Glutathione reductase activity correlates with concentration of extracellular matrix degradation products in synovial fluid from patients with joint diseases.

The mechanisms underlying cartilage matrix degradation in joint diseases is not fully understood but reactive oxygen species are implicated as main causative factors. Comparative studies of glutathione reductase (GR) activity in synovial fluid from patients with rheumatoid arthritis (RA), reactive arthritis (ReA) and osteoarthritis (OA) as well as correlations between GR activity and concentration of the major cartilage components in synovial fluid are presented in this study. We found significantly higher activity of GR in RA (about three-fold) and ReA (about two-fold) than in OA. In RA and ReA patients, GR activity in synovial fluid correlates negatively with the concentrations of collagen and degradation products of sulfated glycosaminoglycans. In OA patients the activity of GR was significantly lower than in RA and ReA, which positively correlated with the concentration of collagen and showed a tendency for positive correlation with the degradation products of sulfated glycosaminoglycans. Our results suggest that in RA and ReA patients increased activity of GR does not prevent the increased degradation of collagen and proteoglycans by ROS.

Association study of LMP gene polymorphisms in Mexican patients with spondyloarthritis.

To evaluate the role of LMP (low molecular weight protein) genes as susceptibility markers for spondyloarthritis (SpA), LMP gene polymorphisms were analyzed in 223 Mexican patients with SpA (81 undifferentiated SpA [U-SpA], 117 with ankylosing spondylitis [AS], 25 with reactive arthritis) and in 139 ethnically matched healthy individuals. LMP genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. The LMP2 and LMP7 allele frequencies were similar in patients and healthy controls. Genotype analysis revealed an increased frequency of LMP2 R/R genotype in the whole group of SpA (pC = 0.003, OR = 2.06, 95%CI = 1.3-3.25) and in the clinical subgroups of AS (pC = 0.039, OR = 1.88, 95%CI = 1.1-3.22) and U-SpA (pC = 0.003, OR = 2.56, 95%CI = 1.37-4.8) compared with healthy controls. Analysis in the LMP7 did not reveal significant differences in patients and healthy controls. The HLA-B27-negative AS subgroup also showed an increased frequency of LMP2 R/R genotype (pC = 0.027, OR = 4.81, 95%CI = 1.21-22.13). The LMP2-R/R AS patients were younger than LMP2-H/R and H/H patients at onset of the disease (16.0 +/- 6.8 years for R/R, 22.0 +/- 11.2 years for H/R and 28.6 +/- 10.9 years for H/H) (p < 0.05). The data suggest that, besides HLA-B27, LMP2 genotypes are also involved in the genetic susceptibility to develop AS in Mexicans. Furthermore, the age at onset of this disease might also be influenced by genotypes of this gene.

Synovial fluid matrix metalloproteinase-3 levels are increased in inflammatory arthritides whether erosive or not.

OBJECTIVE: To study the levels of matrix metalloproteinase-3 (MMP-3) in the knee synovial fluid (SF) of inflammatory arthropathies (rheumatoid arthritis whether erosive or not, reactive arthritis, acute crystal arthritis) and degenerative arthropathies [chronic crystal disease, osteoarthritis and (control) meniscus pathology] and to correlate them with the degree of joint destruction, local inflammatory and immune parameters and systemic markers of inflammation. METHODS: SF levels of MMP-3 (precursor, active and tissue inhibitor of MMP-bound forms), tumour necrosis factor (TNF) alpha, soluble TNF receptors I and II, interleukin (IL)-6 and soluble IL-6 receptor were measured by ELISA in 107 inflammatory and 53 degenerative arthropathies. RESULTS: MMP-3 levels in SF were (i) significantly higher in inflammatory than in degenerative arthropathies; (ii) not related to the degree of joint destruction; (iii) significantly correlated with the levels of all SF markers tested and with erythrocyte sedimentation rate and serum levels of C-reactive protein and fibrinogen. CONCLUSION: Increased MMP-3 levels in SF are found in inflammatory arthropathies and are not specific for erosive joint diseases. MMP-3 in SF is therefore a potential candidate for the assessment of the inflammatory process in joints. However, the exclusive determination of the active form could indicate the degree of joint destruction.

Matrix metalloproteinases, IL-6, and nitric oxide in rat antigen-induced arthritis.

