Impact of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Rheumatic Disease Patients on T Helper Cell Differentiation.

Complex pathogenesis of systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is associated with an imbalance of various Th-cell subpopulations. Mesenchymal stem cells (MSCs) have the ability to restore this balance. However, bone marrow-derived MSCs of SLE and SSc patients exhibit many abnormalities, whereas the properties of adipose derived mesenchymal stem cells (ASCS) are much less known. Therefore, we examined the effect of ASCs obtained from SLE (SLE/ASCs) and SSc (SSc/ASCs) patients on Th subset differentiation, using cells from healthy donors (HD/ASCs) as controls. ASCs were co-cultured with activated CD4+ T cells or peripheral blood mononuclear cells. Expression of transcription factors defining Th1, Th2, Th17, and regulatory T cell (Tregs) subsets, i.e., T-bet, GATA3, RORc, and FoxP3, were analysed by quantitative RT-PCR, the concentrations of subset-specific cytokines were measured by ELISA, and Tregs formation by flow cytometry. Compared with HD/ASCs, SLE/ASCs and especially SSc/ASCs triggered Th differentiation which was disturbed at the transcription levels of genes encoding Th1- and Tregs-related transcription factors. However, we failed to find functional consequences of this abnormality, because all tested ASCs similarly switched differentiation from Th1 to Th2 direction with accompanying IFNγ/IL-4 ratio decrease, up-regulated Th17 formation and IL-17 secretion, and up-regulated classical Tregs generation.

Direct anti-proliferative effect of adipose-derived mesenchymal stem cells of ankylosing spondylitis patients on allogenic CD4+ cells.

OBJECTIVES: T-cell-mediated adaptive immunity contributes to the development and persistence of ankylosing spondylitis (AS). Mesenchymal stromal/stem cells (MSCs) have immunomodulatory potential and are able to inhibit T-cell proliferation, but their functionality in AS patients is relatively unknown. The aim of the study was to assess the direct anti-proliferative effects of MSCs isolated from subcutaneous abdominal adipose tissue of AS patients (AS/ASCs) on allogeneic T lymphocytes, using commercially available ASC lines from healthy donors (HD/ASCs) as a control. MATERIAL AND METHODS: CD3+CD4+ T-cells were isolated from peripheral blood of healthy blood donors, activated with anti-CD3/CD28 beads, and co-cultured for 5 days with untreated and TNF+IFN-γ pre-stimulated HD/ASCs (5 cell lines) and AS/ASCs, obtained from 11 patients (6F/5M). The proliferative response of T-cells was analysed by flow cytometry, while the concentrations of kynurenines, prostaglandin E2 (PGE-2), interleukin 10 (IL-10), and interleukin 1 receptor antagonist (IL-1Ra) were measured spectrophotometrically or using a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: HD/ASCs and AS/ASCs similarly reduced the T-cell proliferation response, i.e. the percentage of proliferating cells, the proliferation, and replication indices, and these effects were dependent mostly on soluble factors. In the co-cultures of activated CD4+ T-cells with HD/ASCs and AS/ASCs significant increases of kynurenines, PGE-2, and IL-1Ra, but not IL-10, production were observed. The release of these factors was dependent either on cell-to-cell contact (IL-10, IL-1Ra) or soluble factors (kynurenines, PGE-2). There was a moderate to strong negative correlation between T-cell proliferative response, and the concentrations of kynurenines, PGE-2, and IL-10, but not IL-1Ra. This association was more evident in the case of TI-treated AS/ASCs than HD/ASCs. CONCLUSIONS: AS/ASCs, similar to HD/ASCs, exert a direct effective anti-proliferative impact on CD4+ T cells, acting via soluble factors that are released in cell contact-dependent (IL-10) and independent (kynurenines, PGE-2) pathways. Thus, our results suggest that AS/ASCs are potentially useful for therapeutic application.

Survival of lymphocytes is not restricted by IDO-expressing fibroblast from rheumatoid arthritis patients.

