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OBJECTIVE: To use the collagenase cleavage site neoepitope, TIINE, a marker of type II collagen breakdown in cartilage, to analyze the mechanism underlying the slowing of joint space narrowing (JSN) in patients with knee osteoarthritis treated with doxycycline. METHODS: The creatinine-adjusted urinary TIINE concentration was determined at baseline and every 6 months thereafter in a subset of patients who completed a 30-month randomized, placebo-controlled study of the effect of doxycycline on radiographic progression of JSN. The subset was selected a priori to permit comparison of 60 radiographic progressors with 60 radiographic nonprogressors. JSN was determined in highly standardized, semiflexed anteroposterior images. RESULTS: The coefficient of variation of TIINE concentrations over the 5 study visits was 30%. At the 5 semiannual followup visits, the mean TIINE concentration for doxycycline-treated patients was higher than that for the placebo group. In both treatment groups, the correlation between TIINE levels and JSN in the index knee was weak (for doxycycline, r(2) = 0.06, P = 0.08; for placebo, r(2) = 0.06, P = 0.05). CONCLUSION: High variability from visit to visit limits the sensitivity of the TIINE assay for detecting changes in individual patients and restricts its utility to group comparisons. The increase in TIINE concentration with treatment indicates that inhibition of collagenase-mediated breakdown of type II collagen in articular cartilage is unlikely to have accounted for the observed reduction of JSN in the index knees of patients in the doxycycline treatment group. [ABSTRACT FROM AUTHOR]

Objective: To determine if anthropometric factors obtainable on routine examination can be used to estimate premorbid knee total subchondral bone area (tAB), cartilage surface area (AC), cartilage thickness (ThC), and cartilage volume (VC)., Method: Young individuals (21-39 years old) without history of knee joint pain, injury or disease were studied. Magnetic resonance imaging of the right knee was used to determine tAB, AC, ThC and VC for knee cartilage. Multilinear regression and curve fitting by variance minimization were used to model the data., Results: VC and AC closely depended on tAB(1.5) in both men and women. This relationship subsumed all dependency on sex, height, weight and body mass index. In females, VC depended on height cubed and tAB on height squared. The relationship was much weaker in males. ThC was poorly related to tAB and VC. Confidence limits for VC standardized to tAB(1.5) were narrower than standardization to tAB or height., Conclusion: The absence of a tight relationship of VC and tAB with height in males suggests that the factors stimulating bone and cartilage growth may be different between sexes. The high correlation between tAB and VC across both sexes suggests, however, that (opposite to measures from routine clinical examination) tAB(1.5) can provide individual reference values for VC, against which changes with age and disease can be estimated with high confidence.

Objective: Females have a higher incidence of knee osteoarthritis (OA) than males, but the reason for this is unclear. Here we examine the hypothesis that women have smaller joint surfaces than men, independent of weight and height, and thus encounter higher articular pressures that might contribute to the higher incidence of OA in the female knee., Methods: Forty healthy women and 57 men (21-39 years) with a body mass index of 16.8-32.8 were studied using magnetic resonance imaging. The right knee was scanned and proprietary software was used to determine the area of subchondral bone (cAB), mean cartilage thickness (ThC) and cartilage volume (VC) for all knee cartilage plates. Multilinear regression was used to correct the data for sex differences in height and weight., Results: cAB, ThC, and VC were larger in men than in women in all knee cartilage plates. Correction for height and weight differences between the sexes reduced but did not eliminate sex differences in these parameters. The cAB was a strong predictor of VC independent of sex, height and weight, but did not predict ThC., Conclusion: Men have greater knee cABs, ThC and VC than females even after correction for height and weight. Nonetheless, estimated tibial and patellar pressures are similar between sexes and thus are unlikely to account for the sex differences in OA incidence.

The SAR of a series of sterically hindered sulfonamide hydroxamic acids with relatively large P1' groups is described. The compounds typically spare MMP-1 while being potent inhibitors of MMP-13. The metabolically more stable compounds in the series contain either a monocyclic or bicyclic pyran ring adjacent to the hydroxamate group. Despite the sparing of MMP-1, pre-clinical and clinical studies revealed that fibrosis in rats and MSS in humans is still produced.

Objective: To develop criteria for the selection and application of molecular markers for the study of osteoarthritis (OA)., Methods: Statistical criteria for marker selection for OA are developed., Results and Conclusions: After studying more than 20 different molecular markers for monitoring OA, procedures for choosing markers for clinical studies have been developed. For a particular study, the process starts with the markers showing 'face-validity' for monitoring OA. They are next required to successfully distinguish OA patients from controls. This necessitates definition of the distribution of marker values in OA patients and controls. So far, they have been consistently log-normal. The difference (Delta) in marker values between OA and controls defines the opportunity for marker improvement. The between-visit standard deviation (S) in patients puts limits on the detection of marker changes. The two variables can be combined to estimate the practicality of a marker using a modified power analysis. The number of patients (N*) required to observe a 50% improvement with an alpha level of P=0.05 and with 80% certainty is estimated as 50(S/Delta)(2). N*, S and Delta should be used to characterize and compare markers. Marker efficiency can be refined by regressing on secondary variables, such as age, sex, BMI, severity, etc. Finally, the use of two or more markers may be required to improve marker prediction of clinical outcome. Correlated markers can be used to reinforce conclusions by essentially adding replicative data. Independent, complementary markers can be used to develop associations with clinical parameters, and perhaps diagnose and monitor disease status, activities that so far have not been possible with single markers.

Objective: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage., Methods: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs])., Results: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis., Conclusion: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.

Monoclonal antibody (MAb) technology was used to examine aggrecan metabolites and the role of aggrecanases and matrix metalloproteinases (MMPs) in proteolysis of the interglobular domain (IGD) and C-terminus of aggrecan. An in vitro model of progressive cartilage degradation characterized by early proteoglycan loss and late stage collagen catabolism was evaluated in conjunction with a broad-spectrum inhibitor of MMPs. We have for the first time demonstrated that IGD cleavage by MMPs occurs during this late stage cartilage degeneration, both as a primary event in association with glycosaminoglycan (GAG) release from the tissue and secondarily in trimming of aggrecanase-generated G1 metabolites. Additionally, we have shown that MMPs were responsible for C-terminal catabolism of aggrecan and generation of chondroitin sulfate (CS) deficient aggrecan monomers and that this aggrecan truncation occurred prior to detectable IGD cleavage by MMPs. The onset of this later stage MMP activity was also evident by the generation of MMP-specific link protein catabolites in this model culture system. Recombinant MMP-1, -3 and -13 were all capable of C-terminally truncating aggrecan with at least two cleavage sites N-terminal to the CS attachment domains of aggrecan. Through analysis of aggrecan metabolites in pathological synovial fluids from human, canine and equine sources, we have demonstrated the presence of aggrecan catabolites that appear to have resulted from similar C-terminal processing of aggrecan as that induced in our in vitro culture systems. Finally, by developing a new MAb recognizing a linear epitope in the IGD of aggrecan, we have identified two novel aggrecan metabolites generated by an as yet unidentified proteolytic event. Collectively, these results suggest that C-terminal processing of aggrecan by MMPs may contribute to the depletion of cartilage GAG that leads to loss of tissue function in aging and disease. Furthermore, analysis of aggrecan metabolites resulting from both C-terminal and IGD cleavage by MMPs may prove useful in monitoring different stages in the progression of cartilage degeneration.

