Characterization of a soluble form of CD58 in synovial fluid of patients with rheumatoid arthritis (RA)

Reduced levels of a soluble form of the adhesion receptor and CD2 ligand CD58 (sCD58) were previously described in RA patients. In order to understand the biological significance of this finding we biochemically characterized sCD58 in RA and asked how well sCD58 binds to CD2. sCD58 concentrations were measured in serum and synovial fluid (SF) samples of RA patients by two ELISAs, one detecting domain 1 of CD58 (CD58-D1), and the other one the complete molecule (CD58-D1 + D2). Small amounts of split sCD58-D1 were found in most RA sera, but not SF. In addition, split sCD58-D2 was detected in SF by affinity chromatography, SDS–PAGE, and Western blotting. Gel filtration gave similar peaks at 95–125 kD for RA sera, SF, and normal serum. Binding of SF-sCD58 to the CD2⁺ Jurkat variant JBB1 or recombinant CD2 was stronger than urinary sCD58 and reached binding of oligomeric recombinant CD58 at low concentrations. In conclusion, sCD58-split products were found in RA sera and SF. At concentrations as they occur in vivo, SF-sCD58 binds to CD2 much more strongly than urinary sCD58. It is conceivable that locally released sCD58 blocks the CD2/CD58 interaction under physiological conditions. Insufficient release of sCD58, e.g. in synovitis, might result in T cell accumulation and perpetuation of inflammation.     

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.     

Soluble cell adhesion molecules and von Willebrand factor in children with Kawasaki disease.

Fifty-nine children with acute Kawasaki disease (KD), a childhood vasculitis, were compared with 35 children with fever due to infection and 48 healthy children. Levels of soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) in the healthy children were double those found in adults. All three soluble cell adhesion molecules and von Willebrand factor (vWF) were higher in the children with KD than in the healthy children, but only sE-selectin, a marker for activated endothelial cells, and sICAM-1 were higher than in the febrile children. The high levels of vWF in KD appear to reflect the prominent acute-phase reaction. This information can help us to understand further the complex interactions between cytokines, circulating inflammatory cells and the vascular endothelium, and may lead to new therapeutic avenues in KD and other inflammatory diseases and vasculitides.     

Chemokines and soluble adhesion molecules in renal transplant recipients with cytomegalovirus infection

Cytomegalovirus (CMV) infection is associated with leucocyte infiltration in various organs, which supports a role for chemokines and adhesion molecules in the pathogenesis of CMV infection. In a prospectively conducted study of renal transplant recipients, 10 patients with CMV disease, five patients with asymptomatic CMV infection and 10 patients who did not have any CMV infection were included. During CMV infection, and in particular during CMV disease, plasma levels of the chemokines IL-8, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) and the soluble adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and L-selectin increased and were positively correlated with the degree of CMV pp65 antigenaemia. Furthermore, a decrease in plasma levels of these chemokines and adhesion molecules was observed following ganciclovir therapy in the patients with CMV disease. This could suggest a role for these molecules in the pathogenesis of CMV infection.     

肺腺癌细胞微环境及转移与黏附分子表达的关系

目的研究肺腺癌细胞生长环境及转移性与黏附分子CD44v6和CD29的表达关系。方法将起源相同、转移性不同的两个肺腺癌细胞系AGZY和Anip分别用简便肿瘤多细胞球体(MTS)培养法培养，并设常规单层贴壁细胞培养对照。通过倒置显微镜、扫描及透射电镜观察MTS形成情况，并用免疫组化法分别对MTS及贴壁细胞上CD44v6和CD29表达进行检测。结果MTS培养成功，贴壁细胞与MTS在细胞结构及细胞连接结构上相似，两种MTS在形态及结构上差异无显著性。免疫组化结果显示，CD29在高转移性的Anip细胞及其MTS上呈阳性表达；在低转移性的AGZY细胞及其MTS上阴性表达。CD44v6在Anip和AGZY细胞及MTS上均呈阳性表达，差异无显著性。贴壁细胞与MTS上两种黏附分子表达均无差异。结论成功建立了一种简易制备MTS的方法。细胞生长方式(单层贴壁与MTS)可能不影响CD44v6和CD29的表达。CD29表达可能与肺腺痛转移性相关；CD44v6表达可能与肺腺癌转移无关。     

Soluble adhesion molecules in sera of patients with leprosy: levels of soluble intercellular adhesion molecule-1 (sICAM-1) rapidly decrease during multi-drug therapy