Objective: Rheumatoid arthritis (RA) is characterized by expansion of fibroblast-like synoviocytes (FLS) in inflamed joints and activation of lymphocytes. Tryptophan (trp) is an essential amino acid indispensable for the biosynthesis of proteins and critical for survival of lymphocytes. Indoleamine 2,3-dioxygenase (IDO) that initiates the degradation of trp and tryptophanyl-tRNA synthetase (TTS) essential for tryptophan synthesis, regulate trp bioavailability. Here, we tested the hypothesis that triggered by cytokines, enhanced IDO activity modulate regulatory function of otherwise non-tolerogenic FLS isolated from RA patients. Materials and methods: IDO and TTS mRNA expression were evaluated by RT-PCR. IDO enzymatic activity was confirmed using HPLC. Resting or PHA-activated PBMC from healthy volunteers and RA patients were co-cultured with IDO expressing untreated (FLSC) or IFNγ-treated (FLSIFNγ) RA FLS. Lymphocyte survival and proliferation were evaluated by flow cytometry analysis and tritiated thymidine incorporation, respectively. Results: RA FLSIFNγ produce functionally active IDO and constitutively express TTS. RA FLSC and FLSIFNγ increased survival of resting lymphocytes in both studied groups, and decreased proliferation of healthy, but not RA, PBMC. Only FLSIFNγ diminished survival of activated CD3+CD4-, but not CD3+CD4+, healthy T cells and similar tendency was observed in rheumatoid cells. Importantly, IDO inhibitor, 1-methyl-DL-tryptophan (1-MT), failed to reverse this effect. PBMC, irrespective of their state (resting versus activated) or origin (healthy or RA), expressed high level of TTS mRNA. Conclusions: We suggest that RA FLS express functionally active IDO but control survival and expansion of healthy cells in IDO-independent mechanism and exert weaker, if any, suppressive effect on rheumatoid cells.

Spondyloarthritis patients with and without intestinal symptoms - searching for discriminating biomarkers.

Spondyloarthritis (SpA) is often complicated with subclinical gut inflammation. This study was aimed at searching for biomarkers discriminating SpA patients with and without intestinal symptoms. A group of 29 SpA patients and 33 healthy volunteers (control) were included in the study. Based on clinical evaluation, the patient cohort was subdivided into two groups: 1) SpA accompanied by various intestinal symptoms suggesting gut inflammation (group 2, n = 14) and 2) without such complications (group 1, n = 15). Serum concentrations of interleukins (IL) (IL-10, IL-17A/F, IL-22, IL-23), tumour necrosis factor (TNF), bone-homeostasis-related factors (osteoprotegerin - OPG and Dickkopf-1 - DKK-1), and the concentrations of selected gut inflammation-associated factors (intestinal fatty acid binding protein - iFABP, claudin 3 - CLDN3 and calprotectin) in samples of sera and/or urine or stool, respectively, were measured by specific ELISA. Serum concentrations of tested factors were similar in SpA patients and control. Faecal calprotectin level was higher in patients but did not discriminate between group 1 and 2. Compared to group 1, group 2 was characterized by elevated erythrocyte sedimentation rate (ESR), higher serum CLDN3 and DKK-1 levels. In SpA patients, serum DKK-1 concentrations correlated with systemic inflammation markers (R = 0.6, p < 0.01), while serum CLDN3 was found to be an independent risk factor (OR = 4.5, p = 0.021) for the occurrence of intestinal symptoms. We conclude that in SpA patients, up-regulated circulating levels of CLDN3 seem to be related to intestinal complication, while the quantity of circulating DKK-1 reflects the intensity of systemic inflammation.

Effects of whole body cryotherapy in patients with rheumatoid arthritis considering immune parameters.

OBJECTIVES: Whole body cryotherapy (WBC) is widely used in inflammatory diseases of the joints, including rheumatoid arthritis (RA), but the mechanism(s) of its action is not fully understood. The aim of the study was to compare the effects of WBC and conventional rehabilitation (CR) on the clinical and immune status of RA patients. MATERIAL AND METHODS: Rheumatoid arthritis patients were classified into 2 groups according to the rehabilitation method used: the study group (CT, n = 25) and control group (CR, n = 25). To measure disease activity, the disease activity score (DAS28) was used, while to assess the morning stiffness and pain intensity, the visual analogue scale (VAS) was applied. Selected laboratory parameters, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels, were also determined. The serum concentrations of pro- (interleukin 6 [IL-6], tumor necrosis factor α [TNF-α], macrophage migration inhibitory factor [MIF]) and anti-inflammatory (IL-10) cytokines were measured to assess the patient's immune status. RESULTS: After rehabilitation disease activity (DAS28), morning stiffness and pain intensity (VAS) decreased in both patient groups and no statistically significant differences were observed between them. However, statistically significant improvement in the CRP serum level was observed in the CT group only. No differences were observed in the serum concentrations of tested cytokines either before and after rehabilitation, or between patient groups. CONCLUSIONS: We report that regardless of the type of therapy, comprehensive rehabilitation improves the patient's clinical status, but has no effect on the levels of circulating cytokines, such as IL-6, IL-10, TNF-α, and MIF, despite significant reduction of a systemic inflammatory marker (CRP), especially in the CT group.

