Study of chemotactic activity developed by neutrophils from rheumatoid arthritis patients

Neutrophils are the predominant cells accumulated in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. Accumulation of neutrophils may be regarded as a possible way by which neutrophils exert cytotoxic functions. The aim of the present study was to analyze the chemotactic response of neutrophils (PMNs) isolated from the peripheral blood or SF of patients with RA by performing the chemotaxis assay, in which N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as chemotactic agent. Our results showed that FMLP induced response of peripheral blood neutrophils from 12 patients with RA was similar with the response of 15 healthy controls. A decreased chemotactic response to FMLP was, however, observed in PMNs isolated from the SF of RA patients as comlipared with peripheral blood cells. Therefore, this defective chemotactic ability of neutrophil, was inversely correlated with the number of infiltrating cells in SF. These results indicate that chemotactic ability of neutrophils may be reduced after migration to the SF. Because PMNs chemotaxis in vivo has likely occurred in the presence of serum or SF, we tried to simulate the same conditions in vitro. Therefore, we analyzed the effect of serum or SF on the RA-PMNs chemotaxis. Heat-inactivated serum produced a marked reduction of chemotactic activity developed by PMNs isolated from patients with RA. Notably, a significant increase of chemotactic activity was observed when FMLP and serum stimuli were used together, as compared with the same stimuli used alone. The results suggested that complement activation might interfere with neutrophils chemotaxis. SF amplifies the chemotactic activity of PMNs isolated from peripheral blood of RA patients, but does not affect the chemotaxis developed by PMNs isolated from SF. The data might suggest that several components of SF (IL-8, leukotrien B4, thrombin, platelet-activating factor, etc.) could serve as a potent stimulus for recruitment of neutrophils from periphery into the RA joint. In conclusion, serum or SF components seem to contribute to chemotaxis of neutrophils and play a role in differential killing of PMNs and incidence of infection.     

Impaired generation of taurine chloramine by synovial fluid neutrophils of rheumatoid arthritis patients

Summary.  Taurine (Tau), a dominant free amino acid present in neutrophil cytoplasm, serves as a scavenger for hypochlorous acid (HOCl) released during these cells activation. The resulting taurine chloramine (Tau-Cl) exerts potent anti-inflammatory properties. In the present study we tested the hypothesis that the formation of Tau-Cl is impaired in neutrophils isolated from rheumatoid arthritis (RA) patients. The inhibition of zymosan-triggered chemiluminescence in the presence of exogenous Tau was used for indirect measurement of Tau-Cl generation. The chemiluminescence of neutrophils isolated from peripheral blood (PB) of healthy volunteers and RA patients was inhibited by Tau with similar potency. By contrast, synovial fluid (SF) neutrophils of these patients were significantly less sensitive for Tau-mediated inhibition. Therefore, our data indicate impaired generation of Tau-Cl in neutrophils isolated from SF of RA patients. Received November 29, 2001 Accepted January 9, 2002 Published online August 30, 2002 Acknowledgements This work was supported by grants from the State Committee for Scientific Research of Poland (No. P05A 104 19) and the Institute of Rheumatology. The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D, Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; PB, peripheral blood; SF, synovial fluid; RA, rheumatoid arthritis     

The levels of neutrophils oxidative burst in rheumatic disorders

We investigated the participation of the polymorphonuclear leukocytes (PMNs) as mediators of tissue destruction in 2 variants of rheumatic disorders that affect primarily the joints: the rheumatoid arthritis (RA) and the osteoarthritis (OA). We noted significant differences in the number of PMNs present at the joint level, which was low in OA and high in RA. The unstimulated and in vitro stimulated with opsonized zymozan (OZ) or ConA PMNs from the peripheral blood of these two groups of patients released a normal level of oxygen free radicals (OFR). On the contrary, the neutrophils isolated from the synovial fluid (SF) of RA patients showed a distinct feature: the unstimulated cells released high amounts of OFR, while those in vitro stimulated with OZ or ConA presented a low level of respiratory burst. In OA both in vitro unstimulated and stimulated with OZ or ConA neutrophils isolated from the SF had diminished values of OFR released. We assume that the chemiluminescence assay could detect distinct differences of the PMNs implications in RA and OA injuries and may be a laboratory test in the differential diagnosis of these rheumatic disorders.     

