1s; cleaves C4 and C2        |
+-----------------------+-----------------------------+-----------------------------------+
| 2. Alternative Pathway| Spontaneous C3 hydrolysis   | C3b deposits on microbe;          |
|                       | ("Tick-over") on microbes  | Factor B cleaved by Factor D      |
+-----------------------+-----------------------------+-----------------------------------+
| 3. Lectin Pathway     | Mannose-Binding Lectin(MBL) | MBL binds microbial mannose;      |
|                       | binding microbial sugars    | MASP-1 & MASP-2 cleave C4 & C2    |
+-----------------------+-----------------------------+-----------------------------------+

Convertase Complexes & Pathway Convergence

• C3 Convertases: The central rate-limiting step of all pathways:
  • Classical & Lectin Pathways: C4b2a cleaves circulating C3 into C3a and C3b.
  • Alternative Pathway: C3bBb (stabilized on microbial surfaces by Properdin).
• C5 Convertases: Addition of a C3b fragment to C3 convertases creates C5 convertase:
  • Classical & Lectin Pathways: C4b2a3b.
  • Alternative Pathway: C3bBb3b.
• The Membrane Attack Complex (MAC / Terminal Pathway):
  • C5 convertase cleaves C5 into C5a (soluble) and C5b.
  • C5b binds to the microbial membrane and sequentially recruits C6, C7, and C8.
  • The C5b-8 complex catalyzes the binding and polymerization of 10 to 16 molecules of C9, forming a hollow transmembrane pore (10 nm diameter) that allows massive influx of water and electrolytes, driving osmotic lysis.

Biological Functions of Complement Cleavage Fragments

• Opsonization: C3b (and its inactive fragment iC3b) deposits covalently onto microbial surfaces, where it is recognized by CR1 (CD35) receptors on macrophages and neutrophils, promoting avid phagocytosis ("C3b binds bacteria").
• Anaphylatoxins: C3a, C4a, and C5a (potency order: C5a > C3a > C4a) bind to GPCRs on tissue mast cells and basophils, stimulating immediate degranulation with histamine release, smooth muscle bronchospasm, and increased vascular permeability.
• Chemotaxis: C5a serves as a potent chemoattractant directing neutrophil migration.

High-Yield Complement Deficiencies

• Early Complement Component Deficiencies (C1q, C1r, C1s, C2, C4): Impair the clearance of circulating apoptotic cells and immune complexes. Patients exhibit severe susceptibility to encapsulated pyogenic bacterial infections and a markedly increased incidence of Systemic Lupus Erythematosus (SLE).
• C3 Deficiency: Severe, recurrent, life-threatening pyogenic infections (encapsulated bacteria) beginning in early infancy; defective opsonization and absent downstream MAC formation.
• Terminal Complement / MAC Deficiencies (C5, C6, C7, C8, C9): Inability to assemble the Membrane Attack Complex. Characterized by severe, recurrent, invasive bacteremia and meningitis caused exclusively by Neisseria meningitidis (meningococcal disease) and disseminated Neisseria gonorrhoeae (gonococcal septic arthritis / tenosynovitis in the lower extremity).
• C1 Esterase Inhibitor (C1-INH) Deficiency: Causes Hereditary Angioedema (HAE). Uncontrolled activation of the kallikrein-kinin system leads to excessive production of the potent vasodilator bradykinin. Patients suffer recurrent, non-pitting episodes of subcutaneous and mucosal edema (laryngeal edema, abdominal colic) without urticaria or pruritus. (ACE inhibitors are strictly contraindicated).
• Paroxysmal Nocturnal Hemoglobinuria (PNH): Acquired somatic mutation in the PIGA gene in hematopoietic stem cells, causing deficiency of GPI anchors required to attach complement regulatory proteins CD55 (Decay-Accelerating Factor / DAF) and CD59 (MAC Inhibitory Protein / MIRL) to the erythrocyte membrane. Results in uncontrolled complement-mediated intravascular hemolysis and venous thrombosis.

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5. Major Histocompatibility Complex: MHC Class I vs. Class II

The Major Histocompatibility Complex (MHC), termed Human Leukocyte Antigen (HLA) in humans, consists of a highly polymorphic cluster of genes located on the short arm of chromosome 6.

+-----------------------------------------------------------------------------------------+
|                            MHC CLASS I vs. MHC CLASS II                                 |
+----------------------------+-----------------------------+------------------------------+
| Parameter                  | MHC Class I                 | MHC Class II                 |
+----------------------------+-----------------------------+------------------------------+
| Genetic Loci               | HLA-A, HLA-B, HLA-C         | HLA-DP, HLA-DQ, HLA-DR       |
| Cellular Distribution      | **All nucleated human cells**| **Professional APCs only**   |
|                            | (absent on mature RBCs)     | (Dendritic, M-phi, B cells)  |
| Molecular Structure        | Heavy alpha chain +         | Heterodimer of               |
|                            | **Beta-2 Microglobulin**    | **Alpha & Beta chains**      |
| Antigen Source             | **Endogenous antigens**     | **Exogenous antigens**       |
|                            | (viral proteins, cytosolic) | (phagocytosed bacteria)      |
| Degradation Machinery      | **Cytosolic Proteasome**    | **Endolysosomal Acid Protease|
| Peptide Loading Site       | Endoplasmic Reticulum (TAP) | Endolysosome (after CLIP off)|
| Presenting Coreceptor      | **CD8+ Cytotoxic T Cells**  | **CD4+ Helper T Cells**      |
| Mathematical Rule          | **Class I x CD8 = 8**       | **Class II x CD4 = 8**       |
+----------------------------+-----------------------------+------------------------------+

