Interleukin-10 expression: is there a neglected contribution of CD8+ T cells in rheumatoid arthritis joints?

OBJECTIVE: To search for RA specific processes among T cell accumulation, T cell activation, or cytokine expression in CD4+ and CD8+ synovial fluid (SF) T cells. METHODS: Flow cytometry of CD4+, CD8+, CD45RA+, CD45RO+, CD69 double or triple stained peripheral blood (PB) and SF T cells. IL-2, IL-10, and IFN-gamma expression was determined in PMA + ionomycin stimulated T cells on the single cell level. Concentrations of secreted IL-2, IL-4, IL-10, and IFN-gamma were quantified in the sera and synovial fluids by enzyme linked immunosorbent assay (ELISA). RESULTS: A preferential recruitment of CD45RO+ memory T cells was found for CD4+ helper T cells, and in similar also for CD8+ suppressor T cells. An elevated CD69 expression was detected in memory, but also in CD45RA+ naive CD4+ and CD8+ SF T cells, whilst IL-2 expression was only demonstrable in a minor proportion of T cells populations. Preferential recruitment of memory T cells, but incomplete activation of naive and memory, CD4+ and CD8+ T cells were in similar found in RA and control patients. In RA but not in the control patients, a relevant proportion of CD4+ and CD8+ PB and SF T cells expressed IL-10 and IFN-gamma. High concentrations of IL-10, that were correlated with the amounts of secreted TNF-alpha, were only detected in RA joints. CONCLUSION: Memory and naive T cell state of CD4+ and CD8+ T cell accumulates in the joints, and early T cell activation occur in similar patterns in RA and control patients. High IL-10 SF concentrations in contrast, and elevated percentages of IFN-gamma and IL-10 expressing CD4+ and CD8+ T cells in the PB and SF were characteristic for RA. Here, CD8+ T cells may contribute to high IL-10 concentrations in RA joints.     

CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid

OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.     

Localized monocyte chemotactic protein-1 production correlates with T cell infiltration of synovium in patients with psoriatic arthritis

OBJECTIVE: To examine normal and psoriatic skin and synovial tissue from patients with psoriatic arthritis (PsA) for evidence of monocyte chemotactic protein-1 (MCP-1) mediated T cell chemotaxis. METHODS: Peripheral blood (PB), synovial fluid (SF), normal and psoriatic skin, and synovial biopsies were obtained from patients with PsA (n = 19) and compared to samples from normal (n = 5) and disease (n = 5) controls (NC, DC). Immune cell populations in PB and SF samples were assessed by immunofluorescent labeling and flow cytometry, levels of soluble MCP-1 were determined by quantitative ELISA, and immunohistochemistry was used to detect T cell subsets and macrophages and MCP-1 protein in frozen skin and synovial tissue sections. RESULTS: CD8+ but not CD4+ T cells were elevated in SF compared to PB, and the majority of these cells expressed CD45RO. Plasma MCP-1 levels in PsA were elevated relative to NC. MCP-1 levels were significantly higher than paired plasma samples in patients with recent onset (< 6 mo) synovitis (n = 10). A positive correlation was observed between synovial T cell numbers and MCP-1 levels in SF. MCP-1 protein was present in all tissues examined, but most intense expression was observed in synovium. CONCLUSION: Elevated concentrations of MCP-1 concomitant with memory T cell infiltration in PsA SF suggests that MCP-1 mediated chemotaxis is involved in the recruitment of T lymphocytes into the synovial compartment of patients with PsA.     

Phenotypes of T lymphocytes from peripheral blood and synovial fluid of patients with rheumatoid arthritis and juvenile rheumatoid arthritis. Evidence in favour of normal helper and suppressor functions of T lymphocytes from patients with juvenile rheumatoid arthritis