The clinicopathological spectrum of leprosy is associated with an altered immunological reaction. The expression of adhesion molecules on endothelial cells directs the cellular traffic to sites of local skin and nerve inflammation. Soluble forms of adhesion molecules, which are released upon cytokine activation, can be detected in the circulation and may reflect ongoing tissue inflammation. We determined the serum levels of sICAM-1, sE-selectin and sL-selectin in 74 patients with leprosy (tuberculoid form, n = 23; lepromatous form, n = 36; acute leprous reaction, n = 16) and 15 healthy age- and sex-matched control donors. Patients with lepromatous leprosy had significantly higher levels of sICAM-1 (564 ± 174 versus 450 ± 92 versus 334 ± 57 ng/ml) and E-selectin (90 ± 31 versus 74 ± 29 versus 50 ± 10 ng/ml) than patients with tuberculoid leprosy and normal donors (P<0.01). No differences between groups were detected for L-selectin. Patients with leprous reactions had similar high levels to lepromatous patients. Twenty lepromatous patients were re-examined after 4 weeks of therapy. A significant decrease in sICAM-1 serum levels was observed after 1 month of anti-mycobacterial treatment, which was accompanied by a reduction of mycobacteria in skin biopsies (P<0.01). Patients with leprous reactions (n = 13) also demonstrated a drop in sICAM-1 after anti-inflammatory therapy. sE-selectin and sL-selectin serum values decreased only in lepromatous patients after therapy. It can be concluded that soluble adhesion molecules like sICAM-1 and sE-selectin are promising activity markers in patients with leprosy, which may be useful for treatment monitoring.     

Characterization of EN4 monoclonal antibody: a reagent with CD31 specificity.

EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells, and the reagent was not assigned to any of the presently known CD. Here, we provide evidence indicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibroblasts transfected with the human CD31 gene. Furthermore, SDS-PAGE analysis of immunoprecipitates of cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31 MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD31 gave the same pattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis or by immunohistochemistry on sections of a variety of human tissues. EN4, however, proved consistently more efficient than the reference anti-CD31 MoAb as judged by both the intensity of fluorescence or of tissue staining. This property has thus allowed a better characterization of the tissue and cellular distribution of CD31.     

Surface molecules involved in the adherence of recombinant interferon-gamma (rIFN-gamma)-stimulated human monocytes to vascular endothelial cells.

During an inflammatory reaction, an increased number of circulating monocytes adhere to the endothelial cells (EC) of the vessel wall. This process is mediated by molecules located on the surface of monocytes and EC. Locally released inflammatory mediators can modulate monocyte-EC interaction. In an earlier study we reported that stimulation of monolayers of cultured venous EC with rIFN-gamma enhanced their adhesiveness for monocytes. The aim of the present study was to investigate the effect of rIFN-gamma on peripheral blood monocytes with regard to the expression of surface molecules and the binding to non-stimulated or cytokine-stimulated EC. Flow cytometric analysis demonstrated that monocytes stimulated with 500 U/ml rIFN-gamma for 24 h showed increased expression of CR3 (CD11b/CD18), p150,95 (CD11c/CD18) and Fc gamma RI (CD64); the expression of LFA-1 (CD11a/CD18), L-selectin, CD14 and VLA-4 (CD49d/CD29) did not change or was slightly reduced. Upon stimulation with rIFN-gamma monocytes showed an enhanced binding to both non-stimulated or rIFN-gamma-stimulated EC. This was even more pronounced when EC had been stimulated with rIL-1 alpha for 24 h. The increased binding of rIFN-gamma-stimulated monocytes to rIL-1 alpha-stimulated EC was further analysed. Studies with MoAbs against adhesion molecules on monocytes revealed that the binding of rIFN-gamma-stimulated monocytes, but not that of non-stimulated monocytes, to rIL-1 alpha-stimulated EC was inhibited by about 30-60% with MoAbs against CD11a, CD11b, CD18, L-selectin or CD14. MoAbs against CD11c or CD49d had little or no effect. From these results, we conclude that exposure of monocytes to rIFN-gamma enhances their adhesiveness to cytokine-stimulated EC by a mechanism which involves CD11a/CD18, CD11b/CD18, CD14 and L-selectin on monocytes.     

Oral administration of muscle derived small molecules inhibits tumor spread while promoting normal cell growth in mice

Tumor metastases are extremely rare in striated muscles. This is surprising given the fact that this tissue constitutes 60% of body weight. The present study focuses on small molecules produced and secreted by muscle cells which possess anti-cancer activity in vivo. Recently we have shown that a low molecular weight fraction (<1000 Dalton) of skeletal muscle cell conditioned medium (muscle factor-MF), markedly inhibits the proliferation of carcinoma, sarcoma or melanoma cell lines in vitro. The MF exerts a cytostatic effect on tumor cell growth and arrests the cells in the G0/G1 of the cell cycle. However, normal cell proliferation, such as bone marrow and fibroblasts, was stimulated following incubation with MF. In this study, the effect of orally administered MF on melanoma and sarcoma growth was examined in mice. The administration of MF to mice inoculated intravenously with melanoma (B16–F10) or sarcoma (MCA-105) cells, resulted in a statistically significant inhibition of metastatic lung foci. In a different model, melanoma was induced in the foot pad and after development of a local lesion, the leg was amputated. A prolonged survival time was observed in the MF treated groups. Since the MF stimulated bone marrow cell proliferation in vitro, we decided to test its efficacy as an inhibitor of the myelotoxic effect exerted by chemotherapy, in vivo. MF, administered after chemotherapy, restored the number of white blood cells and yielded an increased percentage of neutrophils compared with the decline in these parameters after administration of chemotherapy alone. Thus, it is indicated that MF exerted a systemic anti tumor and chemoprotective effect when given orally. It can be concluded that it is bioavailable and is not biodegradable in the digestive system. MF may be considered as a potential therapy for the prevention of tumor spread. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.