The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543.

CP-195543 [(+)-2-(3-benzyl-4-hydroxy-chroman-7-yl)-4-trifluoromethyl-benzoic acid] is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro CP-195543 inhibited [3H]LTB4 binding to high-affinity LTB4 receptors on human neutrophils (HN) and murine spleen membranes with IC50 values of 6.8 nM (Ki = 4.9 nM) and 37.0 nM (Ki = 26.9 nM), respectively. CP-195543 inhibited human and mouse neutrophil chemotaxis mediated by LTB4 with IC50 values of 2.4 nM and 7.5 nM, respectively. Evidence of noncompetitive antagonist effects on the HN high-affinity LTB4 receptor was obtained by Scatchard analysis of [3H]LTB4 binding to and chemotaxis of HN to LTB4. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on HN indicated that CP-195543 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b up-regulation on HN was inhibited competitively by CP-195543 (pA2 = 7.66). In whole blood, CP-195543 also blocked CD11b up-regulation on HN (pA2 = 7.12) and murine neutrophils (pA2 = 7.06) with a similar potency. LTB4-mediated CD11b up-regulation on human monocytes and eosinophils in whole blood were inhibited by CP-195543 with IC50 values of 270 nM and 420 nM, respectively. CP-195543 at 10 microM failed to inhibit HN chemotaxis and CD11b up-regulation mediated through alternative (i.e., complement fragment 5a, interleukin-8, platelet-activating factor) G-protein-coupled chemotactic factor receptors. In vivo, after oral administration, CP-195543 blocked LTB4-mediated neutrophil infiltration in guinea pig and murine skin with ED50 values of 0.1 mg/kg and 2.8 mg/kg p.o., respectively. When administered in osmotic pumps, CP-195543 reduced the clinical symptoms and attendant weight loss in an IL-1-exacerbated murine model of collagen-induced arthritis with half-maximal effects associated with plasma drug levels of 0.4 to 0.5 microg/ml. Collectively these data provide evidence of the in vitro potency and in vivo efficacy of a novel LTB4 antagonist and support its clinical evaluation in a variety of inflammatory diseases in man.

The inhibitory effect of rolipram on TNF-alpha production in mouse blood ex vivo is dependent upon the release of corticosterone and adrenaline.

Intraperitoneal administration of the phosphodiesterase type 4 (PDE4) inhibitor rolipram (1-30 mg/kg) caused a dose-dependent increase in the circulating levels of both corticosterone and adrenaline in male Balb/c mice. These increases were maximal 0.5-1 h after administration of rolipram and had declined to control levels by 4 h. Rolipram (10 mg/kg i.p.) substantially inhibited the production of tumour necrosis factor alpha (TNF-alpha) ex vivo in blood from normal mice but was ineffective in adrenalectomized mice, suggesting that the inhibitory effect of rolipram is dependent on intact adrenal function. The corticosterone antagonist, RU 486, and the beta-adrenoceptor antagonist, propranolol, both partially reversed the inhibitory activity of rolipram while the combination of RU 486 and popranolol abrogated the effect of the rolipram to the same degree as adrenalectomy. These data suggest the release of both corticosterone and adrenaline contribute to the ability of rolipram to inhibit TNF-alpha production in mouse blood ex vivo.

Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin.

The effect of nitric oxide synthase (NOS) inhibitors on plasma extravasation in a rat model of zymosan-induced inflammation has been investigated. Plasma extravasation was determined in response to intradermal test agents over 0 to 45 min or 0 to 4 h by the accumulation of i.v. injected 125I-labeled human serum albumin. Zymosan (1-100 microg/site) produced a dose- and time-dependent plasma extravasation. N(G)-nitro-L-arginine methyl ester (30-300 nmol/site), but not aminoguanidine (AG; 10-300 nmol/site) or L-N6-(1-iminoethyl)lysine (L-NIL; 10-300 nmol/site), significantly (p < 0.01) inhibited zymosan-induced (10 microg/site) plasma extravasation over 0 to 45 min. However, both AG and L-NIL produced significant (p < 0.05) inhibition over 0 to 4 h. The inhibition produced by AG was reversed by i.v. L-arginine or by coinjection of the vasodilator, calcitonin gene-related peptide. Zymosan (10-100 microg/site) induced an increase in dermal blood flow (laser-Doppler flowmetry) and this was inhibited by AG. Neutrophils were depleted selectively with antiserum, but this did not affect plasma extravasation except at the highest dose of zymosan (100 microg/site). Furthermore, zymosan-induced edema was not modified at either time point by pretreatment with the cyclooxygenase inhibitor indomethacin (30 micromol/kg, s.c., -30 min). In conclusion, in this model of dermal inflammation, it is suggested that inducible NOS inhibitors selectively remove an inducible NOS component that, at least in part, acts to increase microvascular blood flow and thus the edema formation observed during 0 to 4 h. There is no evidence of a contributory role for neutrophils or cyclooxygenase products in this model.

