Regulation of CXCL16 expression and secretion by myeloid cells is not altered in rheumatoid arthritis.

OBJECTIVE: Chemokine (C-X-C motif) ligand 16 (CXCL16) is secreted by macrophages and dendritic cells (DCs) to attract memory type T cells. CXCL16 expression is increased in arthritic joints of patients with rheumatoid arthritis (RA) and a role for CXCL16 has been suggested in the pathogenesis of RA. To date, little is known about the regulation of CXCL16 on monocytes/macrophages and DCs. The aim of this study was to elucidate how CXCL16 expression is regulated in healthy donors and patients with RA. METHODS: CD14+cells were isolated from the peripheral blood or synovial fluid of patients with RA and healthy controls, differentiated into different types of dendritic cells or macrophages and stimulated with various cytokines or lipopolysaccharide (LPS). Cell surface proteins, including surface CXCL16, were measured by flow cytometry and soluble CXCL16 was measured by ELISA. RESULTS: Distinct types of dendritic cells constitutively express and secrete CXCL16, which is not affected by maturation. Monocytes rapidly upregulate membrane-bound CXCL16 expression and release soluble CXCL16 upon culture. CXCL16 expression by monocytes is transiently inhibited by the Toll-like receptor (TLR)4 ligand LPS. Th2 type cytokines inhibit soluble CXCL16, whereas T helper (Th)1 cell stimulus enhances its release. In RA monocytes/macrophages, neither CXCL16 expression, nor CXCL16 regulation is different from healthy controls. CONCLUSIONS: Culture of monocytes is the main trigger for CXCL16 surface expression in vitro, which is not altered in RA. Together our data suggest that the increased CXCL16 expression in patients with RA is likely to be caused by increased influx of monocytes rather than intrinsic differences in CXCL16 regulation.

Increased expression of CCL18, CCL19, and CCL17 by dendritic cells from patients with rheumatoid arthritis, and regulation by Fc gamma receptors.

BACKGROUND: Dendritic cells (DC) have a role in the regulation of immunity and tolerance, attracting inflammatory cells by the production of various chemokines (CK). Fc gamma receptors (Fc gamma R) may be involved in regulation of the DC function. OBJECTIVE: To assess the expression of CK by immature (iDC) and mature DC (mDC) and its regulation by Fc gamma R in patients with RA and healthy donors (HC). METHODS: Expression of CK by DC from patients with RA and from HC was determined by real time quantitative PCR and ELISA. DC were derived from monocytes following standardised protocols. To study the potential regulation by Fc gamma R, iDC were stimulated with immune complexes (IC) during lipopolysaccharide (LPS) induced maturation. The presence of CK was studied in synovial tissue from patients with RA, osteoarthritis, and healthy subjects by RT-PCR and immunohistochemistry. RESULTS: iDC from patients with RA had markedly increased mRNA levels of the CK CCL18 and CXCL8. Upon maturation with LPS, expression of CCL18, CCL19, CXCL8, CCL3, and CCL17 increased dramatically, reaching significantly higher levels in patients with RA. Monocytes failed to express these CK, except for CXCL8 and CCL3. IC-mediated triggering of the Fc gamma R on DC from patients with highly active RA down regulated all CK, whereas the reverse was seen when DC from patients with low disease activity and healthy donors were stimulated. CCL18 was significantly increased in RA synovial tissue. CONCLUSION: Increased CK expression by DC was found in patients with RA. This expression is partly regulated by Fc gamma R triggering and results in an inhibitory DC subtype in RA upon Fc gamma R-mediated triggering.

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer.

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans.

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.

The hepatocyte growth factor/Met pathway in development, tumorigenesis, and B-cell differentiation.

This article summarizes the structure, signal transduction and physiologic functions of the HGF/Met pathway, as well as its role in tumor growth, invasion, and metastasis. Moreover, it highlights recent studies indicating a role for the HGF/Met pathway in antigen-specific B-cell development and B-cell neoplasia.

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.

Regulation of adhesion and migration in the germinal center microenvironment.

T cell dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell homing to the GC and interaction with FDC critically depend on integrin-mediated adhesion. We have recently indentified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signalling pathway regulating B cell adhesion (van der Voort et al., 1997, J. Exp. Med. 185, 2121-2131). The c-Met protein is expressed on B cells localized in the dark zone of the GC (centroblasts) and is induced by CD40 plus BCR ligation. Stimulation of c-Met with HGF/SF, which is produced at high levels by tonsillar stromal cells and FDC, leads to receptor phosphorylation and to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Interestingly, these responses to HGF/SF are promoted by heparan-sulfate proteoglycan forms of CD44 (CD44-HS). Like c-Met, CD44-HS is induced on B cells by CD40 ligation. It efficiently binds HGF/SF and strongly promotes signalling through c-Met. We conclude that integrin regulation during antigen specific B cell differentiation involves cross-talk between the HGF/SF-c-Met pathway and CD44-HS.

CD44 isoforms, including the CD44 V3 variant, are expressed on endothelium, suggesting a role for CD44 in the immobilization of growth factors and the regulation of the local immune response.