The "Rule of 8"

A foundational board mnemonic for remembering T-cell receptor engagement: $\text{MHC Class I} \times \text{CD8} = 8$ $\text{MHC Class II} \times \text{CD4} = 8$

Antigen Processing Mechanisms

• MHC Class I Pathway: Endogenous cytosolic proteins (synthesized within the cell by viruses or mutated tumor genes) are ubiquitinated and cleaved into short peptides (8–10 amino acids) by the proteasome. Peptides are actively transported into the lumen of the endoplasmic reticulum (ER) via the Transporter associated with Antigen Processing (TAP1/TAP2). Loaded MHC Class I-peptide complexes are routed through the Golgi to the plasma membrane to engage CD8+ CTLs.
• MHC Class II Pathway: Extracellular pathogens are internalized by professional APCs via endocytosis or phagocytosis into endosomes. In the ER, newly synthesized MHC Class II $\alpha\beta$ heterodimers assemble with an Invariant Chain (Ii / CD74), which physically blocks the peptide-binding groove and directs trafficking into endolysosomes. Within the acidic endolysosome, proteases degrade the invariant chain, leaving a short fragment called CLIP (Class II-associated Invariant chain Peptide) in the binding cleft. The molecular chaperone HLA-DM catalyzes the release of CLIP and facilitates loading of exogenous peptides (13–25 amino acids) into the groove. The mature complex translocates to the cell surface to engage CD4+ T cells.

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6. T-Cell Differentiation: Effector Subsets & Cytokine Networks

Naive CD4+ T helper cells (Th0) emerge from the thymus and encounter their cognate peptide-MHC Class II complex on dendritic cells within secondary lymphoid organs. The specific cytokine milieu dictates the differentiation of Th0 cells into distinct effector lineages:

Table 1: CD4+ Helper T-Cell Subsets & Cytokine Profiles

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7. Humoral Immunity: B-Cell Activation & Immunoglobulin Isotypes

Humoral immunity defends extracellular fluid compartments via secreted antibody molecules produced by terminally differentiated B lymphocytes (plasma cells).

T-Dependent B-Cell Activation & Class Switching

• Signal 1: The B-cell receptor (membrane-bound IgM/IgD monomer) binds native antigen, internalizes it, and presents processed peptides on surface MHC Class II.
• Signal 2 (Co-stimulation): An activated CD4+ helper T cell recognizes the peptide-MHC II complex via its TCR. Concurrently, CD40 Ligand (CD40L / CD154) on the activated T cell binds to the CD40 receptor on the B-cell surface.
• Isotype Switching & Somatic Hypermutation: CD40-CD40L engagement activates the enzyme Activation-Induced Cytidine Deaminase (AID) in germinal centers, initiating DNA double-strand breaks in switch regions to swap heavy chain constant regions (class switching) and introducing point mutations in variable regions (affinity maturation).
• Hyper-IgM Syndrome: An X-linked recessive defect in the gene encoding CD40L (CD154) on activated helper T cells. B cells cannot undergo class switching or affinity maturation. Laboratory results show normal or elevated serum IgM with profound absence of IgG, IgA, and IgE. Patients suffer recurrent severe pyogenic sinopulmonary infections and opportunistic infections (Pneumocystis jirovecii, Cryptosporidium).

Immunoglobulin Isotypes: Structural & Functional Profiles

+-----------------------------------------------------------------------------------------+
|                             HUMAN IMMUNOGLOBULIN ISOTYPES                               |
+---------+-------------+---------------+-------------------------------------------------+
| Isotype | Structure   | Serum %       | Primary Physiological Effector Functions        |
+---------+-------------+---------------+-------------------------------------------------+
| **IgG** | Monomer     | **~75–80%**   | • Only isotype that **crosses the placenta**    |
|         |             | (Most abundant| • Primary antibody of **secondary response**    |
|         |             | in serum)     | • Potent **opsonin** for phagocytosis           |
|         |             |               | • Fixes complement (IgG1, IgG3)                 |
+---------+-------------+---------------+-------------------------------------------------+
| **IgM** | Pentamer    | ~5–10%        | • Primary antibody of **primary response**      |
|         | (with J     |               | • Highest avidity (10 antigen-binding sites)    |
|         |  chain)     |               | • **Most efficient activator of complement**    |
|         |             |               | • Antigen receptor on naive B cells (monomer)   |
+---------+-------------+---------------+-------------------------------------------------+
| **IgA** | Dimer       | ~10–15%       | • Primary isotype in **mucosal secretions**     |
|         | (with J     | (Highest daily|   (tears, saliva, colostrum, GI fluids)         |
|         | chain & SC) | production)   | • Prevents microbial adherence to epithelium    |
|         |             |               | • Most common primary immunodeficiency          |
+---------+-------------+---------------+-------------------------------------------------+
| **IgE** | Monomer     | <0.01%        | • Binds high-affinity **Fc-epsilon-RI** on mast |
|         |             | (Lowest serum |   cells & basophils (Type I hypersensitivity)   |
|         |             |  titer)       | • Defense against parasitic **helminths** (ADCC)|
+---------+-------------+---------------+-------------------------------------------------+
| **IgD** | Monomer     | <0.5%         | • Co-expressed with IgM on mature naive B cells |
|         |             |               | • Serves as cell-surface antigen receptor       |
+---------+-------------+---------------+-------------------------------------------------+