Junctional adhesion molecule C mediates leukocyte adhesion to rheumatoid arthritis synovium

OBJECTIVE: Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint. METHODS: Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed. RESULTS: JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C. CONCLUSION: Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA.     

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. METHODS: Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. RESULTS: Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. CONCLUSION: These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.     

Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1.

Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti-VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation.     

Postanoxic T lymphocyte-endothelial cell interactions induce tumor necrosis factor-alpha production and neutrophil adhesion: role of very late antigen-4/vascular cell adhesion molecule-1

The objective of this study was to define the influence of postanoxic T-lymphocyte-endothelial cell interactions on anoxia-reoxygenation (A/R)-induced neutrophil-endothelial cell adhesion and cell adhesion molecule (CAM) expression on human umbilical vein endothelial cells (HUVECs). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 24 hours of reoxygenation, wherein freshly isolated human T lymphocytes were added at 6 hours during reoxygenation. After an additional 18 hours of incubation (ie, total of 24 hours of reoxygenation), the T-cell/endothelial cell (TC/EC) coculture media were collected and added to naive HUVEC monolayers incubated with neutrophils. Although the A/R-conditioned media per se had no effect on neutrophil adhesion, the media from TC/EC cocultures significantly increased the adhesion response. This enhanced adhesive interaction was associated with significant increases in tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) levels in the TC/EC coculture media and was accompanied by a pronounced increase in endothelial E-selectin expression. Treatment of the TC/EC coculture media with anti-TNF-alpha or anti-IL-8 antibodies reduced the media-induced neutrophil adhesion response. The enhanced neutrophil adhesion and the elevated medium levels of TNF-alpha, but not IL-8, were markedly reduced by inserts that prevented direct TC/EC contact and by monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) or very late antigen-4 (VLA-4). Collectively, these findings show that VLA-4-/VCAM-1-mediated interactions between T lymphocytes and postanoxic endothelial cells stimulates TNF-alpha production, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.     

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium

 Adhesion of CD34⁺ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34⁺ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34⁺ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34⁺ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34⁺ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1β prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34⁺ cells to IL-1β-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34⁺ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against β₂-integrins. Stimulation with the β₁-integrin-activating mAb, 8A2, induced adhesion of CD34⁺ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34⁺ HPCs is necessary for adhesion of CD34⁺ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion. Received: 26 April 1999 / Accepted: 8 February 2000     

MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1

Adhesion molecules expressed by activated endothelial cells play a key role in regulating leukocyte trafficking to sites of inflammation. Resting endothelial cells normally do not express adhesion molecules, but cytokines activate endothelial cells to express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which mediate leukocyte adherence to endothelial cells. We now show that endothelial cells express microRNA 126 (miR-126), which inhibits VCAM-1 expression. Transfection of endothelial cells with an oligonucleotide that decreases miR-126 permits an increase in TNF-alpha-stimulated VCAM-1 expression. Conversely, overexpression of the precursor to miR-126 increases miR-126 levels and decreases VCAM-1 expression. Additionally, decreasing endogenous miR-126 levels increases leukocyte adherence to endothelial cells. These data suggest that microRNA can regulate adhesion molecule expression and may provide additional control of vascular inflammation.     

Serum vascular endothelial growth factor in late rheumatoid arthritis.

OBJECTIVES: To investigate the serum levels of VEGF in patients with rheumatoid arthritis of long duration. METHODS: Serum VEGF levels were measured in 118 patients with long-standing rheumatoid arthritis according to the ACR criteria (mean duration 12 years). The disease activity score was evaluated by the method of van der Heijde et al. RESULTS: Serum levels of VEGF in patients with RA were significantly higher than in healthy controls. VEGF levels showed no correlation with CRP, SAA amyloid protein, or the disease activity score. CONCLUSIONS: Our findings suggest that, contrary to the results reported in patients with early onset RA, where VEGF appears to play an active part in joint inflammation, in long-standing RA elevated VEGF serum levels may be an independent marker although its significance remain to be established.     

