Leukocyte agglomeration in synovial fluid

The agglomeration of leucocytes serves as evidence of leucocytic activation. In rheumatoid arthritis it can be used as a very sensitive criterion of activity. Patients with rheumatoid arthritis with joint effusions showed a high agglomeration of leucocytes in 97%, i.e. a clear activation of leucocytes. In 74.3% the number of punctate leucocyte agglomerated increased. Punctates with a proof of a spontaneous agglomeration of leucocytes showed the most intensive inclination to agglomeration also after incubation. By synovial fluid normal but also leucergic granulocytes could be stimulated to increased agglomeration to leucocytes. In the synovial fluid also factors stimulating the agglomeration of leucocytes are found. They probably come from punctate granulocytes and stimulate also the chemotaxis and activation of the blood granulocytes. Since granulocytes essentially participate in the inflammatory reaction of the rheumatoid arthritis and in the joint destruction evoked by it, the therapeutic aspect should be directed also to the inhibition of such leucocyte-activating factors in the synovia.     

Cytophotometric DNA measurements in mononuclear round cells of synovial fluid]

Cytophotometric investigations were performed on synovial fluid mononuclear round cells in patients with various joint diseases. In nearly all instances only round cells with a diploid DNA-content were found. No increased proliferation could be observed neither in patients with rheumatoid arthritis nor in patients with a high relative number of neutrophilic granulocytes in the synovial fluid.     

Cellular hypersensitivity in rheumatoid arthritis,ankylosing spondylitis,and anterior nongranulomatous uveitis

The humoral and cellular responses to normal human ocular and joint tissue antigens were studied in rheumatoid arthritis, ankylosing spondylitis, and anterior non-granulomatous uveitis. No free-circulating autoantibodies could be detected by hemagglutination, immunodiffusion, and immunofluorescence. Uvea-retina, synovial membrane, and articular cartilage antigen preparations inhibited the migration of leukocytes from patients with rheumatoid arthritis. In ankylosing spondylitis inhibition could be induced only by synovial membrane antigens. Patients with anterior nongranulomatous uveitis were hypersensitive to uvea-retina antigens alone.     

Development of surface antigen during maturation of bone marrow neutrophil granulocytes.

The presence of neutrophil surface antigens on maturing bone marrow granulocytes was examined by indirect immunofluorescence and cellular immunoadsorption using heterologous antibody to mature neutrophil granulocytes. The results show that bone marrow neutrophils possess surface antigens that appear during cell maturation from myeloblasts. Fluorescent antibody capping and patching, indicators of antigen mobility, were more pronounced in mature than in immature cells. Neutrophil surface antigen development could also be demonstrated during granulocyte maturation in vitro.     

HLA-DR antigens and disease patterns of rheumatoid arthritis

HLA-DR antigens were determined in 111 patients with classic or definite rheumatoid arthritis. HLA-DR4 was significantly (P corr. less than 10(-6] increased in patients with rheumatoid arthritis (54%) compared with controls (23.2%). HLA-DR 5 was decreased in rheumatoid arthritis (12.6% vs 26.4% of controls); however, the corrected P value was not significant. There were no significant differences with regard to various clinical, radiological and serological parameters between HLA-DR 4 positive and negative patients. However, a milder course of rheumatoid arthritis was observed in DR 7 positive patients: Patients with this antigen were associated significantly with seronegativity and low titers of IgM-rheumatoid factor. Despite a similar disease duration patients with DR 7 had a significantly lower number of joints with inflammatory arthritis (synovitic swelling with limitation of movement) and developed less frequently severe radiological changes as joint ankylosis than DR 7 negative patients. In addition to the well known association between rheumatoid arthritis and HLA-DR 4, our data indicate that HLA-DR 7 may have a protective effect on the course of rheumatoid arthritis.     

Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: role of C5a in the acute inflammatory phase.

Evidence suggests that complement is activated in rheumatoid joints. A sensitive radioimmunoassay for the activation fragment of C5, C5a, which is a potent chemoattractant for neutrophils, was therefore developed. A mean C5a concentration in 22 rheumatoid joint fluids of about 2.5 x 10(-9) mol/l was found. This concentration of C5a is sufficient to induce two of the characteristic features of the acute inflammatory phases of rheumatoid arthritis: neutrophil accumulation and microvascular plasma protein leakage. In animal models it has been shown that C5a is a potent inducer of inflammatory oedema by a neutrophil dependent mechanism. A striking feature of the acute inflammatory phases of rheumatoid arthritis is the appearance of high numbers of neutrophils in the synovial fluid. It is suggested that C5a might have a role in mediating neutrophil accumulation and, as a consequence, may be important in acute joint swelling and pain.     

