Short Notes on Inflammation & Wound Healing

1. Phagocytosis and Mechanism

Step 1 - Recognition and Attachment

• Mannose receptors - lectins that recognize terminal mannose/fucose on microbial glycoproteins (not present on mammalian cells)
• Scavenger receptors - bind modified LDL particles and microbes
• MAC-1 (CD11b/CD18) - integrin that binds microbes directly

• IgG antibodies (recognized by Fc receptors)
• C3b (complement fragment, recognized by complement receptors)
• Mannose-binding lectin and collectins

Step 2 - Engulfment

Step 3 - Intracellular Killing and Degradation

• NADPH oxidase (phagocyte oxidase) is assembled in the phagosomal membrane
• It oxidizes NADPH and converts O₂ → superoxide (O₂⁻) - this is the respiratory burst
• O₂⁻ is converted to H₂O₂ by spontaneous dismutation
• Myeloperoxidase (MPO) in azurophilic granules converts H₂O₂ + Cl⁻ → hypochlorite (HOCl) - the most potent antimicrobial agent (kills by halogenation and lipid peroxidation)
• Defects in NADPH oxidase → Chronic Granulomatous Disease

• Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO)
• NO + O₂⁻ → peroxynitrite (ONOO⁻), which is microbicidal

• Granules contain elastase, cathepsins, defensins, lysozyme
• Acid proteases degrade bacteria in the acidic environment of the phagolysosome
• During phagocytosis, some granule contents may leak into extracellular space, causing tissue damage

2. Chemotaxis

Chemoattractants

• Bacterial products, especially N-formylmethionyl (f-Met) peptides - short peptides produced only by bacteria (not eukaryotes), recognized by specific receptors

1. Chemokines - especially IL-8 (CXCL8)
2. C5a - complement fragment; most potent chemoattractant
3. Leukotriene B4 (LTB4) - arachidonic acid metabolite

Mechanism of Chemotactic Movement

• All chemoattractants bind seven-transmembrane G-protein-coupled receptors on leukocyte surfaces
• G-protein signaling activates second messengers (IP3, DAG, Ca²⁺)
• Signals cause actin polymerization at the leading edge of the cell
• Myosin at the trailing edge → cell moves forward toward increasing concentration of chemoattractant
• Net result: directed migration toward the source (site of infection/injury)

Leukocyte Selectivity

• Neutrophils respond to C5a, LTB4, IL-8 (CXCL8)
• Monocytes respond to MCP-1 (CCL2)
• Eosinophils respond to eotaxin (CCL11)

3. Role of Prostaglandins in Inflammation

Synthesis Pathway

• Cell injury/inflammation → phospholipases cleave AA from membrane phospholipids
• AA → (via cyclooxygenases COX-1 and COX-2) → cyclic endoperoxides (PGG₂, PGH₂)
• PGH₂ → various prostaglandins via tissue-specific enzymes

Key Prostaglandins and Their Roles in Inflammation

Pathological Roles

• Pain: PGE₂ sensitizes nociceptors to bradykinin and other stimuli (hyperalgesia)
• Fever: PGE₂ acts on thermoregulatory neurons in the hypothalamus, elevating the set point
• Vasodilation + edema: PGI₂ and PGE₂ dilate arterioles and increase postcapillary venule permeability

Pharmacologic Significance

• NSAIDs (aspirin, ibuprofen): Inhibit both COX-1 and COX-2 → block all prostaglandin synthesis → anti-inflammatory, antipyretic, analgesic
• Selective COX-2 inhibitors (coxibs): Spare COX-1 (GI protection) but inhibit inflammation-related prostaglandins
• Corticosteroids: Inhibit phospholipase A₂ → block release of AA → suppress all eicosanoids

4. Granuloma

• The term "granuloma" derives from its granular macroscopic appearance
• It represents a cellular attempt to contain an agent that is difficult to eradicate

Types of Granulomas

• Reaction to inert, non-immunogenic foreign bodies (sutures, talc, silica)
• Too large to be phagocytosed by a single macrophage
• No T-cell immune response is involved
• Foreign material visible in center of granuloma (refractile under polarized light)

• Caused by agents that induce a persistent T cell-mediated immune response
• Agent is persistent microbe (Mycobacterium tuberculosis, fungi, parasites) or chronic stimulus
• Th1 cells produce IFN-γ → activates macrophages (classical/M1 activation)
• In parasitic infections (e.g., schistosomiasis) - strong Th2 response with eosinophils

