The alarmins high mobility group box protein 1 and S100A8/A9 display different inflammatory profiles after acute knee injury.

OBJECTIVE: To compare the concentrations of high mobility group box 1 protein (HMGB1) and S100A8/A9 in synovial fluid between patients with knee injuries and osteoarthritis (OA), and knee healthy subjects. To investigate associations of alarmin levels with different joint injuries and with biomarkers of inflammation, Wnt signaling, complement system, bone and cartilage degradation. METHODS: HMGB1 and S100A8/A9 were measured in synovial fluid by immunoassays in patients with knee injuries, with OA and from knee healthy subjects, and were related to time from injury and with biomarkers obtained from previous studies. Hierarchical cluster and enrichment analyses of biomarkers associated to HMGB1 and S100A8/A9 were performed. RESULTS: The synovial fluid HMGB1 and S100A8/A9 concentrations were increased early after knee injury; S100A8/A9 levels were negatively associated to time after injury and was lower in the old compared to recent injury group, while HMGB1 was not associated to time after injury. The S100A8/A9 levels were also increased in OA. The initial inflammatory response was similar between the alarmins, and HMGB1 and S100A8/A9 shared 9 out of 20 enriched pathways. The alarmins displayed distinct response profiles, HMGB1 being associated to cartilage biomarkers while S100A8/A9 was associated to proinflammatory cytokines. CONCLUSIONS: HMGB1 and S100A8/A9 are increased as an immediate response to knee trauma. While they share many features in inflammatory and immunoregulatory mechanisms, S100A8/A9 and HMGB1 are associated to different downstream responses, which may have impact on the OA progression after acute knee injuries.

Early stage blockade of the alarmin HMGB1 reduces cartilage destruction in experimental OA.

OBJECTIVE: The alarmin HMGB1 is an endogenous molecule that is released into the extracellular space upon trauma or cell activation. Extracellular HMGB1 initiates innate immune responses and besides mediating inflammation, has osteoclast-activating features and mediates pain, all important features in OA. The aim of this study was to examine the involvement of HMGB1 in experimental OA and to explore the effect of local anti-HMGB1-therapy on disease progression. METHOD: OA was induced in mice by surgical destabilization of knee joints and HMGB1 expression and localization was assessed by immunohistochemistry. For therapy evaluation, HMGB1-neutralizing antibodies were injected intraarticularly, alone or encapsulated in an injectable hyaluronan-based delivery vehicle. Human primary chondrocytes were stimulated with rHMGB1 and analyzed by qPCR and cytometric bead-array. RESULTS: HMGB1 immunostaining of mouse OA joints demonstrated intra- and pericellular expression in chondrocytes, overlapping with proteoglycan depleted areas. Intra-articular injection of anti-HMGB1 antibodies had cartilage-protective effects, comparable to treatment with a TNF inhibitor. Direct and vehicle-based delivery had similar ameliorating effects and the effect of a single, early injection could not be enhanced by repeated injections. In vitro stimulation of chondrocytes with rHMGB1 affected chondrocyte function by inducing protein expression of IL6 and IL8 and downregulating mRNA of COL2A1. CONCLUSIONS: Our results suggest that the alarmin HMGB1 might be a new target for OA therapy development as we could observe an aberrant HMGB1 expression in mouse OA joints, stimulation of chondrocytes with rHMGB1 induced cytokine production and decreased matrix production and finally that HMGB1 blockade suppressed disease progression.

An in vivo cross-linkable hyaluronan gel with inherent anti-inflammatory properties reduces OA cartilage destruction in female mice subjected to cruciate ligament transection.