Objective: To study the role of CD44, the principal hyaluronan (HA) receptor, in experimental arthritis., Methods: We generated CD44 gene deficiency in arthritis-susceptible DBA/1LacJ mice to study the role of CD44 directly in collagen-induced arthritis (CIA). Wild-type and CD44-deficient mice were immunized with chicken type II collagen, and the onset and severity of CIA were monitored up to day 64. The immune status of immunized mice was determined at the end of the experiments. Cell transfer experiments were performed to monitor lymphocyte traffic to the inflamed joints., Results: Mice homozygous for the CD44 mutation developed normally and showed no phenotypic defects. Although they showed a normal response to immunization with type II collagen and had Th1/Th2 ratios comparable with those in wild-type animals, CD44-deficient mice exhibited significant reductions in both the incidence and severity of CIA. This was accompanied by altered serum levels of HA, reduced expression of L-selectin, and a delayed entry of intravenously injected CD44-deficient donor lymphocytes into the arthritic joints of recipient mice., Conclusion: While CD44 is not essential for morphogenesis and autoimmunity, this cell surface receptor seems to play an important role in the development of arthritis, most likely by directing leukocyte traffic to the site of inflammation.

Objective: To investigate whether any of 14 serum and urine molecular markers (MMs) used to monitor osteoarthritis (OA) would be associated with particular clinical end-points., Design: Thirty-nine OA patients were bled and urine collected at five time points: at baseline visit and at visits 1, 3, 6 and 12 months later. Twelve clinical measurements were made and the concentrations of each of 14 MMs were determined. Principal component analysis, stepwise linear regression with backward elimination, and logistic regression were used to determine the correlations between MMs and clinical measures., Results: Principal component analysis was used to reduce the 12 clinical measurements into three independent clinical clusters: baseline clinical assessments, changes in clinical assessments and signal joint measurements. The 14 MMs were similarly reduced to five MM clusters. Each of the three clinical clusters was correlated with a single but different MM cluster. Baseline clinical assessments were correlated with bone markers typified by hydroxylysyl pyridinoline (HP) crosslinks, swelling of the signal joint was correlated with inflammation markers, especially CRP, and the change in clinical assessments over the 1 year evaluation was correlated with TGFbeta1. There was no correlation between any of the skeletal markers and the clinical measures, a situation which draws attention to the need for a direct assessment of cartilage damage in OA to validate the use of cartilage markers., Conclusions: This study demonstrates statistical methodology for analysis of clinical trials using multiple MMs and clinical end-points. The patient numbers are sufficient to test hypotheses of relationships of single MMs such as CRP, TGFbeta1 and HP to clinical measures, but larger clinical trials are needed to validate hypotheses., (Copyright 2001 OsteoArthritis Research Society International.)

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.

Objective: Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate changes occurring in both medial and lateral menisci from the knees of anterior cruciate ligament (ACL) transected rabbits at 3 and 8 weeks post-surgery. This study describes both molecular and cellular alterations in menisci during the early stages of OA development., Design: Rabbit meniscal tissues were processed for molecular analysis: DNA and RNA concentrations were assessed, as well as semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules was performed. In situ DNA fragmentation was evaluated using the TUNEL assay., Results: Total RNA yields from the medial meniscus were significantly elevated at both 3 and 8 weeks post-ACL transection, while in the lateral meniscus total RNA levels were unchanged following ACL transection. DNA concentrations were significantly decreased in the medial menisci only at 8 weeks post-ACL transection. Following ACL transection, analysis of in situ DNA fragmentation using the TUNEL assay demonstrated an increase in the number of apoptotic cells in the medial meniscus only, in particular at 3 weeks post-ACL transection, a finding which correlates with declines in DNA content. Analysis of specific mRNA levels by RT-PCR demonstrated complex changes in both menisci following ACL transection. At 3 and 8 weeks post-ACL transection, in both medial and lateral menisci, mRNA levels for type I collagen and TIMP-1 were significantly increased, while mRNA levels for decorin, TNF-alpha and IGF-2 were significantly depressed. In the medial meniscus, significant increases in mRNA levels for type II collagen, biglycan as well as iNOS and PAI-1 were detected at both time periods, while mRNA levels for aggrecan, type III collagen and COX-2 were significantly elevated at 3 weeks post-ACL transection and mRNA levels for MMP-1 were significantly elevated at 8 weeks post-ACL transection. In contrast, mRNA levels for COL2 and aggrecan were unchanged in the lateral meniscus following ACL transection. In the lateral meniscus, at 3 weeks post-ACL transection, type III collagen mRNA levels were dramatically increased while fibromodulin mRNA levels were significantly depressed. In the lateral meniscus, significant increases in mRNA levels for biglycan were detected at 8 weeks post-ACL transection., Conclusion: These results show that after ACL transection complex molecular changes, as well as apoptosis, occur early, particularly in the medial meniscus.

Objective: Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate changes occurring in both medial and lateral menisci from the knees of anterior cruciate ligament (ACL) transected rabbits during the early stages of OA development., Design: Meniscal tissues from control and experimental rabbits were processed for histology and immunohistochemistry for assessment of matrix organization and composition., Results: At 3 and 8 weeks following ACL transection, histological examination demonstrated extensive extracellular matrix deterioration. Altered cell distribution, areas depleted of cells, and areas of cell clusters were found within the medial but not in the lateral meniscus. Immunohistochemistry of both medial and lateral menisci demonstrated significant changes in collagen distribution. Type I and III collagen staining was increased in both medial and lateral menisci. In contrast, type II collagen staining was overtly increased only in the medial meniscus., Conclusion: These results demonstrate that after ACL transection, extracellular matrix deposition as well as altered matrix organization and altered cell distribution occur early in the medial meniscus.

Objective: To determine the in vivo effects of intraarticular MMP-13., Methods: Human recombinant MMP-13 was injected intraarticularly (i.a. ) into the hamster knee joint. MMP-13 activity, collagen and proteoglycan fragments, and hyaluronan were measured in synovial fluid. Antibody 9A4 was used to localize collagen damage. Western blotting was used to determine the size of type II collagen fragments., Results: MMP-13 activity measurements showed greater than 98% of MMP-13 to be cleared instantly from the joint cavity. The remainder was cleared with a t(1/2)of 2 h. Immunohistochemical staining demonstrated collagen cleavage was limited to a thin superficial band on the surface of the articular cartilage whereas collagen damage extended more deeply into the synovial capsule and the menisci. The elevation of proteoglycan and hyaluronan in synovial fluid after MMP-13 was modest. Collagen fragments appeared in synovial fluid within 15 min following MMP-13. They were cleared with a half-life of circa 1.8 h and the predominant fragment was 32 kDa., Conclusions: Activated MMP-13 leads to tissue collagen damage with the release of collagen fragments. These fragments are measurable and could provide a method for assessment of cartilage collagen damage., (Copyright 2000 OsteoArthritis Research Society International.)