OBJECTIVE: Degradation of extracellular matrix by matrix metalloproteinases (MMPs) is believed to be important in processes leading to the progressive destruction of joints in rheumatoid arthritis (RA). We used the model of antigen-induced arthritis (AIA) to study MMP activity and the influence of cytokines on their expression by synoviocytes. MATERIAL AND METHODS: Procedures used were: mBSA-induced arthritis in rats; investigation of synovial fluids and supernatants of cultured synoviocytes at different time points during AIA; estimation of total MMP activities by fluorescence assay; zymographic investigations; IL-6 bioassay with B9 cells; nitric oxide (NO) estimation. RESULTS: Total MMP activity in the synovial fluids of arthritic joints was higher than in the contralateral joints or in the joints of untreated control animals. The maximum was noted between day 7 and day 14 after arthritis induction. Cultured synoviocytes, prepared at different time points after arthritis induction, secreted MMPs into the media with a maximum time lapse of 14 days after arthritis induction. TNF-alpha increased the total MMP activity released. Moreover, TNF-alpha as well as IL-1 beta induced the expression of MMP9. Finally, TNF-alpha increased the levels of IL-6 and NO in the supernatants of synoviocytes; the extent of stimulation was dependent on the course of AIA. CONCLUSIONS: Different MMPs are synthesized in varying concentrations during the course of rat AIA. Cytokines such as TNF-alpha and IL-1 beta differentially influence the activity and expression of MMPs in cultured synoviocytes. The participation of MMPs in tissue degradation during the course of arthritis may be of importance for the development of new therapeutic strategies.

Augmented plasma and tissue kallikrein like activity in synovial fluid of patients with inflammatory articular diseases.

We studied some of the components of the kininogen-kallikrein-kinin system, simultaneously, in plasma and synovial effusions of patients with inflammatory articular diseases. Plasma and tissue kallikrein like activity and kininogen levels were evaluated. Active plasma and tissue kallikreins in plasma and synovial fluid were detected by their amidase activity upon specific chromogenic substrates. Kininogen levels were determined by a bioassay. Both specific amidase activity of plasma and tissue kallikreins were augmented in synovial effusions in relation to their own plasma activity. Kininogen levels in synovial fluid tended to be diminished in relation to plasma, however statistical significance was not reached. The consumption of kininogen is probably related to kinin production. This finding together with increased activities of plasma and tissue kallikreins reinforce the involvement of kinins in pathogenesis of inflammatory articular diseases.

Cathepsin G and elastase in synovial fluid and peripheral blood in reactive and rheumatoid arthritis.

The purpose of the study was to evaluate the involvement of serine proteinases cathepsin G and elastase on pathomechanisms in synovial fluid (SF) of patients with reactive (ReA) and rheumatoid, (RA) arthritis. Cathepsin G, elastase, and their endogenous inhibitors alpha1-antichymotrypsin (alpha1-ACT) and alpha1-proteinase inhibitor (alpha1-PI) were identified immunohistochemically from SF and peripheral blood (PB) of patients with ReA and RA. Cathepsin G and elastase activities in SF and PB were measured spectrophotometrically. Dot-immunostaining was used to identify cathepsin G, elastase, but also alpha1-ACT and alpha1-PI from SF and PB. Cathepsin G and elastase-like activities (IU/I) were slightly elevated in ReA SF compared to the corresponding peripheral blood values (11.4 +/- 9.2 vs 4.8 +/- 1.7, NS, and 5.1 +/- 2.8 vs 2.3 +/- 2.2, NS), which was similar to what was seen in RA (16.4 +/- 6.2 vs 0.53 +/- 0.4, p < 0.05, and 6.51 +/- 1.8 vs 1.22 +/- 0.58, p < 0.05). Although some samples did not contain cathepsin G and/or elastase-like activities, all samples contained immunoreactive enzyme, but also alpha1-ACT and alpha1-PI. In ReA SF, in contrast to monocytes, all polymorphonuclear (PMN) cells contained cathepsin G and elastase. Cathepsin G and elastase activities correlated with each other (r = 0.78, p < 0.05) suggesting PMN / primary granules as their likely source. There was a closer association between the cathepsin G or elastase and SF leukocyte count in ReA than in RA. In ReA and RA SF elevated cathepsin G and elastase activities are detected compared to activity levels in PB suggesting local production mainly from PMNs. The co-existence of highly cellular SF and cathepsin G and elastase activity in the documented presence of endogenous inhibitors in ReA SF together with the, known, usually self-remitting clinical course of ReA, suggest a brisk and even exaggerated local PMN serine proteinase release; sparing of joints does not seem to be due to lack or inhibition of PMN responses but rather to a successful down-regulation or cessation of the responses initially elicited.