Antigenicity and accessory cell function of human articular chondrocytes

It is postulated that chondrocytes may be actively involved in the pathogenesis of inflammatory joint diseases, presumably by providing tissue specific antigens that may initiate or sustain autoimmune reactions. To investigate whether chondrocytes may also function as accessory cells in ongoing immune processes, mixed leukocyte-chondrocyte cultures and antigen presentation assays were studied. Freshly isolated and short term cultured HLA class II antigen (Ia) negative as well as gamma-interferon treated Ia positive chondrocytes were weakly or not stimulatory to allogeneic or autologous resting lymphocytes derived from either normal donors or patients with rheumatoid arthritis. In an antigen presenting system using tetanus toxoid, the majority of chondrocyte preparations tested induced an antigen driven response in HLA matched allogeneic or autologous resting T cells which, however, was much less when compared to blood monocytes. In contrast, using activated T cells derived from tetanus toxoid specific T cell lines, an efficient antigen presenting capacity could be demonstrated in both Ia positive and initially Ia negative chondrocytes. Interestingly, the latter population had acquired Ia antigens upon incubation with the T cell line.     

Differences in the production of and/or the response to interleukin-2 by T lymphocytes from patients with the various connective tissue diseases

Summary The meniscal surfaces from patients with and without inflammatory joint diseases were investigated for the presence of superficially located polymorphonuclear granulocytes (PMNs). In histochemically stained tissue sections as well as in electron microscopic investigations on previously paraffin-embedded menisci, PMNs were observed in cases with inflammatory rheumatoid joint diseases. The inflammatory cells were located in fibrin adhering to the meniscal surface and in the fibrous meniscal tissue just beneath the fibrin. From these observations it is concluded that PMNs in the inflammatory synovial fluid may gain access to the fibrous structures of the joint, thus participating in tissue destruction, as has been assumed from in vitro investigations by other authors.     

Increased expression of the blood group-related Lewis Y antigen on synovial fluid granulocytes of patients with arthritic joint diseases

OBJECTIVE: To evaluate the expression of the carbohydrate structures Lewis Y (LeY), sialyl-LeX (sLeX) and Lewis X (LeX) on paired peripheral blood (PB) and synovial fluid (SF) granulocytes in patients with arthritic diseases. METHODS: Ten patients with rheumatoid arthritis (RA), seven patients with spondyloarthritis (SA) and eight patients with osteoarthritis (OA) were studied. Granulocyte expression of the Le oligosaccharides was analysed by fluorescence-activated cell sorting. RESULTS: SF granulocytes of patients with RA, SA and OA expressed higher levels of the LeY oligosaccharide than PB granulocytes. Increases in LeY on SF granulocytes were similar in all three underlying diseases. No differences in the expression of the Le antigens were detected between PB granulocytes of patients and healthy individuals. Expression of sLeX and LeX showed no variation between SF and PB neutrophils. CONCLUSION: The selective increase in LeY antigen on SF granulocytes in RA, SA and OA suggests a role of the LeY oligosaccharide in granulocyte traffic and inflammatory responses.     

A new acute-phase protein. II. Quantitative determination in the serum of patients with rheumatoid arthritis

An antigen (SF1 or TSGA) originally found in the inflammable synovial fluid of man, which comes from the cytoplasm of neutrophils, possesses the properties of an acute-phase-protein. The production of a specific antiserum against this antigen and the method of its quantitative determination by means of the Mancini-technique are described. In 89 sera of patients with rheumatoid arthritis the antigen was determined. First results of this investigation which speak for a dependence of the antigen concentration in the serum on activity and progression of the disease are reported.     

Polymorphonuclear granulocytes in rheumatic tissue destruction. III. an electron microscopic study of PMNs at the pannus-cartilage junction in rheumatoid arthritis.

Metatarsophalangeal and metacarpophalangeal joints from 3 patients with rheumatoid arthritis were investigated electron microscopically with regard to the occurrence of polymorphonuclear granulocytes (PMNs) at the pannus-cartilage junction. In all 3 cases PMNs could be detected at the junction and within the cartilaginous matrix. PMN cytoplasmic processes surrounded collagenous islands in the cartilage. From the morphological findings it is deduced that PMNs are cells capable of destroying cartilage in inflammatory joint diseases, in particular in rheumatoid arthritis.     

Structured approach to the investigation of anaemia in patients with rheumatoid arthritis.