MORPHOLOGY

1. Epithelioid cells - activated macrophages with abundant pink granular cytoplasm, indistinct cell borders, and elongated "footprint"-shaped nuclei (resemble epithelial cells hence the name)
2. Lymphocytic cuff - aggregates of epithelioid cells surrounded by a collar of lymphocytes (mainly CD4+ T cells)
3. Langhans giant cells - 40-50 μm multinucleated giant cells formed by fusion of multiple activated macrophages; nuclei arranged in a horseshoe or peripheral pattern (key distinction from foreign body giant cells where nuclei are haphazardly scattered)
4. Rim of fibroblasts - in older granulomas; connective tissue rim
5. Caseous necrosis (in tuberculosis) - central zone of necrosis from hypoxia + free radical injury; granular, cheesy appearance grossly; amorphous eosinophilic granular debris with ghost cell outlines on histology

Causes of Immune Granulomas (Mnemonic: BEAST)

• Berylliosis, Brucellosis
• Eosinophilic (Churg-Strauss), Enteropathogenic fungi (Histoplasma, Coccidioides)
• Actinomycosis
• Sarcoidosis, Syphilis, Schistosomiasis
• Tuberculosis (most classic), Toxoplasmosis

5. Chemokines

Classification (Based on Cysteine Residue Arrangement)

Key Members and Their Roles

• IL-8 (CXCL8) - secreted by macrophages, endothelial cells; causes neutrophil activation and chemotaxis; induced by microbial products, IL-1, TNF
• MCP-1 (CCL2) - monocyte chemoattractant protein; recruits monocytes
• Eotaxin (CCL11) - selectively recruits eosinophils (important in allergy/asthma)
• Fractalkine (CX3CL1) - exists as cell-surface bound form (promotes adhesion of monocytes and T cells to endothelium) and soluble form (chemoattractant)

Functions of Chemokines

1. Inflammatory chemokines (inducible): Produced in response to microbes and stimuli
   • Stimulate leukocyte adhesion to endothelium (by increasing integrin affinity)
   • Direct leukocyte migration in tissues toward site of infection/damage
2. Homeostatic chemokines (constitutive): Produced in normal tissues
   • Organize lymphocyte architecture in lymph nodes and spleen (T/B cell zones)
   • Maintain normal tissue compartmentalization

Receptor Mechanism

• Chemokine receptors are G protein-coupled, 7-transmembrane receptors
• Binding triggers activation of Rac, Rho GTPases → actin polymerization → directional migration
• HIV uses co-receptors CXCR4 (naive T cells) and CCR5 (macrophages) as entry co-receptors

6. Steps in Wound Healing

• Healing by first intention (primary union) - clean, surgically apposed wounds; minimal scarring; mainly by epithelial regeneration
• Healing by second intention (secondary union) - larger/infected wounds; combination of regeneration and scar formation; more granulation tissue; wound contraction prominent

Sequential Steps in Wound Healing

Phase 1 - Hemostasis (Minutes to Hours)

• Platelet aggregation and coagulation cascade form a blood clot (hemostatic plug)
• Fibrin clot seals the wound and acts as a provisional scaffold for cell migration
• Platelets release PDGF, TGF-β, which initiate the healing cascade

Phase 2 - Inflammation (Hours to Days 1-3)

• Acute inflammatory response: neutrophils arrive first within 24 hours
• Neutrophils clear bacteria, debris, and clot material
• By day 2-3, monocytes migrate in and differentiate into macrophages (essential orchestrators of repair)
• Macrophages phagocytose debris and release cytokines (TNF, IL-1, IL-6), growth factors (PDGF, FGF, TGF-β, VEGF) that drive subsequent phases

Phase 3 - Proliferation / Granulation Tissue Formation (Days 3-5)

• Angiogenesis: VEGF-A stimulates endothelial cell proliferation and migration; FGF-2 also promotes new vessel formation. New capillaries grow in from wound edges
• Granulation tissue = combination of new capillaries + proliferating fibroblasts + loose ECM; appears pink/granular; rich in ground substance
• Fibroblast proliferation: driven by PDGF, FGF-2, TGF-β; fibroblasts deposit collagen (type III initially, then type I)
• Re-epithelialization: basal keratinocytes migrate from wound edges under the scab to cover the wound surface (within 24 hours in clean incisions)

Phase 4 - Remodeling (Weeks to Months)

• Collagen remodeling: type III collagen gradually replaced by stronger type I collagen
• MMP (matrix metalloproteinase) activity degrades excess ECM; balanced by TIMPs (tissue inhibitors of MMPs)
• Vascularity decreases; granulation tissue matures into an avascular fibrous scar
• Wound contraction: myofibroblasts (fibroblasts with smooth muscle-like properties, expressing α-SMA) contract the wound, reducing its size
• Tensile strength reaches ~70-80% of unwounded skin maximum (never 100%)

