Mechanisms of Transendothelial Migration of Leukocytes

Introduction:

Inflammation is a protective response of body towards any insult and it is the basic mechanism essential for tissue repair after an injury. It consists of a cascade of cellular and microvascular reactions that serve to remove damaged and generate new tissue. Metchnikov laid the basic principles of inflammation by realizing that leukocytes escape from the blood vessels and then take up and digest bacteria that get into the body 1

Molecules involved in transendothelial migration:

The role of majority of following molecules in transendothelial Migration (TEM) of leukocytes has been demonstrated by blocking the respective receptors by antibodies.

Selectins:

Selectins belong to a family of transmembrane molecules, expressed on the surface of leukocytes and activated endothelial cells. They are of 3 types; L-selectin, P-selectin and E-selectin. Smallest of these is L-selectin which is found on most leukocutesP-selectin, the largest selectin, and it is expressed on activated platelets and endothelial cells primarily. E-selectin is expressed on activated endothelium with chemically or cytokine-induced inflammation. These contain an N-terminal extracellular domain with structural homology to calcium-dependent lectins, followed by a domain homologous to epidermal growth factor, and two to nine consensus repeats (CR) similar to sequences found in complement regulatory proteins. Each of these adhesion receptors is inserted via a hydrophobic transmembrane domain and possesses a short cytoplasmic tail. During inflammation the initial attachment of leukocytes from blood stream is because of these molecules which cause slow downstream movement of leukocytes along the endothelium via transient, reversible, adhesive interactions called leukocyte rolling.

Integrins:

They are transmembrane-adhesive heterodimeric glycoproteins, made up of α and β subunits that function as receptors for the extracellular matrix.  β subunit is known as CD18 and α subunit is CD11 found in 4 forms viz. a,b,c and d. So, based upon α subunit variability glycoproteins are classified as

CD11a/CD18, CD11b/CD18, CD11c/CD18, CD11d/CD18.

The principal receptors for ICAM-1 are the β integrins LFA-1 and MAC-1 (CD11a/CD18, CD11b/CD18), and those for VCAM-1 are the integrins α4β1 and α4β7.

Intercellular adhesion molecules (ICAM-1 and ICAM-2):

These molecules perform very important tasks during TEM. They belong to immunoglobulin superfamily and represent endothelial ligands for the leukocyte β-2 integrin LFA-1. ICAM-1 is constitutively expressed on endothelial cells, platelets and most leukocytes whereas ICAM-2 appears to be concentrated at endothelial cell junctions.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) or CD-31:

These molecules help in endothelial cell to cell adhesion via homophilic interactions and they help in transendothelial migration through endothelium-leukocyte heterophilic interactions. Disruption of PECAM-1: PECAM-1 homophilic interactions allows leukocytes to emigrate through the endothelial barrier 2. These belong to immunoglobulin gene superfamily and are expressed on leukocytes, platelets, neutrophils, monocytes, and selected T cell subsets 3.

JAMs ((JAM-A, JAM-B, and JAM-C):

JAMs are members of the immunoglobulin (Ig) gene superfamily. JAM proteins are localized in the intercellular junctions of polarized endothelial and epithelial cells. JAM-A is expressed at epithelial tight junctions and intercellular borders of endothelial cells, as well as on the surfaces of megakaryocytes. JAM-B and JAM-C, are discussed to be involved in leukocyte adhesion, transmigration and interactions between different cell subsets during inflammation 4. Although JAM-A normally engages in homophilic adhesion, during inflammation it can bind to CD11a/CD18 on the leukocyte 5.

VCAM-1 (vascular cell adhesion molecule):

It is an adhesion molecule that is not constitutively expressed on endothelial cells but is upregulated by chemokines 6. This molecule has been shown to interact with monocytes and lymphocytes and participates in leukocyte transmigration during the inflammatory response 7.

VE-cadherin:

It is one of the major component of adherence junctions. VE-cadherin is a transmembrane protein that establishes homotypic calcium-dependent interactions with its extracellular domain. Although many proteins have been implicated in endothelial cell–cell adhesion 8-9. VE-cadherin has a central role in the regulation of the integrity of the endothelial barrier and leucocyte transmigration as evidenced in vitro and in vivo. The juxtamembrane domain binds p120 catenin, while the membrane distal domain binds β-catenin and plakoglobin in a mutually exclusive fashion that depends on cell–cell contact maturation. Finally, α-catenin alternately associates to β-catenin/plakoglobin or to the actin cytoskeleton.

CD99:

CD99 is a 32 kD, highly O-glycosylated molecule that is expressed on the surfaces of most leukocytes and is concentrated at the borders between confluent endothelial cells 10. Similar to PECAM-1, CD99 functions in a homophilic manner in transmigration but CD99 regulates a later step in transmigration as compared to PECAM.

