Changes in cartilage proteoglycan aggrecan after intra-articular injection of interleukin-1 in rabbits: studies of synovial fluid and articular cartilage.

OBJECTIVE: To determine how acute but transient inflammation affects the cartilage proteoglycan aggrecan and the value of analyses of synovial fluid to study this. METHODS: For 96 hours after a single intra-articular injection of rabbit knees with human interleukin-1 alpha (IL-1 alpha) or vehicle, articular cartilage and synovial fluid were examined using a putative indicator of aggrecan synthesis (aggrecan chondroitin sulphate epitope 846), immunoreactive keratan sulphate, and total glycosaminoglycan (GAG) content. Aggrecan extractability (with 0.5 M NaCl) followed by 4 M guanidine hydrochloride extraction permitted analyses of cartilage damage, total content and aggrecan heterogeneity. Aggrecan epitopes as well as GAG were assayed in synovial fluid. Changes were related to total joint leucocyte content in synovial fluid. RESULTS: At 10 ng, IL-1 alpha produced a transient increase in synovial fluid leucocytes at six hours and 24 hours. This accompanied a reduction in content and increased extractability of GAG, which was greatest in the tibial medial compartment of the knee. Further studies of this compartment showed no change in keratan sulphate epitope content, but a transient increase in extractability in 0.5 M NaCl. Epitope 846 content and extractability were unchanged. Total contents and extractability for GAG were inversely correlated in both controls and joints injected with IL-1 alpha. These changes were accompanied by transient increases in GAG, keratan sulphate epitope, and 846 content in synovial fluid. CONCLUSION: According to the aggrecan component measured, damage to the matrix of articular cartilage was sometimes reflected by a transient increased extractability and a net loss of aggrecan. There was always an increased release of GAG, and keratan sulphate, and 846 epitopes into synovial fluid. These studies show that changes in aggrecan epitopes and GAG in synovial fluid reflect changes in cartilage metabolism induced by acute transient inflammation.

Expression of interferon-gamma (IFN-gamma), IL-10, IL-12 and transforming growth factor-beta (TGF-beta) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA).

The expression of immunoregulatory cytokines was investigated in freshly isolated synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with RA, using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. IFN-gamma, TGF-beta, IL-10 and IL-12 (p40) transcripts were detected in SFMC of patients with early disease (<1 year duration) as well as in patients with long standing arthritis (>1 year). The expression of IFN-gamma, IL-10 and IL-12 mRNA was increased in SFMC compared with RA PBMC. In addition, the expression was higher in RA SFMC than in PBMC from health control individuals. Immunoassay analysis of the secreted IL-12 heterodimer demonstrated increased levels in RA SF compared with levels found in serum from RA patients and control individuals. High levels of TGF-beta mRNA were found in SFMC, but a significantly decreased TGF-beta/beta2-microglobulin (beta2-M) ratio was found compared with PBMC from both patients and control individuals. IL-4mRNA could not be detected, either in SFMC or in PBMC. Cytokine expression in RA PBMC did not differ from control PBMC, with the exception of a decreased TGF-beta/beta2-M ratio in RA patients with early disease. Our findings of IFN-gamma mRNA and IL-12, but undetectable levels of IL-4 mRNA, suggest that the synovitis is characterized by a type 1 immune response. The presence of TGF-beta and IL-10 mRNA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level of expression may not be sufficient for down-modulation of immune activation.

Sulphasalazine: mechanism of action in rheumatoid arthritis.

Sulphasalazine a drug used for the treatment of rheumatoid arthritis (RA) shows a wide range of biological activities all of which might contribute to the beneficial clinical effect seen during treatment of RA. This review summarizes some of the biological activities and discusses these in context of possible modes of action of the drug. Sulphasalazine has been described as an antibacterial drug, an anti-inflammatory drug or as an immunomodulator. From the reviewed data it is concluded that the effects of sulphasalazine on various immunological processes, are of outstanding importance for its mode of action.

Role of the bowel flora for development of immunity to hsp 65 and arthritis in three experimental models.