Factors Impairing Wound Healing

Complications

• Hypertrophic scar - excessive collagen within wound boundaries; regresses over time
• Keloid - scar tissue extends beyond original wound margins; predisposition is genetic; commoner in dark-skinned individuals
• Wound dehiscence - surgical wound reopens; risk factors: obesity, malnutrition, infection
• Exuberant granulation (proud flesh) - excessive granulation tissue protrudes above skin; blocks re-epithelialization

Reference: Robbins, Cotran & Kumar - Pathologic Basis of Disease, Chapter 3 (Inflammation and Repair), pp. 82-120.All 6 short notes are complete, sourced directly from Robbins & Cotran - Pathologic Basis of Disease. Here's a quick summary of what's covered:

What's covered in each note:

1. Phagocytosis and Mechanism - 3 steps with full mechanism
2. Chemotaxis - chemoattractants + G-protein mechanism
3. Role of Prostaglandins in Inflammation - synthesis pathway + table of actions + pharmacology
4. Granuloma - 2 types + full morphology + causes
5. Chemokines - classification table + key members + functions
6. Steps in Wound Healing - all 4 phases + first vs second intention + complications

1. Phagocytosis and Mechanism

Step 1 - Recognition and Attachment

• Mannose receptors - recognize terminal mannose/fucose on microbial glycoproteins (absent on mammalian cells)
• Scavenger receptors - bind modified LDL and microbial surfaces
• MAC-1 (CD11b/CD18) - integrin that binds microbes directly

• IgG antibodies (Fc receptors on phagocyte)
• C3b complement fragment (complement receptors)
• Mannose-binding lectin and collectins

Step 2 - Engulfment

• Pseudopods extend around the particle
• Plasma membrane pinches off → forms phagosome
• Phagosome fuses with lysosome → phagolysosome
• Requires actin polymerization and cytoskeletal remodeling

Step 3 - Intracellular Killing

• NADPH oxidase assembles on phagosomal membrane
• Converts NADPH + O₂ → superoxide (O₂⁻) - this is the respiratory burst
• O₂⁻ → H₂O₂ (by spontaneous dismutation)
• MPO (myeloperoxidase) + H₂O₂ + Cl⁻ → HOCl (hypochlorite) - most potent microbicidal agent; kills by halogenation and lipid peroxidation
• Defect in NADPH oxidase → Chronic Granulomatous Disease

• iNOS generates nitric oxide (NO)
• NO + O₂⁻ → peroxynitrite (ONOO⁻) → microbicidal

• Elastase, cathepsins, defensins, lysozyme
• Acid proteases degrade bacteria in acidic phagolysosome
• Leakage into extracellular space → tissue damage

2. Chemotaxis

Chemoattractants

• Bacterial N-formyl methionyl (f-Met) peptides - produced only by bacteria, not eukaryotes

1. C5a - complement fragment; most potent chemoattractant
2. Leukotriene B4 (LTB4) - arachidonic acid metabolite
3. Chemokines - especially IL-8 (CXCL8)

Mechanism

• Chemoattractants bind 7-transmembrane G protein-coupled receptors on leukocytes
• G-protein activation → second messengers (IP₃, DAG, Ca²⁺) → actin polymerization at the leading edge
• Myosin contracts at the trailing edge → net forward movement toward increasing concentration gradient

Leukocyte Selectivity

• Neutrophils: C5a, LTB4, IL-8 (CXCL8)
• Monocytes: MCP-1 (CCL2)
• Eosinophils: Eotaxin (CCL11)

3. Role of Prostaglandins in Inflammation

Synthesis

• Cell injury → phospholipase A₂ cleaves AA from membrane phospholipids
• AA → via COX-1 / COX-2 → cyclic endoperoxides (PGG₂, PGH₂)
• PGH₂ → specific prostaglandins via tissue-specific synthases

Actions in Inflammation

Key Roles in Inflammation

• Fever: PGE₂ acts on hypothalamic thermoregulatory neurons → raises temperature set point
• Pain: PGE₂ sensitizes nociceptors to bradykinin and other stimuli → hyperalgesia
• Vasodilation + edema: PGE₂ and PGI₂ dilate arterioles and increase postcapillary venule permeability

Pharmacological Relevance

• NSAIDs (aspirin, ibuprofen): Block COX-1 and COX-2 → inhibit all prostaglandin synthesis → anti-inflammatory, antipyretic, analgesic
• COX-2 selective inhibitors (coxibs): Spare COX-1 (less GI side effects)
• Corticosteroids: Inhibit phospholipase A₂ → block AA release → suppress all eicosanoids

4. Granuloma

• Represents a cellular attempt to contain an agent that is difficult to eradicate
• Name derived from its granular macroscopic appearance

Types

• Reaction to inert, non-immunogenic foreign bodies (sutures, talc, silica)
• Too large to be phagocytosed by a single macrophage
• No T-cell immune response involved
• Foreign material visible at center (refractile under polarized light)

