CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.

—Apoptosis is important in normal development as well as in diseases such as atherosclerosis. However, the regulation of apoptosis is still not completely understood. We now show that the transcription factor nuclear factor-κB (NF-κB) controls the induction of apoptosis in human and rat vascular smooth muscle cells (SMCs). SMCs in high-density culture exhibited a high NF-κB activity and were insensitive to induction of apoptosis. Inhibition of NF-κB by adenovirus-mediated overexpression of its inhibitor IκBα caused a marked increase in cell death at low but not high cell density. Elevating endogenous IκBα levels by inhibiting its degradation with proteasomal inhibitors resulted in induction of apoptosis in low-density SMCs, as detected by increased binding of annexin V, reduced mitochondrial membrane potential, and increased hypodiploid DNA. In high-density cultures, protection against apoptosis was associated with the expression of inhibitor of apoptosis protein-1 (IAP-1). Transfer of IκBα reduced human IAP-1 mRNA levels, which suggested that IAP-1 is transcriptionally regulated by NF-κB. This was confirmed through identification of a motif with NF-κB–like binding activity in the human IAP-1 promoter region. Moreover, antisense inhibition of IAP-1 sensitized high-density SMCs to the induction of cell death. Together, our data imply that SMCs at high density are protected by an antiapoptotic mechanism that involves increased expression of NF-κB and IAP-1. Interference with pathways that control the susceptibility to programmed cell death may be helpful in the treatment of diseases where dysregulation of apoptosis is involved, eg, atherosclerosis and restenosis.

Interleukin (IL)‐6 is a pleiotropic cytokine involved in the differentiation and proliferation of hematopoietic cells. Hepatocytes respond to IL‐6 with the synthesis and secretion of acute‐phase proteins. In addition, IL‐6 plays a role as a migration factor in vivo. In the present paper, we studied the potential of IL‐6 to mediate migration of human primary T cells and T cell‐derived cell lines. IL‐6 was found to induce migration only in the presence of extracellular matrix, suggesting a cross‐talk between the IL‐6‐ and integrin signal transduction pathways. Furthermore, an IL‐6 gradient is required for chemotactic migration. This activity is not due to the release of secondary chemotactic activities, but is a direct response to IL‐6. T cell migration could also be observed in response to IL‐11, but no migration was found after stimulation with leukemia inhibitory factor or oncostatin M, although these cytokines signal through gp130‐containing receptor complexes. Finally, we present evidence that activation of the mitogen‐activated protein kinase (MAPK) cascade, the phosphatidylinositol 3‐kinase as well as the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is crucial for IL‐6‐induced migration. Selective activation of the JAK/STAT or the MAPK cascade by mutated receptor proteins shows a crucial role of IL‐6‐initiated SH2 domain‐containing tyrosine phosphatase 2/MAPK activity for migration.

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium (1). This cellular infiltrate consists mainly of T lymphocytes, monocytes, and macrophages that are recruited from the circulation into the transplanted tissue by chemotactic molecules known as chemokines. Chemokines belong to a large family of small (8 -10 kDa) inducible chemotactic cytokines, which are characterized by a distinctive pattern of four conserved cysteine residues (2). Currently over 40 chemokines have been identified and classified into two major groups, CXC and CC, dependent on the number and spacing of the first two conserved cysteine residues. The CXC class members include interleukin (IL-8), 1 melanoma growth stimulatory activity, and neutrophil-activating peptide-2, whereas the CC class includes RANTES, monocyte chemotactic protein-1, and MIP-1␣ (macrophage inflammatory protein-1).A number of studies have provided evidence for a role for RANTES in organ transplant rejection, particularly of the kidney. In a model of reperfusion injury in the rat, RANTES levels were increased over normal levels and remained high for more than a week, correlating with the peak of infiltrating macrophages (3). RANTES protein was detected in infiltrating mononuclear cells, tubular epithelium, and vascular endothelium of renal allograft biopsy specimens from patients with cyclosporin nephrotoxicity but not in normal kidney (1). A recent study suggests that RANTES may play a role in graft atherosclerosis (4). Increased levels of RANTES (both mRNA and protein) were detected in mononuclear cells, myofibroblasts, and endothelial cells of arteries undergoing accelerated atherosclerosis compared with normal coronary arteries. In another recent renal transplant study, the chemokine receptor antagonist Met-RANTES when given with low doses of cyclosporin significantly reduced renal injury including interstitial inflammation mainly by reducing the number of infiltrating monocytes (5).Mechanistically this appeared to be achieved by blocking the firm adhesion of these cells to the inflamed endothelium. In summary, these studies ...