A group of 28 patients with rheumatoid arthritis who were severely anaemic were investigated for iron deficiency. On the basis of bone marrow studies, the patients were divided into two groups, those with and those without signs of stainable iron in the marrow. This grouping did not distinguish between the severity of their rheumatoid arthritis measured by clinical parameters. Measurement of the red cell count and biochemical parameters in the peripheral blood showed a statistical difference in red cell size, haemoglobin content, and iron binding capacity between the two groups. The statistical variation of these parameters, however, did not allow these measurements to predict bone marrow iron deficiency in any subject. Investigation of the upper gastrointestinal tract by endoscopy showed that acute macroscopic lesions were infrequently associated with anaemia. It was concluded that anaemia in association with rheumatoid arthritis may mimic iron deficiency anaemia, and that simple investigations of the peripheral blood do not accurately show the iron status of the reticuloendothelial system in the presence of a chronic inflammatory disease. For the investigation of severe anaemia in rheumatoid arthritis, bone marrow assessment of iron status should be performed as the initial investigation. In addition, iron deficient patients require investigation of the lower and the upper gastrointestinal tract.     

Eosinophil cationic protein in inflammatory synovial effusions as evidence of eosinophil involvement.

Eosinophils are seldom noted in inflammatory synovial fluids but are reported to infiltrate the synovial tissue in inflammatory arthritides. To elucidate a possible role for eosinophils in inflammatory joint reactions the concentrations of eosinophil cationic protein (ECP)--a specific granule protein from eosinophils--were measured by radioimmunoassay in 90 synovial fluids from patients with various inflammatory arthritides (rheumatoid arthritis, reactive and crystal arthritides, Reiter's disease and psoriatic arthropathy). In the same specimens lactoferrin was measured as an indicator of neutrophil-involved inflammation. In comparison with the normal circulating levels of ECP and lactoferrin the measured synovial fluid concentrations of both proteins were considerably raised in all patient groups with inflammatory joint diseases in contrast to patients with non-inflammatory arthritides. There was a striking positive correlation between the ECP and lactoferrin synovial fluid concentrations. These data indicate that eosinophil activation is prominent in inflammatory joint reactions and is linked to the activation of neutrophils. The regulation of degranulation or secretion by eosinophils is unknown. Our in-vitro studies showed that peripheral blood isolated neutrophils as well as eosinophils degranulated when exposed to IgG complexes. However, eosinophil degranulation was modest compared with neutrophil degranulation. These data suggest that neutrophil phagocytosis of, for example, immune complexes may be one major mechanism in neutrophil degranulation but that other factors determine the appearance of eosinophil products in inflammatory synovial effusions. The possible modulatory or harmful role of eosinophils in inflammatory joint disease can at present only be speculated on.     

Serum hyaluronic acid and aminoterminal procollagen III peptide in inflammatory and degenerative joint diseases

Serum levels of hyaluronic acid (HA) and the amino-terminal type III procollagen peptide (NP-III-P) were determined simultaneously by specific immunoassays in patients with rheumatoid arthritis (n = 41), osteoarthritis (n = 43), ankylosing spondylitis (n = 7), psoriatic arthritis (n = 6), and reactive arthritis (n = 6). Increased serum levels of both HA and NP-III-P, were found in rheumatoid arthritis and - although less pronounced - in osteoarthritis, differing significantly from age- and sex-matched controls (n = 77). Furthermore, patients suffering from active rheumatoid arthritis showed higher serum levels of both antigens than patients with inactive disease, and significant correlations were found in rheumatoid arthritis between acute phase plasma proteins, HA and NP-III-P, respectively. In contrast, determination of low molecular weight fractions of NP-III-P by Fab- assay proved not to be useful in regard to clinical application. No significant effects of anti-inflammatory treatment were evident in any of the parameters. In rheumatoid patients, the serum concentrations of HA were found to correlate positively with the serum reactivity of NP-III-P related antigens (r = 0.692) and with the excretion of urinary pyridinoline (r = 0.455). Thus, both parameters seem to reflect similar mechanisms of connective tissue activation and may be related to inflammatory activity in joint diseases.     

Protéines S100A8, S100A9 et S100A12 : marqueurs inflammatoires ou acteurs physiopathologiques de la polyarthrite rhumatoïde

Although S100 proteins represent 40% of the neutrophil cytoplasmic proteins, their physiological and pathological functions are still unclear. S100A8, S100A9 and S100A12 protein concentrations are dramatically enhanced in synovial fluid and synovium of patients suffering from rheumatoid arthritis. Their expression seems to correlate with disease activity and joint damage. These proteins are likely involved in rheumatoid arthritis pathogenesis by enhancing extracellular matrix proteolysis, autoimmunity and inducing the pseudotumoral phenotype of the synoviocytes in rheumatoid arthritis. S100A8, S100A9 and S100A12 assessment will probably constitute a relevant tool for rheumatoid arthritis diagnosis and will improve inflammatory arthritides management.     