OBJECTIVE: To explore the possibility of cartilage protection in osteoarthritis (OA) by intraarticular injection of a chemically modified hyaluronan (HA) gel and investigate whether the chemical modifications provide intrinsic anti-inflammatory activity. METHOD: OA was induced in C57BL/6 mice by anterior cruciate ligament transection (ACLT) and HA gel or carbazate-modified component was injected intra-articularly. Assessment of cartilage rescue was performed by histology, immunohistochemistry and TUNEL analysis. Serum levels of proinflammatory cytokines were evaluated with cytometric bead array, measuring IL-1ß, TNF, IFN-γ, KC/CXCL1 and MCP-1. RESULTS: Intraarticular injection of the HA gel showed significantly reduced cartilage destruction and decreased osteophyte formation. Besides the biological and biomechanical effects of HA, we investigated lipid peroxidation products as an alternative inflammatory and potential mechanism contributing to OA. To address this, injection of the carbazate-modified component alone was performed, which also demonstrated a cartilage-saving effect. Besides the cartilage amelioration effects, decreased apoptosis, 4-hydroxynonenal (4-HNE) and MHC class II staining was recorded. No changes in serum levels of proinflammatory cytokines were detected. CONCLUSION: We have shown that the HA gel has an anti-destructive effect on articular cartilage (AC). Our results demonstrated that the carbazate-modified component could suppress apoptotic events, potentially by quenching of ROS/LPO products such as 4-HNE in OA joints. Modification of the HA molecule offers opportunities to introduce (covalent) coupling of additional molecules to the gel, with controlled retention and subsequent release in the joint.

Highly arthritis-susceptible DA rats express IL-1beta in articular cartilage.

There are genetically determined differences in susceptibility to arthritis among inbred rat strains. The aim of the present study was to elucidate phenotypical differences, by determining expression of TNF and IL-1beta, two pivotal mediators of arthritis, in the highly arthritis-prone Dark Agouti (DA) rat compared to that of two arthritis-resistant rat strains, the major histocompatibility complex-homologous Piebald-Viral-Glaxo (PVG.1AV1) rat and the Brown Norway (BN) rat, assessed by immunohistochemistry. We demonstrate a distinct difference in articular cartilage, with chondrocytes expressing IL-1beta, not TNF, in the highly arthritis-prone DA rat as opposed to the two arthritis-resistant BN or PVG.1AV1 rat strains, where no cytokine expression was documented. The results were otherwise congruent among the rat strains. We observed TNF- and IL-1beta-expressing cells within the synovial lining layer in all rat strains. Other tissues studied, auricular cartilage as well as muscle, lung, thyroid gland and kidney tissue, were devoid of cytokine expression. Constitutional expression of IL-1beta in chondrocytes might facilitate initiation and perpetuation of inflammation. This may offer one explanation of why erosive arthritides are so easily induced in the DA rat and also support the hypothesis that articular chondrocytes may themselves play a major role in cartilage matrix degradation.

A novel anti-inflammatory compound, artonkin-4'-O-glucoside, from the leaves of Artocarpus tonkinensis suppresses experimentally induced arthritis.

Artocarpus tonkinenesis (Moraceae) has been used in Vietnamese traditional medicine for the treatment of backache and joint diseases since many 100 years. We have previously shown that a crude extract of A. tonkinensis elicited anti-inflammatory effects in rat collagen-induced arthritis (CIA), with significant improvement of disease symptoms. However, the pharmacological basis of the bioactivity of A. tonkinensis extract is not known. In the present study, we have isolated four individual active components from A. tonkinensis extract by reverse phase high-pressure liquid chromatography. The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and their biological effects investigated. A novel biologically active flavonoid glucoside (5-hydroxy-8-hydroxymethyl-8-methyl-2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenyl]-8H-pyrano[3,2-g]chromen-4-one) with an average molecular mass of 514.49 Da was isolated.We have named the compound artonkin-4'-O-glucoside. The name 'artonkin' for the novel flavonoid part of the compound was coined from the Latin name of its source Artocarpus tonkinensis. The three other active flavonoid glucosides isolated and characterized were alphitonin-4-O-beta-D-glucoside, maesopsin-4-O-beta-D-glucoside and kaempherol-3-O-beta-D-glucoside. All four compounds were found to cause anti-inflammatory effect with different potencies. The anti-inflammatory effects demonstrated in the rat model of arthritis correlate well with the inhibition of mitogen-induced T-cell proliferation. Furthermore, the compounds inhibit production of cytokines, such as tumour necrosis factor-a and interferon-c, in mitogen-stimulated T cells in a concentration-dependent manner. We postulate that the isolated flavonoids suppress T-cell proliferation as well as cytokine expression and thereby contribute to an amelioration of arthritis severity in CIA.