Lymphocytes from peripheral blood (PB) and synovial fluid (SF) from 21 patients with rheumatoid arthritis (RA) and 18 patients with juvenile rheumatoid arthritis (JRA) were studied with respect to T cell phenotypes using monoclonal antibodies in a rosette assay. The percentage of HLA-DR positive T cells was counted in PB and SF using indirect immunofluorescence. Suppressor cell activity of T cells from PB and SF was investigated by measuring the immunoglobulin production by pokeweed mitogen (PWM) stimulated B cells mixed with T cells at various ratios. The mean T4/T8 ratio was significantly lower in SF than in PB of both RA and JRA patients (p = 0.0062 and p less than 0.0001 respectively). The mean percentages of HLA-DR positive T cells were elevated in SF compared with PB in both patients groups (p less than 0.03 and p less than 0.04 in RA and JRA patients respectively). Mean suppressor cell activity and helper cell activity of T cells from SF and PB of JRA patients was normal. Thus there seems to be a dichotomy between the number of T8+ cells and suppressor cell function in mononuclear cells from SF of patients with JRA. This indicates that a considerable proportion of the T8+ cells in the SF do not have suppressor functions.     

Analysis of Th1 and Th2 cytokines expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry

OBJECTIVE: A Th1/Th2 cytokine imbalance with a predominance of Th1 cytokines has been suggested to be of pathogenetic importance in rheumatoid arthritis (RA). To evaluate the role of Th1/Th2 cytokines in RA, we used intracellular cytokine flow cytometry to determine cytokine profiles of CD4+ and CD8+ T cells in 34 peripheral blood (PB) and 10 synovial fluid (SF) samples from patients with RA. Results were compared with 10 PB samples from healthy controls (HC) and 5 SF samples from patients with non-RA synovitis. METHODS: After stimulating cells with PMA and ionomycin or alternatively with anti-CD3/CD28 in the presence of brefeldin A, intracellular levels of Th1 [interleukin 2 (IL-2), interferon-gamma (IFN-gamma)] and Th2 cytokines (IL-4, IL-5, IL-10, IL-13) were determined for CD3+CD8- (i.e., CD4+ Th1 and Th2 cells) and CD3+CD8+ (i.e., CD8+ Tc1 and Tc2 cells) T cells. RESULTS: The percentages of CD4+ and CD8+ Th1 and Th2 cytokines producing T cells (PB) were similar in patients with RA and healthy controls (HC), with a clear predominance of Th1 cytokines expressing, T cells. With regard to T cell subsets, IFN-gamma-producing T cells were significantly more frequently detected in the CD8+ subset [CD8+: median 45.1% (RA; p < 0.001), 38.2% (HC; p = 0.009) vs CD4+: 10.8%(RA), 17.0% (HC)]. Conversely, IL-2 was found in a higher percentage of CD4+ T cells [CD4+: median 33.4% (RA), 17.9% (HC) vs CD8+: 23.6% (RA), 12.3% (HC)]. Patients not in disease remission tended to have more IFN-gamma-producing CD8+ and IL-2-producing CD4+ T cells than patients in remission [CD8+: median 45.9% (IFN-gamma) vs 23.0% (IFN-gamma); CD4+: median 34.1% (IL-2) vs 18.2% (IL-2)1. In all PB samples, the proportion of T cells producing the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 did not exceed 2%. Cytokine profiles did not differ between patients receiving immunosuppressive treatment and patients treated only with nonsteroidal antiinflammatory drugs. In comparison to PB, RA SF analysis revealed a significant increase in the percentage of IFN-gamma-producing CD4+ (p < 0.001) and CD8+ T cells (p < 0.001). In addition, the percentage of IL-10-producing CD4+ (p < 0.001) as well as CD8+ T cells (p = 0.001) was significantly elevated in SF. However, production of the other Th2 cytokines (IL-4, IL-5, IL-13) was similar in SF and PB. CONCLUSION: These data indicate similar cytokine profiles of T cells in PB of RA patients and healthy controls, with a strong predominance of Th1 cytokines producing T cells in the CD4+ and CD8+ T cell subset of both groups. PB cytokine profiles did not significantly differ in patients with active and non-active disease or between patients receiving and those not receiving immunosuppressive medication. In SF, the proportion of Th1 and Tcl cells was significantly elevated compared to PB, emphasizing the local importance of these cells for inflammation. CD8+ T cells (Tc1 cells) mainly contributed to the production of IFN-gamma, indicating an underestimated role of this cell subset for local cytokine production. The upregulation of IL-10-producing Th2 and Tc2 cells in SF may reflect an insufficient effort to down-regulate chronic inflammation in the joint. Modifying this cytokine imbalance in the joints may be a promising therapeutic approach in RA.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation.

