TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines.

OBJECTIVES: To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. METHODS: Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. RESULTS: The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. CONCLUSIONS: These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

Preparation of mononuclear cells from synovial tissue.

In rheumatoid arthritis (RA), the synovium represents the predominant site of inflammation and joint destruction and is regarded as the key organ involved in disease pathogenesis. It has been studied in different ways over the last 30 yr, yielding information about the mechanisms involved in disease and remains the tool most proximal to understanding the pathogenesis of RA. This chapter outlines how both histological and in vitro studies of dissociated tissue played key roles in the development of biological anti-TNF-alpha therapy and provides detailed protocols used routinely in the laboratory to facilitate studies of RA synovium and its composite cell populations.

Presentation of a determinant by MHC class II can be prevented through competitive capture by a flanking determinant on a multideterminant peptide.

Competitive capture is a process by which different determinants of an unfolding antigen compete for binding to the same MHC class II molecule. The "winning" determinant is then dominantly displayed. For self antigens, T cells with specificity for dominantly displayed determinants will be subject to strong tolerance induction. With this in mind we set out to characterize the determinant hierarchy of the junctional region of the Golli-MBP complex. Within this region the MBP 1-9 determinant is known to be a strong inducer of experimental autoimmune encephalomyelitis. We found that the Golli-MBP junctional region contains a triad of three overlapping determinants: LDVM1-5, MBP 1-9, and MBP 7-20. We demonstrate that these three determinants are unique and compete for binding to I-A(u) and that a determinant hierarchy exists with MBP 7-20 being the most dominantly displayed determinant. Because of the prevention of MBP1-9 access to MHC-II, the residual T cell repertoire to this determinant remains complete, thereby permitting its highest affinity members to drive the response, and to convert MBP1-9 into a dominant determinant, despite its poor MHC binding capacity.

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function.

BACKGROUND: Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue. METHODS: Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4+CD45RO+, CCR7-, CD49dhigh population, and that these cells are derived from the effector memory CD4+ T cells in resting blood. RESULTS: After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-alpha production from monocytes stimulated with CD4+CD45RO+ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-alpha production in RA synovial mononuclear cell cultures. CONCLUSION: Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.

T-cell contact-dependent regulation of CC and CXC chemokine production in monocytes through differential involvement of NFkappaB: implications for rheumatoid arthritis.

We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFalpha). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFkappaB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IkappaBalpha, the natural inhibitor of NFkappaB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFalpha, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFkappaB-dependent and NFkappaB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.

A novel mechanism for TNF-alpha regulation by p38 MAPK: involvement of NF-kappa B with implications for therapy in rheumatoid arthritis.

TNF-alpha is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-alpha in primary human cells relevant to inflammation, e.g., macrophages and rheumatoid synovial cells. Using a dominant negative variant (D168A) of p38 MAPK and a kinase inhibitor, SB203580, we confirm in primary human macrophages that p38 MAPK regulates TNF-alpha production using a posttranscriptional mechanism requiring the 3' untranslated region of the gene. However, in LPS-activated primary human macrophages we also detect a second previously unidentified mechanism, the p38 MAPK modulation of TNF-alpha transcription. This is mediated through p38 MAPK regulation of NF-kappaB. Interestingly this mechanism was not observed in rheumatoid synovial cells. Importantly however, the dominant negative mutant of p38 MAPK, but not SB203580 was effective at inhibiting spontaneous TNF-alpha production in these ex vivo rheumatoid synovial cell cultures. These data indicate there are potential major differences in the role of p38 MAPK in inflammatory signaling that have a bearing on the use of this kinase as a target for therapy. These results indicate despite disappointing results with p38 MAPK inhibitors in the clinic, this kinase is a valid target in rheumatoid disease.