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium

Objective. To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. Methods. Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. Results. Treatment of HSE with interleukin-1α (IL-1α) or tumor necrosis factor α (TNFα) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-γ (IFNγ) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFNγ and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFNγ also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNFβ/lymphotoxin. Immunoprecipitation of TNFα + IFNγ–stimulated, ¹²⁵I-labeled HSE cells with anti–ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFNγ alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. Conclusion. These studies indicate that IFNγ plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.     

Follicular non-Hodgkin's lymphoma cell adhesion to normal germinal centers and neoplastic follicles involves very late antigen-4 and vascular cell adhesion molecule-1.

Follicular lymphomas recapitulate the architecture of germinal centers (GCs) of normal secondary lymphoid follicles. Using an in vitro binding assay, it has recently been demonstrated that the normal B lymphocytes bind to GCs. This interaction is mediated by a receptor-ligand pair consisting of the beta 1 integrin very late antigen 4 (VLA-4) on the B cell, and the vascular cell adhesion molecule-1 (VCAM-1) expressed on follicular dendritic cells (FDC). Considering the similarities between follicular lymphomas and normal GCs, the adhesive interaction of follicular non-Hodgkin's lymphoma (NHL) cells and GCs was examined. Cells isolated from 16 of 24 cases of follicular NHL bound to normal GCs. Neoplastic follicles could similarly support the binding of follicular NHL cells. This adhesion was inhibited by monoclonal antibodies (MoAbs) directed against VLA-4 and VCAM-1. This supports the hypothesis that the neoplastic follicles used the identical adhesive interactions responsible, at least in part, for the localization of normal B cells to GCs. Adhesion receptors have an important role in the regulation of normal lymphoid cell proliferation, differentiation, and localization. Therefore, an understanding of the adhesive interaction of follicular NHL cells with GCs may provide insight into the clinical and biologic behavior of these diseases.     

Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species

Lymphocytes bound at endothelial cell junctions extravasate within minutes. Lymphocyte-endothelial cell binding is mediated by receptors such as vascular cell adhesion molecule 1 (VCAM-1). VCAM-1 activates endothelial cell nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in minutes, and this activity is required for VCAM-1-dependent lymphocyte migration. In this report, we examined mechanisms for activation of matrix metalloproteinases (MMPs) during VCAM-1-dependent lymphocyte migration. Lymphocyte binding to VCAM-1 rapidly activated endothelial cell-associated MMPs. Furthermore, inhibition of MMPs on the endothelial cells but not on the lymphocytes blocked VCAM-1-dependent lymphocyte migration across endothelial cells. The activation of endothelial cell MMPs required VCAM-1-stimulated endothelial cell NADPH oxidase activity as determined by scavenging of reactive oxygen species (ROS) and by pharmacologic or antisense inhibition of NADPH oxidase. Exogenous addition of 1 microM H(2)O(2), the level of H(2)O(2) generated by VCAM-1-stimulated endothelial cells, rapidly activated endothelial cell-associated MMPs. In contrast, activation of lymphocyte-associated MMPs was delayed by hours after binding to VCAM-1, and this activation was blocked by inhibition of endothelial cell ROS generation. There was also a delay in H(2)O(2)-induced decrease in lymphocyte-associated tissue inhibitors of metalloproteinases (TIMPs), resulting in an increase in MMP/TIMP ratio. In summary, this is the first report of a mechanism for ROS function in VCAM-1 activation of endothelial cell MMPs during VCAM-1-dependent lymphocyte migration.     

Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin

Recent studies suggested that trafficking of hematopoietic progenitor cells is related to cell cycle status. We studied whether adhesion of progenitor cells to extracellular matrix proteins was modulated by cell cycle transit.Mobilized peripheral blood CD34+ cells were stimulated ex vivo for 48 hours with stem cell factor, flt-3 ligand, and thrombopoietin and fractionated by adhesion to fibronectin or vascular cell adhesion molecule-1 (VCAM-1). Adherent and nonadherent cells were assayed for cell cycle status, long-term culture-initiating cell frequency, and integrin function. Binding to fibronectin, but not to VCAM-1, displayed a cell cycle selectivity as the adherent fraction to fibronectin was enriched in cycling CD34+ cells and in cycling long-term culture-initiating cells compared to the nonadherent fraction. Combined cell cycle and phenotypic analysis showed that the expression of VLA-5 was upregulated during S/G2+M but that of VLA-4 remained constant. The selective binding of cycling CD34+ cells to fibronectin was reverted by anti-VLA-5 but not by anti-VLA-4 blocking antibodies. Also, cycling CD34+ cells preferentially adhered to the VLA-5 binding domain but not to the VLA-4 binding domain of fibronectin. Adhesion of cycling CD34+ cells to fibronectin was a reversible process modulated by cell cycle progression, because adherent cells could exit the cell cycle and return to a nonadhesive state within an additional 48-hour culture period.The results indicate that the enhanced binding capacity of cycling progenitor cells to fibronectin is mediated by VLA-5.     

