Increased CD11/CD18 expression on peripheral blood leucocytes of patients with sarcoidosis.

Sarcoidosis is a multisystem disease of unknown etiology characterized by non-caseating granulomata, formed mainly from macrophages surrounded by lymphocytes and plasma cells. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of LeuCAMs (CD11/CD18) on peripheral blood leucocytes of 11 Caucasian and 10 Afro-Caribbean patients with sarcoidosis compared with age-, sex- and race-matched controls. Whilst the percentages of the cells expressing CD11/CD18 were no different, the density, expressed as mean fluorescence intensity (MFI), was greater for all leucocytes in sarcoids than in normal individuals. The expression of intercellular adhesion molecule-1 (ICAM-1), a ligand for LFA-1 which is expressed on all leucocytes, was not significantly different from normal, whereas HLA-DR was expressed more intensely on sarcoid monocytes (P less than 0.01) and blood lymphocytes (P less than 0.005) than control cells. Our findings are consistent with leucocyte activation although we were unable to confirm reports of elevated tumour necrosis factor-alpha (TNF-alpha) in the patients' plasma using an ELISA. Increased expression of adhesion molecules on peripheral blood leucocytes may play a role in the cellular extravasation, aggregation, and granuloma formation seen in sarcoidosis.     

Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.     

Canine leukocyte cell adhesion molecules (LeuCAMs): Characterization of the CD11/CD18 family

The canine LeuCAM (CD11/CD18) family is characterized using a panel of newly developed anti-canine LeuCAM monoclonal antibodies (mAb) as well as a cross-reactive anti-human CD11b mAb. The new anti-canine LeuCAM mAb were developed by immunizing mice with purified canine CD11/CD18 antigen derived from an anti-canine CD18 affinity column. Six mAb with reactivity to the canine LeuCAM family were cloned and characterized. These 6 included a new anti-canine CD18 mAb, 2 anti-canine CD11a mAb, 2 anti-canine CD11c mAb, and a mAb which recognizes a novel canine CD11/CD18-related macrophage antigen, designated LeuCAMmac, which is described in a separate report. The cell and tissue distribution of canine LeuCAMs was found to be similar to that reported in other species with a few notable exceptions. One prominent exception was the finding that canine CD11c, in contrast to human CD11c, was much more widely distributed on dendritic cells than it was on tissue macrophages. The molecular organization of the canine LeuCAM family was also found to be similar to that reported in other species, with the possible exception of the canine CD11b alpha chain, which may exist as several different species. It is anticipated that these anti-canine LeuCAM mAb will prove useful in immunophenotypic studies of canine hemolymphatic system neoplasia.     

缺血预适应阻抑犬冠状窦血中性粒细胞CD11b/CD18的表达

本文旨在探讨缺血预适应（ＩＰ）心肌保护效应是否与其防止中性粒细胞（ＰＭＮ）ＣＤ１１ｂ／ＣＤ１８分子的表达有关。采用犬衣降支动脉（ＬＡＤ）建立ＩＰ模型,在长缺血前各５ｍｉｎ血和再灌注,反复４次,于基础、缺血前、缺血１ｈ、再灌注０．５和２ｈ分别采集冠状窦血作式细胞仪分析ＰＭＮＣＶＤ１１ＣＤ１８的表达,并一单纯ＩＲ组比较。ＩＲ组在心肌缺因与再灌注各时点ＰＭＮＣＤ１１ｂ／ＣＤ１８表达均显著增加,而ＩＰ组除     

A novel point mutation in CD18 causing the expression of dysfunctional CD11/CD18 leucocyte integrins in a patient with leucocyte adhesion deficiency (LAD)

Leucocyte adhesion deficiency type 1 (LAD-1) is characterized by the incapacity of leucocytes to carry out their adhesion functions via their CD11/CD18 antigens, which are also referred to as the leucocyte integrins. The patients generally suffer from poor wound healing and recurrent bacterial and fungal infections. In severe cases, the infections are often systemic and life-threatening. A LAD patient (AW) of moderate phenotype has been identified but, unlike most other cases, the level of CD11/CD18 antigens on her leucocytes are uncharacteristically high for a LAD patient. Molecular analysis revealed that she is a compound heterozygote for CD18 mutations. She has inherited a D231H mutation from her father and a G284S mutation from her mother. By transfection studies, it was established that the G284S mutation does not support CD11/CD18 antigen expression on the cell surface. In contrast, the D231H mutation does not affect CD18 forming integrin heterodimers with the CD11 antigens on the cell surface. However, the expressed integrins with the D231H mutation are not adhesive to ligands.     