Regulation of natural killer cell activity by macrophages in the rheumatoid joint and peripheral blood

Recently, in another study, we observed that indomethacin, a prostaglandin synthetase inhibitor, significantly increased NK activity in both normal and rheumatoid arthritis (RA) peripheral blood (PB) but not in RA synovial fluid (SF). Because macrophages are a major source of prostaglandins, we examined the effect of macrophage-enriched adherent cells (AC) on NK activity as measured by a 3-hr Cr-release assay with K 562 cells. The removal of AC resulted in increased (p less than 0.01) NK activity in both normal and RA PB. In contrast, the removal of AC from RA SF resulted in a significant decrease (p less than 0.001) of NK activity. By using only nonadherent cells (NAC), NK activity in RA SF and synovial tissue (ST) was significantly reduced when compared to autologous RA PB (p less than 0.001). Enhancement of NK activity of SF NAC by both poly I:C and IL 2 was not dependent on AC. Mixing experiments demonstrated that the addition of synovial AC for 16 hr increased NK activity of synovial NAC to a level similar to that of unseparated mononuclear cells, whereas autologous PB AC suppressed NK activity of PB NAC. PB AC, when added to SF NAC, also increased NK activity. Supernatants from synovial mononuclear cells were stimulatory of synovial NAC NK activity, whereas normal PB mononuclear supernatants were suppressive. These observations document 1) a significant reduction of NAC-mediated NK activity in the rheumatoid joint as compared to PB from the same patient, and 2) that AC modulate NK activity differently in the rheumatoid joint as compared to RA or normal PB.     

A purified green barley extract with modulatory properties upon TNF alpha and ROS released by human specialised cells isolated from RA patients

Based on the finding that reactive oxygen species (ROS) play important roles in mediating proinflammatory cytokine production (e.g. TNF alpha) and that many plant extracts contain substances with antioxidant properties, we examined the antiinflammatory action of a green barley extract, commercially available as "Natural SOD". The results obtained in this study demonstrate that the antiinflammatory properties of "Natural SOD" due to its micromolecular substances, able to scavenge ROS and to down-regulate TNF alpha production, main inflammation mediators produced by specialised cells from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA). We prepared and tested a purified green barley extract (PE) containing micromolecular substances under 1 kDa, able to inhibit TNF alpha releasing--measured by an bioassay--from LPS stimulated human mononuclear cells (MNC) isolated both from PB and SF of RA patients. Luminol-dependent chemiluminescence has been used to measure the scavenging activity of PE on ROS releasing from activated neutrophils isolated from PB of RA patients. PE, containing high concentrations of substances with antioxidant and antiinflammatory properties, could be a more efficient natural drug for human use than "Natural SOD" in the treatment of RA patients.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis.

The cyclic AMP response to prostaglandin E1 (PGE1) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE1 induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10(-6)M) of PGE1 tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE1. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis

Dendritic cells (DCs) comprise heterogeneous subsets of professional antigen-presenting cells, linking innate and adaptive immunity. Analysis of DC subsets has been hampered by a lack of specific DC markers and reliable quantitation assays. We characterised the immunophenotype and functional characteristics of psoriatic arthritis (PsA)-derived and rheumatoid arthritis (RA)-derived myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to evaluate their potential role in arthritis. Circulating peripheral blood (PB) pDC numbers were significantly reduced in PsA patients (P = 0.0098) and RA patients (P = 0.0194), and mDCs were significantly reduced in RA patients (P = 0.0086) compared with healthy controls. The number of circulating mDCs in RA PB was significantly inversely correlated to C-reactive protein (P = 0.021). The phenotype of both DC subsets in PsA PB and RA PB was immature as compared with healthy controls. Moreover, CD62L expression was significantly decreased on both mDCs (PsA, P = 0.0122; RA, P = 0.0371) and pDCs (PsA, P = 0.0373; RA, P = 0.0367) in PB. Both mDCs and pDCs were present in PsA synovial fluid (SF) and RA SF, with the mDC:pDC ratio significantly exceeding that in matched PB (PsA SF, P = 0.0453; RA SF, P = 0.0082). pDCs isolated from RA SF and PsA SF displayed an immature phenotype comparable with PB pDCs. RA and PsA SF mDCs, however, displayed a more mature phenotype (increased expression of CD80, CD83 and CD86) compared with PB mDCs. Functional analysis revealed that both SF DC subsets matured following toll-like receptor stimulation. pDCs from PB and SF produced interferon alpha and tumour necrosis factor alpha on TLR9 stimulation, but only SF pDCs produced IL-10. Similarly, mDCs from PB and SF produced similar tumour necrosis factor alpha levels to TLR2 agonism, whereas SF mDCs produced more IL-10 than PB controls. Circulating DC subset numbers are reduced in RA PB and PsA PB with reduced CD62L expression. Maturation is incomplete in the inflamed synovial compartment. Immature DCs in SF may contribute to the perpetuation of inflammation via sampling of the inflamed synovial environment, and in situ presentation of arthritogenic antigen.     