Subgroups of Sjögren's syndrome patients categorised by serological profiles: clinical and immunological characteristics.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune disease characterised by heterogeneous clinical presentation and presence of various autoantibodies - anti-SSA/Ro of diagnostic value, less specific anti-SSB/La and others. We searched for biomarker(s) and potential therapeutic target(s) of SS subsets that vary in their autoantibody profile. MATERIAL AND METHODS: Eighty-one patients with SS (70 female and 11 male) and 38 healthy volunteers (28 female and 10 male) were included in the study. Patients were categorised according to absence (group 1) or presence of anti-SSA/Ro antibody which occurred either alone (group 2) or together with anti-SSB/La (group 3). Clinical evaluation was performed, and presence of autoantibodies and concentrations of cytokines relevant to SS pathogenesis, i.e. a proliferation inducing ligand (APRIL), B-lymphocyte activating factor (BAFF), interleukin (IL) 4, IL-10, interferon α (IFN-α) and thymic stromal lymphopoietin (TSLP), in sera were determined. RESULTS: Frequency of autoantibodies other than anti-SSA/Ro and anti-SSB/La, the number of autoantibody specificities and anti-nuclear antibody titres were higher in group 2 and/or 3 than in group 1 of SS patients. Moreover, SS patients of groups 2 and 3 developed disease symptoms at younger age, and more often had positive Schirmer's test and skin lesions. In addition, serum concentrations of APRIL, but not other tested cytokines, were significantly higher in the patients of both groups 2 and 3 than those of group 1 and healthy volunteers. CONCLUSIONS: Sjögren's syndrome patients with signs of B-cell epitope spreading are characterised by early disease onset, more frequent xerophthalmia and skin involvement, and up-regulated serum APRIL level. We suggest that therapeutic neutralisation of APRIL may be beneficial for these patients.

Cytokines and integrins related to inflammation of joint and gut in patients with spondyloarthritis and inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel disease (IBD) and spondyloarthritis (SpA) have some overlapping clinical features, i.e. gut and joint inflammation. Cytokines of interleukin 17(IL-17)/IL-23 axis play a pathogenic role in both diseases. Integrins (ITGs) regulate migration of immune cells to inflamed tissues (ITGß7 into gut, ITGß2 into gut and also to other tissues). In this study, we search for differences in the serum concentrations of these cytokines and integrins between patients suffering from SpA or IBD with and without overlapping symptoms. MATERIAL AND METHODS: Patients with SpA (n = 30), IBD (n = 68), and healthy volunteers (n = 28) were included in the study. Fourteen SpA patients reported symptoms characteristic for IBD. Spondyloarthritis symptoms were diagnosed in 50% of IBD patients, while other patients of this group reported arthralgia only. Serum concentrations of IL-17, IL-22, IL-23, ITGß2, and ITGß7 were measured by specific enzyme-linked immunosorbent assay using commercially available sets. The Mann-Whitney and Spearman's rank tests were used for intergroup comparison and correlation assessment, respectively. RESULTS: Comparison of patient groups showed significantly higher serum concentrations of IL-17, IL-22, and ITGß7 in SpA, and up-regulated levels of IL-23 in IBD patients. Similar differences were observed between patient subgroups, both with and without overlapping symptoms. In SpA but not in IBD patients, serum concentrations of ITGß7 inversely correlated (r = -0.552) with C-reactive protein. CONCLUSIONS: Patients with SpA and IBD differ in the circulating concentrations of IL-17/IL-23 axis cytokines and ITGß7, irrespectively of the presence or absence of overlapping symptoms. Therefore, we conclude that observed differences are attributed rather to underlying than concurrent disease.

Secretory activity of subcutaneous abdominal adipose tissue in male patients with rheumatoid arthritis and osteoarthritis - association with clinical and laboratory data.

INTRODUCTION: Adipose tissue exerts widespread effects on the metabolism and immune system, but its activity differs between the genders. In the general population low-grade adipose tissue inflammation contributes to development of diseases of affluence. Little is known about the systemic impact of peripheral fat tissue in osteoarthritis (OA) and rheumatoid arthritis (RA), characterized by chronic, low- and high-grade systemic inflammation, respectively. To clarify this we evaluated the secretory activity of subcutaneous abdominal adipose tissue (SAAT) obtained from male patients affected with RA (n = 21) and OA (n = 13), and assessed its association with body mass and composition, demographic, clinical and laboratory data. MATERIAL AND METHODS: Basal and interleukin (IL)-1ß-triggered secretion of selected adipocytokines from SAAT explants was measured by specific enzyme-linked immunosorbent assays (ELISA). Patients' body composition was evaluated by bioelectric impendence technique. RESULTS: Rheumatoid SAAT secreted more adiponectin and macrophage migration inhibitory factor (MIF) than respective osteoarthritis tissue. In both RA and OA patient groups, stimulation of SAAT explants with IL-1ß (1 ng/ml/100 mg tissue) significantly up-regulated release of pro-(IL-6, IL-8, tumor necrosis factor - TNF) and anti-inflammatory (IL-10) cytokines but had no effect on the secretion of adiponectin, leptin, MIF and hepatocyte growth factor (HGF). Compared with RA, patients with OA were more obese. In RA patients SAAT-released adiponectin and TNF inversely correlated with body mass index (BMI) and visceral fat rating (FVSC). In addition, SAAT-secreted adiponectin and leptin positively correlated with DAS28 and disease duration, respectively. In the OA group tissue-released TNF positively correlated with patients' age. CONCLUSIONS: We conclude that in RA male patients adipocytokines originating from SAAT are of clinical importance because: (i) adiponectin and TNF may contribute to maintenance of normal body composition and mass, (ii) in addition adiponectin may play a pathogenic role. Moreover, in both RA and OA male patients secretory activity of SAAT may vary with time.