An infectious aetiology in rheumatoid arthritis (RA) has for long been suggested, although no conclusive evidence for this is at present available. Lately a large interest has been devoted to the involvement of heat shock proteins (hsps) in autoimmune disorders due to their conserved structure and immunogenic properties. Immunity to hsps has been observed both in human autoimmune conditions and in experimental models of autoimmune disease. We have studied the role of the bacterial flora and hsp immunity in the arthritic response in three experimental models of arthritis; type II collagen arthritis (CIA), adjuvant arthritis (AA) and oil induced arthritis (OIA); by using germ free and conventional DA rats. A high incidence of severe arthritis developed in all the models evaluated irrespectively of whether the animals were in the conventional or germ free state. This confirms earlier results which show a minor effect of the bacterial flora in CIA and AA in high responder strains. These results also show that a severe OIA can develop in germ free animals. Despite the severe arthritic response induced, no serum antibody levels to hsp 65 could be detected in the germ free animals, which was in contrast to the conventional animals where a positive anti-hsp 65 serum response was seen in 35-80% of the animals with CIA, AA or OIA. These results show that development of a humoral response to hsp 65 in these models of arthritis is dependent on the presence of a bacterial flora. Further, the lack of humoral immunity in germ free animals despite a severe arthritic response indicates that humoral immunity to hsp 65 is not involved in development of disease in these three models of experimental arthritis.

Sulphasalazine inhibition of human granulocyte activation by inhibition of second messenger compounds.

The effects of sulphasalazine on the production of second messenger compounds in human granulocytes have been characterised by various stimuli. The increases in cytosolic calcium, inositol trisphosphate, diacylglycerol, and phosphatidic acid (all important mediators of intracellular signal transduction) triggered by stimulation were inhibited by sulphasalazine. The metabolites 5-amino-salicylic acid and sulphapyridine were less potent inhibitors than the mother compound. It is concluded that sulphasalazine inhibits the synthesis of phosphoinositide derived second messenger compounds at the level of phospholipase C or its regulatory guanosine 5'-triphosphate (GTP) binding protein. Inhibition of phosphatidic acid synthesis was either due to the same mechanism, or to interaction with a phospholipase D regulating GTP binding protein.

Sulfasalazine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells.

Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicyclic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-gamma-secreting cells (IFN-gamma-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-gamma secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-gamma-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-gamma-sc method may be relevant for the evaluation of new immunotherapeutic strategies in inflammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.

Effects of sulfasalazine and a sulfasalazine analogue on the formation of lipoxygenase and cyclooxygenase products.

A sulfasalazine analogue, 5'-(2,4-dichlorobenzoyl)2'-hydroxyphenylacetic acid (CL 42A), potently inhibited the formation of 5-lipoxygenase products (leukotrienes B4 and C4 and 5-hydroxyeicosatetraenoic acid) by human leukocytes. Half-maximal inhibition of leukotriene production was obtained with 5 and 10 microM CL 42A after stimulation with serum-treated zymosan or ionophore A23187, respectively. CL 42A was equipotent to nordihydroguaiaretic acid and about 50 times more potent than sulfasalazine and benoxaprofen in studies on the inhibition of LTB4 formation in leukocyte suspensions stimulated with serum-treated zymosan. Furthermore, CL 42A had no inhibitory effect on the production of 15-hydroxyeicosatetraenoic acid after incubation of human leukocytes with ionophore A23187 in the presence of exogenous arachidonic acid. Sulfasalazine inhibited the synthesis of 5-lipoxygenase products (5-hydroxyeicosatetraenoic acid and leukotriene B4: IC50 250 microM, leukotriene C4: IC50 100 microM) in a concentration-dependent manner but had no effect on 15-hydroxyeicosatetraenoic acid formation. The metabolites of sulfasalazine, sulfapyridine and 5-aminosalicylic acid, and the isomer, 4-aminosalicylic acid, were all less potent than sulfasalazine as inhibitors of leukotriene formation. Both CL 42A (IC50 20 microM) and sulfasalazine (IC50 500 microM) inhibited the synthesis of thromboxane B2 and hydroxyheptadecatrienoic acid in human platelet suspensions after arachidonic acid stimulation. However, while CL 42A inhibited cyclooxygenase, the inhibitory effect of sulfasalazine was exerted mainly on thromboxane synthase. The platelet formation of 12-hydroxyeicosatetraenoic acid was not inhibited by CL 42A whereas sulfasalazine had a weak inhibitory effect.

Endotoxin-induced shock in the rat. A role for C5a.