Protein-protein interactions (PPI) are a ubiquitous mode of transmitting signals in cells and tissues. We are testing a stepwise, generic, structure-driven approach for finding low molecular weight inhibitors of protein-protein interactions. The approach requires development of a high-affinity, single chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To this end, we developed a single chain antibody (sc1E3) against hIL-1beta that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-1R) for hIL-1beta and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1beta than the sIL-1R in that it failed to bind to either murine IL-1beta or human/murine IL-1alpha proteins. Additionally, failure of sc1E3 to bind to several hIL-1beta mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with other surface plasmon resonance and isothermal titration calorimetry measurements, shows that sc1E3 can achieve comparable affinity of binding hIL-1beta as the receptor through interactions at a smaller interface. This stable single chain antibody based heterodimer has simplified the complexity of the IL-1/IL-1R PPI system and will facilitate the design of the low molecular weight inhibitors of this interaction.

Objective: To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis., Design: Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology., Results: The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis., Conclusions: The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

High-affinity IgG autoantibodies (aAb) to IL-1alpha are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1alpha, we immunised mice with recombinant murine IL-1alpha. Unprimed and Bacille Calmette-Guérin (BCG)-primed BALB/cA mice were vaccinated with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding of IL-1alpha to the murine T-cell line NOB-1 by simple competition and neutralised IL-1alpha, but not IL-1beta-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1alpha can be induced in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

To determine whether the collagen network is compromised by collagenase during acute inflammation, a monoclonal antibody (9A4) was developed with specificity for the C-terminal neoepitope sequence generated by collagenase-cleavage of type II collagen (Gly-Pro-Pro-Gly-Pro-Gln-Gly-COOH). 9A4 was shown to detect the collagen collagenase-cleavage neoepitope with a K = 1.7 x 10(-7) M (type II) and K = 2 x 10(-6) M (type I). It does not recognize uncleaved native or denatured collagen. Articular cartilage from control animals is unstained by 9A4. During acute inflammation elicited in hamsters by intra-articular LPS, positive staining for the 9A4 neoepitope indicated the collagen was damaged. Wheel running exercise was used to apply stress to control cartilage and cartilage from animals with damaged collagen. After 6 months of running, the cartilage from normal animals was unaffected. By contrast, in the group with damaged collagen, the cartilage was fibrillated in all animals and in half of those, the cartilage failed and bony eburnation resulted.

Quinolone-induced changes were studied in the knee joints of 4-wk-old female hamsters given intraperitoneal doses of either nalidixic acid (400 mg/kg body weight) or vehicle on days 0 and 1. After euthanasia on day 4, synovial fluid was collected for cytologic evaluation and for analysis of concentrations of hyaluronan, proteoglycans, total protein, and collagen as hydroxyproline. Slides of formalin-fixed decalcified tissues were stained with hematoxylin-eosin or safranin O for histologic scoring of lesion severity. Nine of 10 hamsters treated with nalidixic acid had fissures within articular cartilage of the femur and reduced safranin O staining of matrix indicative of loss of proteoglycans. Synovial membranes from affected joints, however, were not inflamed. Synovial fluid cell counts and cytomorphology were unaffected by treatment. In synovial fluid from 5 of 10 treated hamsters, proteoglycans were elevated by more than 2 SDs above the control group, and individual animal levels correlated with the histologic severity score (r2 = 0.36; p = 0.02). The hyaluronan content of the synovial fluid from treated hamsters was mildly but significantly elevated (p = 0.005), and the histologic severity score again correlated with individual animal levels (r2 = 0.42; p = 0.01). Hydroxyproline was unaffected by treatment. Although synovial fluid changes and histologic changes were correlated on a group basis, interanimal variability was significant and the magnitude of biochemical changes were far smaller than those that occur during inflammation. Changes in synovial fluid composition are not sufficiently robust to predict cartilage changes in individual animals.

Adult articular cartilage is divided by the tidemark into a deep calcified layer and a more superficial uncalcified layer. Histologic examination of articular cartilage from the knee joint of golden Syrian hamsters 123 days of age or older revealed defects at the tidemark in the tibia. Defects ranged from small separations of the calcified and uncalcified layers along the tidemark to progressively larger defects apparently formed by dissolution. These larger defects appeared as cavities in the noncalcified cartilage, had smooth rather than rough edges, frequently contained coalesced debris, and often resulted in a bulge in the articular surface. Occasionally, these large defects broke through the articular surface. Defects were not observed in tibial cartilage of younger (<90 days old) hamsters or in femoral cartilage from hamsters of any age. Exercise neither protected against nor increased the severity of the defects. Collagen cross-linking by pyridinoline was examined as a function of age and increased from 1,090 to 3,062 micromoles of pyridinoline/mole of hydroxyproline over the period of 1-9 months of age but was not correlated with defect formation. With increasing age, these focal tidemark defects could lead to osteoarthrosis-like cartilage lesions.

Objective: It has been reported that osteoarthritis can occur in hamsters. The present study was undertaken to determine the effects of exercise on the composition of articular cartilage and synovial fluid and on the development of cartilage degeneration in these animals., Methods: Young (2.5-month-old) group-housed hamsters were compared with 5.5-month-old hamsters that had undergone 3 months of daily wheel running exercise (6-12 km/day) or 3 months of sedentary, individually housed living. The condition of the femoral condyles was determined by scanning electron microscopy in 12 exercising hamsters, 12 sedentary hamsters, and 6 of the young controls. The content of proteoglycan, hyaluronic acid, hydroxyproline, and proline in synovial fluid and patellar cartilage was measured., Results: By scanning electron microscopy, the femoral articular cartilage was smooth and undulating in young controls and older exercising hamsters. In contrast, the femoral condyles were fibrillated in all 12 of the sedentary hamsters. There was no difference in the patellar cartilage collagen content between the 3 groups, but proteoglycan content and synthesis were lower in the patellar cartilage of the sedentary group. Synovial fluid volume was also decreased in the sedentary group compared with the young controls or the older exercising hamsters., Conclusion: A sedentary lifestyle in the hamster leads to a lower proteoglycan content in the cartilage and a lower synovial fluid volume. These changes are associated with cartilage fibrillation, pitting, and fissuring. Daily exercise prevents early cartilage degeneration and maintains normal articular cartilage.