Pyrrolidine dithiocarbamate (PDTC) has been found to induce or inhibit apoptosis in different cell types. Here we show that PDTC dose-dependently reduced the viability of rat smooth muscle cells (rSMC), human fibroblasts, and endothelial cells at low but not at high cell density. Endothelial cells were least sensitive, fibroblasts showed a medium sensitivity, and rSMC showed a high sensitivity to PDTC-mediated cell death. An early reduction in the mitochondrial membrane potential indicated a rapid onset of apoptosis in rSMC. Apoptosis was further confirmed by annexin V staining and DNA fragmentation analysis. Gel shift analysis demonstrated increased nuclear factor (NF)-kappaB activity in high-density rSMC compared with low-density cells. NF-kappaB has recently been shown to regulate the induction of anti-apoptotic proteins. Although PDTC is widely used as an inhibitor for NF-kappaB and a radical scavenger, our data show that PDTC rather enhanced NF-kappaB activity and, alone or in combination with menadione, induced oxygen radical generation. Notably, PDTC failed to reduce rSMC viability in medium without Cu(2+) or Zn(2+), and addition of Cu(2+) or Zn(2+) resulted in a dose-dependent increase in PDTC-induced cell death. Addition of both Cu(2+) and Zn(2+) showed synergistic effects. Our results indicate that the induction of apoptosis by PDTC requires Cu(2+) and Zn(2+) and is dependent on cell type and density. Such differential effects may have implications for studies of PDTC as an anti-atherosclerotic or immunomodulatory drug.

Inflammation is associated with atherosclerosis of coronary arteries. Chemokines have an important role in inflammation. The CCR2 chemokine receptor mediates leukocyte chemoattraction, which is involved in the pathogenesis of coronary heart disease. We prospectively studied 1960 consecutive patients aged under 65 years and referred for a first-time left ventricular catheter. Left heart catheters were analyzed by two independent cardiologists for the presence of myocardial infarction (regional wall motion abnormality) and moderate or severely reduced left ventricular function on cineventriculography and presence of coronary atherosclerosis on angiography. Genotyping for CCR2 V64I polymorphism was performed. The presence of the rare allele of the CCR2 gene was significantly associated with a higher prevalence of myocardial infarction on cinventriculography (32.0% vs. 24.2%, moderately or severely reduced left ventricular function (14.0% vs. 9.5%) and NYHA class III or IV (16.7% vs. 12.2%). The association of the CCR2 genotype with heart failure was not independent of the presence of myocardial infarction in multivariate analysis. There was no association of the CCR2 genotype with coronary atherosclerosis. The CCR2 genotype seems to predispose patients for myocardial infarction before the age of 65 years. The higher prevalence of heart failure in gene carriers with the rare alle might be a consequence of myocardial infarction. If the CCR2 genotype is associated with higher mortality in the general population must be investigated in further studies.

Binding of the β2 integrin LFA-1 (αLβ2) to junctional adhesion molecule-A (JAM-A) has been shown to enhance leukocyte adhesion and transendothelial migration. This is mediated by the membrane-proximal Ig-like domain 2 of JAM-A; however, the location of the JAM-A binding site in LFA-1 has not been identified. We have deleted the I domain in the αL subunit of LFA-1 and expressed this αL mutant in αl-deficient Jurkat J-β2.7 cells to demonstrate that the I domain of LFA-1 is crucial for their adhesion to immobilized JAM-A. This was substantiated by blocking the stimulated adhesion of wild-type Jurkat T cells or monocytic Mono Mac 6 cells to JAM-A using the I domain-directed mAb TS1/22 or the small molecule antagonist BIRT 377, which stabilizes the low-affinity conformation of the I domain. The immobilized LFA-1 I domain locked in the open high-affinity conformation was sufficient to support binding of transfected Chinese hamster ovary cells expressing JAM-A. Solid-phase binding assays confirmed a direct interaction of recombinant JAM-A with the immobilized locked-open I domain. These data provide the first evidence that the I domain of LFA-1 contains a functional binding site for JAM-A.