OBJECTIVE. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). METHODS. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. RESULTS. RA SF and ST T cells were found to be markedly enriched in CD45RAdim, CD45RO+, CD45RBdim mature memory cells, whereas in the PB, CD45RAbright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RBdim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3-stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RBdim phenotype and B cell help, CD45RBdim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RBdim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RBdim T cells with mitomycin C. In contrast, healthy control sorted CD45RBbright or sorted CD4+, CD45RO+, CD45RBbright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. CONCLUSION. RA synovium is enriched in differentiated CD45RBdim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.     

Coexistent naïve phenotype and higher cycling rate of cord blood T cells as compared to adult peripheral blood

OBJECTIVE: Umbilical cord blood (UCB) T cells are predominantly CD45RA(+), secrete less cytokines, and have diminished cytotoxicity compared to adult peripheral blood (PB). We hypothesized that the functional impairment of bulk UCB cells results from the relative dominance of immature lymphocyte subsets. In this study we established the physiologic ranges of lymphocyte subsets in UCB, and contrasted those with adult PB. MATERIALS AND METHODS: Four-color FACS was utilized to characterize surface and intracellular protein expression on lymphocyte subsets from fresh unmanipulated UCB and adult PB. RESULTS: We found that UCB contain significantly higher absolute numbers of T cells, NK cells, and B cells than adult PB (p<0.0001). UCB also contains more "na?ve" cells not only among CD4(+) and CD8(+) T cells but also among B lymphocytes (p=0.003). Most UCB T cells are CD45RA(+)/CD62L(+) "recent thymic emigrants" with smaller TCRgammadelta (p<0.0001) and CD25(+) subsets (p=0.0068). Fewer UCB T cells display HLA-DR and CCR-5 activation markers (p<0.0001) while the CD8(+)/CD57(+)/CD28(-) "suppressor," CD8(+)/CD45RA(+)/CD27(-) "cytotoxic," and skin homing CLA(+) T-cell subsets are absent altogether. Compared with adult PB, more cord blood T cells progress through cell cycle (p<0.0001) and enter apoptosis (p=0.0003). Unlike in adult PB, the majority of proliferating Ki-67(+) T cells in UCB retain a CD45RA(+)/RO(-), CD69(-), CD25(-), HLA-DR(-) "resting" phenotype (p=0.0002). CONCLUSION: Most T and B lymphocytes express a nai;ve phenotype in cord blood while "suppressor" and "cytotoxic" T-cell subsets are absent. Cycling UCB T cells retain a nai;ve immunophenotype that may represent homeostatic expansion rather than antigen-driven proliferation.     

Selective lymphocyte chemokine receptor expression in the rheumatoid joint.

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.     

CCR3, CCR5, interleukin 4, and interferon-gamma expression on synovial and peripheral T cells and monocytes in patients with rheumatoid arthritis

OBJECTIVE: To characterize cytokine and chemokine receptor profiles of T cells and monocytes in inflamed synovium and peripheral blood (PB) in patients with rheumatoid arthritis (RA) and other arthritides. METHODS: We studied PB and synovial fluid (SF) samples taken from 20 patients with RA and 9 patients with other arthritides. PB cells from 8 healthy adults were used as controls. CCR3, CCR5, and intracellular interferon-g (IFN-g) and interleukin 4 (IL-4) expression in CD8+ and CD8- T cell populations and in CD14+ cells were determined with flow cytometry. RESULTS: Expression of CCR5 and CCR3 by CD8-, CD8+ T cells and CD14+ monocytes was increased in SF compared to PB cells in patients with RA and other arthritides. The number of CD8+ T cells spontaneously expressing IL-4 and IFN-g was higher in SF than in PB in RA patients. Spontaneous CCR5 expression was associated with intracellular IFN-g expression in CD8+ T cells derived from SF in RA. In CD8- T cells the ratio of CCR5+/CCR3+ cells was increased in patients with RA compared to patients with other arthritides. The number of PB CD8- T cells expressing IFN-g after mitogen stimulation was higher in controls than in patients. In PB monocytes the ratio of CCR5+/CCR3+ cells was increased in patients with RA compared to patients with other arthritides and controls. CONCLUSION: T cells and monocytes infiltrating joints in RA and in other arthritides showed increased activation of both type 1 and type 2 immune markers. More pronounced type 1 immune response in joints, shown as an increased CCR5/CCR3 ratio, was found in the patients with RA versus those with other arthritides. Monocytes but not T cells in PB showed increased activation in RA.     