The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation

This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 μg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 μg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 μg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.     

Profilographic studies with 99mTc-pertechnetate in joint diseases in children

The inflammatory activity of joints can be stated after intravenous administration of 99mTc-pertechnetate by demonstration of an enrichment of isotope. In 85 out of 111 patients (77%) a good accordance between profilogram and clinical finding could be seen. "False positive" results could be found in juvenile rheumatoid arthritis, ulcerative colitis, reactive arthritis and juvenile gout. In some cases of juvenile rheumatoid arthritis the clinical finding is later obvious than the demonstration by nuclear medicine. "False negative" results mainly can be seen at the wrists of patients with juvenile rheumatoid arthritis and tendosynovitis as well as in already existing radiological changes of the bones. Differential diagnostical evidences of profilography cannot be expected with the investigation technique used here. It, however, may contribute to assure the diagnosis in non-inflammatory joint diseases ("psychogenic rheumatism", lymphatic oedema).     

Rheumatoid arthritis and enteric bacteria

Factors in the etiopathogenesis of rheumatoid arthritis (RA) include the genetic back-ground, environmental factors and perpetuation of the inflammatory process. This review focuses on enteric bacteria as initiating or perpetuating factors in the etiopathogenesis of RA. Based on the hypothesis that entrobacterial antigens that originated from intestinal flora induce rheumatoid inflammation in the joints, animal models of arthritis due toEnterobacteriaceae, studies on humoral and cellular responses to entrobacterial antigens in RA, etiology of RA involving both genetic and environmental factors, pathogenesis of rheumatoid inflammation accompanied by joint destruction, and clinical trials with basic therapeutic considerations are reviewed. The results of immunological studies on RA suggest that some patients with RA are sensitized to antigens common amongEnterobacteriaceae (bacterial outer membrane proteins of 35 and 38 kDa). A bacterial outer membrane protein of 38 kDa was identified as OmpC having amino acid homology (NYGVV) with HLA-DR4. This OmpC peptide elicited peripheral blood T cell proliferative responses in patients with RA. The presentation of this enterobacterial peptide by the RA-associated DR motif to CD4⁺ T cells could lead to initiation of disease. We now consider that in some patients, RA may be based on autoimmunity from molecular mimicry by entrobacterial antigens of HLA-DR4.     

Effect of steroid treatment on the migration behaviour of neutrophils in patients with early rheumatoid arthritis

The influence of methylprednisolone on the migratory characteristics of neutrophil granulocytes was investigated in 10 patients with early rheumatoid arthritis (RA) and compared to 12 controls. The migration of neutrophils was measured with a whole-blood membrane filter assay with and without stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Total migration index (TMI), distribution characteristics (DC) and the product of TMI and DC (neutrophil migratory activity; NMA) served to characterize the migratory behaviour of neutrophils. The data demonstrated an increased polymorphonuclear leucocyte (PMN) migration in patients with early RA, indicating a bystander role of PMNs in inflammatory joint injury. Treatment with methylprednisolone reduced significantly the penetration depth (DC) of neutrophils, but did not influence the number of migrating cells (TMI). The unstimulated NMA was significantly reduced due to the marked DC reduction, whereas steroids did not influence the stimulated NMA of neutrophils. A significant reduction in PMN penetration depth was demonstrated only after a steroid therapy of at least 10 days, suggesting that a longer period of steroid therapy is necessary to provide effective inflammatory control. Received: 24 March 1997 / Accepted: 8 September 1997     

Pathogenesis of bone erosions in rheumatoid arthritis

Focal marginal joint erosions represent the radiographic hallmark of rheumatoid arthritis (RA). These bone changes are characteristically localized to the joint margins, but in addition, regions of focal bone resorption can be detected in the subchondral bone adjacent to the bone marrow space into which the synovial inflammatory tissues have extended. Because progressive destruction of the periarticular bone contributes significantly to joint dysfunction and disability in patients with RA, there is considerable interest in developing a better understanding of the pathologic mechanisms involved in this process and in developing therapies that can arrest these events. Previous analysis of joint tissues from patients with RA have provided morphologic evidence that osteoclasts are the cell types that mediate the focal bone resorption associated with the rheumatoid synovial lesion. Additional recent data from animal models have helped to further implicate these cells in the pathogenesis of focal bone erosions. Furthermore, analysis of RA synovium and joint tissues from animal models of inflammatory arthritis, as well as cell and tissues culture studies, have helped to define the cytokines and inflammatory mediators that are involved in the recruitment and activation of bone resorbing cells associated with focal bone erosions. These findings provide a rational framework for developing targeted therapies that can specifically inhibit or slow the progressive focal bone destruction associated with the rheumatoid synovial lesion.