Th17 and Th22 cells in psoriatic arthritis and psoriasis

Introduction

The aim of this study was to characterize interleukin 17 (IL-17) and interleukin 22 (IL-22) producing cells in peripheral blood (PB), skin, synovial fluid (SF) and synovial tissue (ST) in patients with psoriasis (Ps) and psoriatic arthritis (PsA).

Methods

Flow cytometry was used to enumerate cells making IL-22 and IL-17, in skin and/or SF and PB from 11 patients with Ps and 12 patients with PsA; skin and PB of 15 healthy controls and SF from rheumatoid arthritis (RA) patients were used as controls. Expression of the interleukin 23 receptor (IL-23R) and chemokine receptors CCR4 and CCR6 was examined. Secretion of IL-17 and IL-22 was measured by ELISA. ST was analysed by immunohistochemical staining of IL-17 and IL-22.

Results

Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were seen in PB of patients with PsA and Ps. IL-17 secretion was significantly elevated in both PsA and Ps, whilst IL-22 secretion was higher in PsA compared to Ps and healthy controls. A higher proportion of the CD4+ cells making IL-17 or IL-22 expressed IL-23R and frequencies of IL-17+, CCR6+ and CCR4+ T cells were elevated in patients with Ps and those with PsA. In patients with PsA, CCR6+ and IL-23R + T cells numbers were elevated in SF compared to PB. Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were demonstrated in Ps skin lesions. In contrast, whilst elevated frequencies of CD4+ IL-17+ cells were seen in PsA SF compared to PB, frequencies of CD4+ IL-22+ T cells were lower. Whereas IL-17 expression was equivalent in PsA, osteoarthritis (OA) and RA ST, IL-22 expression was higher in RA than either OA or PsA ST, in which IL-22 was strikingly absent.

Conclusions

Elevated frequencies of IL-17 and IL-22 producing CD4+ T cells were a feature of both Ps and PsA. However their differing distribution at disease sites, including lower frequencies of IL-22+ CD4+ T cells in SF compared to skin and PB, and lack of IL-22 expression in ST suggests that Th17 and Th22 cells have common, as well as divergent roles in the pathogenesis of Ps and PsA.     

A comparison of lymphocyte subpopulations simultaneously on local and systemic levels in acute rheumatoid arthritis patients

Rheumatoid arthritis (RA) is one of the most destructive inflammatory and autoimmune joint diseases, most frequently accompanied by extraarticular complications. The pathophysiologic mechanism and the importance of cell subpopulations in the initiation and perpetuation of synovitis are not sufficiently understood. In this study the frequency of lymphocyte subpopulations simultaneously in the synovial fluid (SF), the synovial membrane (SM) and peripheral blood (PB) of acute RA patients is determined, using flow cytometry procedures. The changes in the distribution of T lymphocyte subpopulations were significant on local levels in acute RA patients, resulting in a decreased CD4/CD8 ratio in SF, but an increased CD4/CD8 ratio in SM, compared to the ratio found in PB. The differences observed in the frequency of cells positive on natural killer (NK) cell markers suggest the role of CD16-CD56+ NK cell population in SF of RA patients. Significant differences in the observed frequency of lymphatic subpopulations suggest certain specificities of local immunological events in SM and SF in acute RA. These results confirm the T-lymphocyte hypothesis in initial pathogenic events in RA.     

