Transendothelial Migration (Diapedesis) — Carranza's Clinical Periodontology

Overview of the Process

Steps of Transendothelial Migration

1. Margination & Rolling

• In inflamed gingival vessels, blood flow slows (due to vasodilation).
• Leukocytes move from the axial stream to the periphery (margination).
• Selectins (E-selectin and P-selectin on endothelium; L-selectin on leukocytes) mediate loose, reversible attachments.
• This results in rolling of leukocytes along the endothelial surface.

2. Activation

• Chemoattractants and cytokines (e.g., IL-8/CXCL8, C5a, LTB₄, fMLP from bacteria) bind to receptors on rolling leukocytes.
• This triggers intracellular signaling, activating integrins on the leukocyte surface.

3. Firm Adhesion

• Activated integrins (CD11b/CD18 = Mac-1; CD11a/CD18 = LFA-1) on leukocytes bind to ICAMs (ICAM-1, ICAM-2) on endothelial cells.
• This causes firm, irreversible adhesion and leukocyte arrest.

4. Crawling

• The firmly adherent leukocyte crawls along the endothelial surface, mediated by Mac-1 and ICAM-1, to find an optimal site for transmigration (usually a tricellular junction or bicellular junction).

5. Transendothelial Migration (Diapedesis)

• Paracellular route (predominant): leukocyte squeezes through the junctions between endothelial cells.
  • Involves loosening of endothelial tight junctions (VE-cadherin, JAM-A, JAM-B, JAM-C, PECAM-1/CD31).
  • PECAM-1 (CD31) is crucial — it is expressed at both the leukocyte and endothelial cell surfaces and mediates homophilic binding guiding the leukocyte through.
• Transcellular route (minor): leukocyte passes directly through the endothelial cell body via vesicle-vacuole organelles (VVOs).

6. Penetration of the Basement Membrane

• After crossing the endothelium, the leukocyte must traverse the subendothelial basement membrane (composed of collagen IV, laminin, fibronectin).
• Matrix metalloproteinases (MMPs) — particularly MMP-2 and MMP-9 — are released to locally degrade this barrier.
• The leukocyte then undergoes chemotaxis in the extracellular matrix toward the source of infection/inflammation.

Key Molecules Involved

Relevance to Periodontitis

• In gingivitis and periodontitis, the junctional epithelium and gingival sulcus are continuously populated by PMNs migrating from gingival capillaries via this process.
• PMNs represent the primary defense against periodontal pathogens; impaired TEM (e.g., in Chediak-Higashi syndrome, leukocyte adhesion deficiency/LAD) leads to severe, aggressive periodontitis.
• Leukocyte Adhesion Deficiency (LAD): deficiency of CD18 (β₂ integrin) impairs firm adhesion → leukocytes cannot migrate → extreme susceptibility to Papillon-Lefèvre-like aggressive periodontal destruction.
• The continuous stream of PMNs moving through the junctional epithelium into the gingival crevicular fluid (GCF) constitutes a major component of the innate defense in the periodontium.
• Paradoxically, excessive or unregulated leukocyte migration contributes to tissue destruction through release of proteases, reactive oxygen species (ROS), and inflammatory mediators.

In the Context of the Gingival Sulcus

• The junctional epithelium is uniquely permeable to leukocyte trafficking.
• PMNs migrate through intercellular spaces of the junctional epithelium and accumulate in the gingival crevicular fluid.
• The density of PMN migration correlates with the degree of gingival inflammation.
• Bacterial products (particularly LPS from gram-negative anaerobes like P. gingivalis, T. forsythia) are potent stimulators of TEM via upregulation of endothelial adhesion molecules and production of chemoattractants.

Transendothelial Migration — Steps per Carranza's Clinical Periodontology

Step 1: Rolling

• Leukocytes (primarily neutrophils/PMNs) in the blood marginate and begin loosely interacting with the vascular endothelium.
• Mediated by selectins:
  • P-selectin (stored in Weibel-Palade bodies, rapidly expressed on endothelium after stimulation)
  • E-selectin (synthesized de novo after cytokine activation)
  • Both bind to PSGL-1 (P-selectin glycoprotein ligand-1) on the leukocyte surface.
• The interaction is weak and transient → the leukocyte rolls slowly along the vessel wall rather than being carried by the blood flow.