Arthritogenicity of collagen type II is increased by chlorination.

During inflammation, activated neutrophils, monocytes and macrophages produce and release myeloperoxidase (MPO). MPO converts hydrogen peroxide to hypochlorous acid, a highly reactive and oxidizing agent. Proteins subjected to hypochlorous acid become chlorinated. We analysed how chlorination of the cartilage antigen collagen type II (CII) affects its immunogenic and arthritogenic properties by studying immune responses to chlorinated CII in comparison to immune responses to CII and by studying the development of arthritis in rats immunized with CII-Cl. CII-Cl immunization of LEW.1AV1 rats caused a 100% incidence of arthritis with a mean maximum score of 9.2 (maximal score possible 16). The same dose of non-chlorinated CII did not induce arthritis at all. Rats immunized with CII-Cl developed high anti-CII-Cl IgG titres and also developed IgG antibodies recognizing the non-chlorinated form of CII. Analysis of cytokine mRNA expression in lymph nodes 10 days after immunzation revealed an increased expression of interferon (IFN)-gamma mRNA and interleukin (IL)-1beta mRNA in CII-Cl-immunized rats compared to CII-immunized rats. Thus, chlorination of CII increased its immunogenicity as well as its arthritogenicity. As neutrophils, monocytes and macrophages are abundant cells in arthritic joints of patients with rheumatoid arthritis, chlorination might be a mechanism by which immunoreactivity to CII is induced and by which chronic joint inflammation is supported.

Morphological characterization of receptor activator of NFkappaB ligand (RANKL) and IL-1beta expression in rodent collagen-induced arthritis.

Bone loss represents a major unsolved problem in rheumatoid arthritis (RA). The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the development and activation of osteoclasts, which are key mediators of bone erosions. This study was performed to determine temporal and spatial expression of RANKL compared with the potentially destructive cytokine interleukin-1beta (IL-1beta), related to progression of synovitis and joint destruction in collagen-induced arthritis (CIA), a model of RA. CIA was induced in dark agouti (DA) rats, and tissue specimens were obtained for immunohistochemical analyses at various time points before and after disease onset. Arthritis was monitored visually, and joint pathology was examined histologically. No disease-preceding expression of RANKL was detected. However, a marked increase of both RANKL- and IL-1beta-expressing cells correlated with the progression of synovial inflammation and clinical disease severity. Abundant and concomitant expression of these cytokines was detected at sites of bone erosion, where a colocalization by osteoclast-like multinuclear tartrate-resistant acid phosphatase (TRAP)+ cells was noted. In contrast to the paucity of RANKL expression in cartilage, an abundant expression of IL-1beta was demonstrated, particularly in superficial cartilage layers. These data support the hypothesis that RANKL and IL-1beta are central contributors to joint destruction in CIA.

Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1.

The nuclear protein high-mobility group box chromosomal protein 1 (HMGB1) was recently described to act as a pro-inflammatory cytokine and as a late mediator of severe sepsis and septic shock. The protein is released from monocytes in response to endotoxin and activates monocytes and endothelial cells through nuclear factor kappa B. We have previously demonstrated that the B-box of HMGB1 mediates a pro-inflammatory effect on endothelial cells including the upregulation of cell-adhesion molecules and release of interleukin (IL)-8 and granulocyte colony-stimulating factor. Here, we report that HMGB1 is released from human umbilical vein endothelial cells (HUVEC) in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-alpha. A nuclear relocation of HMGB1 to the cytoplasm was seen at 4 h. Subsequently, high amounts of HMGB1 could be seen in the supernatants from stimulated cells after 16 h. It was also observed that the pro-inflammatory activity of HMGB1 is sensitive to dexamethasone. Interestingly, the HMGB1-induced TNF-alpha release from monocytes could be inhibited by either the A-box of the protein or the p38 inhibitor CNI-1493, but neither had any inhibitory effects on the HMGB1-dependent upregulation of cell-adhesion molecules on HUVEC. Altogether, these results suggest that HUVEC may be an important source of HMGB1 secretion in response to systemic infection and that endothelial cells and monocytes may use different signalling pathways.