Phenotypic characteristics of dissociated mononuclear cells from rheumatoid synovial membrane

The phenotypic characteristics of enzymatically dissociated synovial membrane mononuclear cells from 8 patients (14 samples) with rheumatoid arthritis (RA) were assessed by fluorescence activated flow cytometry and compared to peripheral blood (PB) mononuclear cells from 18 patients with RA and 14 normal controls. There was no significant difference between the percentage of CD4+ and CD8+ lymphocytes in synovial membrane compared to RA and normal PB. Double labelling experiments revealed similar percentages of CD4+ CDw29+ (helper-inducer) and CD4+ CD45R+ (suppressor-inducer) cells in RA and normal PB. In contrast, SM CD4+ CDw29+ cells were present in significantly higher proportions than in RA and normal PB (p less than 0.001). Conversely, synovial membrane CD4+ CD45R+ cells were present in significantly lower proportions than in RA and normal PB (p less than 0.001). A similar pattern of CDw29 and CD45R antigen expression was noted on CD8+ lymphocytes reflecting increased killer-effector (p less than 0.001) and decreased suppressor-effector (p less than 0.001) cells, respectively. Other experiments revealed a significant increase in the percentage of synovial membrane CD20+ cells (B lymphocytes) and HLA-DR+ cells compared to RA PB (p less than 0.02 and p less than 0.001) and normal PB (p less than 0.01 and p less than 0.005), and similar proportions of CD14+ cells (monocytes/macrophages). Our results suggest that RA synovial membrane contains populations of T and B lymphocytes that differ quantitatively and qualitatively from those in PB. These may account for some of the abnormalities in intraarticular humoral and cellular immune responses in patients with RA.     

Effect of didanosine, stavudine, and hydroxyurea therapy on apoptosis in CD45RA+ and CD45RO+ T lymphocyte subpopulations

The effect of aggressive antiretroviral therapy on spontaneous apoptosis (AP) in CD4+ and CD8+ lymphocytes expressing CD45RO (memory cells) and CD45RA (naive cells) and their relationship to cellular activation and viral load were examined. Ten patients receiving simultaneous treatment with d4T, ddI, and HU were evaluated. Flow cytometric analysis showed significant levels of AP (measured by TUNEL assay) among memory and naive T cells and an enhanced expression of CD38 and HLA-DR activation markers. The percentage of apoptotic CD4+CD45RO+ and CD4+CD45RA+ cells decreased, respectively, from 34 +/- 3.3 and 29 +/- 3.6 prior to treatment to 20.5 +/- 4 and 22 +/- 3.8 at week 8 into therapy. The percentage of apoptotic CD8+CD45RO+ and CD8+CD45RA+ cells similarly decreased, respectively, from 20 +/- 2.5 and 24 +/- 3 prior to treatment to 14.5 +/- 2.7 and 16 +/- 3 at week 8 into treatment. The percentage of CD4+ cells expressing the activation markers CD38 and HLA-DR decreased from 27 +/- 6 to 13 +/- 2 and from 26 +/- 4 to 13.5 +/- 3, respectively. The percentage of CD8+ cells expressing either CD38 or HLA-DR fell from 22 +/- 3 to 10 +/- 2 for the former and from 39 +/- 5 to 22 +/- 4 for the latter. This was associated with a significant decrease in viral load (mean, 1.4 log10), and a decline in circulating plasma TNF-alpha and sIL-2R levels from 50.5 +/- 10 to 21 +/- 6 and 92.5 +/- 11 to 68 +/- 9, respectively. These data indicate that short-term therapy with ddI, d4T, and HU in combination diminished AP, immune activation, and partially restored naive and memory T cell subpopulations.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation

Objective. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). Methods. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. Results. RA SF and ST T cells were found to be markedly enriched in CD45RA^dim, CD45RO+, CD45RB^dim mature memory cells, whereas in the PB, CD45RA^bright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RB^dim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3—stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RB^dim phenotype and B cell help, CD45RB^dim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RB^dim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RB^dim T cells with mitomycin C. In contrast, healthy control sorted CD45RB^bright or sorted CD4+, CD45RO+, CD45RB^bright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. Conclusion. RA synovium is enriched in differentiated CD45RB^dim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.     