Administration of endotoxin from gram-negative bacteria to rats results in systemic hypotension, an increased hematocrit, and decreased numbers of circulating leukocytes (polymorphonuclear), monocytes, and platelets. These potentially lethal physiologic changes may be partially attributed to complement activation and generation of anaphylatoxins by the endotoxin (LPS). We demonstrated an elevation in the plasma levels of both C3a and C5a in LPS-treated rats. Injection of 5 micrograms C5ades Arg (rat) into rats produced effects similar to those induced by LPS, including decreased mean arterial pressure (systemic hypotension) and decreased numbers of circulating polymorphonuclear leukocytes, monocytes, and platelets. Unlike the response to LPS, C5a did not increase the hematocrit, indicating little effect on vascular permeability at the doses used. When LPS-treated animals were pretreated with F(ab')2 fragments of rabbit anti-rat C5a, no changes were measured in the circulating cell counts compared with LPS alone; however a significant improvement in the mean arterial pressure and a decrease in hematocrit was observed. We conclude that LPS-induced (septic) shock in the rat may result, in part, from the effects of complement activation and particularly from the generation of C5a. The influence of C5a on the LPS effect in the rat appears to enhance both the hypotensive (mean arterial pressure) and vascular permeability (hematocrit) responses. These results appear to support and confirm earlier observations that anti-human C5a increased survival in a septic-shock monkey model by eliminating circulating C5a and presumably thereby reducing the effects of endotoxin on blood pressure. Our results demonstrate that C5a plays a significant role in the hemodynamic changes associated with endotoxin-induced shock. Neutralization of C5a with specific antibodies may reduce the hypotensive response to endotoxin sufficiently to prevent lethal septic shock both in animals and in man.

Inhibitory effects of sulfasalazine and related compounds on superoxide production by human polymorphonuclear leukocytes.

The inhibitory effects of sulfasalazine, some sulfasalazine-related compounds and indomethacin on superoxide production by human polymorphonuclear (PMN) leukocytes were studied. The inhibition of the chemotactic peptide (FMLP)-induced superoxide production, which is membrane receptor-mediated, was strongly dependent on the concentration both of the secretory stimulus and of the test compounds, indicating an interaction between the receptor and the test compound. Furthermore, a positive correlation was found between the lipophilicity of the compound and the degree of inhibition. However, when the receptor was by-passed by direct activation of the receptor-linked G protein by the use of fluoride ions as secretory stimuli, the test compounds still inhibited superoxide production. On the other hand, superoxide production by cells stimulated with phorbol ester was not inhibited by the test compounds. Furthermore, the production of phosphatidic acid was decreased in the presence of sulfasalazine, indicating impaired phosphoinositide metabolism. The inhibition of this metabolism was not due to increased intracellular concentrations of cyclic AMP, although sulfasalazine did inhibit cyclic nucleotide phosphodiesterase. We conclude that sulfasalazine attenuates superoxide production by PMN leukocytes at a post-receptor site of action at a step before the activation of protein kinase C, possibly by interfering with the phosphoinositide metabolism but independent of cyclic AMP.

Serum hyaluronate levels reflect disease activity in experimental arthritis models.

Serum hyaluronate (HA) levels were measured in rats subjected to adjuvant or type II collagen induced arthritis. As the arthritic lesions developed, both models showed an increase in serum HA levels of approximately 5 times, from a baseline level of 61-126 ng/ml (range). Furthermore a positive correlation was found between HA level and arthritic score. The increase in HA was not related to metabolic impairment, as the half life of serum HA in adjuvant arthritic rats was similar to that of normal rats. Serum HA may thus serve as a useful variable for evaluation of the severity of experimental arthritis.

Elevated levels of serum hyaluronate and correlation with disease activity in experimental models of arthritis.

The serum hyaluronate (HA) concentration was measured in groups of rats immunized for adjuvant or type II collagen arthritis. Serum HA increased as the arthritic lesions developed, correlating with the severity of the disease. This increase in HA was not related to metabolic impairment, because rats with adjuvant arthritis metabolized intravenously administered tritiated HA at a rate similar to that of normal rats. Serum HA levels may be useful as an indicator of synovitis in experimental and possibly in clinical arthritis. Further, this model could serve as an experimental approach for studies of HA metabolism in chronic joint inflammation.

An intravital microscopic model for mast cell-dependent inflammation in the hamster cheek pouch.