Vascular cell adhesion molecule 1 induces T-cell antigen receptor-dependent activation of CD4+T lymphocytes.

Effective stimulation of CD4+ T cells in an immune response depends on activation signals transduced via not only the CD3-T-cell receptor (TCR) complex but also those generated by accessory cell-surface proteins, including some that mediate adhesion between T cells and antigen-presenting cells (APC). Three members of the Ig superfamily, CD54 [intercellular cell adhesion molecule 1 (ICAM-1)], CD58 [lymphocyte function-associated antigen 3 (LFA-3)], and B7, expressed on the surface of APC, have been shown to mediate both adhesion and signaling during T cell-APC interactions. Recently another member of the Ig superfamily, [vascular cell adhesion molecule 1 (VCAM-1; INCAM110)], has been identified. VCAM-1 mediates adhesion between endothelial cells and activated lymphocytes and certain tumor cells. Here, using a soluble VCAM-1 fusion protein with receptor globulin (Rg), we examined the role of VCAM-1 in T-cell activation. We observed that CD4+ T cells, which are inefficiently stimulated by immobilized anti-TCR-1 or anti-CD3 monoclonal antibody (mAb) alone, can be induced to proliferate when exposed to immobilized VCAM-1-Rg in conjunction with either immobilized anti-TCR-1 or immobilized anti-CD3 mAb. The costimulatory effects of VCAM-1-Rg on CD4+T cells is inhibited by mAb to either the CD29 (integrin beta 1)-CD49d [very late activation antigen 4 alpha (VLA-4 alpha)] complex on the surface of CD4+ T cells or to VCAM-1. Stimulation of CD4+ T cells with immobilized VCAM-1-Rg and anti-TCR or -CD3 mAb results in the synthesis of both interleukin 2 (IL-2) receptors and IL-2. In addition, anti-CD25 (anti-IL-2 receptor a) mAb significantly inhibited the VCAM-1-Rg/anti-TCR or -CD3 mAb-driven activation of CD4+ T cells, indicating that endogenously produced IL-2 is in part responsible for the observed T-cell proliferation. Collectively, these results suggest that VCAM-1 can play an important costimulatory role during the activation of CD4+ T cells.     

趋化素样因子1在类风湿关节炎滑膜中的表达研究

目的研究趋化素样因子1(CKLF1)在类风湿关节炎(RA)滑膜中的表达,探讨CKLF1在RA发病过程中的可能的病理作用。方法取35例RA,20例骨关节炎(OA)及20例半月板损伤患者手术切除的膝关节滑膜标本。采用反转录-聚合酶链反应(RT-PCR)方法检测CKLF1在上述3种病变滑膜中的mRNA水平表达水平。采用免疫组织化学方法检测CKLF1在滑膜组织中的蛋白水平表达特征。结果RA滑膜组织中CKLF1在mRNA水平的表达(0．41±0．17)明显高于OA(0．07±0．06)和半月板损伤患者(0．16±0．10)(P<0．05)。免疫组织化学结果显示,OA病例中仅有2例显示少量滑膜衬里层细胞着色,半月板损伤病例仅有5例显示少量滑膜衬里层细胞着色。在RA滑膜中20例染色强阳性,10例染色阳性,5例染色阴性。阳性染色颗粒位于细胞胞质内,阳性细胞定位在滑膜衬里层的成纤维细胞、滑膜下层大量的浆细胞以及增生的血管上皮细胞。结论CKLF1基因在RA滑膜中有高水平的表达特征,为进一步深入研究RA的发病机制提供新的研究思路。     