Selective lymphocyte chemokine receptor expression in the rheumatoid joint

Objective

In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX₃CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF).

Methods

Using flow cytometry, immunohistochemistry, and 2‐color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB.

Results

The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX₃CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2‐color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up‐regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX₃CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down‐regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint.

Conclusion

These results identify CCR4, CCR5, CXCR3, and CX₃CR1 as critical chemokine receptors in RA.

     

Cytokine flexibility of early and differentiated memory T helper cells in juvenile idiopathic arthritis

OBJECTIVE: It has been suggested that CD45RO+CD27+ T cells represent recently activated memory cells, whereas CD45RO+CD27- T cells are activated memory T cells in the process of differentiating into effector cells. We investigated (1) CCR7 and CCR5 expression and (2) modulation of cytokine expression in "early" (CD27+) and "differentiated" (CD27-) memory CD4+ T cells from peripheral blood and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). METHODS: SF CD4+CD45RO+CD27+ and CD27- memory T cells from 6 patients with JIA were tested by flow cytometry for intracellular interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) after in vitro priming with CD3 and CD28 mAb in the presence of IL-4, and subsequent culture with IL-2. RESULTS: SF CD4+CD45RO+CD27+ cells contained higher proportions of CCR7+ (median 46% vs 23%) and lower proportions of CCR5+ (73% vs 90%) cells than paired CD27- T cells. Both CD27+ and CD27- memory T helper cells from SF displayed a higher IFN-gamma/IL-4 ratio than their peripheral blood counterparts. No significant difference was observed in the percentage of IFN-gamma-expressing cells between CD27+ (32%, range 4-47%) and CD27- (29.4%, range 5-52%) memory T helper cells from SF. CONCLUSION: Irrespective of their differentiation stage, both CD27+ and CD27- SF memory T helper cells were found to switch from a proinflammatory to an antiinflammatory pattern of cytokine production.     

T lymphocytes in the synovial fluid of patients with active rheumatoid arthritis display CD134-OX40 surface antigen.

OBJECTIVE: To assess the percentage of T lymphocytes, bearing CD134, a member of the TNF receptor superfamily, primarily found on autoreactive CD4+ T cells in the peripheral blood (PB) and synovial fluid (SF) of rheumatoid arthritis (RA) patients. METHODS: The surface expression of CD134 on SF and PB mononuclear cells was performed by flow cytometry in 25 RA patients and correlated to the disease activity. RESULTS: CD134 expression on CD3+, CD4+, CD8+ and CD25+ cells was higher in SF than in PB of RA patients (P < 0.001). No differences were observed in the percentage of CD134+/CD4+ T lymphocytes in the PB of RA patients and controls. Patients with active RA had significantly higher percentage of CD3+/CD134+, CD4+/CD 134+, CD8+/CD134+ and CD25+/CD 134+ than those with inactive disease. CONCLUSION: These findings suggest that CD134+ T cells are involved in the immunopathological process of RA synovitis, maybe mirroring some other autoimmune disease in which autoreactive T cell infiltrating the target tissues largely coexpress CD134.     