Objective: To examine the effect of tenidap on the expression of collagenase- and interleukin 1beta (IL-1beta) genes in experimental canine osteoarthritis (OA)., Methods: The anterior cruciate ligaments of the right stifle joints of experimental dogs were sectioned by a stab wound incision and tissues samples were taken. Four dogs had received no treatment, 4 were treated with oral omeprazole (20 mg/day), and another 4 were treated with tenidap (3 mg/kg/bid) and omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were sacrificed at the end of this period. Tissues from 4 healthy dogs were used as controls. IL-1beta and collagenase-1 gene expression were measured in synovial membrane (synovial lining cells and mononuclear cell infiltrate) and collagenase-1 expression in cartilage using in situ hybridization techniques. Results were calculated as the percentage of cells expressing the gene, and expressed as cell score., Results: The collagenase-1 cell score in the full thickness samples was significantly higher in OA cartilage than in normal cartilage, both in condyles and plateaus (p < 0.0002). Tenidap treated dogs showed a significantly lower cell score in femoral condyles and tibial plateaus in the superficial (p < 0.0002), deep (p < 0.005, p < 0.002, respectively), and full thickness (p < 0.0002) layers compared to OA dogs. No staining for collagenase-1 was observed in normal membrane. In OA synovial membrane, the collagenase-1 cell score was high in both the synovial lining cells and mononuclear cell infiltrate. Tenidap treated dogs showed a significantly lower score compared to OA tissue in both the synovial lining cells (p < 0.03) and the mononuclear cell infiltrate (p < 0.03). The relative decrease in the collagenase-1 cell score in the tenidap treated dogs was more pronounced in the mononuclear cell infiltrate. Staining for IL-1beta was observed in only a few lining cells in normal synovial membrane from unoperated dogs. In OA synovial membrane from untreated dogs, staining for IL-1beta was intense and was found in all dog specimens. The cell score was significantly higher in OA lining cells (p < 0.03) and mononuclear cell infiltrate (p < 0.03) compared to normal. In tenidap treated dogs, the score for IL-1beta was significantly lower than in OA, both in synovial lining cells (p < 0.03) and mononuclear cell infiltrate (p < 0.03)., Conclusion: Tenidap significantly reduced in vivo expression of collagenase-1 and IL-1beta in experimental OA. These data are an extension of our previous study and showed that tenidap exerts its protective effects on OA lesions, likely by reducing the catabolic pathways of the disease.

Objective: To analyze the in vivo compartmental expression of collagenases 1 and 3 (MMP-1 and MMP-13) in the Hartley guinea pig model of spontaneously occurring osteoarthritis (OA) for the purpose of elucidating their roles in the pathogenesis of OA., Methods: Competitive reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry quantification of messenger RNA (mRNA) and protein levels in medial and lateral tibial cartilage obtained from the knee joints of 2-month-old (no OA) and 12-month-old (OA) guinea pigs., Results: The patterns of mRNA expression of collagenases 1 and 3 varied with the age of the animal and the compartment of the knee. We also found focal areas of collagenase 1 and collagenase 3 proteins localized to the extracellular matrix of OA lesion sites, coincident with three-quarter/one-quarter collagen cleavage. Collagenase 3 protein was also abundant throughout the medial tibial cartilage of 2-month-old animals., Conclusion: This represents the first description of bona fide collagenase 1 in a rodent species. Recent evidence, however, based on analysis of mitochondrial DNA homologies, suggests that the guinea pig is not a member of the order Rodentia and may be more closely allied with lagomorphs. This taxonomic controversy leaves open to question the issue of the expression of collagenase 1 in other rodents, such as mice and rats. The presence of active collagenases 1 and 3 at OA lesion sites is consistent with an important role of these enzymes in the cartilage degradation of OA in guinea pigs. The expression of collagenase 3 in medial tibial cartilage from 2-month-old guinea pigs may signify a role of this enzyme in cartilage remodeling with growth and development, or it may represent an early molecular manifestation of OA.

Objective and Design: The limitation of activity and its modification by therapy in an experimental arthritis was studied., Subjects: Female hamsters in groups of six per treatment were used., Treatment: An acute arthritis was induced by intraarticular injection of 0.1 microgram lipopolysaccharide (LPS) in hamsters with free access to running wheels. Tenidap at 100 mg%, and piroxicam and indomethacin at 30 mg% were administered in the hamster's normal diet., Methods: Activity was monitored and analysed by computer. Plasma blood levels of drugs were determined by high pressure liquid chromatographic (HPLC) analysis., Results: Hamsters normally run 10-15 km/day. That distance was reduced to less than 2 km/day after arthritis induction. Speed of movement, essentially the equivalent of walking time, was reduced 40% by the arthritis. However, the time spent in movement (activity time) was more severely affected by arthritic disease. Therapy gave a modest 1.3-fold increase in speed of movement, but a highly significant 2-fold increase in activity time., Conclusions: The effects of arthritis on activity in this animal model suggest that time spent in movement (activity time) should be considered as an outcome measure in clinical studies. These observations may also help explain why the modest disease improvements obtained with cyclooxygenase inhibition are valued. From a patient perspective, a doubling of activity time is a highly significant improvement in quality-of-life.

Objective: To study, in vivo, the therapeutic effectiveness of tenidap, an antirheumatic drug, on the progression of lesions in an experimental osteoarthritis (OA) dog model. The action of tenidap on the activity and expression of metalloproteases in cartilage, as well as on the bioactivity of interleukin-1 (IL-1) in synovial fluid, was determined., Methods: The anterior cruciate ligament of the right stifle joint of 20 mongrel dogs was sectioned through a stab wound. Dogs were divided into 3 groups: group I (n = 7) received no treatment, group II (n = 6) was treated with oral omeprazole (20 mg/day), and group III (n = 7) received oral omeprazole (20 mg/day) and a therapeutic dosage of oral tenidap (3 mg/kg twice daily). Four weeks following surgery, the untreated dogs (group I) were killed, and drug treatments were begun for the other dogs (groups II and III). These dogs received medications for 8 weeks (weeks 4-12) and then were killed. Evaluations were made of the incidence and size of osteophytes as well as of the size and grade of cartilage erosions on both the condyles and plateaus. Histologic examination of the severity of the cartilage lesions and synovial inflammation was also performed. Activity levels of collagenase, stromelysin, and gelatinase as well as collagenase-1, collagenase-3, and stromelysin-1 messenger RNA were determined in the cartilage. The level of IL-1 activity in the synovial fluid was also measured., Results: Among the dogs with OA, lesions were more severe at 12 weeks than at 4 weeks. Group III (tenidap-treated) dogs had a slightly reduced incidence of osteophytes compared with the group II (12-week OA) dogs (71% versus 100%), and the size of the osteophytes was significantly diminished (mean +/- SEM 1.75 +/- 0.69 mm versus 4.38 +/- 0.64 mm). Macroscopically, tenidap decreased the size (condyles 6.00 +/- 2.18 mm2 versus 21.08 +/- 6.70 mm2, plateaus 15.50 +/- 4.77 mm2 versus 35.0 +/- 3.64 mm2) and the grade (condyles 0.57 +/- 0.20 versus 1.17 +/- 0.21, plateaus 1.07 +/- 0.22 versus 2.00 +/- 0.25) of the cartilage lesions compared with the 12-week OA dogs. At the histologic level, the severity of cartilage lesions was also decreased in the tenidap-treated dogs versus the 12-week OA dogs, both on the condyles (3.43 +/- 0.54 versus 5.55 +/- 0.38) and on the plateaus (3.39 +/- 0.35 versus 5.54 +/- 0.60). All 3 OA groups showed a significant and similar level of synovial inflammation. Tenidap markedly decreased collagenase, stromelysin, and gelatinase activity, as well as the level of expression of collagenase-3 in the cartilage. Interestingly, the activity level of IL-1 in synovial fluid was also significantly reduced in the tenidap-treated dogs., Conclusion: Tenidap markedly reduced the severity of OA lesions, indicating the effect of this drug in decreasing the progression of disease. It appears that the drug acts by reducing the activity and/or expression of metalloproteases in cartilage, a process known to play a major role in the pathophysiology of OA lesions. This effect could be mediated by the suppressive effect of tenidap on IL-1 activity.