Regulation of synovial B cell survival in rheumatoid arthritis by vascular cell adhesion molecule 1 (CD106) expressed on fibroblast-like synoviocytes

OBJECTIVE: B lymphocytes accumulate in the inflamed joints of patients with rheumatoid arthritis (RA) and are responsible for production of high amounts of (auto)-antibodies. The aim of this study was to determine the capacity of fibroblast-like synoviocytes (FLS) to contribute to the accumulation of synovial fluid (SF) B cells by extending their life span. METHODS: Highly purified SF B cells were cultured with FLS in the presence or absence of blocking antibodies directed against cell adhesion molecules, and cell viability was determined after various time intervals by trypan blue, annexin V, propidium iodide, or Hoechst staining. Phenotypic characterization of peripheral blood and SF B cells and FLS was carried out by flow cytometry. RESULTS: Synovial B cells, which consist predominantly of memory B cells and plasma cells (PC), undergo spontaneous cell death by apoptosis upon removal from their in vivo environment, despite expression of Bcl-2. Coculture with FLS rescued synovial B cells from apoptosis in a cell contact-dependent manner. Blocking studies using monoclonal antibodies demonstrated a role for the molecular interaction of SF B cells with vascular cell adhesion molecule 1 (VCAM-1; CD106) in FLS-induced survival. The ability of FLS to induce SF B cell survival was not related to the rheumatoid origin since FLS from non-RA patients had similar properties. CONCLUSION: These findings indicate a crucial role for FLS in the survival of synovial B cells at the site of inflammation in RA through the interaction with VCAM-1 expressed on FLS. Consequently, memory B cells and PC accumulation arise and persist not only as a result of maturation and recruitment of these cells, but also by active prevention from cell death by the microenvironment.     

Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds

Background—Inflammatory boweldiseases (IBD) are characterised by an intense infiltration ofleucocytes that is mediated by adhesion molecules expressed on thesurface of activated endothelial cells.
Aims—To determine whether drugsused in the treatment of IBD, specifically dexamethasone (DEX),5-aminosalicylic acid (5-ASA), methotrexate (MTX), and 6-mercaptopurine(6-MP), alter the expression of endothelial cell adhesion molecules (ECAMs).
Methods—The expression ofP-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1), andvascular CAM 1(VCAM-1) in different vascular beds of C57Bl/6J mice wasmeasured using the dual radiolabelled monoclonal antibody technique.
Results—Lipopolysaccharide (LPS)elicited a profound increase in the expression of all ECAMs in themesentery, small intestine, caecum, and distal colon. The LPS inducedincrease in CAM expression was not significantly affected by priortreatment with either MTX or 6-MP. However, pretreatment with eitherDEX or 5-ASA significantly attenuated LPS induced increases inexpression of P- and E-selectin, and VCAM-1 in the majority of tissuesevaluated. DEX also blunted the LPS induced increase in ICAM-1expression in the caecum and distal colon. DEX, but not 5-ASA, largelyabolished the rise in plasma tumour necrosis factor α elicited by LPS.
Conclusions—These findings suggestthat DEX and 5-ASA may exert their beneficial therapeutic action inIBD, at least in part, by inhibiting the expression of ECAMs whichmediate leucocyte adhesion and transmigration in the microvasculature.

     

Treatment of refractory rheumatoid arthritis with a monoclonal antibody to intercellular adhesion molecule 1

Objective. To assess the safety and efficacy of a monoclonal antibody (MAb) to intercellular adhesion molecule 1 (ICAM-1; CD54) in rheumatoid arthritis (RA). Methods. A phase I/II, open-label, dose-escalation study of 32 patients. Results. During treatment, a peripheral CD³⁺/CD4+ lymphocytosis was noted, and several patients demonstrated transient cutaneous anergy, which suggests that therapy modified T cell recirculation. Thirteen of the 23 patients who received 5 days of treatment demonstrated clinical improvement through day 29, and 9 of 23 through day 60. Adverse effects were minor and transient. Conclusion. Anti—ICAM-1 MAb therapy was well tolerated, resulted in a transient alteration in T lymphocyte recirculation, and effected clinical improvement in some RA patients.