Levels of soluble CD27 in sera and synovial fluid and its expression on memory T cells in patients with juvenile idiopathic arthritides

OBJECTIVE: CD27 is a member of tumour necrosis factor receptor family. Its expression is predominantly confined to mature lymphocytes and is strongly enhanced after cell activation. Shedding of the CD27 from the surface of activated cells is related to their effector phase. The aim of the present study was to evaluate the levels of soluble CD27 in sera and synovial fluids, together with its expression on circulating and synovial fluid (SF) memory T cells, in children with JIA. METHODS: Sera from 40 patients with active JIA were studied for soluble CD27. Paired SF samples were available in 20 patients. Sera from 12 age-matched patients affected with various acute infectious diseases and 12 age-matched healthy subjects were used as controls. In 8 JIA patients freshly isolated circulating and SF lymphocytes were stained for CD27 in CD4+CD45 RO+ T cell subpopulation and analyzed by cytometry. RESULTS: Soluble CD27 serum levels were significantly higher in patients with polyarticular JIA and acute systemic infectious diseases than in patients with active oligoarticular or healthy controls. Both polyarticular and oligoarticular JIA patients showed increased levels of soluble CD27 in SF when compared with paired serum samples (p = 0.01). In all the patients tested a significant enrichment of CD27- T cells was seen in the SF (median 39.5%, range 18-56%) when compared to paired CD4+CD45RO+ peripheral lymphocytes (median 19.5%, range 5-43%; p = 0.01). CONCLUSIONS: A clear enrichment of CD4+ memory SF T cells with a CD27-phenotype is observed when compared to correspondent circulating T lymphocytes. This issue is conceivably related to re-activation and recruitment of memory T cells to the site of inflammation, and to the subsequent expansion of a subpopulation of "effector" memory T cells.     

Selective recruitment of polarized T cells expressing CCR5 and CXCR3 to the inflamed joints of children with juvenile idiopathic arthritis

OBJECTIVE: To study the expression of chemokine receptors CCR5 and CXCR3 and the Th1/Th2 cytokine balance in children with oligoarticular or polyarticular juvenile idiopathic arthritis (JIA). METHODS: Using 3-color immunofluorescence, we studied the expression of CCR5 and CXCR3 on, and T cell cytokine production by, paired samples of synovial fluid (SF) and peripheral blood (PB) T cells from 20 patients with oligoarticular- or polyarticular-onset JIA. Chemokine and cytokine phenotypes were also compared within the CD45RO+,CD3+ subsets. CCR5 genotypes were confirmed by polymerase chain reaction typing and sequencing. RESULTS: In the majority of samples, the number of T cells that were CCR5+ and CXCR3+ was higher in SF than in PB, and this difference was significant. One child was homozygous for the null A32 CCR5 allele; 4 others had lower expression of CXCR3 in SF than in blood. All samples showed strongly Th1-type cytokine production by synovial T cells compared with that by PB T cells. Both features were also markedly polarized within the synovial CD45RO+ subset compared with PB CD45RO+ T cells. CONCLUSION: The high expression of CCR5 and CXCR3 and high interferon-gamma:interleukin-4 ratios suggest a type 1 phenotype of SF T cells in JIA. The difference between CD45RO+ T cells from SF and from PB suggests that specific activation events have occurred in synovial T cells. We suggest that the highly activated, Th1-type phenotype of T cells within the chronically inflamed joints of children with JIA may reflect specific recruitment events that contribute to the polarization of these cells.     

Accessory molecules expressed on the peripheral blood or synovial fluid T lymphocytes from patients with Sj?gren's syndrome or rheumatoid arthritis.

We previously demonstrated that the cells expressed by activation antigens were increased in several T cell subsets from the peripheral blood (PB) and joint fluid (JF) of patients with Sj?gren's syndrome (SS) or rheumatoid arthritis (RA). In the present report, we further determined by three-color flow cytometry the homing receptors (Leu8) for peripheral lymph nodes expressed on naive (CD45RA+) or memory (CD45RA-) CD4+ cells and on the two subsets of CD8+ cells (CD11b+ and CD11b-) in the PB and JF from SS and RA patients. In addition, the activation antigens (HLA-DR) and two adhesion molecules, including the alpha-chain of the leukocyte function associated antigen-1 (LFA 1 alpha: CD11a) and its ligand (intercellular adhesion molecule-1: ICAM-1: CD54) expressed on T cells, were compared. We found that CD45RA-CD4+ cells were markedly increased in JF, while CD45RA+CD4+ cells were almost absent. Leu8+ cells were decreased in both CD45RA-CD4+ and CD8+CD11b-cells (cytotoxic T cells) in the JF, and were also decreased in the CD8+CD11b-subset in the patients' PB. Furthermore, activated (DR+) T cells were markedly increased in JF, and the cells expressed more adhesion molecules in both the PB and JF from patients, compared with the DR-T cells. The DR+ T cells therefore are considered to be memory T cells, which are more efficient for cell-to-cell interactions. These observations also suggest that the Leu8- and DR+ T cells with increased adhesion molecules might preferentially migrate into inflammatory tissues, and that naive T cells are being further converted to to memory T cells by in vivo stimulation within the tissues.     