In vitro pharmacologic measures of drug specificity are well established, i.e. drug interaction with a specific target such as an enzyme, receptor, or ion channel. However, in vitro measures of drug selectivity, defined as effects on secondary targets, are lacking. Two-dimensional gel electrophoresis (2-D gel) was examined as a measure of drug selectivity by comparing the effects of three drugs, tenidap, piroxicam, and dexamethasone, on the synthesis of intracellular proteins in lipopolysaccharide (LPS)-stimulated murine macrophages. A set of 902 35S-methionine-labeled proteins were separated consistently, identified by their coordinates of apparent isoelectric point and molecular weight, and quantified. LPS altered the concentrations of 45 proteins. Tenidap, at 10 microM, affected a total of five proteins (suppressed three; stimulated two), whereas piroxicam, at 10 microM, suppressed two proteins. Dexamethasone at 0.01 microM suppressed eight proteins and stimulated one. Thus, none of the drugs reversed the LPS-induced changes. Two of the eight proteins suppressed by dexamethasone were also suppressed by tenidap and were identified as proIL-1 alpha and proIL-1 beta. Since the subset of affected proteins provided a unique protein "fingerprint" for each drug, the three drugs were mechanistically differentiated by 2-D gel analysis. Compared to LPS (5% affected proteins), all three drugs were selective (< or = 1% affected) with piroxicam > tenidap > dexamethasone. With identification of affected proteins, this technique can provide a useful in vitro assessment of drug selectivity.

Tenidap is a new anti-rheumatic agent which has clinical properties characteristic of a disease modifying drug combined with acute antiinflammatory and analgesic activity. This paper details tenidap's cyclooxygenase (COX) inhibitory activity and the resulting pharmacological properties in experimental animals. Tenidap inhibited calcium ionophore-stimulated prostaglandin D2 synthesis by rat basophilic leukemia cells (COX-1) with an IC50 of 20 nM. In two different in vitro human test systems, tenidap inhibited COX-1 activity more potently than COX-2, although the relative potency ratio (COX-1/COX-2) differed markedly between the two systems. Tenidap inhibited the COX pathway when added to human blood in vitro (IC50, 7.8 mu M) and when administered orally to monkeys, rats and dogs (at 5, 2.5 and 10 mg/kg p.o., respectively) and COX activity measured ex vivo in blood collected 2 to 4 hours post dose. After oral administration to rats, tenidap inhibited carrageenan-induced paw edema with an ED50 of 14 mg/kg and inhibited the glucocorticoid-resistant UV erythema in guinea pigs with an ED50 of 1.4 mg/kg. It retained antiinflammatory activity in adrenalectomized rats indicating that this property is independent of adrenal stimulation. Oral administration of tenidap inhibited the development of adjuvant-induced polyarthritis in the rat and exhibited antinociceptive activity in the murine phenylbenzoquinone and rat acetic acid abdominal constriction tests. These data indicate that tenidap is an effective antiinflammatory and analgesic agent in animal models. These cyclooxygenase-dependent pharmacologic activities do not explain tenidap's disease modifying anti-arthritic properties but add a useful symptom modifying component to its clinical profile.

Objective: To examine the effects of tenidap and diclofenac on osteoarthritic lesions and metalloprotease activity in experimental osteoarthritis (OA)., Methods: The anterior cruciate ligament of the right stifle joint of 25 mongrel dogs was sectioned by a stab wound. Seven dogs received no treatment, 6 were treated with oral omeprazole (20 mg/day), another 6 were treated with diclofenac (0.25 mg/kg/twice daily) plus omeprazole (20 mg/day), and 6 received oral tenidap (3 mg/kg/twice daily) plus omeprazole (20 mg/day). The dogs received medication for 8 weeks; all dogs were killed at the end of this period. Eight normal dogs were used as controls. Lesions were evaluated macroscopically for the incidence and size of osteophytes and the area and grade of cartilage erosions on the condyles and plateaus, along with histologic evaluation of the severity of the cartilage lesions and synovial inflammation. Stromelysin and collagenase activities and the collagenase messenger RNA (mRNA) level were measured in cartilage and synovial membrane., Results: Compared with the untreated or omeprazole-treated OA groups, the dogs treated with tenidap exhibited significant reduction in the incidence (P < or = 0.001) and size (P < or = 0.0001) of osteophytes. Tenidap also significantly decreased the size and grade of cartilage macroscopic lesions, as well as the histologic severity of cartilage lesions on both condyles and plateaus. The histologic severity of synovial inflammatory reaction was also significantly reduced (P < or = 0.003) in the tenidap group. Tenidap markedly decreased stromelysin and collagenase activity in both cartilage (stromelysin P < or = 0.003; collagenase P < or = 0.01) and synovial membrane (stromelysin P < or = 0.003; collagenase P < or = 0.005). Moreover, tenidap also decreased the collagenase mRNA level in cartilage (P < or = 0.005) and synovial membrane (P < or = 0.002). Diclofenac slightly reduced the incidence and size of osteophytes and cartilage lesions, but these changes were not statistically significant. Diclofenac had no effect on the severity of synovial inflammation, metalloprotease activity, or collagenase expression., Conclusion: This study showed that tenidap had a more potent anti-osteoarthritic effect than diclofenac in this model. The effect of the drug in suppressing metalloprotease synthesis, a process known to play a major role in the pathophysiology of osteoarthritic lesions, may explain its mechanism of action.