Increased expression and occupancy of receptors for tumour necrosis factor on blood monocytes from tuberculosis patients.

Blood monocytes from tuberculosis patients release high amounts of tumour necrosis factor-alpha (TNF-alpha). Because the biological efficiency of TNF-alpha would depend on the expression of TNF-alpha receptors on target cells, we thought to analyse the capacity of blood monocytes from a group of patients with pulmonary tuberculosis to bind 125I-TNF-alpha. We report a slight but not significant enhancement in specific binding of 125I-TNF-alpha on monocytes of 15 consecutively studied patients compared with 10 controls. Per cent cell surface bound and internalized 125I-TNF-alpha was identical in the two groups. To evaluate the receptor occupancy by endogenously generated TNF-alpha, similar experiments were performed after cell exposure to low-pH glycine buffer. Under these conditions, specific binding of 125I-TNF-alpha was significantly higher on tuberculosis monocytes compared with control monocytes. Moreover, the occupancy of TNF-alpha receptors by endogenously generated TNF-alpha that was found to be significantly higher on tuberculosis monocytes than on control monocytes, was directly related to the enhanced capacity of mononuclear cells to generate TNF-alpha in vitro. It normalized after 3 months of antituberculous therapy. Scatchard analysis of the binding data revealed that tuberculosis infection caused a significant increase in high affinity 125I-TNF-alpha binding to monocytes without any significant change in the dissociation constant. Collectively, these results indicate an up-regulation of TNF-alpha generation and binding to blood monocytes in patients with pulmonary tuberculosis. They provide support to the hypothesis that TNF-alpha is of critical importance in the pathogenesis of this infection.     

Characterization of four CD18 mutants in leucocyte adhesion deficient (LAD) patients with differential capacities to support expression and function of the CD11/CD18 integrins LFA-1, Mac-1 and p150,95.

Leucocyte adhesion deficiency (LAD) is a hereditary disorder caused by mutations in the CD18 (beta2 integrin) gene. Four missense mutations have been identified in three patients. CD18(A270V) supports, at a diminished level, CD11b/CD18 (Mac-1, alphaMbeta2 integrin) and CD11c/CD18 (p150,95, alphaXbeta2 integrin) expression and function but not CD11a/CD18 (LFA-1, alphaLbeta2 integrin) expression. Conversely, CD18(A341P) supports a limited level of expression and function of CD11a/CD18, but not of the other two CD11/CD18 antigens. CD18(C590R) and CD18(R593C) show a decreasing capacity to associate with the CD11a, CD11c and CD11b subunits. Transfectants expressing the CD11a/CD18 with the C590R and R593C mutations are more adhesive than transfectants expressing wild-type LFA-1, and express the reporter epitope of the monoclonal antibody 24 constitutively. Thus, the four mutations affect CD18 differently in its capacities to support CD11/CD18 expression and adhesion. These results not only provide a biochemical account for the clinical diversity of patients with leucocyte adhesion deficiency, but also offer novel insights into the structural basis of interaction between the alpha and beta subunits, which is an integral component in our understanding of integrin-mediated adhesion and its regulation.     

Anti-CD11b/CD18 antibodies reduce inflammation in acute colitis in rats.