Endothelium plays a central role in the regulation of site and inflammation specific leukocyte migration. Some of the mediators involved in leukocyte migration, such as chemokines, can bind to heparan sulfate on the endothelium resulting in immobilization near their sites of production. Because CD44 variants expressing V3 have been shown to carry heparan sulfate side chains and to interact through these side chains with heparan sulfate binding growth factors, we investigated the expression of CD44 variants on endothelium. We found a strong expression of V5, V7-8 and V10 CD44 variants and a weaker expression of V3 and V6 CD44 variants on endothelium by using immuno-histochemistry and by FACS analysis. Expression of CD44 V3 variants was confirmed at both the protein and mRNA levels by Western blotting and by reverse transcriptase-PCR respectively. Expression of CD44 variants was unaffected by IL-1beta, IL-8, TNFalpha, IFNgamma or IL-4 treatment, indicating either constitutive expression of these variants or involvement of other cytokines in their regulation.

CD44 splice variants as prognostic markers in colorectal cancer.

BACKGROUND: Splice variants of CD44 play a causal role in the metastatic spread of pancreatic carcinoma in the rat. In previous studies we have shown that homologues of these CD44 isoforms (CD44v6) are overexpressed during colorectal tumorigenesis in man and that CD44v6 overexpression is associated with an unfavorable prognosis in this disease. In the present study we have assessed the prognostic significance of CD44 variants containing exon v5. In addition, we have used a panel of different antibodies against CD44v6 and applied a combined scoring system to improve its value as prognosticator. METHODS: Expression of CD44 variants was studied by immunohistochemistry on frozen tissue sections, and the prognostic value of the CD44 variant expression was assessed using univariate and multivariate analysis. RESULTS: Our studies show that expression of CD44v6, but not CD44v5, has significant prognostic value. Analysis of CD44v6 expression by means of a combined scoring system, on the basis of a panel of three different monoclonal antibodies (mAbs), makes CD44v6 a highly significant prognostic marker that is independent of Dukes stage, tumor grade, or tumor localization. CONCLUSION: Assessment of CD44v6 expression by a combination of mAbs yields an independent prognosticator that may be of value in identifying patients with a high propensity to develop distant metastasis.

Paracrine regulation of germinal center B cell adhesion through the c-met-hepatocyte growth factor/scatter factor pathway.

T cell-dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38(+)CD77(+) tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.

Preferential expression of the mucosal homing receptor integrin alpha 4 beta 7 in gastrointestinal non-Hodgkin's lymphomas.

Recent studies have identified the integrin alpha 4 beta 7 as a mucosal homing receptor that mediates lymphocyte migration to the intestinal mucosa by binding to MAdCAM-1, a vascular recognition molecule (addressin) selectively expressed on mucosal endothelium. In the present study, we have assessed the expression of alpha 4 beta 7 on B- and T-cell non-Hodgkin's lymphomas of different primary localization and on related normal lymphocytes. Among B-lineage lymphomas, expression of alpha 4 beta 7 was present in the majority of cases of malignant lymphomatous polyposis of the intestine and low-grade lymphoma of the mucosa-associated lymphoid tissue/monocytoid B-cell lymphoma and in some cases of precursor B-cell lymphoma. CLL/small lymphocytic lymphoma, (nodal) mantle cell lymphoma, follicular center cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma were virtually always alpha 4 beta 7 negative, as was the case when localized in the mucosa-associated lymphoid tissue. The normal B cells of the follicle mantles and part of the B cells of the extrafollicular B-cell compartment of lymphoid tissues expressed moderate levels of alpha 4 beta 7. By contrast, follicular center cells were alpha 4 beta 7 negative. Among T-lineage lymphomas, expression of alpha 4 beta 7 was also strongly related to the primary localization; in mucosal, nodal, and cutaneous T cell lymphomas the percentage of positive cases was 56%, 17%, and 0%, respectively. All cases of precursor T-cell lymphoma were alpha 4 beta 7 negative. High expression of alpha 4 beta 7 was found on a subset of peripheral blood memory T cells as well as on lymphocytes in the intestinal mucosa. We conclude that non-Hodgkin's lymphomas that are related to mucosa-associated B- and T-lymphocyte populations selectively express the mucosal homing receptor alpha 4 beta 7. The presence of this receptor underscores their distinctive character and may play an important role in determining their characteristic mucosal dissemination pattern.

Binding of cell-surface expressed CD44 to hyaluronate is dependent on splicing and cell type.

CD44 is a major cell-surface receptor for hyaluronate (HA). By alternative RNA-splicing a large number of CD44 variants are generated. To explore the role of CD44 splicing in the regulation of cell binding to HA, three different isoforms of CD44 were transfected in the CD44 negative B-cell lymphoma line Namalwa and in the fibroblastoid cell line COS7. We observed that whereas the standard form of CD44 (CD44s) mediated adhesion of Namalwa to HA, Namalwa transfected with CD44v3-10 or CD44v8-10 was unable to bind to either immobilized or soluble HA. After stimulation of CD44 with an activating anti-CD44 mAb or with phorbol ester, the binding of CD44s to HA was 5- to 10-fold higher than that of the other two isoforms. By contrast, COS7 cells transfected with CD44s, CD44v8-v10, or CD44v3-v10 bound equally effectively to HA. Hence, in addition to alternative splicing, cell type determines CD44 binding to HA.