Serum procalcitonin, interleukin-6, soluble intercellular adhesin molecule-1 and IgG to short-chain exocellular lipoteichoic acid as predictors of infection in total joint prosthesis revision

The diagnosis of prosthetic joint infection and its differentiation from aseptic loosening remains problematic. The definitive laboratory diagnostic test is the recovery of identical infectious agents from multiple intraoperative tissue samples; however, interpretation of positive cultures is often complex as infection is frequently associated with low numbers of commensal microorganisms, in particular the coagulase-negative staphylococci (CNS). In this investigation, the value of serum procalcitonin (PCT), interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) as predictors of infection in revision hip replacement surgery is assessed. Furthermore, the diagnostic value of serum IgG to short-chain exocellular lipoteichoic acid (sce-LTA) is assessed in patients with infection due to CNS. Presurgical levels of conventional serum markers of infection including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell count (WBC) is also established. Forty-six patients undergoing revision hip surgery were recruited with a presumptive clinical diagnosis of either septic (16 patients) or aseptic loosening (30 patients). The diagnosis was confirmed microbiologically and levels of serum markers were determined. Serum levels of IL-6 and sICAM-1 were significantly raised in patients with septic loosening (P=0.001 and P=0.0002, respectively). Serum IgG to sce-LTA was elevated in three out of four patients with infection due to CNS. In contrast, PCT was not found to be of value in differentiating septic and aseptic loosening. Furthermore, CRP, ESR and WBC were significantly higher (P=0.0001, P=0.0001 and P=0.003, respectively) in patients with septic loosening. Serum levels of IL-6, sICAM-1 and IgG to sce-LTA may provide additional information to facilitate the diagnosis of prosthetic joint infection.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Chemokine and cell adhesion molecule mRNA expression and neutrophil infiltration in lipopolysaccharide-induced hepatitis in ethanol-fed rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.

Expression of E-Selectin and Vascular Cell Adhesion Molecule-1 in So-Called 'Negative' Appendices: First Results to Support the Pathological Diagnosis in Borderline Cases.

Background: Since the rate of histologically 'negative' appendices still ranges between 15 and 20%, appendicitis in 'borderline' cases remains a challenging disease. As previously described, cell adhesion molecule expression correlates with different stages of appendicitis. Therefore, it was of interest to determine whether the 'negative' appendix correlated with the absence of E-selectin or vascular cell adhesion molecule-1 (VCAM-1). Methods: Nineteen grossly normal appendices from a series of 120 appendectomy specimens from patients with suspected appendicitis were analysed in frozen sections for the expression of E-selectin and VCAM-1. As control, 5 normal appendices were stained. Results: This study showed a coexpression of E-selectin and VCAM-1 in endothelial cells in early and recurrent appendicitis. In patients with symptoms for less than 6 h, only E-selectin was detected. Cases with fibrosis and luminal obliteration were only positive for VCAM-1. In cases of early appendicitis with symptoms of less than 6 h duration, a discordance between histological and immunohistochemical results was found. Conclusions: This report indicates that E-selectin and VCAM-1 expression could be useful parameters in the diagnosis of appendicitis in borderline cases.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis

Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding a4b1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic a4b1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by a4b1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 Tcell survival. Conclusions: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to a4b1 expressing proinflammatory T lymphocytes in CLD.

Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E−/−), reconstituted with minimal numbers (≤5 × 104) of wild-type BM cells, poorly survived the procedure compared with wild-type recipients. Excess mortality in P/E−/− mice, after a lethal dose of irradiation, was likely caused by a defect of HPC homing. Indeed, we observed that the recruitment of HPC to the BM was reduced in P/E−/− animals, either splenectomized or spleen-intact. Homing into the BM of P/E−/− recipient mice was further compromised when a function-blocking VCAM-1 antibody was administered. Circulating HPC, 14 hr after transplantation, were greatly increased in P/E−/− mice treated with anti-VCAM-1 compared with P/E−/− mice treated with just IgG or wild-type mice treated with either anti-VCAM-1 or IgG. Our results indicate that endothelial selectins play an important role in HPC homing to the BM. Optimal recruitment of HPC after lethal doses of irradiation requires the combined action of both selectins and VCAM-1 expressed on endothelium of the BM.

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): A strategy for vascular immunotargeting of drugs

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.