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane,synovial fluid and peripheral blood

Introduction

CD4⁺CD25^+/highCD127^low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.

Methods

Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.

Results

CD4⁺ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO⁺RA^-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.

Conclusions

Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L^-CD69⁺), whereas peripheral Tregs are resting central memory cells (CD62L⁺CD69^-).     

The influence of synovial fluid on lymphocyte functions in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic recurrent and systemic inflammatory disease affecting around 1% of the population, that primarily involves the joints. In this study, we determined the Th1/Th2 lymphocytes ratio at the site of rheumatoid inflammation and the influence of the synovial fluid (SF) on the secretory and proliferative function in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC), obtained from patients with RA. Our results showed significant differences concerning the mononuclear cells and the CD4/CD8 ratio in synovial fluid and peripheral blood of patients. In SF prevailed Th1 cells, while in peripheral blood we found another cytokine profile of T lymphocytes. Also synovial fluid lymphocytes had a low PHA-stimulated blastogenic response. Patients plasma and synovial fluid showed an inhibitory effect on prolipheration indexes.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis

The cyclic AMP response to prostaglandin E₁ (PGE₁) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE₁ induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10⁻⁶M) of PGE₁ tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE₁. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

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.     

RANTES and chemotactic activity in synovial fluids from patients with rheumatoid arthritis and osteoarthritis

A massive accumulation of inflammatory cells in synovial tissues is a major pathological feature of rheumatoid arthritis (RA). Neutrophiles dominate synovial fluid while rheumatoid synovium is infiltrated with mononuclear cells. Mechanisms regulating influx of particular subpopulations of leukocytes into articular cavity and synovium compartment are not completely defined. An increasing amount of data supports a crucial role of a C-C chemokine RANTES in the RA pathogenesis. Our objective is to evaluate chemotactic activity for neutrophils (NCA), lymphocytes (LCA), and monocytes (MoCA) in SFs obtained from patients with RA and osteoarthritis (OA). We also aimed to characterise the relation between chemotactic activity, RANTES, and percentage distribution of leukocytes in SF. SFs from 11 patients with RA and 6 with OA were included in the study. Modified microchamber Boyden method was employed to assess chemotactic activity. Cytological and biochemical analysis of SF was performed. RANTES was measured with ELISA. Rheumatoid SFs were rich in cells with predominance of neutrophiles while osteoarthritic fluids were lymphocytic. RA SFs were also characterised by increased lactoferrin level. Both NCA and LCA were higher in SF from patients with RA (62 +/- 12 and 24 +/- 6 cells/HPF, resp) as compared to patients with OA (23 +/- 6; P < .05 and 6 +/- 2 cells/HPF; P < 0.05). The chemoattractive effect of RA SF was more pronounced on neutrophiles than on lymphocytes. RA SF expressed high RANTES levels (145+/- 36 pg/mL), while OA SF was characterised by only trace amount of this chemokine (2 +/- 1 pg/mL). We found positive correlation of RANTES with chemotactic activity for mononuclear cells (LCA + MoCA; R = 0.61; P < .05). Surprisingly, RANTES correlated also positively with neutrophiles number (R = 0.77; P < 0.001). Rheumatoid SF possesses strong chemotactic potency for leukocytes. RANTES is overexpressed in RA SF and is a potential mediator influencing intensity and composition of cellular infiltration in joints affected with inflammatory arthritis.     

Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue

Introduction

We evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed.

Methods

Peripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA.

Results

No differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability or positivity for autoantibodies.

Conclusions

The presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.     