Different expression of chemokines in rheumatoid arthritis and osteoarthritis bone marrow.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to joint destruction. In addition to involvement of the joints, there is growing evidence that inflammatory/autoimmune processes take place in bone marrow, beginning the disease onset. Activated T and B cells accumulate in bone marrow, where also effective antigen presentation takes place. An increased number of activated T cells was observed in RA in comparison to osteoarthritis (OA) bone marrow. In the present study we analyzed the levels of chemokines that may be responsible for accumulation/retention of T-cells in the bone marrow of RA and OA patients. MATERIAL AND METHODS: Bone marrow samples were obtained from RA and OA patients during total hip replacement surgery, and bone marrow plasma was obtained by gradient centrifugation. Levels of the chemokines CX3CL1, CCL5, CCL2, CXCL12 and CXCL1 were measured in bone marrow plasma by specific ELISAs. Comparison between the groups of patients and statistical significance were analyzed by the two-tailed Mann-Whitney U test. RESULTS: Increased levels of CX3CL1 (818 ±431 pg/ml vs. 502 ±131 pg/ml, p < 0.0007) and CCL5 (5967 ±1680 pg/ml vs. 4878 ±2360 pg/ml, p < 0.05) respectively in bone marrow plasma from RA in comparison with OA patients were observed. In contrast, similar levels of CCL2, CXCL12 and CXCL1 in RA and OA bone marrow suggest that these cytokines do not play a significant role in the observed T cell accumulation in RA bone marrow. CONCLUSIONS: CX3CL1 and CCL5 overproduced in RA bone marrow may contribute to the accumulation of T cells observed in RA bone marrow.

Prospective assessment of cytokine IL-15 activity in patients with refractory atrial fibrillation episodes.

AIMS: Inflammatory state is considered a risk factor of atrial fibrillation (AF) occurrence. The aim of this study was a prospective evaluation of the inflammation parameters in patients with different forms of AF without structural heart disease. METHODS AND RESULTS: One hundred fifty-eight patients with paroxysmal/persistent AF (87; 55.1% men, mean age 65.8±9.6 years) without structural heart disease were enrolled in the study. Inflammatory parameters: WBC, ESR, hs-CRP, IL-6, IL-15 and TNF-alpha were measured at baseline and after one year follow-up. Despite frequent AF episodes median values of WBC, ESR and C-reactive protein at baseline and after follow up were within normal ranges. There were no significant differences between WBC, ESR and hs-CRP regarding AF types. In patients who developed permanent AF form (n=14) hs-CRP concentrations were higher at baseline: 0.35 (IQR1: 0.09 IQR: 0.61) vs 0.15 (IQR1: 0.07 IQR: 0.29), p<0.01. Nevertheless, after one year's observation these differences were not significant. Among all cytokines were studied only IL-15 was significantly correlated with the number of AF episodes (r=0.26), mean (IQ1-IQ3): 10 (3-30) vs 60 (50-100), p=0.00681. CONCLUSION: Basic inflammatory markers were not changed in patients with refractory atrial fibrillation episodes in prospective one year's observation. Only cytokine IL-15 was correlated to numbers of AF episodes. It's potential role as a marker of arrhythmia deserves further evaluation.

Inflammatory bowel disease-related arthritis - clinical evaluation and possible role of cytokines.