CD99L2 (CD99-like molecule 2):

It represents a protein of unknown function with moderate sequence homology to CD99 which is expressed on leukocytes and endothelial cells 11. Similar to PECAM, homophilic interaction between CD99 at the endothelial cell border and CD99 on monocytes 12 and neutrophils 13 is required for transmigration.

Steps in Transendothelial migration of Leukocytes 14:

  • Tethering and rolling,
  • Adhesion strengthening under chemoattractant signals,
  • Shedding of L-selectins and intraluminal crawling,
  • Arrest of crawling,
  • Transendothelial migration
  • Interstitial migration 

Diagrammatic representation of transendothelial migration

Transendothelial migration

  1. Inflammation is a protective response in response of body to any insult, which is manifested by release of variety of inflammatory mediators from resident leukocytes and mast cells. They secrete mediators like IL-1β, TNF-α and complement components like C3a and C5a.
  2. These mediators stimulate endothelial cells to express P-selectin and E-selectin on their luminal surfaces 15. Initial tethering and rolling are mediated by P-, E- and L-selectins 16. The initial rolling brings the leukocyte into proximity with endothelial cells, where it can be activated by luminal surface-bound chemokines or lipid chemoattractants (for example, platelet activating factor – PAF) 17. Recent works suggest that not only selectins but also integrins such as lymphocyte function-associated antigen-1 (LFA-1) and macrophage receptor-1 (MAC-1) support leukocyte rolling 18.
  3. After initial tethering and rolling the intercellular adhesion molecule-1 – ICAM-1, vascular cell adhesion molecule – VCAM-1 are expressed. Binding to activated integrins probably contributes to adhesion stabilization and cell motility. In addition to mediating adhesion, integrins also generate intracellular signals that regulate various cellular functions 19-20.
  4. Subsequently, leukocytes crawl inside the blood vessels in a MAC-1- and ICAM-1-dependent manner, seeking the preferred sites of diapedesis 21. After crawling, leukocytes migrate to a nearby endothelial border and squeeze between the tightly opposed endothelial cells to the underlying basement membrane in a next process – transendothelial migration (also called diapedesis) 22. Platelet–endothelial-cell adhesion molecule-1 (PECAM-1, CD31) and CD99 act at sequential steps as the leukocyte crosses the endothelial barrier 23. Two main routes of leukocyte transendothelial migration are described in the literature: i) The transcellular route, when leukocytes emigrate through the body of endothelial cells, and 

    ii) The paracellular route, when leukocytes emigrate through junctions between adjacent endothelial cells 24-25

    In transcellular migration of leukocyte ICAM-1 on endothelial cell engages with LFA-1 on leukocyte and induces formation of micro-villi like endothelial cell projections embracing the migrating leukocyte in a cup like structure 26-27

     

    Most of the recently published studies identify, however, the paracellular migration route as the main mechanism by which leukocytes emigrate from the intravascular compartment into the interstitium 26. Many cell contact proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1), members of the junctional adhesion molecule (JAM) family (JAM-A, JAM-B, and JAM-C), CD99, and ICAM-2 etc. are involved in paracellular migration of leukocyte into extra cellular matrix.

  5. Ultimately the leukocyte enters the extracellular matrix and under the chemo-attractant gradient reaches the site of inflammation, where it performs its various functions.

Conclusion:

The microbial etiology of periodontal diseases is well established. Neutrophils provide the first line of defence when a micro-organism enters the junctional epithelium and then connective tissue. A thorough understanding of neutrophil function is very important to understand the etiopathogenesis of periodontal diseases. 

References:

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  2. Johnson-Leger C, Aurrand-Lions M, Imhof BA. The parting of the endothelium: miracle, or simply a junctional affair? J Cell Sci 2000; 113(Pt 6):921-933.
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  10. Schenkel, A.R. et al. (2002) CD99 plays a major role in the migration of monocytes through endothelial junctions. Nat. Immunol. 3, 143–150.
  11. Suh YH, Shin YK, Kook MC, Oh KI, Park WS, Kim SH et al. Cloning, genomic organization, alternative transcripts and expression analysis of CD99L2, a novel paralog of human CD99, and identification of evolutionary conserved motifs. Gene 2003; 307:63-76.
  12. Schenkel AR, Mamdouh Z, Chen X, Liebman RM, Muller WA. CD99 plays a major role in the migration of monocytes through endothelial junctions. Nat Immunol. 2002; 3:143–150.
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  19. Giagulli C, Ottoboni L, Caveggion E, Rossi B, Lowell C, Constantin G et al. The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion. J Immunol 2006; 177(1):604-611. 
  20. Shattil SJ. Integrins and Src: dynamic duo of adhesion signaling. Trends Cell Biol 2005; 15(8):399-403.
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