IL-1 is a potent cytokine that promotes host defense and inflammation. These processes may be modulated by an IL-1 receptor antagonist (IL-1Ra) that binds to and blocks IL-1 receptors. The objective of this study was to define the cellular origin and regulation of IL-1Ra production during bacterial infection. Oral infection of mice with Yersinia enterocolitica resulted in expression of IL-1Ra mRNA and synthesis of IL-1Ra in Peyer's patches (PP), the local site of infection, as well as in noninfected organs such as spleens. By immunostaining, recruited circulating neutrophils were identified to be the primary source of IL-1Ra in tissues. Only approximately 20% of the IL-1Ra-staining cells were accounted for by inflammatory macrophages. Strikingly, neutralization of IL-6 by anti-IL-6 antiserum caused a suppression of both IL-1Ra mRNA in PP and synthesis of IL-1Ra in circulating neutrophils. Confirmatory evidence that IL-6 participates in the generation of IL-1Ra was obtained when rIL-6 induced, and anti-IL-6 antiserum blocked, IL-1Ra expression in cultures of macrophage and polymorphonuclear leukocytes (PMN). These findings suggest that IL-6 induced induction of IL-1Ra may provide a negative feedback loop, facilitating resolution of the inflammatory response locally and presumably at remote sites of infection.

The secretion of IL-1 from murine macrophages in vitro is an inefficient process that is distinct from those of other cytokines such as IL-6. We have therefore studied this process in vivo to see if these differences are maintained. Intraperitoneal injection of LPS in mice induced production and release of IL-6 into the extracellular fluid (peritoneal lavage). Although induction of intracellular IL-1 alpha and IL-1 beta was readily detected, these cytokines were not detected extracellularly. Injection of ATP 2 h after LPS led to the rapid extracellular release of IL-1 beta, IL-1 alpha, lactate dehydrogenase, and beta-N-acetylglucosaminidase. Western blot analysis revealed that a large proportion of the IL-1 beta was released as the 17-kDa form, whereas IL-1 alpha was unprocessed. Adenosine 5'-O-(3-thiotriphosphate) was also effective in causing IL-1 release but not UTP or ADP. This suggests that the ATP-mediated release of IL-1 is a receptor-mediated phenomenon that is associated with cell lysis.

The effect of running activity on normal and inflamed knees was determined by light microscopic (LM) and scanning electron microscopic (SEM) observations on hamster articular cartilage. Animals were split into two groups; one housed in standard cages and one given free access to running wheels. Twenty-one days prior to analysis, half of each group was given an intra-articular injection of lipopolysaccharide (LPS) to cause an inflammation, the other half were uninjected. No remarkable changes were observed by LM in either the control running or nonrunning groups. In contrast, cartilage proteoglycan depletion, and pannus and synovial hyperplasia were equally observed in both groups of LPS-injected animals. SEM observations on the patellae from control animals found them to be free from damage to the articular cartilage. The joints of both the LPS nonrunning and running animals contained synovial hypertrophy with villus projection from the synovial lining. However, only the LPS-injected running hamsters had cartilage fraying over large areas of the articular surface, as well as areas in which the villus projections had been flattened. These results demonstrated that mechanical stress applied to a proteoglycan-depleted cartilage enhances the breakdown of the collagen matrix as judged by fibrillation, and may aggravate the inflammation by crushing the swollen synovial lining where it encroaches on the joint space.

Since 1973, assessment of serum concentrations of C-reactive protein (CRP) has been advocated as a objective measure of disease activity in rheumatoid arthritis (RA). Our review of clinical experience with CRP measurement suggests it has at least two important roles to play in the management of RA. First, persistently elevated CRP levels have prognostic value. In general, such elevated levels are found in those patients who are at greater risk for continuing joint deterioration and therefore may need more aggressive treatment and supportive care. Second, in general, improvement in CRP levels is an objective indication that a drug has produced a beneficial effect and thus may be useful to the physician for monitoring effects of therapy. Since CRP may be elevated in a number of conditions besides RA, a diagnosis of RA must be made before using CRP as a prognostic factor.

Interleukin 1 (IL-1) gene expression was investigated in mice following oral infection with Yersinia enterocolitica 08. In Peyer's patches (PP), the primary site of bacterial invasion, induction of IL-1 alpha mRNA was delayed when compared to IL-1 beta mRNA. As shown by in situ hybridization. IL-1 alpha and IL-1 beta mRNA were found to be expressed within different cell types. These results indicate that expression of the two forms of IL-1 is regulated in a cell-specific manner at the transcriptional level. Moreover, IL-1 (alpha and beta) mRNA was increased in other organs such as spleen and lung. In spleens, IL-1 beta mRNA was found within the red pulp, and IL-1 alpha mRNA was located to the marginal zone confirming that differential expression of IL-1 alpha and IL-1 beta mRNA does not represent a tissue-specific event. However, as revealed by immunohistochemistry and measuring IL-1 activity in tissue homogenates, synthesis of IL-1 proteins was not detectable in spleens, unless mice were challenged with LPS. Because IL-1 synthesis was inducible in spleen cells following actinomycin D treatment, the results indicate that at distant sites of infection IL-1 (alpha and beta) mRNA is expressed but not translated into protein. It is concluded that cell-specific transcription of IL-1 alpha and IL-1 beta as well as dissociation between IL-1 mRNA and protein synthesis are two mechanisms effective in regulating the production of IL-1 during infection.

The reparative responses of articular cartilage after an arthritic insult have not been studied extensively to this day. In the present study, we injected interleukin-1 (IL-1) into knee joints of mice to provoke a mild and transient arthritic insult, and characterized both the catabolic and the subsequent recovery phase. In the catabolic phase, which lasted 2 days after IL-1 injection, proteoglycan (PG) breakdown was profoundly accelerated and PG synthesis was markedly inhibited. Sulfation and polysaccharide synthesis were not affected, yet the number of chondroitin sulfate chains was decreased. The general chondrocyte protein synthesis was not inhibited by IL-1. IL-1 injected every other day for a total of three injections prolonged this catabolic phase and resulted in frank loss of articular cartilage proteoglycans. In the recovery phase, started 3 days after IL-1, PG synthesis was enhanced (1.7 times the normal) and proteoglycans had normal hydrodynamic properties. Remarkably, PG degradation was significantly decreased (approximately 50% of the normal). Zymographic analysis demonstrated enhanced expression of gelatinolytic activities in the extracts of the articular tissues shortly after IL-1 exposure and decreased levels in the recovery phase. We found that the overshoot of PG synthesis and impaired degradation act together to facilitate full cartilage repair 7 days after the last of the three IL-1 injections.