Shift toward T lymphocytes with Th1 and Tc1 cytokine-secterion profile in the joints of patients with osteoarthritis

Osteoarthritis (OA), although a heterogeneous disease, is generally believed by rheumatologists to be primarily a disease generated by biomechanical alterations. In order to determine the role of T cells, the pattern of T lymphocyte cytokines and to characterize the mononuclear cells from both peripheral blood (PB) and synovial fluid (SF) from patients with OA flow cytometry with a panel of monoclonal antibodies recognising CD4, CD8 and intracellular cytokines (IL2, IL4, IL10, gamma-IFN) was employed. The coexpression of CD4 and CD8 markers only on the SF T cells surface after in vitro stimulation by phorbol-miristate acetate and ionomicine, but not on PBL or in vitro unstimulated SFL was noted. The intracellular IFN-gamma was detected in enhanced levels in both CD4+ and CD8+ SF T cells, while the IL2 contents was not different in PB and SF samples. The Th2/Tc2 cytokines (IL4 and IL10) were detected in low amounts in both PBL and SFL. This study shows the role of some T cell populations and cytokines in the generation of joint destruction in osteoarthritis.     

B-cell subsets in the joint compartments of seropositive and seronegative rheumatoid arthritis (RA) and No-RA arthritides express memory markers and ZAP70 and characterize the aggregate pattern irrespectively of the autoantibody status

The aim of the present study was to determine whether different subsets of B cells characterize synovial fluid (SF) or synovial tissue (ST) of seropositive or seronegative rheumatoid arthritis (RA) with respect to the peripheral blood (PB). PB, SF and ST of 14 autoantibody (AB)-positive (rheumatoid factor [RF]-IgM, RF-IgA, anti-citrullinated peptide [CCP]), 13 negative RA and 13 no-RA chronic arthritides were examined for B-cell subsets (Bm1-Bm5 and IgD-CD27 classifications), zeta-associated protein kinase-70 (ZAP70) expression on B cells and cytokine levels (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, IL-6, IL-8 and monocyte chemotactic protein [MCP]-1). Synovial tissues were classified as aggregate and diffuse patterns. No differences were found in B-cell percentages or in subsets in PB and SF between AB(+) and AB(-) RA and no-RA. In both AB(+) and AB(-) RA (and no-RA), the percentage of CD19(+)/ZAP70(+) was higher in SF than in PB (AB(+): P = 0.03; AB(-): P = 0.01; no-RA: P = 0.01). Moreover, SF of both AB(+) and AB(-) RA (and no-RA) patients was characterized by a higher percentage of IgD-CD27(+) and IgD-CD27(-) B cells and lower percentage of IgD(+)CD27(-) (P < 0.05) B cells compared to PB. In SF, ZAP70 positivity is more represented in B cell CD27(+)/IgD(-)/CD38(-). The aggregate synovitis pattern was characterized by higher percentages of Bm5 cells in SF compared with the diffuse pattern (P = 0.05). These data suggest that no difference exists between AB(+) and AB(-) in B-cell subset compartmentalization. CD27(+)/IgD(-)/ZAP70(+) memory B cells accumulate preferentially in the joints of RA, suggesting a dynamic maturation of the B cells in this compartment.     

IL-9, a local growth factor for synovial T cells in inflammatory arthritis

ObjectiveThe regulatory role of the T_h9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis.MethodsCD3⁺ T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3⁺ T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9.ResultsSF of PsA and RA was enriched with IL-9 producing CD3⁺ T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3⁺ T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p < .005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway.ConclusionIL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.     

Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effects of IL-10.

IL-10 down-regulates the APC function of many dendritic cells (DC), including human peripheral blood (PB) DC. In rheumatoid arthritis (RA), synovial fluid (SF) DC express markers of differentiation and are effective APC despite abundant synovial IL-10. The regulation of DC responsiveness to IL-10 was therefore examined by comparing the effect of IL-10 on normal PB and RA SF DC. Whereas IL-10 down-modulated APC function and MHC class II and B7 expression of PB DC, IL-10 had no such effect on SF DC. Since SF DC have differentiated in vivo in the presence of proinflammatory cytokines, PB DC were cocultured in the presence of IL-10 and either GM-CSF, IL-1beta, TNF-alpha, IL-6, or TGF-beta. GM-CSF, IL-1beta, and TNF-alpha were all able to restore APC function. Whereas the effects of IL-10 on PB DC were shown to be mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1 protein was demonstrated in PB and SF DC whole cell lysates, suggestive of predominant intracellular localization of the receptor. Thus, DC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R1 expression or signaling.