Differential bone turnover in an angulated fracture model in the rat.

We have developed a simple rat model of angulated tibial fracture which elicits substantial differences in bone formation and resorption within the same bone. In 35 rats the right mid-tibia was manually fractured and fixed with an intramedullary 17-gauge cannula needle. Twenty tibias were fixed in anterior angulation (27 +/- 5 degrees) and 15 in posterior angulation (31 +/- 5 degrees). Serial X-rays were taken over a 12-week period. All fractures healed completely within five weeks. In both groups, bone thickness was already significantly greater on the concave side than on the convex side at week 3 and remained so until the end of the experiment. The thickness on the convex side decreased dramatically within 3 to 5 weeks and gradually thereafter. For morphological analysis of bone mineralization, 3 rats from each group were given calcein and alizarin red injected at different time points up to 14 weeks. Maximum new bone formation was noted within the first 3 weeks. Over the ensuing weeks, new bone formation remained intense on the concave side, but it was virtually absent on the convex side. These results show that angulated fracture deformity reproducibly exhibits differential bone turnover, which can be exploited in research on local regulatory factors. To exemplify the utility of the model, an immunohistochemical study on two local markers was done. Callus tissue of five rats in the anterior angulation group at week 3 post-fracture was stained for the cytokine IL- 1beta, a stimulator of bone resorption, and the neuropeptide CGRP, an inhibitor of resorption, showing clear differences in positive staining between the concave and convex sides. Our in-vivo model offers a means of analyzing morphologically and quantitatively the differential expression and action of factors involved in local bone turnover.

Corticosteroid treatment of experimental arthritis retards cartilage destruction as determined by histology and serum COMP.

OBJECTIVE: To examine if changes in serum cartilage oligomeric matrix protein (COMP) correlate with the development of cartilage damage, as measured by histological grading, in corticosteroid-treated animals with collagen-induced arthritis (CIA). METHODS: DA rats with established CIA were treated with corticosteroids (betamethasone, 0.1 mg/kg body weight) or placebo (saline) intraperitoneally once daily after reaching an arthritis score exceeding 1. The treatment continued throughout the study. Arthritis progression was monitored by clinical scoring of paws, serial measurements of serum COMP and fibrinogen, and histological grading of paws. RESULTS: Corticosteroid treatment reduced clinical signs of arthritis compared with placebo (arthritis score reduced, P < 0.01 at day 25). Corticosteroid treatment also reduced fibrinogen levels compared with placebo (P < 0.01). The morphological changes in the joint were less severe in the corticosteroid-treated animals (median cartilage score 4 in the placebo group, 0 in the corticosteroid-treated group; P < 0.01). The levels of COMP remained unchanged during treatment in the corticosteroid-treated arthritic animals, whereas an increase in levels of COMP was observed in rats treated with placebo (P < 0.01). There was a correlation between serum COMP and the extent of cartilage destruction at day 25 after immunization (r = 0.77, P < 0.001). CONCLUSIONS: Corticosteroids given therapeutically to arthritic rats diminish joint destruction histologically, and stable serum COMP levels reflect this effect.

HMGB1 is a potent trigger of arthritis.

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial inflammation and structural damage of joints. Although the cause of rheumatoid arthritis (RA) remains unknown, the excessive production of proinflammatory cytokines such as tumour necrosis factor (TNF) and interleukin-1 (IL-1) by intra-articular macrophages occupies a critical pathogenic role in the development and progression of the disease. High mobility group box chromosomal protein 1 (HMGB1) is a recently identified mediator of interest in human and experimental arthritides. HMGB1 can either be actively secreted from macrophages or passively released from necrotic cells of all kinds. Activated macrophages and unprogrammed cell death caused by ischaemia or activated complement are all prominent features of chronic arthritis, contributing to the persistent synovial inflammation. HMGB1 is cytoplasmically and extracellularly overexpressed in inflammatory synovial tissue in human RA as well as experimental collagen-induced arthritis. Elevated levels of HMGB1 are also present in synovial fluid samples from RA patients. Synovial tissue from rats with experimental arthritis exhibits aberrant deposition of HMGB1 preceding the onset of clinical signs of arthritis, and the expression becomes prominent after the onset of clinical disease. The synovial levels of HMGB1 are comparable with those of TNF and IL-1beta at the peak of manifest disease. HMGB1-targeted intervention with either neutralizing antibodies or the antagonistic A box domain of HMGB1 ameliorates collagen-induced arthritis both in mice and rats, and inhibits the local overexpression of IL-1beta in the joints. It is thus conceivable that therapeutic HMGB1 blockade may contribute to future treatment of human chronic arthritis.

