The murine CD99-related molecule CD99-like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response

CD99, a glycoprotein found on the surfaces of leukocytes and concentrated at the borders of endothelial cells, plays a major role in the migration of leukocytes across endothelial cells into sites of inflammation, and has other roles in thymocyte development. The human and mouse genomes encode only two proteins related to CD99. One of these, XGA, is a red blood cell surface antigen. The function of the other, CD99-like 2 (CD99L2), is not known. We cloned mouse CD99L2 and used CD99L2 isolated from transfected cells to raise specific antibodies. Similar to human CD99, CD99L2 was expressed at the borders between transfected cells as well as on mouse leukocytes and vascular endothelial cells in situ. Transfection of L cell fibroblasts with CD99L2 imparted to them the ability to adhere to each other in a divalent cation-dependent, homophilic manner. Anti-CD99L2 antibody blocked influx of neutrophils and monocytes into a site of inflammation in vivo.     

CD99 is a key mediator of the transendothelial migration of neutrophils

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.     

Eosinophil tissue recruitment to sites of allergic inflammation in the lung is platelet endothelial cell adhesion molecule independent

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

Background

CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration.

Methods and results

Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6 dyne/cm²) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated.

Conclusion

CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.     

Genetic evidence for functional redundancy of Platelet/Endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.     

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.     

Cutaneous injection of human subjects with macrophage inflammatory protein-1 alpha induces significant recruitment of neutrophils and monocytes

Macrophage inflammatory protein (MIP-1 alpha), a member of the CC chemokine subfamily, has been shown to attract T cells and monocytes in vitro and to be expressed at sites of inflammation. Although the in vitro activities of MIP-1 alpha have been well documented, the in vivo biological activities of MIP-1 alpha in humans have not been studied. To address this, we challenged human subjects by intradermal injection with up to 1000 pmol of MIP-1 alpha and performed biopsies 2, 10, and 24 h later. Although no acute cutaneous or systemic reactions were noted, endothelial cell activation, as indicated by the expression of E-selectin, was observed. In agreement with its in vitro activity, monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration was observed, peaking at 10-24 h. Surprisingly, in contrast to its reported lack of in vitro neutrophil-stimulating activity, a rapid infiltration of neutrophils was observed in vivo. This neutrophil infiltration occurred as early as 2 h, preceding the appearance of other cells, and peaked at 10 h. Interestingly, we found that neutrophils in whole blood, but not after isolation, expressed CCR1 on their cell surface. This CCR1 was thought to be functional as assessed by neutrophil CD11b up-regulation following whole-blood MIP-1 alpha stimulation. These studies substantiate the biological effects of MIP-1 alpha on monocytes and lymphocytes and uncover the previously unrecognized activity of MIP-1 alpha to induce neutrophil infiltration and endothelial cell activation, underscoring the need to evaluate chemokines in vivo in humans.     

Transgenic mice expressing different levels of soluble platelet/endothelial cell adhesion molecule-IgG display distinct inflammatory phenotypes.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), expressed on the surfaces of leukocytes and concentrated in the junctions between endothelial cells plays an important role in transendothelial migration of neutrophils and monocytes. Soluble recombinant PECAM-IgG injected i.v. into mice blocks acute leukocyte emigration by 80%. To study the role of PECAM in models of chronic inflammation, we generated transgenic mice constitutively expressing soluble full-length murine PECAM as an IgG chimera. Three founder lines expressed this transgene and constitutively secreted murine PECAM-IgG into the plasma where it was maintained at characteristic concentrations for each line. All mice had similar hematologic profiles to wild-type littermates and were healthy when maintained in the standard laboratory animal facility. Both the leukocytes and the endothelium of mice of all transgenic lines expressed the same levels of endogenous PECAM-1 as wild-type littermates. Similarly, there were no detectable differences in the expression of several other common leukocyte and endothelial cell adhesion molecules. Mice that produced moderate (10-20 microg/ml) concentrations of PECAM-IgG demonstrated a severely blunted acute inflammatory response, despite mobilizing appropriate numbers of circulating leukocytes. Surprisingly, mice that constitutively produced high (400-1,000 microg/ml) concentrations of PECAM-IgG were unresponsive to its anti-inflammatory effects. This is the first demonstration that a soluble form of a cell adhesion molecule can be stably expressed and retain efficacy in vivo over prolonged periods. This approach is applicable to many other extracellular molecules. However, the plasma concentrations of such constitutively produced inhibitors may greatly influence the resulting phenotype.     

The anti-inflammatory peptides, antiflammins, regulate the expression of adhesion molecules on human leukocytes and prevent neutrophil adhesion to endothelial cells.

Antiflammin-1 and antiflammin-2 are nonapeptides corresponding to the region of highest similarity between glucocorticoid-inducible proteins lipocortin-1 and uteroglobin. We have studied whether antiflammins could affect expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and binding of neutrophils (PMNs) to HCAEC. Although neither antiflammin-1 nor antiflammin-2 affected expression of adhesion molecules on resting PMNs, monocytes, and lymphocytes in whole blood, they attenuated changes in L-selectin and CD11/CD18 expression evoked by platelet-activating factor or interleukin-8 with IC(50) values of 4-20 micromol/l. The maximum inhibition was similar to those seen with human recombinant lipocortin-1 (100 microgram/ml). Unlike dexamethasone (100 nmol/l), the antiflammins had little effect on LPS-stimulated expression of E-selectin and ICAM-1 on HCAEC. Consistently, culture of HCAEC with dexamethasone, but not with antiflammins, decreased PMN binding to endothelial cells. Preincubation of PMNs with antiflammins markedly decreased their adhesion to LPS-activated HCAEC. Inhibition of adhesion was additive with function blocking anti-E-selectin and anti-L-selectin antibodies, but was not additive with anti-CD18 antibody. These results show that antiflammins inhibit PMN adhesion to HCAEC by attenuating activation-induced up-regulation of CD11/CD18 expression on leukocytes, and suggest that antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.     