Topical antigen challenge in cheek pouches of immunized hamsters led to an acute inflammatory reaction which was characterized by intravital microscopy. The response consisted of short-lasting arteriolar spasm, followed by leakage of plasma, vasodilation, and accumulation of leucocytes. Several observations indicated that the reaction was due to mast cell activation. Thus, a very similar inflammatory response was seen after challenge with compound 48/80, and both antigen and compound 48/80 degranulated the numerous mast cells present in the cheek pouch. In addition, fluorescein-labelled antigen bound specifically to mast cells in cheek pouches of immunized animals, also suggesting the presence of mast cell-fixed antigen-specific antibodies, possibly immunoglobulin E. However, although antigen and compound 48/80 caused similar microvascular responses, cross-desensitization experiments indicated that the two stimuli activated mast cells via different mechanisms. The histamine antagonist mepyramine, which abolished plasma leakage induced by exogenous histamine, substantially inhibited the increase of microvascular permeability evoked by antigen or compound 48/80, but did not appear to affect the vasospasm and leucocyte accumulation. It is concluded that the hamster cheek pouch may be a most useful tool for investigation of dynamic microvascular events during allergic mast cell-dependent inflammation.

Histopathological lung changes in immune complex mediated anaphylactic shock in humans elicited by dextran.

Severe dextran-induced anaphylactic reaction (DIAR) is being recognized as a form of immediate IgG mediated immune complex reaction. Support for this pathogenesis is found in the correlation between the titer of dextran-reactive antibodies of IgG class and the severity of the reaction. Autopsy records were reviewed in 27 certified cases of fatal DIAR. The most frequent macroscopic findings were dilatation of the right side of the heart and acute pulmonary stasis. Autopsy lung specimens were collected from 17 of these patients. In 15 of the 17 lung specimens pulmonary microemboli were found. The microemboli had the appearance of hyaline eosinophilic globules, and the lung vasculature also contained leukocytes, platelets and disintegrated erythrocytes. These findings show similarity to the findings in a monkey model of known IgG mediated anaphylaxis, and give further support to the proposed pathogenesis of severe DIAR.

Oxygen radicals are not a prerequisite for neutrophil-mediated increased vascular permeability.

The neutrophil granulocyte is considered to play a key role in the inflammatory process, contributing to the increased microvascular permeability and tissue damage seen at inflammatory sites. The mechanism underlying this process is unknown, but studies in vitro using cultured endothelium and blood polymorphonuclear granulocytes (PMNs) point to an involvement of oxygen-free radicals. In this study, we have used the hamster cheek pouch microcirculatory model to evaluate the impact of radical scavenging enzymes, superoxide dismutase (SOD), and catalase on the microvascular inflammatory reactions induced by leukotriene B4 (LTB4), which is known to induce PMN-dependent plasma leakage in this model. The variables studied were leukocyte adhesion in postcapillary venules, macromolecular permeability as leakage of fluorescent dextran, and emigration of PMNs. SOD and catalase were given in high doses as intravenous infusion and also as superfusion over the cheek pouch. All studied variables increased dramatically upon superfusion of LTB4 (4 nM) over the cheek pouch preparation and remained elevated throughout the 30-minute exposure period. Treatment with SOD and/or catalase did not influence this inflammatory response to LTB4. Nor did SOD and catalase influence the plasma leakage and emigration of PMNs caused by superfusion of the synthetic chemotactic peptide, formylmethionylleucylphenylalanine (400 nM) which from other animal models is known to induce a PMN-dependent plasma leakage. Although free radicals might contribute to the tissue damage seen at some inflammatory sites, we conclude that they are not crucially involved in the interaction between the PMN and the microvascular endothelium induced by the inflammatory mediators LTB4 and formylmethionylleucylphenylalanine, and are thus not a prerequisite for PMN-dependent microvascular permeability.

Leucocytes mediate the hCG-induced increase in testicular venular permeability.

Human chorionic gonadotrophin (hCG) treatment of adult male rats induces microvascular changes in the testis consisting of abolished vasomotion, an accumulation of leucocytes in postcapillary venules and increased vascular permeability. To study the role of leucocytes, rats were made leucopenic with a specific antineutrophil serum (ANS). Testicular interstitial fluid volume was decreased in leucopenic rats. Leucopenic rats also failed to show an hCG-induced increase in venular permeability as in saline-treated rats. The normally pulsatile blood flow pattern (vasomotion) persisted in leucopenic rats but was abolished after hCG treatment both in saline-treated and leucopenic rats. Plasma testosterone concentration after hCG treatment was not affected by elimination of circulating polymorphonuclear (PMN) leucocytes. It is concluded that PMN leucocytes mediate in part the hCG-induced increase in testicular venular permeability but not the hCG-induced inhibition of vasomotion.

