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.

Orthopaedic surgery of the lower limbs in 49,802 rheumatoid arthritis patients: results from the Swedish National Inpatient Registry during 1987 to 2001.

OBJECTIVES: To analyse changes in the rates of hospital admission and use of orthopaedic surgery to the lower limbs in Swedish patients with rheumatoid arthritis between 1987 and 2001. METHODS: Data for all rheumatoid patients admitted to hospital between 1987 and 2001 were abstracted from the Swedish National Hospital Discharge Register (SNHDR). The data in the register are collected prospectively, recording all inpatient admissions throughout Sweden. The SNHDR uses the codes for diagnoses at discharge and surgical procedures according to the Swedish version of the International Classification of Diseases (ICD). RESULTS: In all, 49,802 individual patients with rheumatoid arthritis were identified, accounting for 159,888 inpatient visits. Hospital admissions for rheumatoid arthritis decreased by 42% (p<0.001) during the period 1987 to 2001. Twelve per cent of all admissions were for a rheumatoid arthritis related surgical procedure to the lower limbs; those admissions decreased markedly (by 16%) between 1987 and 1996, and by 12% between 1997 and 2001, as did the overall number of rheumatoid arthritis related surgical procedures to the lower limbs during both time periods. Between 1997 and 2001, 47% of all rheumatoid arthritis related surgical procedures were total joint arthroplasties. There was an overall trend towards reduced length of hospital stay after orthopaedic surgery to the lower limbs during the study period. CONCLUSIONS: Rates of hospital admission and rheumatoid arthritis related surgical procedures to the lower limbs in Swedish patients with rheumatoid arthritis decreased between 1987 and 2001. This may reflect trends in disease severity, management, and health outcomes of this disease in Sweden.

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.

CNI-1493, an inhibitor of proinflammatory cytokines, retards cartilage destruction in rats with collagen induced arthritis.

OBJECTIVE: To investigate if administration of CNI-1493, an inhibitor of the synthesis of proinflammatory cytokines and NO, protects against development of joint destruction in collagen induced arthritis (CIA) in rats. METHODS: In a placebo controlled experiment, CNI-1493 was given once daily intraperitoneally after onset of clinical arthritis in DA rats. Disease progression was studied by clinical scoring of arthritis, serial measurement of serum levels of COMP, and histological examination of joints. RESULTS: Clinical signs of arthritis were significantly reduced in the CNI-1493 treated group of rats in comparison with the placebo treated group. Histological examinations of paws demonstrated a significant reduction of cartilage destruction in the CNI-1493 treated group, but marked destruction of cartilage in the placebo group. Serum levels of COMP increased in the placebo group, whereas in the CNI-1493 treated group levels were low and decreased significantly during the observation time. CONCLUSIONS: Treatment with CNI-1493 provides efficient protection against synovial inflammation and cartilage destruction when used therapeutically in CIA. The protective effect against cartilage destruction can be monitored by measuring serum COMP. These observations make CNI-1493 an attractive candidate for therapeutic studies in human arthritis, and COMP an attractive serum marker for monitoring joint protective effects.

Successful treatment of collagen-induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity.

OBJECTIVE: Extracellular high mobility group box chromosomal protein 1 (HMGB-1) is a recently identified, endogenous, potent tumor necrosis factor- and interleukin-1 (IL-1)-inducing protein detectable in inflamed synovia in both human and experimental disease. In the present study, we examined clinical effects in collagen-induced arthritis (CIA) using therapeutic administration of neutralizing HMGB-1 antibodies or truncated HMGB-1-derived A-box protein, a specific, competitive antagonist of HMGB-1. METHODS: CIA was induced in DBA/1j mice or dark agouti rats, and animals were examined daily for signs of arthritis. Treatment with polyclonal anti-HMGB-1 antibodies or the A-box protein was initiated at the onset of disease and was administered intraperitoneally twice daily for 7 days. Animals were killed 8 days after initiation of therapy, and immunohistochemical analysis of synovial tissue specimens was performed. RESULTS: Systemic administration of anti-HMGB-1 antibodies or A-box protein significantly reduced the mean arthritis score, the disease-induced weight loss, and the histologic severity of arthritis. Beneficial effects were observed in both mice and rats. Immunohistochemical analysis revealed pronounced synovial IL-1beta expression and articular cartilage destruction in vehicle-treated mice. Both these features were significantly less manifested in animals treated with anti-HMGB-1 antibodies or A-box protein. CONCLUSION: Counteracting extracellular HMGB-1 with either neutralizing antibodies or a specific HMGB-1 antagonist may offer a new method for the successful treatment of arthritis. Inflammation and tissue destruction were suppressed in CIA after HMGB-1 blockade.

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. METHODS: Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. RESULTS: Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. CONCLUSION: The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.

IFN-alpha beta-dependent, IFN-gamma secretion by bone marrow-derived macrophages controls an intracellular bacterial infection.

Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-gamma. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-gamma at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-gamma mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-gamma mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10(-/-) BMMs. Such IL-10(-/-) BMMs contained less bacteria than the wild-type controls, whereas IFN-gammaR(-/-) BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS(-/-) BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-alphabeta, but was independent of IFN-gamma. Interestingly, IFN-alphabeta are also required for increased IFN-gamma mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-alphabetaR(-/-) BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-alphabeta-dependent IFN-gamma and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.

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.

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.

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.