Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-gamma or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-gamma producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-gamma alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-gamma- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-gamma and IL-4 producers.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4⁺ memory T cells, whereas CD4⁺ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4⁺ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α. A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.     

Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-γ producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-γ production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-γ production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-γ production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.     

Interleukin-15 and interferon-γ participate in the cross-talk between natural killer and monocytic cells required for tumour necrosis factor production

We have characterized the lymphocyte subset and the receptor molecules involved in inducing the secretion of TNF by monocytic cells in vitro. The TNF secreted by monocytic cells was measured when they were co-cultured with either resting or IL-15-stimulated lymphocytes, T cells, B cells or natural killer (NK) cells isolated from the peripheral blood of healthy subjects and from the synovial fluid from patients with inflammatory arthropathies. Co-culture with IL-15-activated peripheral blood or synovial fluid lymphocytes induced TNF production by monocytic cells within 24 hours, an effect that was mainly mediated by NK cells. In turn, monocytic cells induced CD69 expression and IFN-γ production in NK cells, an effect that was mediated mainly by β₂ integrins and membrane-bound IL-15. Furthermore, IFN-γ increased the production of membrane-bound IL-15 in monocytic cells. Blockade of β₂ integrins and membrane-bound IL-15 inhibited TNF production, whereas TNF synthesis increased in the presence of anti-CD48 and anti-CD244 (2B4) monoclonal antibodies. All these findings suggest that the cross-talk between NK cells and monocytes results in the sustained stimulation of TNF production. This phenomenon might be important in the pathogenesis of conditions such as rheumatoid arthritis in which the synthesis of TNF is enhanced.     

TH-17 cells in rheumatoid arthritis

Introduction  

The aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.     

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression,but little interleukin-22 and interleukin-23R expression

Introduction  

Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity.     

Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-γ and counteraction by interferon-γ

Introduction  

Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Since interferon (IFN)-γ inhibits Th17 cell development, IFN-γ receptor knockout (IFN-γR KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-γ on the effector mechanisms of IL-17 in an in vitro system was also investigated.     

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fcγ receptors (FcγR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil FcγRs were examined with a specific FcγR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of FcγRIIa and FcγRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative FcγR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.     

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-gamma mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4(-) cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-gamma than CD4+/CCR4(-) cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.     

RETRACTED ARTICLE: Mast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium

Introduction  

Mast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.     

Rheumatoid arthritis synovium contains plasmacytoid dendritic cells

We have previously described enrichment of antigen-presenting HLA-DR⁺ nuclear RelB⁺ dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123⁺HLA-DR⁺ plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c⁺ myeloid DCs and CD123⁺ pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB^-CD123⁺ DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB⁺CD123^- DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-γ, IL-10, and tumor necrosis factor α were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB⁺ synovial DCs. pDCs may contribute significantly to the local inflammatory environment.     

Cytokine, activation marker, and chemokine receptor expression by individual CD4(+) memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-gamma, tumor necrosis factor (TNF)-alpha, LT-alpha, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4(+) memory T cells, whereas CD4(+) cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4(+) cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-alpha. A correlation between expression of IL-10 and CCR7, LT-alpha and CCR6, IFN-gamma and CCR5, and TRANCE and CXCR4 was also detected.     

Inhibition of nuclear factor kappa B (NFκB) activity in oral tumor cells prevents depletion of NK cells and increases their functional activation

The aim of this study is to identify candidate factors which may be responsible for the functional inactivation and depletion of NK cells by tumor cells. Inhibition of NFκB activity by an IκB super-repressor in HEp2 cells, a cell line commonly used as an oral tumor model, blocked tumor-induced NK cell death, and increased the function of NK cells significantly. Increased expression of CD69 early activation antigen on NK cells as well as augmented proliferation and secretion of IFN-γ by NK cells were observed when these cells were co-incubated with IκB super-repressor transfected HEp2 cells (HEp2-IκB_(S32AS36A)). More importantly, the secretion of IL-6 was significantly inhibited when NK cells were co-cultured with HEp2-IκB_(S32AS36A) cells. In addition, the survival and function of cytotoxic effector cells remained significantly elevated in the presence of IFN-γ-treated HEp2-IκB_(S32AS36A) cells when compared to either untreated or IFN-γ-treated vector-alone transfected HEp2 cells. Similar findings to those obtained using purified peripheral blood NK cells were also observed when non-fractionated peripheral blood mononuclear cells were used in the co-cultures of immune effectors with HEp2 cell transfectants. Addition of recombinant human IL-6 to the co-cultures of immune effectors with the NFκB knockdown HEp2 tumor cells substantially decreased the levels of secreted IFN-γ. Thus, the results presented in this paper suggest that the inhibition of NFκB function in oral tumors may serve to activate and expand the function and numbers of NK cells. Moreover, NFκB-mediated increase in IL-6 secretion by oral tumors may in part be responsible for the observed inactivation and death of the immune effectors.This work was supported by RO1-DE12880 from NIDCR-NIH.     

Regulation of pathogenic IL-17 responses in collagen-induced arthritis: roles of endogenous interferon-gamma and IL-4

Introduction  

Interleukin (IL)-17 plays an important role in the pathogenesis of rheumatoid arthritis and the mouse model collagen-induced arthritis (CIA). Interferon(IFN)-γ and IL-4 have been shown to suppress Th17 development in vitro, but their potential immunoregulatory roles in vivo are uncertain. The goals of this study were to determine the relationship between Th17 responses and disease severity in CIA and to assess regulation of IL-17 by endogenous IFN-γ and IL-4.     

Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis

Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.     

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo

Background We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)⁺ T cells is reflected by changes of selected immune parameters measured in peripheral blood. Methods ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)⁺ T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. Results Prior to infusion, scFv(G250)⁺ T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX⁺ RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-γ were produced, whilst production of TNF-α, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-γ was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-γ increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. Conclusion ScFv(G250)-receptor-mediated functions of the scFv(G250)⁺ T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-γ levels.     

Interferon-γ inhibits interleukin-1β-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis

Introduction  

The first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-γ (IFN-γ) during early inflammatory arthritis.     

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions

The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product.