OBJECTIVES: In inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation, rheumatic abnormalities ranging from arthralgia to spondyloarthritis (SpA) are the most common extraintestinal manifestations. The pathogenesis of IBD-related arthritis is unclear. In this study, we search for clinical and immunological differences between patients with IBD-associated spondyloarthritis and IBD patients without SpA symptoms. MATERIAL AND METHODS: Patients with an established diagnosis of IBD, suffering from Lesniowski-Crohn disease (L-CD, n = 24) or ulcerative colitis (UC, n = 27), were enrolled in the study. Clinical evaluation of patients, based on medical history, blood tests, physical and radiological examinations, allowed two subgroups of patients to be established. One subgroup comprised patients fulfilling criteria for both IBD and SpA (IBD + SpA, n = 29), while the other included IBD patients with arthralgia only (IBD, n = 22). Serum concentrations of interleukins (IL-6, IL-10, IL-21, IL-22, IL-23) and interferon γ (IFN-γ) were measured by specific enzyme-linked immunosorbent assays (ELISA). RESULTS: Patients with IBD + SpA were characterized by shorter disease duration (3 vs. 9 years), higher frequency of HLA-B27 positivity (60.7% vs. 4.5%) and uveitis (20.7% vs. 0%), compared with the IBD subgroup. The serum concentrations of C-reactive protein (CRP) and tested cytokines did not differ between IBD + SpA and IBD patients, or between L-CD and UC groups. However, in the IBD + SpA subgroup there was weak to moderate positive correlation between serum concentrations of CRP and several cytokines (IL-6, IL-21, IFN-γ), and additional moderate positive correlation between serum concentrations of IL-23 and clinical activity of SpA. By contrast, in IBD subgroup a strong inverse correlation between serum concentrations of Interleukin 23 and CRP was found. CONCLUSIONS: IBD-related spondyloarthritis occurs relatively early, affects mostly HLA-B27(+) individuals, and is often accompanied by ocular involvement. In these patients several circulating cytokines are associated with systemic inflammation. IL-23 seems to be protective in IBD while detrimental in IBD-related spondyloarthritis.

The relationship between the presence of autoantibodies, indicators of local and systemic inflammation, the serum concentration of B-cell activating factor (BAFF) and the intensity of salivary gland infiltration in patients with primary Sjögren's syndrome - a preliminary study.

OBJECTIVES: The aim of this study was to find markers related to activation of B cells, which show a correlation with the systemic inflammation markers - erythrocyte sedimentation rate and C-reactive protein and with the intensity of in situ inflammation. MATERIAL AND METHODS: Forty-one primary Sjögren's syndrome (pSS) patients (33 female, 8 male) of the mean age 52.9 ±15 years were included. A group of 20 healthy volunteers was applied as a control. Erythrocyte sedimentation rate (ESR), concentration of gamma-globulins, C-reactive protein (CRP) and rheumatoid factor (RF) were measured by routine laboratory tests. Titres of antinuclear antibodies (ANAs) were determined by the indirect immunofluorescence method, while anti-SS-A/SS-B antibodies were detected by both the dot-blot method and an enzyme immunoassay. The concentrations of BAFF in sera were measured by sandwich ELISA. Biopsies of minor salivary glands were taken and the focus score (FS) was calculated. Correlations between quantitative variables were assessed using the Spearman correlation coefficient (r). RESULTS: Serum concentrations of BAFF was significantly higher in the pSS patients than in the control group. The study revealed a statistically significant correlation between ANAs titre and the FS (r = 0.421). Anti-SS-A/Ro and anti-SS-B/La antibodies positively correlated with ESR. There was also a positive correlation between the gamma globulin level and the titres of all tested autoantibodies. CONCLUSIONS: The positive correlation between ANAs and FS confirms the importance of these autoantibodies in the local inflammatory process. The positive correlation between anti-SS-A/SS-B antibodies and ESR suggests involvement of these antibodies in generalization of the inflammatory response. In the pSS group serum concentrations of BAFF were statistically significantly higher than healthy volunteers. All presented results confirm the role of activity of B cells in the course of pSS.

Taurine and inflammatory diseases.

Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in humans and plays an important role in several essential biological processes such as bile acid conjugation, maintenance of calcium homeostasis, osmoregulation and membrane stabilization. Moreover, attenuation of apoptosis and its antioxidant activity seem to be crucial for the cytoprotective effects of taurine. Although these properties are not tissue specific, taurine reaches particularly high concentrations in tissues exposed to elevated levels of oxidants (e.g., inflammatory cells). It suggests that taurine may play an important role in inflammation associated with oxidative stress. Indeed, at the site of inflammation, taurine is known to react with and detoxify hypochlorous acid generated by the neutrophil myeloperoxidase (MPO)-halide system. This reaction results in the formation of less toxic taurine chloramine (TauCl). Both haloamines, TauCl and taurine bromamine (TauBr), the product of taurine reaction with hypobromous acid (HOBr), exert antimicrobial and anti-inflammatory properties. In contrast to a well-documented regulatory role of taurine and taurine haloamines (TauCl, TauBr) in acute inflammation, their role in the pathogenesis of inflammatory diseases is not clear. This review summarizes our current knowledge concerning the role of taurine, TauCl and TauBr in the pathogenesis of inflammatory diseases initiated or propagated by MPO-derived oxidants. The aim of this paper is to show links between inflammation, neutrophils, MPO, oxidative stress and taurine. We will discuss the possible contribution of taurine and taurine haloamines to the pathogenesis of inflammatory diseases, especially in the best studied example of rheumatoid arthritis.

