Luminal Surface Molecules
ICAM-1 is involved in the firm adhesion of leukocytes to the apical surface of endothelial cells through interactions with leukocyte CD11a/CD18 and/or CD11b/CD18. Dimers of ICAM-1 on the endothelial surface (i.e., in cis
) are the preferential ligands for CD11/18 (4
). Once adherent, ICAM-1 becomes enriched under the leukocyte as it migrates to the endothelial cell border and continues to surround it during transmigration (6
). The actin cytoskeleton is involved in this process; specifically, Src-dependent phosphorylation of the actin-binding molecule cortactin is required for ICAM-1 clustering (7
VCAM-1 is involved in the firm adhesion of monocytes and lymphocytes bearing CD49d/CD29. VCAM-1 clustering has been observed in the steps leading up to diapedesis. Both ICAM-1 and VCAM-1 are enriched over actin-rich docking structures that form prior to TEM (9
). Engagement of VCAM-1 activates intracellular calcium release and the small GTPase Rac-1, which in turn activates the endothelial NADPH oxidase Nox2 (11
). Reactions downstream of this process affect the adherens junctions (see section entitled Loosening the Junctions, below).
ICAM-2, another CD11a/CD18 ligand, is constitutively expressed on endothelial cells, where it is concentrated at cell borders but retains considerable surface expression. Antibodies against ICAM-2 do not seem to have a major effect on TEM in vitro. Compared with ICAM-1, ICAM-2 seems to play a lesser role (12
). However, in some inflammatory models in vivo, blocking antibodies or genetic deletion of ICAM-2 inhibits the transmigration of neutrophils (13
Junctionally Enriched Molecules
Junctional adhesion molecule (JAM)-A is concentrated at endothelial cell borders. Although it normally engages in homophilic adhesion, during inflammation it can bind to CD11a/CD18 on the leukocyte (15
). On the one hand, blocking JAM-A on human endothelial cells in vitro using antibodies generally has no reported effect on TEM (6
), with one notable exception (15
). On the other hand, in vivo studies show decreased inflammation (18
) and TEM (14
) when JAM-A is blocked. JAM-C is likewise concentrated at endothelial cell borders. It can engage in homophilic adhesion with JAM-C or heterophilic adhesion with JAM-B or CD11b/CD18. The latter interaction is implicated in TEM both in vitro (19
) and in vivo (20
). For an extensive review of the roles of JAM family members in the inflammatory response, see Reference 21
Endothelial cell–selective adhesion molecule (ESAM) is molecularly related to the JAMs but has a long cytoplasmic domain. As its name implies, its distribution is limited mostly to endothelial junctions, but it is also expressed on activated platelets (22
). It binds homophilically; no ligand on leukocytes has been described. ESAM-deficient mice have no defect in lymphocyte extravasation, but they do show a transient decrease in neutrophil emigration in response to thioglycollate broth injected intraperitoneally. Compared with wild-type mice, there was markedly reduced neutrophil emigration into the peritoneal cavity at 2 h, but normal levels of emigration were observed by 4 h (23
Platelet/endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31) is an immunoglobin superfamily member that is concentrated at the borders of endothelial cells and expressed diffusely on platelets and leukocytes. Homophilic interaction between leukocyte PECAM and endothelial PECAM is required for TEM (24
). Blockade with monoclonal antibody against the amino-terminal homophilic interaction domain, soluble PECAM-Fc chimeras, and genetic deletion of PECAM inhibit TEM in vitro and in vivo (reviewed in Reference 3
). When PECAM is transfected into cells that normally lack it, expression of PECAM allows them to support TEM (26
). This gain of function has not been demonstrated with other adhesion molecules. When PECAM-PECAM interactions are blocked, leukocytes are arrested tightly adherent to the apical surface of the cell (27
) and actively migrate along the junctions as if searching for a place to transmigrate (17
). In vivo, at sites of inflammation leukocytes can get to the postcapillary venules at the site of inflammation but are unable to transmigrate efficiently. They are observed in vastly increased numbers apparently adherent to the endothelial cell luminal surface (28
), which is reminiscent of the block to TEM observed in vitro (24
This phenotype is observed with human cells and in all mouse strains examined except for C57BL/6 (29
). Interestingly, this mouse strain seems to be unique. Genetic deletion of PECAM or administration of blocking antibody or mouse PECAM-Fc to these mice has no effect in a variety of inflammatory models (29
). Even the closely related C57BL/10 strain responds to anti-PECAM therapy (30
). The ability to circumvent the need for PECAM in the thioglycollate peritonitis model of inflammation has been linked to a small locus at the proximal end of chromosome 2 (30
). Therefore, earlier studies carried out in C57BL/6 mice that found no role or only a minor role for PECAM in inflammation need to be reevaluated. See Reference 3
for a detailed discussion of the role of PECAM in various in vivo models.
There is a role for leukocyte PECAM in traversing the basal lamina (33
). C57BL/6 mice in which PECAM has been knocked out (31
) or blocked with antibody (14
) are defective in terms of their ability to migrate across this extravascular barrier.