Arthritis refers to a heterogeneous class of diseases characterized by impairment of movement. Yet animal models of arthritis have traditionally been based on the utilization of animals housed without the capability of extended free movement and without adjunctive measurement of mobility. To define the determinants of mobility impairment, we have established a lipopolysaccharide (LPS)-induced arthritis model in the hamster that prominently features monitoring of mobility and compares mobility changes with histological and biochemical changes during arthritis. Intraarticular LPS induces a dose-dependent inhibition of the hamster's mobility as measured by decreased daily distance on a running wheel (normal distance 9 to 12 km/day). At low concentrations of LPS (0.1 and 1 microgram/knee), daily distances returned to normal after 4 and 6 days, respectively. At higher concentrations, the mobility was still markedly suppressed after 6 days, and, at 100 micrograms/knee, irreversible chondrocyte loss was observed on the femoral condylar margins. Further studies were therefore conducted using 1 microgram LPS/knee. Histological and biochemical changes were examined to determine which resolved at the time of restoration of mobility. At the time of restoration of mobility, the synovial capsule was still edematous and heavily infiltrated with leukocytes; proteoglycan loss from the medial femoral condyle was still increasing. Plasma keratan sulfate failed to correlate with either proteoglycan loss or mobility changes. Proteoglycan synthesis, which was maximally suppressed the second day after LPS, was enhanced over controls at the time of restoration of mobility, suggesting the onset of repair. These results suggest a possible association of mobility inhibition with local cytokine synthesis. This model provides an approach to define the causes of mobility impairment.

The mechanism of action for the mild analgesics is controversial. While some have proposed that they inhibit prostaglandin synthesis in the central nervous system to interfere with nociceptive mediators in the brain, others have proposed that they act directly on nociceptive neural pathways to produce analgesia. This class of drugs also possesses antipyretic activity. We examined the antipyretic effect of one such drug, dipyrone, because this might elucidate the mechanism of its analgesic activity. In rats implanted with a femoral vein catheter and a cannula guide tube aimed towards the organum vasculosum laminae terminalis (OVLT) in the brain, an i.v. injection of 2 micrograms/kg interleukin-1 beta (IL-1 beta) produced a fever of 0.38 +/- 0.07 degrees C while an injection of 20 ng prostaglandin E1 (PGE) into the OVLT produced a fever of 1.18 +/- 0.18 degrees C. Dipyrone (25 mg/kg, i.v.) decreased the IL-1 beta fever but had no effect on the PGE fever. After pretreatment with the immunoadjuvant, zymosan, the IL-1 beta fevers were enhanced to equal those induced by PGE. Only 0.1 micrograms/kg, i.v. IL-1 beta raised body temperature by 1.20 +/- 0.10 degrees C. An increased dose of dipyrone (50 mg/kg, i.v.) was required to attenuate this IL-1 beta fever; however, the PGE fever remained unaffected by this treatment with dipyrone. Thus, dipyrone treatment blocks IL-1 beta fever where synthesis of prostaglandin is a crucial step in the febrile process, but it has no effect on PGE fever where synthesis is bypassed. This suggests that dipyrone, probably through its active metabolites, inhibits prostaglandin synthesis to induce antipyresis and, by analogy, analgesia as well.

The destruction of joints caused by rheumatoid arthritis and osteoarthritis is characterized by an imbalance of enzyme catalysed cartilage breakdown and regeneration. A complex cytokine network perpetuates joint conditions by direct regulation of metalloproteases, by indirect recruitment of cells that secrete degradative enzymes, and by inhibition of reparative processes. The destructive action of cytokines such as interleukin-1, interleukin-6 and tumour necrosis factor-alpha can be modulated at multiple points associated either with cytokine production or with cytokine action. Potential agents for cytokine reduction include selective anti-cytokine antibodies, anticytokine receptor antibodies, cytokine receptor antagonist proteins, and soluble and chimeric cytokine receptor molecules. Pharmacologic regulation of IL-1 and TNFalpha remain primary targets for treatment of arthritis, and results of early clinical trials are promising. However, the results of long-term clinical trials will be required to support the value of anti-cytokine therapy in treatment of arthritis.

Many studies in animals have examined biochemical, immune and histological changes during arthritis; however, the study of the effects of arthritis on mobility has been largely neglected. Interleukin-1, administered by the intraarticular route into hamster knee joints, resulted in inhibition of spontaneous wheel running activity; however, the effect was transient, lasting only through the evening following IL-1 administration. A further injection of IL-1 2 days later showed still greater inhibition of running. The effect again did not extend beyond the first evening after injection. IL-1alpha and IL-1beta showed equivalent effects on mobility, and no evidence was seen for cooperative interaction between them. A 50% inhibition of running occurred at a dose of approximately 10 ng/knee of IL-1alpha. The effect appeared not to be systemic since intraperitoneal injection required microgram amounts of IL-1 for an equivalent inhibition. At the time mobility had been restored to normal, histological examination showed the continued presence of inflammatory cells, soft tissue swelling and cartilage proteoglycan loss. These results suggest a lack of correlation between inhibition of mobility and histopathological changes in cartilage and soft tissue.

Mobility is impaired during arthritis. In order to study the causes of the mobility impairment, we have examined hamsters with a LPS arthritis in which running is inhibited over a 4-5 day period. Parameters have been examined to determine which correlate with running impairment. Knee diameter, as well as cell infiltration into the surrounding synovial tissue and the accompanying edema, failed to resolve by the time normal mobility was reestablished. Other factors must be primary determinants of mobility.

One of the early events in murine antigen-induced arthritis is the generation of IL-1 in the inflamed joint. We investigated the role of IL-1 in the acute phase of the arthritic process by selective blockage of IL-1 bioactivity by treatment with neutralizing antibodies. Pretreatment with anti-IL-1 antibodies moderately suppressed joint swelling. The decrease in chondrocyte proteoglycan synthesis seen in the acute phase of arthritis was prevented by treatment with anti-IL-1 antibodies. IL-1 does not appear to be a major contributor to the accelerated breakdown of articular cartilage in this model. The major impact of anti-IL-1 antibodies was the prevention of proteoglycan synthesis inhibition which clearly reduced articular cartilage depletion by maintaining normal proteoglycan synthesis.

In situ production of interleukin 1 alpha (IL-1 alpha) and IL-1 beta was investigated in Peyer's patches (PP) of mice undergoing an acute bacterial infection with Yersinia enterocolitica O8. Synthesis of IL-1 beta, as determined by immunohistochemistry, was found primarily in monocytes migrating into the inflamed PP. In comparison, synthesis of IL-1 alpha was temporarily delayed by at least 24 h and was only found in mature macrophages, which did not produce detectable levels of IL-1 beta. This indicates a transition from IL-1 beta to IL-1 alpha production during maturation of monocytes into inflammatory macrophages, and further emphasizes a dichotomy between IL-1 alpha and IL-1 beta.