High mobility group 1 B-box mediates activation of human endothelium.

OBJECTIVES: Severe sepsis and septic shock is a consequence of a generalized inflammatory systemic response because of an invasive infection that may result in acute organ dysfunction. Mortality is high despite access to modern intensive care units. The nuclear DNA binding protein high mobility group 1 (HMGB1) protein has recently been suggested to act as a late mediator of septic shock via its function as a macrophage-derived pro-inflammatory cytokine (J Exp Med 2000; 192: 565, Science1999; 285: 248). We investigated the pro-inflammatory activities of the A-box and the B-box of HMGB1 on human umbilical venular endothelial cells (HUVEC). DESIGN: The HUVEC obtained from healthy donors were used for experiments. Recombinant human full-length HMGB1, A-box and B-box were cloned by polymerase chain reaction (PCR) amplification from a human brain quick-clone cDNA. The activation of HUVEC was studied regarding (i) upregulation of adhesion molecules, (ii) the release of cytokines and chemokines, (iii) the adhesion of neutrophils to HUVEC, (iv) the activation of signalling transduction pathways and (v) the involvement of the receptor for advanced glycation end-products (RAGE). RESULTS: The full-length protein and the B-box of HMGB1 dose-dependently activate HUVEC to upregulate adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and to release IL-8 and G-CSF. The activation of HUVEC could be inhibited to 50% by antibodies directed towards the RAGE. HMGB1-mediated HUVEC stimulation resulted in phosphorylation of the ELK-1 signal transduction protein and a nuclear translocation of p65 plus c-Rel, suggesting that HMGB1 signalling is regulated in endothelial cells through NF-kappaB. CONCLUSIONS: The HMGB1 acts as a potent pro-inflammatory cytokine on HUVEC and the activity is mainly mediated through the B-box of the protein. HMGB1 may be a key factor mediating part of the pro-inflammatory response occurring in septic shock and severe inflammation.

Antigen inhibition of collagen-induced arthritis is associated with up-regulation of IL-4 mRNA and induction of Ox40 on T cells in draining lymph nodes.

The addition of a foreign antigen to an inoculum completely inhibits the development of collagen-induced arthritis (CIA). However, the mechanism of this phenomenon, antigen -inhibition, is incompletely understood. Previous studies have demonstrated that the inhibition of arthritis is not mediated through suppression of the antibody response to cartilage antigens. In this paper we investigated cytokine mRNA levels in lymph nodes cells recovered 3, 7 or 16 days from animals immunized with either collagen II in IFA or OVA + collagen II in IFA. At day 7, but not at other time-points, IL-4 mRNA was up-regulated in the lymph nodes of OVA-inhibited non-arthritic animals compared to control animals which all developed arthritis. No significant differences between the two groups could be detected when expression of IFN-gamma, IL-2, TNF-alpha, IL-1beta or IL-10 mRNA was analysed. Flow cytometry analysis of draining lymph node cells demonstrated that the T cell marker Ox40 was up-regulated in the OVA-inhibited group. Our results indicate that the complete inhibition of CIA caused by addition of OVA to the collagen II inoculum is due to the presence of a TH2 environment resulting from an increased production of IL-4 mRNA and a parallel increase in Ox40+ T cells.

Serum concentrations of cartilage oligomeric matrix protein, fibrinogen and hyaluronan distinguish inflammation and cartilage destruction in experimental arthritis in rats.