The influx of monocytes and neutrophils into the inflamed tissue could be an important aspect in the pathogenesis of inflammatory bowel disease (IBD). A membrane protein involved in the monocyte/neutrophil adherence to endothelium is CD11b/CD18 or alpha M beta 2 (complement receptor type 3 = CR3). In the present study the role of CD11b/CD18 in experimental IBD was studied by treatment with ED7 and OX42, two MoAbs against CD11b/CD18. Colitis was induced in rats by a single, rectal administration of 30 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol 30%. Two hours before and 3 days after induction of colitis, the animals were given an i.v. dose of 0.5 mg of either ED7 or OX42 in 1 ml PBS. Controls received PBS or an irrelevant MoAb. Four days after the last treatment with the antibodies, the rats were killed, and macroscopic damage scores of the colon were determined. Macrophages and granulocytes were studied by immunohistochemistry and quantified by Interaktives Bild Analysen System (IBAS), and myeloperoxidase (MPO) activity in colonic tissue was measured. After treatment with ED7 and OX42 the mean damage score of the colon was reduced from 4.2 in IBD animals to 1.0 and 1.3, respectively. Smaller areas of ulcerations and a decrease in the number of ulcerations were observed compared with PBS-treated rats. Furthermore, the amount of infiltrating monocytes and leucocytes in the submucosa was enormously reduced, as well as MPO activity in the colonic tissue. These results show that treatment with MoAbs against CD11b/CD18 reduces clinical signs of experimental IBD in rats by a partial blockade of infiltrating macrophages and granulocytes.     

In vitro production of tumour necrosis factor and prostaglandin E2 by peripheral blood mononuclear cells from tuberculosis patients.

We investigated the production of tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy controls. PBMC from tuberculosis patients generated constitutively more TNF-alpha than did control PBMC. This production was significantly higher for patients with high-grade fever and cachexia. The increase of TNF-alpha production by PBMC from tuberculosis patients was associated with a comparatively weaker elevation of PGE2 synthesis which did not parallel fever or weight loss. In vitro treatment of control PBMC with the tuberculin purified protein derivative (PPD) promoted an increased TNF-alpha production which was similar to that of untreated PBMC from tuberculosis patients. Thus, the increased TNF-alpha production in tuberculosis could be explained by the in vivo exposure of PBMC to mycobacterial antigens. In contrast, the concentration of PGE2 was weaker in the medium of untreated PBMC from tuberculosis patients than in the medium of PPD-treated control PBMC, suggesting that PGE2 synthesis by PBMC was limited in tuberculosis by unidentified factors.     

CD123+CD11c-pDC在初发类风湿关节炎患者外周血及关节液中的表达

目的:探讨外周血及关节液中浆细胞样树突状细胞(Plasmaetoid dendritic cell,pDC)的数量在初发类风湿关节炎(RA)患者中的表达,进一步明确pDC在类风湿关节炎发病机制中的作用.方法:采集RA组患者(30人),骨关节炎(0A)组(25人),健康对照组(25人)抗凝外周血及关节液,流式细胞术检测CD123+ CD11c-pDC的表型及数量.结果:活动性RA患者外周血中CD123+ CD11c-pDC(1.69％±0.97％)与OA组(5.38％±2.31％)相比含量减少,差异具有统计学意义(P＜0.05),与健康对照组(5.63％±2.33％)相比含量减少,差异具有统计学意义(P＜0.05),后两者相比差异无统计学意义(P＞0.05).关节液中两组CD123+ CD1 1c-pDC含量差异无统计学意义(P＞0.05),pDC与DAS28评分及疾病的活动度指标RF、ESR、CRP无相关性.结论:初发RA患者体内外周血中CD123+ CD1 1c pDC含量减少与机体免疫功能紊乱有关,这对进一步阐明RA的发病机制及探索新的靶向治疗起到一定的积极作用.     

A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50

We have made a monoclonal anti-CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM-1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM-1 and ICAM-3. The binding to ICAM-2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand-specific manner by engagement of CD44.     