We have used neutralizing antibodies raised against murine recombinant interleukin 1 (IL-1) to demonstrate a role for IL-1 in the cartilage destruction and inflammation of antigen induced arthritis. Ex vivo production of IL-1 was demonstrated in tissue cultures of joint cross sections shortly after arthritis induction. Neutralizing antimurine IL-1 antibodies identified the activity to be about 80% IL-1 alpha 24 h after onset of arthritis. In animals receiving a single injection of anti-IL-1 antisera at Day -3, cartilage proteoglycan synthesis suppression during the first 2 days of arthritis was prevented. Normal proteoglycan synthesis was maintained until Day 4 when anti-IL-1 antisera was given at Days -2, 0, and 2 or arthritis. Dose response experiments showed that the reduction in inflammation was insufficient to account for the clearcut reduction in cartilage proteoglycan synthesis inhibition. Our results demonstrate that IL-1 plays a role in cartilage pathology in murine antigen induced arthritis.

Interleukin-6 (IL-6) is considered an important multifunctional cytokine involved in the regulation of a variety of cellular responses, including the induction of acute-phase protein synthesis, lymphocyte activation, and hematopoiesis. In vitro studies have identified many cells that can produce IL-6, but the cellular sources under physiologic conditions have yet to be identified. Using immunoaffinity purified goat anti-murine IL-6, the authors performed immunohistochemical studies to localize cells expressing IL-6 in selected organs of normal and endotoxin challenged NIH-Swiss outbred mice. In the blood, findings were correlated with cell-associated bioactivity using the standard B9 cell proliferation assay. In normal mice, constitutive expression was seen in granulocytes, monocytes and their precursors as well as in bone marrow and splenic stromal macrophages. Hepatic macrophages were negative, as were lymphocytes, megakaryocytes, erythroid precursors, and endothelial cells. In the absence of significant serum levels of IL-6, cell-associated IL-6 bioactivity was detected in circulating polymorphonuclear leukocytes (PMNs), but not lymphocytes. After endotoxin challenge, there was a threefold increase in PMN IL-6 content from 1 to 3 hours followed by almost complete depletion at 6 hours. This correlated well with a threefold increase of IL-6 mRNA in the bone marrow followed by a decrease at 6 hours. This pattern also correlated with serum levels of IL-6, which peaked at 3 hours and dropped significantly by 6 hours. By 24 hours, cell-associated IL-6 showed recovery with no increase in serum levels. In vivo findings showing IL-6 expression in bone marrow macrophages support in vitro studies suggesting a role for IL-6 in hematopoiesis. Furthermore, PMNs as well as macrophages are likely important sources of IL-6 during inflammatory and septic states.

The potential role of interleukin-1 in postoperative adhesion formation was examined. Cecal abrasion gave a consistently higher adhesion score when compared with sham laparotomy, on the basis of adhesion number, density, and vascularity, and so was chosen for use in further studies. The extent of serosal bleeding during cecal abrasion did not affect adhesion scores. Intraperitoneal injection of 10 micrograms murine recombinant interleukin-1 alpha in cecally abraded animals on the day of surgery and on the following 4 days resulted in a significant increase in adhesion scores when compared with those of cecally abraded animals injected with vehicle alone. Adhesions enhanced with murine recombinant interleukin-1 alpha, which were thicker and more vascular, were equivalently enhanced at doses from 10 to 10,000 ng, implying maximal response over that range. Rats not operated on and receiving recombinant interleukin-1 alpha 2 weeks after injury had increased adhesion formation. These results demonstrate that interleukin-1 alpha may be an important short-term mediator of postsurgical adhesion formation.

Tenidap is a new antiarthritic drug of novel chemical structure. This study shows the effects of tenidap on the in vitro synthesis of interleukin 1 (IL-1). IL-1 production by murine peritoneal macrophages was induced either by stimulation with lipopolysaccharide (LPS) or by phagocytosis of zymosan. With either stimulus, tenidap inhibited IL-1 production as measured by a quantitative competitive IL-1 receptor binding assay. Approximately 20 ng/mL of IL-1 was produced by 10(6) macrophages in response to LPS and about half that amount was produced in response to zymosan. Fifty percent inhibition of IL-1 production by tenidap was found at 3 microM for both stimuli. Using goat anti-IL-1 alpha and Western blot analysis, the appearance of intracellular 34 kDa pro-IL-1 alpha was inhibited by tenidap down to 3 microM. Tenidap decreased [35S]Met incorporation into cellular protein at 30 microM but not at 10 or 3 microM, indicating selectivity for IL-1 inhibition relative to total protein synthesis. Because tenidap inhibited IL-1 induction by both zymosan and LPS, it must act subsequently to receptor triggering. As the appearance of IL-1 was inhibited both intracellularly and extracellularly, the primary drug effect cannot be on secretion.

Continuous infusion of murine recombinant interleukin 1 alpha (rIL-1 alpha) produces weight loss, appetite suppression, reduction in horizontal locomotor activity (crossovers) and vertical locomotor activity (rears), and an increase in drinking behavior in the rat. The role of prostaglandins (PG) in the elicitation of these effects was studied. Infusion of rIL-1 alpha produced a transient increase in serum (PGs) which peaked at 24 to 48 h. This increase was completely inhibited by piroxicam. However, inhibition of circulating PG by piroxicam did not block the reductions in appetite, crossover, and rears induced by rIL-1 alpha; it restored normal drinking behavior and only partially restored body weight. Continuous intraperitoneal infusion of PGE2 at 24 micrograms/day exposed the animals to serum levels of PGE2 comparable to those produced by infusion with rIL-1 alpha. Yet, at the point of maximum weight loss induced by rIL-1 alpha (72 h), PGE2 infusion resulted in only a quarter of the weight loss. Compared with rIL-1 alpha, continuously infused PGE2 produced significantly smaller reductions in appetite, crossovers, and rears, and had no effect on drinking behavior. From these observations, we conclude that the rIL-1 alpha-induced increase in drinking behavior was fully dependent on products of the cyclooxygenase pathway, but not necessarily PGE2. However, because of the failure of piroxicam to fully reverse rIL-1 alpha effects on eating, mobility, and weight loss, there must also be a significant PG-independent component to account for the full range of rIL-1 alpha effects.

Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.

Murine interleukin-6 (mIL-6) was expressed in Escherichia coli in the insoluble fraction of cell lysates. Approximately equal amounts of two polypeptide species, reactive with anti-IL-6 antibodies, were produced. The two forms of mIL-6 were isolated and found to have identical N-terminal sequences initiated by Met-Phe-Pro-Thr-Ser-Gln-. Peptide mapping after endoproteinase glu-C digestion led to isolation and characterization of the C-terminal peptides from each of the two forms and allowed the source of the heterogeneity to be identified as a C-terminal addition of three amino acids, Gln-Lys-Leu, to authentic mIL-6. Inspection of the nucleotide sequence of the plasmid containing the mIL-6 gene and expression of the plasmid in other strains suggested that the addition of three amino acids was caused by a readthrough of the termination codon arising from an unexpected suppressor mutation in the original host strain. Although the C-terminus of IL-6 is critical for the activity of this cytokine, the IL-6 variant with extended C-terminus was fully active in two separate bioassays. This suggests that the additional amino acids do not disrupt the structure or function of this important region of the molecule.