Immunological changes after highly active antiretroviral therapy with lopinavir-ritonavir in heavily pretreated HIV-infected children

We evaluated the effect of salvage antiretroviral therapy with lopinavir/ritonavir (LPV/r) on the immune system of heavily antiretroviral pretreated HIV-infected children. We carried out a longitudinal study in 20 antiretroviral experienced HIV-infected children to determine the changes in several immunological parameters (T cell subsets, thymic function) every 3 months during 18 months of follow-up on salvage therapy with LPV/r. Statistical analyses were performed with the Wilcoxon test, taking as a reference the basal value at the entry in the study. HIV-infected children showed an increase of CD4+ T cells, a decrease in CD8+ T cells, and an increase in T cell rearrangement excision circle (TRECs) levels. The percentage of HIV children with undetectable viral load (VL < or = 400 copies/ml) increased significantly (p = 0.007) and the percentage with SI viral phenotype decreased significantly (p = 0.002) at the end of the study. Thus, the viral phenotype changed to NSI/R5 after salvage therapy with LPV/r. Interestingly, we observed a significant decrease of memory (CD4+ CD45RO+) and a moderate decrease of activated (CD4+ HLA-DR+, CD4+ HLA-DR+CD38, CD4+, CD45RO+HLA-DR+) CD4+ T cells during the follow-up. On the other hand, memory (CD8+ CD45RO+ and CD8+ CD45RO+CD38+), activated (CD8+ HLA-DR+CD38+, CD8+ HLA-DR+, CD8+ CD38+), and effector (CD8+ CD57+, CD8+ CD28(-)CD57+) CD8+ T cells had a very significant decrease during follow-up. Our data indicate an immune system reconstitution in heavily pretreated HIV-infected children in response to salvage therapy with LPV/r as a consequence of a decrease in immune system activation and an increase in thymic function.     

Enrichment of differentiated CD45RBdim,CD27 – memory T cells in the peripheral blood,synovial fluid,and synovial tissue of patients with rheumatoid arthritis

Objective. To delineate in greater detail the phenotype of T cells that reside in the synovial tissue (ST) and synovial fluid (SF) of patients with rheumatoid arthritis (RA), in order to determine their precise differentiation status, and to determine whether the accumulation of these specific T cell subsets in these synovial compartments could be related to their capacity for transendothelial migration. Methods. Lymphocytes from normal subjects or from the peripheral blood (PB), ST, and/or SF of RA patients were phenotypically analyzed by flow cytometry. Normal PB CD4+ T cells were also characterized using an in vitro assay of transendothelial migration. Results. ST and SF were found to be enriched with memory (CD45RA-, CD45RO+, CD11a^bright, CD44^bright) and activated (CD69+) T cells. Moreover, ST and SF cells from RA patients were enriched in differentiated CD4+, CD45RB^dim, CD27- T cells, a subset of mature memory T cells that develops after prolonged antigenic stimulation. In addition, PB of some RA patients contained an increased number of CD4+, CD45RB^dim, CD27- T cells. The CD4+, CD11a^bright, CD44^bright memory T cells, which included the CD45RB^dim, CD27- more mature memory cells, exhibited an enhanced capacity for transendothelial migration that is likely to contribute to their enrichment in the rheumatoid synovium. Conclusion. RA patients manifest an increased number of mature memory T cells in the SF and ST, and some also have an increased number of these cells in PB that is likely to reflect chronic antigenic stimulation. The enrichment of these cells in the SF and ST reflects, in part, an enhanced capacity to migrate from the vascular space into inflamed tissue.