Step 2: Complement Activation

• Bacterial products (e.g., LPS from P. gingivalis) in the gingival tissues activate the complement cascade.
• This generates C3a and C5a (anaphylatoxins).
• C5a is a potent chemoattractant and activates endothelial cells and resident mast cells/macrophages.
• This step amplifies the inflammatory signal, preparing both the endothelium and the leukocyte for the next steps.

Step 3: Signalling the Endothelium

• Inflammatory mediators (IL-1β, TNF-α, C5a, histamine from mast cell degranulation) signal the endothelial cells.
• Endothelial cells become activated:
  • Upregulate expression of E-selectin, ICAM-1, VCAM-1
  • Increase surface expression of P-selectin
  • Alter their cytoskeleton and become more permissive to leukocyte transmigration
• This step essentially "primes" the endothelium to receive and direct migrating leukocytes.

Step 4: Increased Rolling

• Following endothelial activation and upregulation of adhesion molecules, more leukocytes are captured from the bloodstream and begin rolling.
• Rolling velocity decreases as selectin density on the endothelium increases.
• This represents amplification of Step 1 — a positive feedback that recruits larger numbers of PMNs to the site.

Step 5: Chemokine Signal

• Chemokines (particularly IL-8/CXCL8) produced by endothelial cells, macrophages, and fibroblasts in the inflamed tissue are presented on the endothelial surface bound to heparan sulfate proteoglycans.
• Rolling leukocytes encounter these surface-bound chemokines → chemokine receptors (e.g., CXCR1, CXCR2 on neutrophils) are activated.
• Intracellular signaling (via Gi-coupled receptors → PI3K → inside-out integrin activation) causes a conformational change in leukocyte integrins from low-affinity to high-affinity state.

Step 6: Rolling Arrest (Firm Adhesion)

• Activated high-affinity integrins on the leukocyte (LFA-1/CD11a:CD18 and Mac-1/CD11b:CD18) bind firmly to their ligands on the endothelium:
  • ICAM-1 and ICAM-2 (for LFA-1)
  • ICAM-1 (for Mac-1)
• This results in complete arrest of the leukocyte — it stops rolling and adheres firmly to the endothelial surface.
• This is an irreversible step under physiological shear conditions.

Step 7: Zipper Phase (Transendothelial Crossing)

• The arrested leukocyte spreads on the endothelial surface and probes for a transmigration site (usually an intercellular junction).
• The "zipper" refers to the sequential, step-by-step opening and re-closing of endothelial junctions as the leukocyte passes through:
  • PECAM-1/CD31 homophilic interactions guide the leukocyte through the junction
  • JAM-A (Junctional Adhesion Molecule-A), CD99, and VE-cadherin are displaced and re-assembled behind the leukocyte like a zipper
• The basement membrane is locally degraded by MMP-2 and MMP-9.
• The leukocyte fully enters the perivascular connective tissue and follows the chemokine gradient toward the source of infection.

Summary Table

Periodontal Relevance

Transendothelial Migration — Simple Explanation

Step 1: Rolling 🎲

• Blood vessels are like highways — blood flows fast.
• When bacteria cause inflammation, the vessel wall puts out "sticky patches" (selectins).
• Neutrophils passing by loosely stick to these patches and start rolling slowly along the vessel wall — like a ball rolling on velcro.
• They haven't stopped yet, just slowing down.

Step 2: Complement Activation 🔔

• Meanwhile, bacterial toxins in the gum tissue activate the complement system (a group of proteins in the blood that act like an alarm system).
• This produces C5a — a powerful chemical signal that works like a distress flare, telling nearby cells "there's an infection here!"
• It also activates the vessel wall to prepare for the next steps.

Step 3: Signalling the Endothelium 📣

• The alarm signals (C5a, IL-1, TNF-α) reach the endothelium (the inner lining of blood vessels).
• The endothelial cells wake up and put out more sticky molecules on their surface (ICAM-1, E-selectin).
• Think of it as the vessel wall opening the gate and putting out a "soldiers, stop here!" sign.

Step 4: Increased Rolling 📈

• Because of all those extra sticky molecules, more and more neutrophils get captured and start rolling.
• This is just amplification — the body is sending a bigger army to the site.

