Selective eosinophil transendothelial migration triggered by eotaxin via modulation of Mac-1/ICAM-1 and VLA-4/VCAM-1 interactions.
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We have recently cloned eotaxin, a highly efficacious eosinophilic chemokine involved in the development of lung eosinophilia during allergic inflammatory reactions. To understand more precisely how eotaxin facilitates the specific migration of eosinophils, we have studied which adhesion receptors are essential for eotaxin action both in vivo and in vitro. Experiments using mice genetically deficient in adhesion receptors demonstrated that molecules previously reported to be involved in both leukocyte tethering/rolling (P-selectin and E-selectin) and in sticking/ transmigration (ICAM-1 and VCAM-1) are required for eotaxin action in vivo. To further elucidate the mechanism(s) involved in this process, we have used an in vitro transendothelial chemotaxis model. mAb neutralization studies performed in this system suggest that the integrins Mac-1 (CD11b/18), VLA-4 (alpha4beta1) and LFA-1 (CD11a/18) are involved in the transendothelial chemotaxis of eosinophils to eotaxin. Accordingly, the expression of these integrins on eosinophils is elevated by direct action of this chemokine in a concentration-dependent manner. Taken together, our results suggest that eotaxin-induced eosinophil transendothelial migration in vivo and in vitro relies on Mac-1/ICAM-1 and VLA-4NCAM-1 interactions, the latter ones becoming more relevant at later time points of the eotaxin-induced recruitment process. (+info)
Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells.
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Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells. (+info)
Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.
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Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro. (+info)
Expression of cell adhesion molecules in dilated cardiomyopathy: evidence for endothelial activation in inflammatory cardiomyopathy.
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BACKGROUND: Dilated cardiomyopathy (DCM) is pathogenically linked to inflammatory cardiomyopathy (InfCM), which is characterized by intramyocardial infiltration. The transendothelial migration of immunocompetent cells is mediated by cell adhesion molecules (CAMs). METHODS AND RESULTS: We investigated the expression pattern of CAMs (immunoglobulin superfamily, 32 selectins, and beta1- and beta2-integrins) in endomyocardial biopsies from DCM patients (n=152; left ventricular ejection fraction <40%) using immunohistochemistry. Whereas few specimens obtained at autopsy (controls; n=14) presented enhanced expression regarding single endothelial CAMs (human leukocyte antigen [HLA] class I, 7%; HLA-DR, 14%; CD29, 14%), none demonstrated concurrent abundance of >3 CAMs (inflammatory endothelial activation), nor did any control tissue prove positive for InfCM (>7.0 CD3+ lymphocytes per 1 mm2). In comparison, 64% (n=97) of the DCM biopsies were evaluated positive for InfCM and 67% (n=101) for inflammatory endothelial activation, respectively. Whereas expression of HLA class I, HLA-DR, intercellular cell adhesion molecule-1, and CD29 was distributed homogeneously within a patient's serial sections, immunoreactivity of vascular cell adhesion molecule-1, lymphocyte function antigen-3, and the selectins was accentuated on single vascular endothelia. Sixty-six percent of the DCM biopsies presented CD29 abundance also within the extracellular matrix and the sarcolemma. CD62P and CD62E were present in 16% and 40% of the DCM patients, respectively. Endothelial CAM representatives correlated with one another (P<0.05), except for CD62P with HLA. Endothelial CAM expression correlated with intramyocardial infiltrates phenotyped by the corresponding counterreceptors. CONCLUSIONS: Inflammatory endothelial activation is present in 67% of DCM patients. Because CAM expression correlates with the immunohistological diagnosis of InfCM and counterreceptor-bearing intramyocardial infiltrates, evaluation of endothelial CAMs might be of diagnostic significance in InfCM. (+info)
Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin.
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Selectins support the capture and rolling of leukocytes in venules at sites of inflammation and in lymphocyte homing. Gene-targeted mice with null mutations at the L-, E-, or P-selectin locus develop normally and show mild (E-/-) to moderate (P-/-, L-/-) defects in inflammatory cell recruitment. Mice lacking both P- and E-selectin (E/P-/-) have severe neutrophilia and spontaneous skin infections that limit their life span. Other combinations of selectin deficiency have not been investigated. We have generated novel mice lacking L- and P-selectin (L/P-/-), L- and E-selectin (L/E-/-), or all three selectins (E/L/P-/-) by bone marrow transplantation. L/P-/- mice (only E-selectin present) show an absence of leukocyte rolling after trauma and severely reduced rolling (by approximately 90%) in inflammation induced by TNF-alpha. Residual rolling in L/P-/- mice was very slow (3.6 +/- 0.2 micrometers/s after TNF-alpha). L/E-/- mice (only P-selectin present) showed rolling similar to that of L-/- at increased velocities (15.1 +/- 0.3 micrometer/s). The number of adherent leukocytes after 2 or 6 h of TNF-alpha treatment was not significantly reduced in L/E-/- or L/P-/- mice. E/L/P-/- mice showed very little rolling after TNF-alpha, all of which was blocked by mAb to alpha4 integrin. Adherent and emigrated neutrophils were significantly reduced at 6 h after TNF-alpha. We conclude that any one of the selectins can support some neutrophil recruitment but eliminating all three selectins significantly impairs neutrophil recruitment. (+info)
Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells.
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Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4(+) or CD8(+) T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti-E- and -P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon gamma-inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand. (+info)
Leukocyte adhesion: High-speed cells with ABS.
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In order to decide where to exit blood vessels and enter tissues, leukocytes roll along endothelial surfaces. Recent studies suggest that an 'automatic braking system' (ABS), involving selectin cell-adhesion molecules, enables leukocytes to roll at a fairly constant velocity despite large variations in blood flow rate. (+info)
Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene.
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Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2+/-3 versus 133.4+/-3 seconds, P<0.00005). To determine the contribution of endothelium- versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (Deltableeding time, -24.6+/-9.1 and -3.4+/-5.3 seconds, respectively; P<0.04). Platelet recruitment was studied by measuring serotonin release from a second recruitable population of platelets that were added to stimulated platelets at the peak of NO production. There was 40.3+/-3.7% and 52. 0+/-2.1% serotonin release for platelets added to wild-type or eNOS-deficient platelets, respectively (P<0.05). In summary, mice that lacked eNOS had markedly decreased bleeding times even after endothelial NO production was controlled. These data suggest that the lack of platelet-derived NO alters in vivo hemostatic response by increasing platelet recruitment. Thus, these data support a role for platelet-derived NO production in the regulation of hemostasis. (+info)