In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis.

We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lactoferrin and TIMP-1 of saliva (n = 10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment.

Preliminary study of the tryptase levels in the synovial fluid of patients with inflammatory arthritis.

This study examined levels of tryptase, a specific mast cell product, in synovial fluid. Samples of synovial fluid from eight patients with rheumatoid arthritis and ten with other arthritides were measured in solid-phase immunoradiometric assays. Elevated concentrations of tryptase were present in samples from three patients with rheumatoid arthritis, one with psoriasis, and one with Reiter's syndrome. The data support the theory that mast cell activation is involved in the pathogenesis in some inflammatory joint diseases, but activation does not appear to be disease specific.

Reduction of matrix metalloproteinase 8-neutrophil collagenase levels during long-term doxycycline treatment of reactive arthritis.

The aim of this work was to determine whether human polymorphonuclear neutrophilic interstitial collagenase (matrix metalloproteinase 8 [MMP-8]) levels are reduced during long-term doxycycline treatment in humans with reactive arthritis. Serum MMP-8 levels were reduced (mean +/- standard error of the mean, 678.9 +/- 185.6 versus 491.2 +/- 144.8 ng of MMP-8 per ml), but not statistically significantly. However, the reduction of salivary MMP-8 levels was statistically significant (3,729 +/- 1,905.3 versus 1,866 +/- 780.0 ng of MMP-8 per ml, P < 0.05). This study demonstrated that a 2-month regimen of doxycycline can reduce MMP-8 levels in serum and especially in body fluids (i.e., saliva) containing inflammatory exudates and thus may contribute to reduced tissue destruction.

Granzyme A-immunoreactive cells in synovial fluid in reactive and rheumatoid arthritis.

Perforin and granzyme A co-localize in the cytotoxic granules of killer cells like cytotoxic T lymphocytes (CTL). Perforin is the cytolytic pore-forming protein, whereas the function of the homodimeric serine protease granzyme A and other members of the granzyme family is still unclear. Granzyme A-immunoreactive cells formed 8 +/- 2% of the resting peripheral blood lymphocytes of healthy individuals. In contrast, granzyme A-positive cells formed 15% of peripheral blood mononuclear cells in patients with reactive or rheumatoid arthritis. However, 29 +/- 4% (p < 0.05 compared to normal peripheral blood) and 25 +/- 4% (p < 0.05) of all lymphocytes in synovial fluid in reactive and rheumatoid arthritis, respectively, were granzyme A-positive. This suggests involvement of cell-mediated cytolytic mechanisms in the articular pathogenic mechanisms. This involvement, however, does not differentiate between reactive and rheumatoid arthritis.

Immunohistology of joint inflammation induced in rats by cell wall fragments of Eubacterium aerofaciens.

After a single intraperitoneal injection of cell wall fragments of Eubacterium aerofaciens, a main resident from the human intestinal flora, an acute arthritis develops within 2 days which is followed by a chronic arthritis that lasts at least 90 days. In an earlier report the histological appearance of the joint inflammation during this period has been described. In this study we investigated in more detail the cell types that are involved in the development of arthritis by using cell-type-specific monoclonal antibodies in an immunohistological assay. In the acute phase of arthritis, T-helper cells appeared in the synovial tissue together with ED1-positive (ED1+) and ED3-positive (ED3+) macrophages. After a temporary decline at day 12 all macrophage subsets, as well as T-helper cells, reappeared or increased again at day 33. Later, in the chronic phase (days 47-90), an increased number of ED1-positive (ED1+) cells in the synovial tissue and a decreased number of ED2-positive (ED2+) cells in the synovial lining was the most prominent finding when compared with control rats. These results indicate that, apart from T lymphocytes, macrophages also play an important role in the development and continuation of chronic arthritis in this model.

Cytidine deaminase and lactoferrin in inflammatory synovial fluids. Indicators of local polymorphonuclear cell function?