Step 5: Chemokine Signal 📍

• The vessel wall now releases chemokines (especially IL-8), which are like GPS coordinates for neutrophils.
• These chemokines sit on the vessel wall surface and bind to receptors on the rolling neutrophil.
• When the neutrophil detects these signals, it activates its gripping molecules (integrins) — like a soldier going from relaxed hands to gripping tightly.

Step 6: Rolling Arrest ✋

• The now-activated integrins on the neutrophil grip tightly to ICAM-1 on the vessel wall.
• The neutrophil comes to a complete stop — it's fully stuck to the vessel wall.
• This is called arrest — rolling is over, the soldier has found its exit point.

Step 7: Zipper Phase 🤐

• The stopped neutrophil now needs to squeeze through the vessel wall into the gum tissue.
• It finds the tiny gap between two endothelial cells and pushes through.
• As it passes, the junction opens in front and closes behind it — just like a zipper opening and closing.
• The neutrophil then follows the chemical trail (chemokine gradient) straight toward the bacteria in the gum tissue.

The Big Picture

Bacteria in gums
      ↓
Alarm signals released
      ↓
Neutrophils in blood slow down → roll → stop → squeeze through vessel wall
      ↓
Neutrophils enter gum tissue and attack bacteria

One Important Point

Transendothelial Migration — Flowchart

(Carranza's Clinical Periodontology)

┌─────────────────────────────────────────────────────────┐
│                  BACTERIAL INFECTION                    │
│         (LPS, fMLP from periodontal pathogens)          │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│              STEP 1: ROLLING                            │
│  • Inflammation → P-selectin & E-selectin on endothelium│
│  • Binds PSGL-1 on neutrophil surface                   │
│  • Neutrophil slows down and rolls along vessel wall    │
│  • Weak, reversible attachment                          │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│         STEP 2: COMPLEMENT ACTIVATION                   │
│  • Bacterial toxins activate complement cascade         │
│  • C3 → C3a + C3b                                       │
│  • C5 → C5a (anaphylatoxin) + C5b                       │
│  • C5a = powerful alarm signal & chemoattractant        │
│  • Activates mast cells → histamine release             │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│         STEP 3: SIGNALLING THE ENDOTHELIUM              │
│  • C5a + IL-1β + TNF-α + Histamine signal vessel wall   │
│  • Endothelial cells activated                          │
│  • ↑ ICAM-1, VCAM-1, E-selectin expressed              │
│  • Endothelium "primed" → ready to receive neutrophils  │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│           STEP 4: INCREASED ROLLING                     │
│  • More adhesion molecules on endothelium               │
│  • More neutrophils captured from bloodstream           │
│  • Rolling velocity ↓ (slower rolling)                  │
│  • Positive feedback → larger army recruited            │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│           STEP 5: CHEMOKINE SIGNAL                      │
│  • IL-8 (CXCL8) released by endothelium & macrophages  │
│  • IL-8 presented on endothelial surface (heparan SO₄)  │
│  • Binds CXCR1 / CXCR2 on rolling neutrophil           │
│  • Inside-out signaling → PI3K pathway activated        │
│  • Integrins change shape: LOW affinity → HIGH affinity │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│           STEP 6: ROLLING ARREST                        │
│  • LFA-1 (CD11a/CD18) + Mac-1 (CD11b/CD18) activated   │
│  • Firmly grip ICAM-1 on endothelial surface            │
│  • Neutrophil comes to COMPLETE STOP                    │
│  • Irreversible firm adhesion                           │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│             STEP 7: ZIPPER PHASE                        │
│  • Neutrophil crawls to intercellular junction          │
│  • PECAM-1 (CD31) guides entry between cells            │
│  • JAM-A, CD99, VE-cadherin open sequentially           │
│  • Junction opens ahead → closes behind (like zipper)  │
│  • MMP-2 & MMP-9 digest basement membrane              │
│  • Neutrophil fully crosses into connective tissue      │
│  • Follows IL-8 gradient → moves toward bacteria        │
└─────────────────────┬───────────────────────────────────┘
                      │
                      ▼
┌─────────────────────────────────────────────────────────┐
│                NEUTROPHIL IN TISSUE                     │
│  ✔ Phagocytoses bacteria                                │
│  ✔ Releases antimicrobial enzymes (elastase, MMP-8)     │
│  ⚠ Releases ROS → oxidative tissue damage              │
│  ⚠ Chronic activation → bone & collagen destruction    │
└─────────────────────────────────────────────────────────┘