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM- 1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

Granulocyte-macrophage colony-stimulating factor and other cytokines regulate surface expression of the leukocyte adhesion molecule-1 on human neutrophils, monocytes, and their precursors.

There is increasing evidence that cytokines such as granulocyte-macrophage (GM)-CSF can profoundly affect the adhesion, aggregation, and mobility of neutrophils both in vitro and in vivo. However, the mechanisms whereby these factors might alter the adhesive properties of neutrophils are incompletely understood. A new family of cellular adhesion molecules has recently been identified by cDNA cloning. The members of this family include human leukocyte adhesion molecule-1 (LAM-1), the human endothelial-leukocyte adhesion molecule, and the mouse leukocyte homing receptor for high endothelial venules, MEL-14. LAM-1 is the human homologue of murine MEL-14, and is believed to mediate binding of leukocytes to human high endothelial venules. LAM-1 can be identified by mAb TQ-1, Leu 8, or anti-LAM1.1. The expression and regulation of LAM-1 on granulocytes, monocytes, and their precursors was investigated using flow cytometry and the anti-LAM-1.1 mAb. Neutrophils, eosinophils, monocytes, marrow myeloid cells, granulocyte/macrophage colony-forming unit, and burst-forming unit for erythroid cells were LAM-1+ by flow microfluorimetry. The regulation of LAM-1 expression was tested by treating various cell populations with cytokines or other stimuli for 0-90 min. Exposure of neutrophils, monocytes, and marrow myeloid cells to GM-CSF induced rapid and complete loss of LAM-1 from the cell surface, but had no effect on LAM-1 expression by lymphocytes. The loss of LAM-1 was temporally correlated with up-regulation of CD11b (Mo1), an adhesion molecule involved in neutrophil aggregation. Several other factors known to activate neutrophils also caused down-regulation of LAM-1 and up-regulation of CD11b, including TNF, FMLP, and leukotriene B4. Interestingly, granulocyte-CSF and IFN-gamma had minimal effects on neutrophil LAM-1 expression. Similar results were observed on monocytes and myeloid precursor cells. Thus, exposure of neutrophils to GM-CSF results in a profound change in surface expression of adhesion molecules, with coordinated up-regulation of CD11b and down-regulation of LAM-1. These changes in adhesion proteins are likely to alter aggregation and mobility of both mature myeloid cells and their precursors in patients receiving certain types of cytokine therapy.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

CD157 is part of a supramolecular complex with CD11b/CD18 on the human neutrophil cell surface

CD157 is a GPI-anchored cell surface glycoprotein expressed by human peripheral blood neutrophils. Cross-linking of CD157 induces intracellular Ca2+ mobilization and re-shaping in neutrophils, thus regulating their adhesive and migratory properties. Results obtained by immunolocalization and confocal microscopy indicate that CD157 lies in close proximity to the CD11b/CD18 complex which is strongly expressed on the activated neutrophil cell membrane where it plays a predominant role in adhesion. This study analyses the physical association between CD157 and CD18 in human neutrophils by co-immunoprecipitation experiments. The anti-CD157 monoclonal antibody RF3 co-precipitates CD18, and the anti-CD18 antibody TS1/18 co-precipitates CD157 from human neutrophil lysates. These results confirm that CD157 physically interacts with CD11b/CD18 complex in human neutrophils.     

Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. We show here that monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocytes, and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an 125I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.     

Endothelial CD47 Promotes Vascular Endothelial-Cadherin Tyrosine Phosphorylation and Participates in T Cell Recruitment at Sites of Inflammation In Vivo

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein γ (SIRPγ) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47(-/-) mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-α-induced inflammation. Reconstitution of CD47(-/-) mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47(-/-) endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(αi)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(αi), had no inhibitory effect, whereas G(αi) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(αi). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM.     

Deletions in the cytoplasmic domain of platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) result in changes in ligand binding properties

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a member of the immunoglobulin superfamily present on platelets, endothelial cells, and leukocytes that may function as a vascular cell adhesion molecule. The purpose of this study was to examine the role of the cytoplasmic domain in PECAM-1 function. To accomplish this, wild- type and mutated forms of PECAM-1 cDNA were transfected into murine fibroblasts and the functional characteristics of the cells analyzed. Wild-type PECAM-1 localized to the cell-cell borders of adjacently transfected cells and mediated heterophilic, calcium-dependent L-cell aggregation that was inhibitable by a polyclonal and two monoclonal anti-PECAM-1 antibodies. A mutant protein lacking the entire cytoplasmic domain did not support aggregation or move to cell-cell borders. In contrast, both forms of PECAM-1 with partially truncated cytoplasmic domains (missing either the COOH-terminal third or two thirds of the cytoplasmic domain) localized to cell-cell borders in 3T3 cells in a manner analogous to the distribution seen in cultured endothelial cells. L-cells expressing these mutants demonstrated homophilic, calcium-independent aggregation that was blocked by the polyclonal anti-PECAM-1 antibody, but not by the two bioactive monoclonal antibodies. Although changes in the cytoplasmic domain of other receptors have been shown to alter ligand-binding affinity, to our knowledge, PECAM-1 is the first example of a cell adhesion molecule where changes in the cytoplasmic domain result in a switch in the basic mechanism of adhesion leading to different ligand-binding specificity. Variations in the cytoplasmic domain could thus be a potential mechanism for regulating PECAM-1 activity in vivo.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.