Articular adipose tissue resident macrophages in rheumatoid arthritis patients: potential contribution to local abnormalities.

OBJECTIVES: The objectives of this study were to characterize macrophages resident in inflamed articular adipose tissue (AAT) and non-inflamed subcutaneous adipose tissue (ScAT) of RA patients and to evaluate the basal and cytokine-triggered secretory activities of these tissues. METHODS: Tissues were obtained from patients undergoing knee joint replacement surgery. The number of total CD68(+), CD14(+) and CD163(+) macrophages was evaluated by immunohistochemistry. The concentrations of select factors were measured in supernatants from untreated and cytokine-treated tissue explant cultures using ELISA. IL-1ß and TNF were applied as the stimuli. RESULTS: Paired samples of AAT and ScAT, obtained from the same patients, contained a similar number of macrophages, displaying an M2-skewed phenotype. Both tissues released equivalent amounts of IL-1ß, TNF, IL-10 and macrophage migration inhibitory factor (MIF). However, AAT secreted more chemokines (CCL2, CCL5), cytokines [IL-6, IL-8, IL-1 receptor antagonist (IL-1Ra)], hepatocyte growth factor (HGF) and MMP-3 than ScAT. Basal secretion of adipocytokines was not patient specific. Except for HGF and MIF, cytokine treatment up-regulated the release of these factors from both tissues, but also upon stimulation AAT produced more IL-6, IL-8 and IL-1Ra than ScAT. CONCLUSION: The secretory activity, reflecting cell activation status but not phenotype or the number of macrophages, discriminates rheumatoid AAT from ScAT. By releasing various factors possessing chemotactic, proinflammatory, anti-inflammatory and tissue degrading activities, AAT resident macrophages may drive and control local pathological processes.

Significance of bone marrow edema in pathogenesis of rheumatoid arthritis.

Assessing the pathology of the synovium, its thickening and increased vascularity through ultrasound and magnetic resonance examinations (more often an ultrasound study alone) is still considered a sensitive parameter in the diagnosis of rheumatoid arthritis and in monitoring of treatment efficacy. Magnetic resonance studies showed that, aside from the joint pannus, the subchondral bone tissue constitutes an essential element in the development of rheumatoid arthritis. Bone marrow edema correlates with inflammation severity, joint destruction, clinical signs and symptoms of rheumatoid arthritis, and thus is considered a predictor of rapid radiological progression of the disease. The newest studies reveal that bone marrow edema may be a more sensitive indicator of the response to therapy than appearance of the synovium. Bone marrow edema presents with increased signal in T2-weighted images, being most visible in fat saturation or IR sequences (STIR, TIRM). On the other hand, it is hypointense and less evident in T1-weighted images. It becomes enhanced (hyperintense) after contrast administration. Histopathological studies confirmed that it is a result of bone inflammation (osteitis/osteomyelitis), i.e. replacememt of bone marrow fat by inflammatory infiltrates containing macrophages, T lymphocytes, B lymphocytes, plasma cells and osteoclasts. Bone marrow edema appears after a few weeks from occurrence of symptoms and therefore is considered an early marker of inflammation. It correlates with clinical assessment of disease activity and elevated markers of acute inflammatory phase, i.e. ESR and CRP. It is a reversible phenomenon and may become attenuated due to biological treatment. It is considered a "herald" of erosions, as the risk of their formation is 6-fold higher in sites where BME was previously noted.

Comparison of rheumatoid articular adipose and synovial tissue reactivity to proinflammatory stimuli: contribution to adipocytokine network.

OBJECTIVES: (1) To compare spontaneous and stimuli-induced adipocytokine secretion by articular adipose tissue (AAT) and synovial membrane (SM) explants obtained from patients with rheumatoid arthritis (RA). (2) To investigate the biological activity of AAT and SM released factors. METHODS: Tissues were obtained from patients undergoing joint replacement surgery. Tissue explants were treated with proinflammatory cytokines relevant to RA pathogenesis (interleukin 1ß (IL-1ß), tumour necrosis factor (TNF), interferon γ, IL-15, IL-17, IL-23). Selected adipocytokine (TNF, IL-6, IL-8, IL-1ß, IL-1Ra, adiponectin, leptin) concentrations were measured in culture supernatants using ELISA. The biological activity of tissue-conditioned media was evaluated by measuring production of selected factors (IL-6, IL-8, Dickkopf-1, osteoprotegerin) by fibroblast-like synoviocytes (FLS). RESULTS: Spontaneous cytokine release from AAT was ≤12% of that produced by SM, while leptin was secreted in similar amounts. AAT was highly reactive to proinflammatory cytokines (IL-1ß>TNF). AAT treated with IL-1ß released four times more leptin, similar amounts of IL-6 and IL-8 and about 20% of TNF, as compared with SM. Upon activation, the IL-1 receptor antagonist (IL-1Ra)/IL-1ß ratio was higher in AAT than in SM cultures. Irrespective of activation status, SM produced twice as much adiponectin as AAT. Conditioned media from AAT and SM cultures similarly upregulated IL-6, IL-8, Dickkopf-1 and osteoprotegerin production by rheumatoid FLS. CONCLUSION: Rheumatoid AAT is highly reactive tissue which upon stimulation secretes considerable amounts of proinflammatory (IL-6, IL-8, TNF) and anti-inflammatory (IL-1Ra) cytokines and classical adipokines. This tissue releases biologically active factors that intensify pathogenic activities of rheumatoid FLS. Thus, AAT should be considered an important contributor to the pathological processes taking place in the RA joint.

Intra-articular adipose-derived mesenchymal stem cells from rheumatoid arthritis patients maintain the function of chondrogenic differentiation.

OBJECTIVES: To evaluate the chondrogenic potential, phenotype and percentage of IA adipose-derived mesenchymal stem cells (ADSCs) from RA patients in comparison with OA patients. The effect of TNF treatment on ADSC differentiation was also examined. METHODS: Adipose tissue was obtained from RA and OA patients. ADSCs were isolated and cultured until passage 4. After that period, the phenotype and percentage of these cells were analysed by flow cytometry. Passage 4 cells were cultured in chondrogenic medium with or without TNF. After 3 weeks of differentiation the expression of Sox9, aggrecan (Acan) and collagen 2a (Col2a) mRNA was assessed by RT-PCR and GAG deposition by alcian blue staining. RESULTS: The phenotype and percentage of ADSCs were similar in both RA and OA. The results of alcian blue staining showed effective chondrogenesis in RA and OA ADSCs. TNF inhibited GAG deposition in both RA and OA samples similarly. Sox9, Acan and Col2a mRNA expression was significantly increased in chondrogenic-medium-treated cells (P<0.05) and decreased after TNF exposure (P<0.01). No statistically significant differences between RA and OA were observed. CONCLUSION: ADSCs from RA and OA patients are similar with regard to their phenotype, percentage in IA tissue and chondrogenic potential, which is reduced after exposure to TNF.

Associations of IL-18 with Altered Cardiovascular Risk Profile in Psoriatic Arthritis and Ankylosing Spondylitis.

OBJECTIVE: To investigate the associations of IL-18 serum levels with serum lipids, cardiovascular risk, and disease activity in patients with ankylosing spondylitis (AS) and psoriatic arthritis (PsA) with axial (axPsA) and peripheral (perPsA) joint involvement. METHODS: 155 adult patients (PsA 61/AS 94) were enrolled in the study. Standard disease activity indices, BASDAI, and ASDAS, were calculated for AS and PsA and DAPSA for PsA. Sera from peripheral blood samples were obtained after night fasting. Serum concentrations of cytokines (IL-18, IL-17) were measured by ELISA, while lipid profile with total cholesterol (TC), triglycerides (TG), low-density cholesterol-(LDL), high-density cholesterol (HDL), and C-reactive protein (CRP) concentrations were determined using routine procedures. The atherogenic index was calculated using the standard formula AI = TC/HDL. RESULTS: Patients with PsA and peripheral joint involvement (perPsA) had significantly higher IL-18 serum levels than axial PsA and AS patients (medians 160 vs. 116 vs. 80 pg/mL). In patients with PsA and in the subgroup with PsA+ ischemic heart disease (IHD), IL-18 positively correlated with atherogenic index (AI) (rho = 0.46 and rho = 0.67, respectively) and TG serum concentrations (rho = 0.4 and rho = 0.675), while negatively with HDL levels (rho = -0.37 and rho = -0.608). In PsA + IHD subgroup IL-18 serum levels correlated positively also with disease activity (DAPSA) (rho = 0.613). Importantly, in patients with perPsA, characterized by the highest IL-18 serum levels, cardiovascular risk, and frequency of both hypertriglyceridemia and IHD, positive correlations between IL-18 and IL-17 (rho = 0.47, p = 0.002), TG (rho = 0.45 p = 0.01) levels and AI (rho = 0.63 p = 0.021) were found. Whereas linear regression models revealed that IL-17, TG concentrations and the tender joint count had an impact on IL-18 Conclusions: We confirmed that patients with perPsA are characterized by a more pronounced proinflammatory and proatherogenic cardiovascular risk profile than patients with axPsA and AS. Importantly our study indicates that in PsA, but not in AS, elevated serum concentration of IL-18 is associated with higher disease activity and proatherogenic lipid profile, leading to a higher cardiovascular risk. Thus, our results point out IL-18 as a critical contributor in these pathological processes and possible therapeutic targets.

Elevated number of recently activated T cells in bone marrow of patients with rheumatoid arthritis: a role for interleukin 15?

OBJECTIVES: (1) To compare the absolute T-cell numbers in bone marrow (BM) isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA); (2) to measure the levels of soluble interleukin 15 (IL-15) and IL-7; (3) to analyse the expression of activation markers on T cells; (4) to analyse influence of IL-15 stimulation on T-cell proliferation. METHODS: BM samples were obtained from patients undergoing joint replacement surgery. Concentrations of IL-15 and IL-7 were measured using specific ELISAs. The absolute number of T lymphocytes, their activation status and proliferation were evaluated by flow cytometry. RESULTS: BM from patients with RA contained double the number of CD3 T cells in comparison with OA (6.1 vs 2.7 × 10(6) cells/ml, p<0.008). Ratio CD3CD4:CD3CD8 was increased in RA BM, clearly indicating accumulation of CD3CD4 cells. T cells obtained from patients with RA expressed higher level of early activation markers than from OA. Elevated levels of IL-15 were found in BM plasma from patients with RA in comparison with patients with OA (1304.5±956.3 pg/ml and 760±238.7 pg/ml respectively, p<0.01). These data were confirmed by immunohistochemistry of RA BM from regions proximal and distal to the joint. Although both CD3CD4 and CD3CD8 cells proliferated after IL-15 stimulation in vitro, CD3CD4 cells from patients with RA proliferated more vigorously than those from patients with OA, reflecting the composition of T-cell subsets in BM. CONCLUSION: These results suggest that locally overproduced IL-15 may be responsible for the activation and proliferation of T cells in situ, reflected by significantly increased number of activated T cells in RA BM, possibly contributing to the pathogenesis of RA.

Impact and Possible Mechanism(s) of Adipose Tissue-Derived Mesenchymal Stem Cells on T-Cell Proliferation in Patients With Rheumatic Disease.

Objectives: Systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are chronic wasting, incurable rheumatic diseases of autoimmune background, in which T cells play a critical pathogenic role. Autologous adipose tissue-derived mesenchymal stem cells (ASCs) may represent an alternative therapeutic option for SLE and SSc patients, but the biology of these cells is poorly understood. Methods: Herein, we evaluated the anti-proliferative impact of ASCs of healthy donors (HD/ASCs, 5 reference cell lines), SLE patients (n = 20), and SSc patients (n = 20) on T lymphocytes. To assess the direct and indirect pathway of ASCs action, peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells of HD were activated and co-cultured in cell-to-cell contact (C-C) and transwell (T-W) conditions with untreated or cytokine (TNF + IFNΥ, TI)-licensed ASCs, then analyzed by flow cytometry to rate the proliferation response of CD8+ and/or CD4+ T cells. The concentrations of kynurenines, prostaglandin E2 (PGE2), interleukin 10 (IL-10), and transforming growth factor ß (TGFß) were measured from culture supernatants. Specific inhibitors of these factors (1-MT, indomethacin, and cytokine-neutralizing antibody) were used to assess their contribution to anti-proliferative ASCs action. Results: All tested ASCs significantly decreased the number of proliferating CD4+ and CD8+ T cells, the number of division/proliferating cell (PI), and fold expansion (RI), and similarly upregulated kynurenines and PGE2, but not cytokine levels, in the co-cultures with both types of target cells. However, TI-treated SLE/ASCs and SSc/ASCs exerted a slightly weaker inhibitory effect on CD4+ T-cell replication than their respective HD/ASCs. All ASCs acted mainly via soluble factors. Their anti-proliferative effect was stronger, and kynurenine levels were higher in the T-W condition than the C-C condition. Blocking experiments indicated an involvement of kynurenine pathway in inhibiting the number of proliferating cells, PI, and RI values as well as PGE2 role in decreasing the number of proliferating cells. TGFß did not contribute to ASCs anti-proliferative capabilities, while IL-10 seems to be involved in such activity of only SLE/ASCs. Conclusion: The results indicate that SLE/ASCs and SSc/ASCs retain their capability to restrain the expansion of allogeneic CD4+ and CD8+ T cells and act by similar mechanisms as ASCs of healthy donors and thus may have therapeutic value.