Key Molecules — Quick Reference

Host-Microbe Interaction in Periodontics

1. The Microbial Side — The Biofilm

Dental Plaque as a Biofilm

• Periodontal pathogens don't act as single organisms — they live in a structured subgingival biofilm.
• The biofilm has a defined microbial succession (Socransky's complexes):
  • Yellow, Purple, Green complexes → early colonizers (Streptococcus, Actinomyces)
  • Orange complex → bridge organisms (Fusobacterium nucleatum, Prevotella intermedia)
  • Red complex → the most pathogenic (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola)

Key Virulence Factors of Periodontal Pathogens

2. The Host Side — Innate and Adaptive Immunity

A. Innate Immune Response (First Line)

• Host cells (epithelial cells, macrophages, dendritic cells) express Toll-like receptors (TLRs):
  • TLR2 → recognizes lipoteichoic acid (gram-positive), P. gingivalis fimbriae
  • TLR4 → recognizes LPS (gram-negative)
  • TLR9 → recognizes bacterial DNA
• TLR activation → NF-κB pathway → production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-8)

• The primary defense in the gingival sulcus
• Recruited via TEM (as discussed previously)
• Kill bacteria via:
  • Phagocytosis + oxidative burst (ROS)
  • Degranulation (elastase, MMP-8, cathepsin G)
  • Neutrophil extracellular traps (NETs)
• Impaired neutrophil function = severe periodontitis (LAD, Chediak-Higashi)

• Activated by bacteria via alternative and lectin pathways
• C3b → opsonization (tags bacteria for phagocytosis)
• C5a → potent chemotaxis of neutrophils
• P. gingivalis hijacks this system via C5aR1 signaling to subvert killing

B. Adaptive Immune Response (Second Line)

• Th1 cells → IFN-γ → activates macrophages → promotes bacterial killing
• Th2 cells → IL-4, IL-13 → promotes B cell response
• Th17 cells → IL-17 → recruits neutrophils; important in bone loss
• Tregs → IL-10, TGF-β → anti-inflammatory, protective against bone loss

• Produce antibodies against periodontal pathogens (IgG, IgA)
• Advanced periodontitis lesions are dominated by plasma cells
• Antibodies can opsonize bacteria but also form immune complexes that trigger further inflammation

3. The Central Concept: Dysbiosis & Immune Subversion

The Keystone Pathogen Hypothesis (Hajishengallis)

• P. gingivalis, even in low numbers, acts as a keystone pathogen
• It doesn't cause disease alone — it manipulates the microbial community and the immune response:
  • Impairs complement-mediated killing (via C5aR1/TLR2 crosstalk)
  • Degrades IL-8 → reduces neutrophil recruitment locally
  • Upregulates C5aR1 → stimulates cAMP → blocks phagocytosis
  • Result: bacteria survive, but inflammation persists and damages tissue

Polymicrobial Synergy and Dysbiosis (PSDH Model — Hajishengallis & Lamont)

• Shift from a symbiotic microbiome to a dysbiotic one
• Not one single pathogen causes disease — the whole community becomes pathogenic
• Accessory pathogens + keystone pathogen + pathobionts = dysbiotic biofilm
• Host cannot clear this biofilm → chronic, unresolved inflammation

4. How Inflammation Causes Tissue Destruction

Bacteria (biofilm)
        ↓
TLR activation → NF-κB → IL-1β, TNF-α, IL-6, IL-17
        ↓
Neutrophils & Macrophages recruited
        ↓
Release of MMPs (MMP-1, 8, 13) → collagen destruction
Release of ROS → oxidative tissue damage
        ↓
Osteoclast activation (RANKL ↑, OPG ↓)
        ↓
ALVEOLAR BONE LOSS

RANKL/OPG Axis — Key to Bone Loss

• Th17 cells and fibroblasts produce RANKL (Receptor Activator of NF-κB Ligand)
• RANKL binds RANK on osteoclast precursors → osteoclast differentiation → bone resorption
• OPG (osteoprotegerin) is the decoy receptor that blocks RANKL
• In periodontitis: RANKL ↑, OPG ↓ → net bone loss

5. Balance vs. Dysregulation

6. Summary Concept

Periodontal disease = Dysbiotic biofilm + Susceptible host + Impaired resolution of inflammation