Immune-complex-induced inflammatory reaction studied by intravital microscopy: role of histamine and arachidonic acid metabolites.

An immune-complex-induced inflammatory reaction was elicited in the hamster cheek pouch microvasculature of ovalbumin (OA)-immunized animals by exposure to 1 or 100 micrograms/ml OA. The low antigen dose caused arteriolar constriction, transient platelet aggregation, and a reversible increase in vascular leakage at postcapillary venules. With the high antigen dose, the constriction and platelet aggregation were more pronounced and the vascular leakage was prolonged. This antigen dose also caused a massive PMNL accumulation in small venules, which coincided with the prolonged vascular leakage. Histamine was released in the reaction as pretreatment with mepyramine largely inhibited the leakage response to 1 microgram/ml OA. With 100 micrograms/ml OA, only the initial phase of vascular leakage was inhibited by mepyramine, leaving the prolonged vascular leakage and PMNL accumulation unaltered. Pretreatment with methylprednisolone 16-18 h prior to the experiments reduced both phases of vascular leakage as well as the PMNL accumulation. Pretreatment with the combined cyclo- and lipoxygenase inhibitors BW755C or nordihydroguaiaretic acid (NDGA) reduced the initial vasoconstriction induced with 100 micrograms/ml OA, thereby augmenting the initial vascular leakage. Despite this, the prolonged phase of vascular leakage was reduced in the NDGA-treated animals. Cyclooxygenase products were not found to play a crucial role in mediating the vascular response; on the contrary, indomethacin pretreatment slightly potentiated the vascular leakage induced by the low antigen dose.

Effect of anti-inflammatory drugs on xanthine oxidase and xanthine oxidase induced depolymerization of hyaluronic acid.

The inhibitory effect of various anti-inflammatory drugs on the xanthine oxidase derived depolymerization of hyaluronic acid was studied. The depolymerization was assayed by repeated viscosity measurements. By using a low xanthine oxidase activity, the decrease in viscosity with time followed first order reaction kinetics and was therefore suitable for kinetic analysis. The xanthine oxidase activity was monitored by assay of O2-consumption with a Clark-electrode and by assay of urate production. We present evidence that salicylic, acetylsalicylic, gentisic and azodisalicylic acid and sulfasalazine inhibit the production of oxygen-derived free radicals by xanthine oxidase. We found that sulfapyridine, 5-aminosalicylic acid, allopurinol, mannitol, glucuronic acid and N-acetylglucosamine in addition to the earlier studied drugs, paracetamol, ibuprofen, benoxaprofen and gentisic acid exert their effect via scavenging of free radicals. These drugs had very little effect on the enzyme activity.

Microvascular effects of anaphylatoxins C3a and C5a.

The direct microvascular effects of human C3a and C5a were investigated by using hamster cheek pouches prepared for intravital microscopy. Topical application of 10 nM C3a resulted in pronounced microcirculatory alterations, characterized by vasoconstriction, platelet aggregation, and an increase in macromolecular leakage at postcapillary venules, as assessed by extravasation of intravascular fluorescein-labeled dextran (m.w. 150,000). Exposure of the preparation to 500 nM COOH-terminal octapeptide analogs of C3a resulted in a microvascular response almost identical to that of C3a, supporting the view that the active site of this anaphylatoxin resided within the COOH-terminal portion. Changes similar to those caused by C3a were also induced by 20 or 100 nM C5a and, in addition, the higher concentration of C5a caused accumulation of polymorphonuclear leukocytes (PMNL) in small venules and somewhat prolonged vascular leakage from venules exhibiting PMNL accumulation. Histamine was found to partially mediate the vascular leakage induced by C3a and the initial (first 5 min) permeability response to the high concentration of C5a, whereas the subsequent leakage induced by the latter anaphylatoxin was unaffected by mepyramine pretreatment. In neutropenic and mepyramine-treated animals exposed to the high concentration of C5a, a partial reduction of both the early and the subsequent vascular leakage was seen, indicating that accumulated PMNL play a role in the prolonged phase of leakage. The pronounced microvascular alterations induced by low concentrations of C3a and C5a strengthen the view that these anaphylatoxins act as mediators of inflammatory reactions in which the complement system is activated.