• Caused by persistent agents inducing T cell-mediated immune response
• Agents: M. tuberculosis, fungi, parasites, sarcoidosis, berylliosis
• Th1 cells produce IFN-γ → activates macrophages (classical M1 activation)
• In schistosomiasis: Th2 response with eosinophils

MORPHOLOGY (H&E)

1. Epithelioid cells - activated macrophages; abundant pink granular cytoplasm; indistinct cell borders; elongated "footprint" nuclei; resemble epithelial cells
2. Lymphocytic cuff - collar of CD4+ T lymphocytes surrounding the epithelioid cell aggregate
3. Langhans giant cells - 40-50 μm multinucleated cells formed by fusion of activated macrophages; nuclei arranged in horseshoe/peripheral pattern
4. Rim of fibroblasts - in older granulomas; connective tissue encapsulation
5. Caseous necrosis (in TB) - central zone of necrosis; granular, cheesy appearance grossly; amorphous eosinophilic granular debris on histology

Common Causes

• Tuberculosis (most classic - with caseation)
• Sarcoidosis (non-caseating)
• Fungal infections (Histoplasma, Coccidioides)
• Schistosomiasis
• Berylliosis, Silicosis
• Crohn disease
• Syphilis

5. Chemokines

Classification (Based on Cysteine Residue Arrangement)

Key Members

• IL-8 (CXCL8) - secreted by macrophages and endothelial cells; activates and recruits neutrophils; induced by microbes, IL-1, TNF
• MCP-1 (CCL2) - recruits monocytes to sites of inflammation
• Eotaxin (CCL11) - selectively recruits eosinophils (allergy, asthma)
• Fractalkine (CX3CL1) - membrane-bound form promotes adhesion; soluble form is chemoattractant

Functions

• Produced in response to microbes and inflammatory stimuli
• Increase integrin affinity on leukocytes → promote firm adhesion to endothelium
• Direct leukocyte migration in tissues toward infection/damage site

• Produced in normal tissues to maintain tissue architecture
• Organize T and B lymphocytes into discrete zones in lymph nodes and spleen

Clinical Note

• HIV uses CXCR4 (T cell co-receptor) and CCR5 (macrophage co-receptor) as entry co-receptors
• Individuals with CCR5 mutations are resistant to HIV infection

6. Steps in Wound Healing

• First intention (primary union): clean, well-apposed wound; minimal scar; mainly epithelial regeneration (e.g., surgical incision)
• Second intention (secondary union): large/infected wound; more granulation tissue, wound contraction, significant scarring

The 4 Phases of Wound Healing

Phase 1 - Hemostasis (Minutes to Hours)

• Vessel injury → platelet aggregation → blood clot (fibrin) formation
• Fibrin clot seals wound and acts as provisional scaffold
• Platelets release PDGF, TGF-β → initiate repair cascade

Phase 2 - Inflammation (Hours - Day 1-3)

• Neutrophils arrive within 24 hours → clear bacteria and debris
• By day 2-3, monocytes arrive → differentiate into macrophages
• Macrophages are the critical orchestrators of repair:
  • Phagocytose debris and dead neutrophils
  • Release PDGF, FGF, VEGF, TGF-β → drive proliferative phase

Phase 3 - Proliferation / Granulation Tissue (Days 3-5 onward)

• Angiogenesis: VEGF-A drives endothelial cell proliferation and new capillary sprouting; FGF-2 also contributes
• Granulation tissue forms = new capillaries + proliferating fibroblasts + loose ECM; soft, pink, granular
• Fibroblast proliferation: PDGF, FGF-2, TGF-β → fibroblasts deposit type III collagen initially
• Re-epithelialization: keratinocytes migrate from wound edges under the scab to resurface the wound

Phase 4 - Remodeling (Weeks to Months)

• Type III collagen gradually replaced by stronger type I collagen
• MMPs (matrix metalloproteinases) degrade excess ECM, balanced by TIMPs
• Vascularity decreases; granulation tissue → avascular fibrous scar
• Wound contraction: myofibroblasts (α-SMA positive) contract the wound
• Final tensile strength = 70-80% of normal (never reaches 100%)

Factors Impairing Wound Healing

Complications of Wound Healing

• Hypertrophic scar - excess collagen within wound margins; regresses over months
• Keloid - scar extends beyond original wound margins; does not regress; genetic predisposition; commoner in dark-skinned individuals
• Proud flesh (exuberant granulation) - excessive granulation tissue above skin level; blocks re-epithelialization; needs cautery/excision
• Wound dehiscence - wound re-opens; risk factors: obesity, malnutrition, infection

Reference: Robbins, Cotran & Kumar - Pathologic Basis of Disease, Chapter 3: Inflammation and Repair