OBJECTIVES: We investigated if changes in serum/plasma fibrinogen (FIB), hyaluronan (HA) and cartilage oligomeric matrix protein (COMP) levels can be used to differentiate between inflammation and cartilage involvement during arthritis. METHODS: Collagen-induced arthritis (CIA), oil-induced arthritis (OIA) and for comparison, experimental autoimmune encephalitis (EAE) induced in DA rats were investigated. RESULTS: Elevations of FIB concentrations were apparent at days 4-7 post-immunization in both arthritis models reaching a maximum on day 20-21, i.e. before peak arthritis. Elevations of HA in both models were seen shortly before macroscopically apparent arthritis, and peaked at or just before maximal arthritis, i.e. later in CIA than in OIA. COMP levels increased only after onset of arthritis and peaked late in disease (days 34-37), being significantly higher in the more destructive CIA compared with the less destructive OIA. During EAE flares, only FIB levels increased. CONCLUSIONS: FIB is a general inflammation marker, HA appears to be a marker for synovitis and changes in COMP levels appear to reflect the cartilage destruction process.

Dynamics of early synovial cytokine expression in rodent collagen-induced arthritis : a therapeutic study using a macrophage-deactivating compound.

This study was performed to elucidate pathophysiological events before and during the course of collagen-induced arthritis in Dark Agouti rats, a model for rheumatoid arthritis. Kinetic studies of local cytokine responses were determined using immunohistochemical techniques, quantified by computer-assisted image analysis. We recently reported that the macrophage-pacifying agent CNI-1493 successfully ameliorated collagen-induced arthritis. In the present trial, we investigated the potential of CNI-1493 to down-regulate pro-inflammatory cytokines. Synovial cryosections were analyzed at various time points for the presence of interleukin (IL)-1beta, tumor necrosis factor (TNF), and transforming growth factor (TGF)-beta. Unexpectedly, an early simultaneous TNF and IL-1beta expression was detected in resident cells in the lining layer, preceding disease onset and inflammatory cell infiltration by >1 week. The predominant cytokine synthesis by synovial (ED1+) macrophages coincided with clinical disease. TNF production greatly exceeded that of IL-1beta. CNI-1493 treatment did not affect the early disease-preceding TNF and IL-1beta synthesis in the lining layer. However, after disease onset, CNI-1493 intervention resulted in a pronounced reduced IL-1beta and in particular TNF expression. Furthermore, CNI-1493 significantly up-regulated synthesis of the anti-inflammatory cytokine TGF-beta and thereby shifted the balance of pro-inflammatory and anti-inflammatory cytokines in the arthritic joint in a beneficial way.

Parasite-mediated down-regulation of collagen-induced arthritis (CIA) in DA rats.

Microbial infection can impact on the course of autoimmune disease, both in disease-inducing and disease-protecting capacities. Here we investigated if infection with Trypanosoma brucei brucei (Tbb), the protozoan causative agent of African Sleeping Sickness, could ameliorate the course of CIA in the Dark Agouti rat, an experimental model which shares many features with human rheumatoid arthritis. Infection of animals with living, but not inoculation with dead Tbb resulted in complete or significant reduction of clinical arthritic symptoms. Infection prior to collagen immunization was more effective than a later treatment, and this effect was related to the level of parasitaemia. Using reverse transcriptase-polymerase chain reaction we detected an increase in interferon-gamma mRNA in the draining lymph nodes of Tbb-treated animals relative to controls at day 28 after disease induction. Transforming growth factor-beta could be detected in the lymph nodes in four out of six animals that had received Tbb. In the joints, immunohistochemistry revealed reduced production of tumour necrosis factor-alpha in Tbb-treated animals relative to controls. The most striking difference between Tbb-infected and control groups, as measured by ELISA, was the down-regulation of anti-collagen II IgG antibody responses in parasite-infected animals. We conclude that live parasites can exert an immunomodulatory and protective effect in CIA in which several mechanisms may work in parallel, although the almost complete down-regulation of the anti-collagen antibody response may alone explain the protective effect in CIA. The described model may be useful in further attempts to use the mechanisms involved in parasite immune defence to prevent and treat certain autoimmune conditions.

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

Role of selectins in experimental Staphylococcus aureus-induced arthritis.