Evidence for cell surface association of CD2 and LFA-1 (CD11a/CD18) on T lymphocytes

Previous studies have reported an association of the cell surface adhesion molecule CD2 with the T cell receptor and with CD45 on mouse and human T lymphocytes. In this study the association of CD2 with cell surface molecules was investigated using cell surface biotinylation of T lymphocytes, coupled with immunoprecipitation using two CD2-specific monoclonal antibodies (mAb) (RM2–5 and 12–15) and analysis by SDS-PAGE. Although both CD2 mAb immunoprecipitated CD2 from lysates of murine lymphocytes, it was found that mAb 12–15, but not RM2–5, co-precipitated two other molecules of 95 and 180 kDa. Subsequent studies revealed that the 95- and 180-kDa molecules were associated with a subspecies of CD2 (? 5%) on thymocytes, the antigen-specific T cell line D10, and splenic T cells but not B cells. Two lines of evidence were obtained consistent with the 95- and 180-kDa molecules being the β and α chains of LFA-1. Firstly, an analysis of 12–15 mAb immunoprecipitates on 4–12% gels under reducing and nonreducing conditions shows that the 95- and 180-kDa molecules have a molecular weight and migration pattern identical to LFA-1. Secondly, depletion of LFA-1 from lysates with LFA-1 mAb abolished the ability of CD2 mAb 12–15 to co-precipitate the 95- and 180-kDa molecules, thereby identifying these as the β and α chains of mouse LFA-1, respectively. These results provide evidence for the first time for an association of LFA-1 and CD2 on mouse T lymphocytes, and suggest that the association occurs with an immunologically distinct subspecies of CD2 molecules.     

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.     

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the α chain of the β₂ integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD 18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activites of murine myeloid cells.     

严重烧伤病人PMNCD11b/CD18受体表达及特异性iRNA对其调节作用

本文探讨了严重烧伤对病人外周血中性粒细胞 (PMN)CD11b/CD18受体表达的影响及特异性免疫核糖核酸 (iRNA)对其调节作用。结果发现 :(1)严重烧伤病人PMNCD11b/CD18受体表达率明显下降 ,至伤后第 10天时 ,分别只有正常的6 7 1%和 6 8 9% ,且其下降程度与烧伤面积成正比 ;(2 )伤后早期应用特异性iRNA可明显提高烧伤病人PMNCD11b/CD18受体表达率 ;(3)临床观察发现 ,治疗组伤后创面细菌培养阳性率 ,创面脓毒症及菌血症的发生率明显低于对照组 (P均 <0 0 5 ) ,创面愈合时间明显短于对照组 (P <0 0 1)。该结果为临床应用特异性iRNA防治烧伤后感染提供了实验依据。     

Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis.

Studies have implicated tumour necrosis factor-alpha (TNF-alpha) in type-II collagen (CII)-induced arthritis (CIA), a well established animal model of human rheumatoid arthritis. Precisely how TNF is involved in CIA is not yet clear. In this study the effects of TNF on CIA were examined, independent of its potential effects on the immune response, by performing peri-articular injection of TNF in combination with passive immunization of rats. A sub-arthritic dose (5 mg) of affinity-purified anti-CII IgG, which alone was insufficient to induce spontaneous clinical arthritis, was used throughout the study. Obvious clinical arthritis that persisted for several days was rapidly induced by injections of 100 ng TNF into hindpaws of rats that were passively immunized shortly before the TNF injection. Injections of TNF in non-immunized control rats did not induce clinical arthritis, nor did buffer-only injections in passively immunized controls. The clinical arthritic response was a local phenomenon, limited only to the TNF-injected hindpaws. No swelling was observed in the opposite, buffer-injected hindpaws, indicating the effects of TNF were not systemic. Depletion of peripheral blood phagocytes with anti-rat neutrophil antiserum before passive immunization completely abolished the ability of TNF to induce clinical arthritis, identifying phagocytic cells as the essential target cells in evoking this arthritic response. A role for complement activation was also demonstrated in this model through the use of a soluble recombinant version of CD35, the cell surface complement receptor type-1 (sCR1, BRL55730), which significantly reduced TNF-induced arthritis in phagocyte-replete rats.     

Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease.