MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.

Synergy between adiponectin and interleukin-1beta on the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in fibroblast-like synoviocytes

To determine whether adiponectin may have synergistic effects in combination with the proinflammatory cytokine interleukin (IL)-1beta regarding the production of proinflammatory mediators during arthritic joint inflammation, synovial cells from rheumatoid arthritis (RA) patients were treated with adiponectin, IL-1beta, and their combination for 24 h. Culture supernatant was collected and analyzed by enzyme-linked immunosorbent assay for levels of IL-6, IL-8, prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Adiponectin-mediated intracellular signaling pathways were investigated to elucidate the molecular mechanisms underlying their synergy. The association of proinflammatory mediators with adiponectin was investigated in the synovial fluid of arthritis patients. Adiponectin functioned synergistically with IL-1beta to activate IL-6, IL-8, and PGE2 expression in RA fibroblast-like synoviocytes; Levels of VEGF, MMP-1, and MMP-13 were not synergistically stimulated. Adiponectin and IL-1beta each increased the expression of both adiponectin receptor 1 and IL-1 receptor 1. However, adiponectin and IL-1beta did not synergistically support the degradation of IkappaB-alpha or the nuclear translocation of NF-kappaB. Synergistically increased gene expression was significantly inhibited by MG132, an NF-kappaB inhibitor. Supporting the in vitro results, IL-6 and IL-8 levels were positively associated with adiponectin in synovial joint fluid from patients with RA, but not osteoarthritis (OA). In conclusion, adiponectin and IL-1beta may synergistically stimulate the production of proinflammatory mediators through unknown signaling pathways during arthritic joint inflammation. Adiponectin may be more important to the pathogenesis of RA than previously thought.

Aspectos fisicoquímicos e citológicos do líquido sinovial da articulação temporomandibular de equinos em diferentes idades / Cytological and physicochemical aspects of the temporomandibular joint synovial fluid of horses in different ages

A análise do líquido sinovial é de grande importância para identificar alterações citológicas e químicas de afecções inflamatórias supurativas e não supurativas, hemorragias, neoplasia ou doenças infecciosas. O objetivo deste estudo foi analisar os aspectos fisicoquímicos e citológicos do líquido sinovial da articulação temporomandibular em 24 equinos hígidos, sendo 12 machos e 12 fêmeas, que foram divididos em quatro grupos, sendo um grupo controle (Gc) com a idade variando de cinco a 13 anos, grupo 1 (G1) com a idade variando entre cinco e sete anos, grupo 2 (G2) com idade variando entre oito e dez anos e grupo 3 (G3) com idade que varia entre 11 e 13 anos. O líquido sinovial foi avaliado quanto ao seu volume, pH, densidade, glicose, proteínas totais, hemácias, células nucleadas, neutró filos, linfócitos e macrófagos. Os animais do presente estudo foram tratados para alterações dentárias leves e não foi possível detectar alterações fisicoquímicas e citológicas no líquido sinovial entre os diferentes grupos.

The synovial fluid analysis is very important to identify cytology and chemical modifications of suppurative and nonsuppurative inflammatory diseases, bleeding, neoplasm or infectious diseases. The aim of this study was to analyze the physicochemical and cytological aspects of temporomandibular joint synovial fluid in 24 healthy horses, 12 males and 12 females, who were divided into four groups: control group (Gc) with age varied between five to 13 years, group 1 (G1), with ages varied between five and seven years, group 2 (G2) age varied between eight and ten years and group 3 (G3) with age varied between 11 and 13. The synovial fluid was evaluated for volume, pH, density, glucose, total protein, red blood cells, nucleated cells, neutrophils, lymphocytes and macrophages. The animals in this study were treated for minor dental abnormalities and it was not possible to detect changes in physicochemical and cytologic synovial fluid between the different groups.

Laboratory tests in pediatric rheumatology.

Abstract Laboratory tests in rheumatology are important tools that help to support the diagnosis of autoimmune diseases, evaluate the disease activity, monitor the side effects of therapy, and also assist the physician to exclude rheumatologic mimics. Few relevant tests should be ordered after a detailed clinical review of the patient has been carried out and a provisional clinical diagnosis has been reached. There is no test that can rule in or rule out any rheumatologic disease and therefore, there is no role of a detailed “Rheumatology panel” of investigations. In this review, routine blood investigations, acute phase reactants, auto antibodies, HLA B27 and complements have been discussed.

Approach to a child with monoarthritis.

Abstract Arthritis in childhood is common. The pattern, presentation and duration of arthritis help differentiate between the various possible diagnoses. When only one joint is involved, i.e., monoarthritis, it may be difficult to make a diagnosis as there are many possibilities both acute and chronic in nature. A detailed history and clinical examination is important to reach a correct diagnosis and the single most important investigation when a child presents acutely is a joint aspiration to rule out septic arthritis that may destroy the joint in hours. Inflammatory markers, antinuclear antibody testing, test for tuberculosis and imaging (in specific cases) play an important role in the diagnosis of a child that presents with a chronic monoarthritis. In this article we provide a clinical approach to the diagnosis of monoarthritis in a child.

Induction of metalloproteinases expression by TLR ligands in human fibroblast like synoviocytes from juvenile idiopathic arthritis patients.

Background & objective: Juvenile idiopathic arthritis (JIA) is characterized by chronic synovitis, cartilage damage and bone erosion. Both genetic and environmental factors and microbes probably play a role in pathogenesis. Microbes are recognized by Toll like receptors (TLRs) and activate innate immune response. We studied the ability of bacterial and viral products to produce matrix metalloproteinases (MMPs) and cytokines by fibroblast like synoviocytes (FLS) from patients with JIA. Methods: FLS were cultured from synovial fluid (SF) of patients with JIA and subsequently stimulated for 48 h by different TLR ligands [peptidoglycan (PG) for TLR2, poly(I-C) for TLR3, lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, imiquimod for TLR7 and CpG DNA for TLR9]. Later the production of IL6, IL8, MMP-1, MMP-3, tissue inhibitors of metalloproteinase (TIMP1) was measured in the culture supernatants by ELISA. Expression of TLR2, TLR4, TLR7 and TLR9 was studied in FLS derived from JIA patients by RT-PCR. Results: IL6, IL8, MMP3 and MMP1 production was induced on stimulation of FLS with TLR2 ligand, TLR3 ligand, TLR4 ligand, TLR5 ligand but not with TLR7 ligand and TLR9 ligand. There was no effect of these ligands on the production of TIMP thus the balance was tilted in favour of MMPs after TLR ligation. TLR2, TLR4 and low expression of TLR9 was found but, no expression of TLR7 was found in FLS from JIA patients. Interpretation & conclusion: TLR pathway stimulation by microbial products or endogenous ligands could be involved in the production of MMPs in JIA and may contribute to disease pathology. Thus it may be beneficial to inhibit TLR pathway to reduce cartilage destruction.

Influência do exercício na indução da apoptose e necrose das células do líquido sinovial de eqüinos atletas / Exercise induced apoptosis and necrosis in the synovial fluid cells of athletic horses

Examinaram-se os efeitos do estresse mecânico na resposta inflamatória e adaptativa dos tecidos articulares de cavalos atletas. O líquido sinovial foi colhido das articulações metacarpofalangeanas de eqüinos atletas antes, 3, 6 e 24 horas após o exercício, assim como de um grupo controle (cavalos não exercitados). A porcentagem de apoptose/necrose, o TNF-a e a PGE2 foram determinados pelo ensaio de AnexinaV/Iodeto de Propídeo, bioensaio (L929) e ELISA, respectivamente. Os resultados mostraram que a contagem total de células nucleadas foi sempre menor no grupo controle em relação ao grupo atleta (P<0,05). Observaram-se aumentos na porcentagem de células em apoptose (P<0,05) e necrose (P<0,05), concentração de PGE2 (P<0,05) e proteína sinovial (P<0,05), e diminuição da concentração de TNF-a (P<0,05) após 3 horas do término do exercício. O grupo atleta apresentou grau moderado de inflamação articular após o exercício intenso. Esta resposta dos tecidos articulares frente ao insulto mecânico do exercício, com maior intensidade às 3 horas após término da atividade esportiva e retornando à normalidade 24 horas após, revela a capacidade da adaptação articular ao estresse físico, em eqüinos atletas.

The effects of biomechanical stress on inflammatory and adaptative responses of articular tissues in athletic horses were investigated. Synovial fluid was collected from the metacarpophalangeal joints of athletic horses before exercise and 3, 6, 24 hours after exercise, and as well as from the control group (without exercise). Apoptosis/necrosis percentage, TNF-a and PGE2 were determined by annexin V/PI assay, bioassay (L929) and ELISA, respectively. The results showed that total leukocyte count was higher in the athletic group when is compared with the control group. Three hours after the exercise was done there were increases of cellular apoptosis (P>0.05) and necrosis (P<0.05) percentage, PGE2 concentration (P<0.05) and protein concentration (P<0.05), and the TNF-a level has dropped. The athletic group showed moderate level of joint inflammation after the strenuous exercise. This articular tissue response to biomechanical insult due to the exercise, with high intensity after 3 hours after training associated with normality after 24 hours, reveals the articular adaptation to physical stress in athletic horses.

Polarized light microscopy [PLM]: a role in filling the gaps of undiagnosed arthritis

Joint arthritis is a major clinical problem for any rheumatologic clinic. Diagnosis of these types of arthritis usually depends upon certain clinico-investigatory criteria usually settled by international organizations. Even the use of these criteria does not always revealed a solid diagnosis in many occasions. Moreover, there are reported literatures about presence of coexistence between different types of arthritis. Lack of diagnosis may result in poor outcome of management and sometimes worsen the prognosis of the case. This study aimed t. To evaluate synovial fluid analysis in diagnosis of effusion-associated arthritis to reach a final diagnosis in undiagnosed cases and to role out the importance of Polarized Light Microscopy [PLM] in diagnosis of the coexistence of two or more types of arthropathies. The present study is a cross-sectional descriptive hospital-based study, conducted in the department of Rheumatology and Rehabilitation in Assiut University Hospital. Sixty-one patients with established joint effusion [acute or chronic] were included in the study. The patients were grouped according to the type of rheumatological disease into 6 groups each of them represented one of the rheumatological diseases. Twelve cases were diagnosed as RA, 16 as OA, 9 as gout, 1 as pseudogout, 4 as SLE, and 4 as SPA. The final diagnosis could not be reached in 15 of them. The seventh group was the undiagnosed group. All the allocated participants were subjected to synovial fluid [SF] examination, macroscopically using [PLM] and microscopically, for leukocytic count and crystals. Monosodium urate [MSU] and Calcium pyrophosphate dihydrate [CPPD] crystals were identified. Of [SF] analysis were correlated with the preliminary clinical diagnosis which revealed that out of 61 examined cases combined arthritis was diagnosed in 10 cases [16.4%]. These 10 cases were combined OA and CPPD in 5 cases, combined RA and CPPD in 2 cases, and combined RA, MSU and CPPD in one case. Additionally, combined SLE and CPPD was diagnosed in one case and combined SPA and MSU in another one. Consequently, [PLM] examination allowed us to reduce the undiagnosed cases from 24.6% to 16.4%. Examination of SF for MSU and CPPD crystals was worth looking and can change the management strategy. PLM remained the only practical way of identifying these particles in the clinical setting

Serum and synovial fluid concentrations of soluble granzyme B in early rheumatoid arthritis

Cytotoxic cells possess specialized granules which contain perform and a group of serine proteinases termed granzymes. Granzyme B is a serine protease that have been identified in synovial fluid and tissue of rheumatoid arthritis [RA] patients, where they may play an important role as mediators of granule-mediated apoptosis, extracellular proteolysis, and cytokine induction. To assess plasma and synovial fluid soluble granzyme B concentrations in early RA patients and its association to seropositivity, ESR, C-reactive protein [CRP], disease activity and the rate of radiological progression. This was in an attempt to throw light on its possible role as predictive factor for disease severity and joint outcome. This study included 80 early RA patients presenting with knee effusion and 30 matched osteoarthritis [OA] patients presenting with traumatic non-hemorrhagic knee synovial effusion as a control group. Serum and synovial fluid granzyme B concentrations were determined with ELISA. Assessment of disease activity. Rheumatoid factor [RF], CRP and ESR were measured. Radiographs of the hands, wrists and forefeet were taken for all RA patients at baseline and after one year. Radiographical damage and scoring were evaluated. There was a highly significant increase in serum and synovial fluid concentrations of soluble granzyme B in RA patients as compared to OA control group. Also, there was a highly significant increase in synovial fluid concentrations of granzyme B as compared to serum concentrations in RA patients. There was a highly significant increase in serum and synovial fluid concentrations of granzyme B in seropositive RA patients as compared to seronegative patients. There were highly significant elevations in the mean serum and SF granzyme B concentrations at the entry of the study in patients showing radiological progression than non-progressive group. There was a statistically significant positive correlation between serum granzyme B concentration and CRP and a highly significant positive correlation as compared with disease duration, number of swollen joints, Ritchie articular index [RAI], general health status, ESR, disease activity score, RF, radiological progression or granzyme B synovial fluid concentration. High soluble granzyme B concentrations are found in early rheumatoid arthritis patients. Increase in serum concentrations of granzyme B in seropositive RA patients as compared to seronegative patients and increased concentrations associated with the development of radiographic erosions in those patients raise the attention to the importance of granzyme R as a marker for prediction of radiographic joint damage. So granzyme B concentration can be considered as a useful prognostic marker in early rheumatoid arthritis patients

Características físico-químicas e citológicas do líquido sinovial da bainha tendínea digital de eqüinos / Physical, biochemical and cytological characteristics of the equine digital flexor tendon sheath synovial fluid

Foram estudadas as características físico-químicas e citológicas do líquido sinovial da bainha tendínea digital de nove eqüinos hígidos. Verificou-se que o líquido é viscoso, amarelo claro, límpido, livre de partículas e que não coagula à temperatura ambiente. Sua concentração média de ácido hialurônico foi 60,20mg/dl, a taxa de glicose, similar à plasmática e sua concentração protéica não ultrapassou 1,74g/dl, com relação média albumina:globulina de 0,94. O número médio de células nucleadas foi de 313 células/æl, com predominância de grandes células mononucleares e linfócitos. Houve correlação significativa (r = - 0,649, P<0,01) entre o aumento da concentração de ácido hialurônico e a diminuição percentual de linfócitos. As mensurações das características pesquisadas no líquido sinovial da bainha tendínea digital de eqüinos são de execução simples e passíveis de implantação na rotina de atendimentos clínico-cirúrgicos.

Serology and immunoglobulin profile in rheumatoid arthritis.

One hundred and twenty cases of clinically diagnosed rheumatoid arthritis, 80 non-rheumatoid cases suffering from various other diseases and 40 healthy individuals were investigated for the presence of rheumatoid factor, quantitation of serum immunoglobulin, demonstration of ANA and LE cell phenomenon. Microlatex agglutination test of serum for rheumatoid factor showed 56.6% positivity in rheumatoid group and 3.7% positivity in non-rheumatoid group. All three serum immunoglobulins (IgG, IgM, IgA) were raised in serum in significant titre in cases of rheumatoid arthritis, whereas only IgA lever was elevated in the group of non-rheumatoid diseases. ANA and LE cell phenomenon were observed in 11.7% and 4.4% cases of rheumatoid arthritis who had severe underlying disease. In non-rheumatoid group, only one of 6 cases of systemic lupus erythematosus showed rheumatoid factor and that too in an insignificant titre (less than 1:20). Synovium and synovial fluid contained plenty of plasma cells and lymphocytes. It has been observed that RF appears first in synovial fluid and it may take several months to a year to attain detectable level in serum.

The clinical significance of cytoplasmic inclusions(CPI) in synovial fluid examination

The clinical significance of cytoplasmic inclusions(CPI) in synovial fluid(SF) examination was evaluated. We examined SF specimens collected from major rheumatology clinics in the Philadelphia area during the period of January to December 1995. Among 759 patients in the initial study group, 419 cases with established diagnoses and full synovial analyses were included. Their diagnoses and SF analysis results including leukocyte counts, differential counts and wet preparations were collected and analysed. Ninety seven of the 419 SF specimens were found to have CPI. CPI were found in SF from almost all rheumatic diseases. They were most likely to be found in inflammatory arthropathy including rheumatoid arthritis(RA, 46%), juvenile rheumatoid arthritis(JRA, 78%) and psoriatic arthritis(55%). On the contrary, CPI were least common in crystal-induced arthropathy among the inflammatory arthropathy. CPI were found 8 out of 98 gout cases(8%) and 2 among 53 calcium pyrophosphate dihydrate(CPPD) deposition disease(4%). In noninflammatory arthropathy, CPI were found in only 6 cases(6%) out of the 103 osteoarthritis(OA). In RA cases with non-inflammatory SF, 4 of the 20 SF(20%) had CPI while only 6% of OA SF had CPI. OA SF with CPI were all noninflammatory SF. In summary, CPI were a common finding on SF examination. CPI were more likely to be found in inflammatory arthropathy than noninflammatory. Among inflammatory arthropathy, CPI can favor non-crystal arthropathy than crystal arthropathy. Awareness of the presence of CPI is suggested as an addendum to routine SF analysis. Renewed investigation of the several types of CPI may add further to the understanding of joint disease.

Discrepancy in T cell clonal expansions in synovial fluid and peripheral blood from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease involving the synovial membrane of peripheral joints. T cells specific for self antigens may play a critical role. Identification of T cell receptors (TCR) of such specific T cell clones is very important for treatment, prevention and identification of relevant autoantigens. To identify specific T cells, TCR V beta family repertoire and the clonal expansion of T cells were analyzed in this study. The percentage of V beta 5+ or V beta 8+ cells in the synovial fluid mononuclear cells (SFMCs) was similar to that in the peripheral blood mononuclear cells (PBMCs). However, the percentage of DR+ T cells in the SFMCs was higher (p< 0.01). Analyzing the clonality of T cells in 8 V beta families (V beta 1, V beta 5, V beta 8, V beta 14, V beta 16, V beta 17, V beta 18, V beta 20), clonal expansions in CD8+ T cells from the SFMCs were found more frequently than in the PBMCs. The patterns of clonal expansions were discrepant between the SFMCs and the PBMCs even in the same patient, which suggests several inflamed tissue specific T cell clonal expansions in the SFMCs. These T cell clones might be activated by autoantigens which are not identified yet and responsible for the RA pathogenesis.

Estudio anatomopatologico de las afecciones de la sinovial y tejidos periarticulares / Study anatomopatologico of the afections of the synovial and tissue periarticulares

Se presenta una revision de la patologia de la sinivial y tejidos periarticulares en 446 casos clasificandolos en lesiones inflamatorias, tumorales, pseudotumorales y lesiones diversas. Se destaca el predominio de las afecciones inflamatorias (46%) y entre ellas la alta frecuencia de lesiones inespesificas mas en las dos terceras partes. Se mensiona asimismo la frecuencia importante (23%) en nuestro medio de inflamacion tuberculosa, enpacientes generalmente jovenes

La bioquímica y su aplicación en ortopedia: revisión I; la célula sinovial / Biochemistry and its application in orthopedics: a review I; the sinovial cell

Se presenta una breve revisión sobre algunos aspectos del exoplasma y el endoplasma del sinoviocito, en la que se atiende a elementos bioquímicos, que son de interés en Ortopedia, porque pueden ser útiles en el diagnóstico, el pronóstico y la terapéutica

Ferritin measurement in synovial tissue and fluid in patients with inflammatorys joint disease

Se dosificó mediante ensayo inmunorradiométrico la concentración de ferritina (FET), una proteína que almacena hierro, en el líquido sinovial (LS) de 41 pacientes con sinovitis clínicamente activa de distinta etiología. De acuerdo al diagnóstico los enfermos se distribuyeron en cuatro grupos: el grupo de artropatía degenerativa (DA) formado por cuatro enfermos con osteoartosis; siete pacientes con gota y uno con pseudogota integraron el grupo de artropatía por depósito de cristales (AC); el grupo de artropatía inmune (AI) constituido por trece enfermos con artritis reumatoide y siete con lupus eritematoso sistémico; y un grupo de artropatías misceláneas (AM) formado por nueve pacientes. El grupo AD mostró nivele sde FET significativamente menores a los encontrados en los grupos AC, AI y AM. En cambio, no se observaron diferencias significativas en los valores de FET en LS entre los grupos AC, AI y AM. Además, se cuantificó FET en nueve muestras de suero y en cinco de tejido sinovial (TS). Los niveles de FET en LS correlacionaron en forma significativa con la cantidad de FET en TS y con el número de leucocitos en el LS, pero no existió correlación entre la FET en LS y en suero. Estos resultados indican que la dosificación de FET en LS tiene poca utilidad en el diagnóstico diferencial de las artropatías inflamatorias, y sugiere que probablemente los leucocitos y/o las células sinoviales sean la fuente de FET en el LS