The selectin family of adhesion molecules mediates the initial attachment of leukocytes to venular endothelial cells at sites of tissue injury and inflammation. To assess the role of selectin family in Staphylococcus aureus-triggered septic arthritis, we used several approaches. First, treatment with fucoidin, a carbohydrate molecule capable of binding to and blocking selectin functions, was used. In addition, we used P-selectin gene-targeted mice as well as mice pretreated with monoclonal antibody blocking L-selectin function. The P-selectin-deficient and fucoidin-treated animals initially exhibited a less severe septic arthritis both clinically and histopathologically. In the later stages of the disease no significant differences with respect to arthritis were evident. Pretreatment with L-selectin blocking antibody did not influence the severity of arthritis. High numbers of staphylococci were recovered from the kidneys of selectin-deficient mice, indicating a less efficient clearance of bacteria. Our results demonstrate a dual role for selectins in S. aureus-induced arthritis: on the one hand, blockade of these selectins leads to less severe arthritic lesions in the initial stage of the disease; on the other, delayed recruitment of phagocytes decreases the clearance of bacteria.

Anti-inflammatory effects of a new tumour necrosis factor-alpha (TNF-alpha) inhibitor (CNI-1493) in collagen-induced arthritis (CIA) in rats.

A recently developed compound, a multivalent guanylhydrazone (CNI-1493) that inhibits TNF-alpha production by suppressing TNF-alpha translational efficiency, was administered in an experimental model of collagen type II-induced arthritis in DA rats. CNI-1493 was injected daily intraperitoneally either before the onset of arthritis or after the establishment of clinical disease. Prophylactic treatment with CNI-1493 significantly prevented or delayed the onset and suppressed the severity of arthritis in a dose-dependent manner. Therapeutic intervention with CNI-1493 in established joint disease also resulted in a significant reduction of clinical signs of arthritis in treated animals. No severe side-effects were noted when animals were treated with daily CNI-1493 doses up to 5 mg/kg. An immunohistochemical study was performed which demonstrated that CNI-1493 led to a reduced expression of TNF-alpha at the site of disease activity. Thus, CNI-1493 with documented inhibitory effects on TNF-alpha synthesis, has proven successful in ameliorating the course of arthritis in CIA. We believe that the use of a compound such as CNI-1493 with a defined mode of action provides a useful tool for dissecting and understanding important pathogenic mechanisms operating in the development of chronic arthritis.

Polyclonal Th1 cells transfer oil-induced arthritis.

T-cells play a critical role in oil-induced arthritis (OIA) in DA rats. The present study focuses on the involvement of CD4/CD8 T cells in OIA by using adoptive transfer. Mitogen-activated T cells from DA rats previously injected with incomplete Freund's adjuvant (IFA) were depleted of CD4+ T cells or CD8+ T cells before transfer to irradiated naive receipients. The results indicate that CD4+ T cells are essential for the induction of passively induced OIA. However, in vitro blocking experiments with monoclonal antibodies (mAb) to the CD4 molecule of the T cells before transfer did not affect the passive OIA. Neither was passive OIA inhibited by treating the CD4+ T cells with mAb to intracellular adhesion molecule-1 (ICAM-1) in order to block cell-cell interactions or migration. The arthritogenic CD4+ T cells were sensitive, however, to in vitro treatment with mAb to the interleukin-2 receptor, which inhibited the disease or delayed the onset of passive OIA in recipients. The arthritogenic CD4+ T cells were also analysed for expression of specific T-cell receptor (TCR) variable (V) beta chains, critical for recognition of autoantigen, by utilizing V beta gene-specific polymerase chain reaction (PCR). The results show a heterogeneous expression of V beta segments of the TCR, indicating a polyclonal origin of the pathogenic cells. Moreover, an investigation of the T helper (Th)1/Th2 status of the CD4+ T cells, defined by cytokine expression, was made at the mRNA level by using in situ hybridization. High numbers of interleukin-2 (IL-2) mRNA expressing cells and also interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha)-expressing cells could be identified. We conclude from this study that non-immunogenic IFA triggers polyclonal, IL-2-dependent Th1 cells which induce arthritis. The contribution of the CD4 or ICAM-1 molecules for arthritis induction seem to be of minor importance.