Cytidine deaminase (CD) is a cytoplasmatic enzyme present predominantly in polymorphonuclear cells (PMNC) in inflamed joints. Lactoferrin is situated in the secondary granules of PMNC and is released by secretory/phagocytic stimuli, whereas CD is released mainly upon cell lysis. To study the release of these molecules in arthritic conditions we measured CD and lactoferrin levels in synovial fluid (SF) drawn from patients with rheumatoid arthritis (RA), crystal pyrophosphate disease (CPPD), psoriatic arthropathy, reactive arthritis, spondylarthropathy, and osteoarthrosis. CD activity was highest in SF from RA and CPPD followed by psoriatic arthropathy, reactive arthritis and spondylarthropathy. Lactoferrin concentrations were highest in CPPD followed by RA, reactive arthritis, psoriatic arthropathy, and spondylarthropathy. Both CD and lactoferrin levels were low in osteoarthrosis SF. Although SF CD activity and lactoferrin levels correlated well in all diagnostic groups, the ratio between CD and lactoferrin was higher for RA, psoriatic arthropathy, and spondylarthropathy compared to reactive arthritis and CPPD. This suggests predominant release by PMNC lysis in the more chronic arthritis groups and more degranulation in the more episodic CPPD and reactive arthritis groups. CD activity and lactoferrin levels correlated significantly with SF cell counts in the RA and psoriatic arthropathy groups.

Production of angiotensin converting enzyme by rheumatoid synovial membrane.

Vascular proliferation and mononuclear cell infiltration are prominent changes observed in synovium from actively inflamed joints of patients with rheumatoid arthritis. Angiotensin converting enzyme (ACE) is a halide activated peptidase produced mainly by endothelial cells and by activated monocytes. It has been proposed that levels of ACE activity in synovial fluid might reflect changes in membrane vascularity, the degree of monocyte infiltration, or the thickness of the lining layer. In this study, ACE activity in serum and synovial fluid samples from 18 patients with inflammatory arthritis was measured and compared with levels in 12 control subjects with non-inflammatory arthritis. Although serum levels were similar in the two groups, ACE activity in synovial fluid was significantly increased in the group with inflammatory arthritis compared with controls (mean (SE) 37 (5) v 19 (3)). Staining of synovial membranes from patients with rheumatoid arthritis with a monoclonal antibody to ACE localised ACE to the endothelium and to mononuclear cells of macrophage origin. ACE activity was then measured in supernatants of synovial membrane from patients with rheumatoid arthritis after one and seven days of culture. A significant increase in ACE activity was observed after seven days of culture (mean (SE) day 1, 17 (5) v day 7, 25 (3)). Levels of ACE activity, however, did not correlate with the lining layer thickness, with the number of macrophages per square millimetre, nor with the number of blood vessels per square millimetre of synovial tissue. No correlation was observed either between levels of ACE in the supernatant of synovial membrane and levels of interleukin 1 or interleukin 6. In conclusion, ACE is produced by the synovial membrane of patients with rheumatoid arthritis and is localised to monocytes and endothelial cells. Levels of activity do not directly reflect membrane vascularity, monocyte or macrophage number, or the thickness of the lining layer.

The anticollagenolytic potential of lymecycline in the long-term treatment of reactive arthritis.

OBJECTIVE: We sought to determine the antiinflammatory properties of lymecycline in the long-term treatment of reactive arthritis (ReA). METHODS: Quantitative assay of collagenase activity by densitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Therapeutic levels of lymecycline do not directly inhibit the activity of human neutrophil interstitial collagenase, but can prevent the oxidative activation of latent human neutrophil collagenase. CONCLUSION: This non-antimicrobial, anticollagenolytic property of lymecycline may contribute to its therapeutic efficacy in the treatment of patients with ReA.

Caeruloplasmin concentration and oxidase activity in polyarthritis.

Caeruloplasmin (Cp) concentration and oxidase activity have been shown to be elevated in rheumatoid arthritis (RA) and psoriatic arthritis, but normal in Reiter's syndrome, Behcet's syndrome and juvenile seronegative polyarthritis. Synovial fluid Cp was significantly depressed in comparison with serum Cp in RA. During second-line therapy in RA, Cp concentration and activity fell significantly (P less than 0.001), but the change in Cp did not correlate with plasma viscosity.

Elastase and collagenase activities in synovial fluid of patients with arthritis.

The activity of elastase and collagenase was measured in the synovial fluid (SF) of 24 patients with inflammatory and 6 patients with traumatic joint effusions. The enzyme activity was compared with local and systemic variables of the disease. Elastase and collagenase activity could be detected in 79 and 83%, respectively, of the arthritic SF, whereas no enzyme activity was found in the traumatic joint effusions. The SF enzyme activity showed no correlation with clinical, laboratory or radiographic variables of arthritis in the joint from which the fluid was obtained. Our results show that proteolytic enzyme activity in SF may help to distinguish inflammatory from noninflammatory joint effusions but does not reflect the severity of the arthritic process.