CD99 is unrelated to any other molecule in the human genome except the closely related paralog CD99-like 2 (CD99L2), which may have arisen from a common ancestral gene (34
). The gene encoding CD99 is in the pseudoautosomal region of the human X chromosome (35
). In mice, the region of the genome syntenic to the pseudoautosomal region of the human X chromosome is on chromosome 4 (36
), where mouse CD99 is encoded. Similar to PECAM interactions, homophilic interaction between CD99 at the endothelial cell border and CD99 on monocytes (37
) and neutrophils (38
) is required for transmigration. However, CD99 regulates a later step in transmigration than PECAM does. Leukocytes in which PECAM has been blocked can still be prevented from transmigrating if anti-CD99 is added after the anti-PECAM block has been removed. Conversely, when CD99 interaction is first blocked, leukocytes cannot be inhibited from transmigrating by anti-PECAM antibody after the anti-CD99 block is removed (37
). In support of this mechanism, confocal images of leukocytes blocked in the act of transmigration by anti-CD99 show their leading edge under the endothelial cytoplasm, their cell body lodged at the border between endothelial cells, and their trailing uropod on the apical surface (37
). As long as the block continues, the leukocytes migrate along the junctions over the surface of the endothelium in this manner, unable to finish transmigration (38
). There is indirect evidence that CD99 cannot function unless PECAM acts first (26
). Blocking antibodies against mouse CD99 inhibit inflammation in several animal models. Migration of T lymphocytes into skin (39
) and migration of neutrophils and monocytes into the peritoneal cavity (40
) are blocked by interference with CD99 function.
The CD99L2 molecule is ancestrally related to CD99. It is encoded by a gene on the X chromosome, as is CD99, but unlike CD99, the gene encoding CD99L2 is not in the pseudoautosomal region (36
). CD99L2 expression in mice seems similar to that of CD99; that is, it is expressed on the vascular endothelium of all tissues examined (41
) and at the borders of endothelial cells. It is expressed to varying degrees on all circulating blood cells. Only polyclonal antibodies against murine CD99L2 have been tested in vivo. They block neutrophil and monocyte influx in the thioglycollate peritonitis model (41
). The incomplete blockade of inflammation observed when interfering with either CD99 or CD99L2 may be due to partial redundancy of the function of these molecules.
Vascular endothelial cell–specific cadherin (VE-cadherin) is the major adhesion molecule of the endothelial adherens junction. It negatively regulates transmigration. Antibodies against VE-cadherin enhance early migration into a site of inflammation in vivo (43
). In vitro studies show that VE-cadherin is transiently removed from the site of transmigration at the cell junction (44
). Mutation of the cytoplasmic tail of VE-cadherin so that it cannot interact with p120 or β-catenin, or overexpression of p120 to outcompete the kinases that would phosphorylate it (see the section entitled Loosening the Junctions, below), prevents clearance of VE-cadherin from the cell border and blocks transmigration (46
Why So Many Molecules?
Antibodies against the following molecules block TEM, implicating their role in transmigration: poliovirus receptor (48
), MUC18 (49
), activated leukocyte cell adhesion molecule (50
), integrin-associated protein (51
), and nepmucin (52
). There have been numerous recent discoveries of molecules on endothelial cells or leukocytes that are implicated in diapedesis. When added to the well-characterized molecules discussed in the previous section, these discoveries raise the question of why so many molecules are required for TEM. Are they simply an artifact of clogging up the junction with antibody or turning the cell junctions into immune complexes? This conjecture is unlikely, as most of these studies used control antibody, Fab or F(ab′)2
fragments, soluble recombinant adhesion molecules, small interfering RNA knockdown, or genetic deletion to buttress their claims.
The process of diapedesis itself can be further broken down into a series of molecularly defined steps controlled by specific molecules acting in sequence. Sequential blocking experiments demonstrated that PECAM regulates a step in diapedesis that is “upstream” of the step regulated by CD99 (37
). As mentioned above, images of the blocked cells show that blocking PECAM arrests leukocytes before the start of transmigration; blocking CD99 arrests leukocytes during the process of transmigration. Sequential blockade analysis has not been performed with other pairs of molecules, but images of leukocytes blocked by antibodies in vivo in C57BL/6 mice show that ICAM-2 arrests neutrophils on the apical surface of the endothelium, anti-JAM-A arrests them at the cell junctions, and anti-PECAM arrests them between the endothelial cell and basal lamina (14
). These findings beg the questions of whether each molecule controls its own defined step in the sequence, whether multiple molecules control each step, and how many steps there are. Until sequential blockade studies can be performed with each of these molecules, these questions will remain unanswered. The answers are likely to be different for different leukocyte types, vascular beds, and inflammatory stimuli, as well as the amount of time elapsed after the initiation of the stimulus. However, it seems unlikely that there is a separate unique step in diapedesis controlled by each molecule reported to be important for transmigration.
What if most of the endothelial molecules that are reported to control transmigration were part of a large, multimolecular transmigration complex, or a series of multimolecular transmigration complexes (one for each successive step in diapedesis) that combine to make a platform to support transmigration, analogous to the way that multiple transcription factors and coactivators combine to make DNA accessible to transcription? In that case, loss of or interference with any one of the molecules could make the complex less efficient at supporting diapedesis and could account for the published results.