[Non-lymphoid mononucleated cells in the synovial fluid in arthrosis and various inflammatory arthropathies. Morphologic, quantitative and cytoenzymologic study]. / Les cellules mononucléées non lymphoïdes du liquide synovial dans l'arthrose et diverses arthropathies inflammatoires. Etude morphologique, quantitative et cyto-enzymologique.

This paper describes a morphologic, quantitative, cytochemical study of mononuclear non lymphoid cells in knee synovial fluid in osteoarthritis and various arthritides. Morphologic criteria allow to identify among these cells various synoviocytic and monocytic subtypes with in both types, phagocytic subtypes. Quantitative study shows in arthritides an important afflux of monocytes and a hyperexfoliation of synoviocytes. In fluids with intermediate cellularity, Monocytes/Synoviocytes ratio allows the differential cytodiagnosis between osteoarthrosis and arthritis. All monocytic subtypes and especially the phagocytic one are highly significantly increased in arthritides. Synoviocytic subtypes show a lower increase, except the phagocytic one, which is not changed. Giant multinuclear synoviocytes are found in every type of disease and cannot constitute a cytodiagnosis marker. Alcian Blue and hyaluronidase treatment show hyaluronate in a few percentage of Synoviocytes. Cytoenzymologic study shows that synoviocytes and monocytes are positive in all tested hydrolases: beta Glucuronidase, Acid Phosphatase, alpha Naphthyl Acetate Esterase, these activities being always higher in synoviocytes. With peroxidase, synoviocytes are always negative, so this reaction although it marks only a minority of monocytic population can be used as an extra cytologic criterion for discrimination of mononuclear cells in synovial fluid. In these four enzymes there is no significant quantitative difference at cellular level between osteoarthrosis and arthritides. Lysosomal enzymatic activity in both monocytic and synoviocytic cells confirms their heterophagic properties. However synoviocytic heterophagy seems to be a physiological process not or few affected by inflammatory events. On the opposite, monocytic heterophagy and then macrophagic transformation of monocytes appears as a major aspect of intrasynovial inflammatory reaction. If a large majority of exfoliated synoviocytes comes from A type synovial lining cells and if they belong to Mononuclear Phagocyte System, why do they so weakly, or not, participate as phagocytes to inflammatory reaction.

The clinical significance of serum alkaline phosphatase isoenzymes in locomotor diseases.

AP isoenzymes were estimated in 292 patients with locomotor diseases and in 124 healthy controls. The diagnostic usefulness of AP determination is increased by estimation of isoenzymes. Investigations were made to study the biological profile of organ specific AP activities: 1. Rheumatoid arthritis and Reiter's syndrome - the total AP and L-AP activities were increased. 2. Ankylosing spondylitis treated by physiotherapy - the total AP, B-AP and I-AP activities were increased. After drug therapy an increase occurred also in L-AP activity while I-AP activity showed no significant change. 3. Progressive OA of hip and knee showed increased levels of total AP and B-AP activities. 4. Degenerative diseases of the spine, chiefly cases of discopathy, showed significantly reduced levels of AP and B-AP activities. 5. In osteoporosis there was an increase in total AP, L-AP, B-AP and I-AP activities. 6. In the active generalised form of Paget's disease, increased levels were found of total AP, B-AP, I-AP and L-AP activities. 7. In neoplastic diseases the isoenzymes can help to reveal metastatic dissemination and thus aid preoperative evaluation. 8. In gout and hyperuricemic syndromes there was a relative increase of B-AP activity and non-significant fall of L-AP activity. Increased levels of L-AP occured in patients with gallbladder disease, after immunosuppressive therapy or after infectious hepatitis. A fall of L-AP levels was found after Corticotrophin and after intraarticular administration of Kenalog. Increased B-AP activities occurred after total hip replacement, in acute or chronic pyelonephritis and in active osteonecrosis and osteoporosis. Anabolic therapy caused a significant fale of B-AP activity to fall significantly. Reduced B-AP levels were also found after antibiotic therapy. Increased I-AP activity was found in cases of osteoporosis, and in secondary amyloidosis; reduced I-AP activity was seen in mucous colitis. The activity of I-AP is assumed to increase as a result of the changed intestinal calcium and phosphorus regulation occurring in association with the enhanced bone tissue metabolism. From this point of view an order of significance is given for the activity of bone pathology in the separate diagnostic groups of locomotor diseases.