The phosphodiesterase type 4 (PDE4) inhibitor CP-80,633 elevates plasma cyclic AMP levels and decreases tumor necrosis factor-alpha (TNFalpha) production in mice: effect of adrenalectomy.

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo.

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.

Regulation of tumour necrosis factor production by adrenal hormones in vivo: insights into the antiinflammatory activity of rolipram.

1. The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNF alpha) was examined in male Balb/c mice. 2. Intraperitoneal injection of 0.3 mg E. coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNF alpha without stimulating TNF alpha production in the peritoneal cavity. Systemic production of TNF alpha in response to LPS was increased in adrenalectomized animals and in normal animals treated with the beta-adrenoceptor antagonist, propranolol. The glucocorticoid antagonist, RU 486, did not modify systemic TNF alpha production. These results indicate that systemic TNF alpha production is regulated by adrenaline but not by corticosterone. 3. When mice were primed with thioglycollate, TNF alpha was produced in the peritoneal cavity in response to low dose LPS (1 micrograms). The levels of TNF alpha in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNF alpha in the peritoneal cavity is not regulated by adrenaline or corticosterone. 4. The phosphodiesterase type IV (PDE-IV) inhibitor, rolipram, inhibited both the systemic production of TNF alpha in response to high dose endotoxin (ED50 = 1.3 mg kg-1) and the local production of TNF alpha in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg-1). In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNF alpha (ED50 = 3.3 mg kg-1) while the ability of rolipram to inhibit the local production of TNF alpha in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg-1). The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNF alpha production while propranolol was without effect. 5. Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo. 6. In summary, these data indicate that systemic production of TNF alpha in response to high dose endotoxin is controlled differently from the local production of TNF alpha in response to low dose endotoxin. The systemic production of TNF alpha is regulated by catecholamines, but not by corticosterone, while the local production of TNF alpha in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone. 7. These data also show that the ability of rolipram to inhibit the local production of TNF alpha is dependent on the release of corticosterone from the adrenal glands.

Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis.

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.

Resident joint tissues, rather than infiltrating neutrophils and monocytes, are the predominant sources of TNF-alpha in zymosan-induced arthritis.

Intraarticular injection of zymosan (1 mg) into the knee joints of male Wistar rats led to infiltration of neutrophils and monocytes, joint swelling and elevation of tumour necrosis factor (TNF-alpha) in joint lavage fluids. Neutrophil infiltration was maximal at 5 h after induction of arthritis but had declined by 24 h post injection. Monocyte infiltration was maximal around the same time as that of the neutrophil infiltration but was sustained through 24 h. Joint swelling was apparent at 1 h but was not maximal until 6 h after induction of zymosan-induced arthritis. The maximum levels of TNF-alpha (150-200 pg of mouse equivalents per joint) in joint fluid preceded the infiltration of neutrophils and monocytes and was maximal at 1-2 h after injection of zymosan but had declined to below 100 pg per joint at 5 h (the time of maximal leukocyte infiltration) and was below the lower limit of detection at 24 h after induction of arthritis. Depletion of neutrophils and monocytes with a rabbit anti-rat leukocyte antibody reduced leukocyte infiltration and the later phase of joint swelling but did not reduce the levels of TNF-alpha in joint fluid. These data indicate that in zymosan-induced arthritis TNF-alpha is produced from the resident joint tissues such as the synovial lining cells rather than infiltrating neutrophils or monocytes.

The in vitro and in vivo pharmacologic activity of the potent and selective leukotriene B4 receptor antagonist CP-105696.

CP-105696, (+)-1-(3S,4R)-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl] cyclopentane carboxylic acid, is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro, CP-105696 inhibited [3H]LTB4 (0.3 nM) binding to high-affinity LTB4 receptors on human neutrophils with an IC50 value of 8.42 +/- 0.26 nM. Scatchard analyses of [3H]LTB4 binding to these high-affinity receptors indicated that CP-105696 acted as a noncompetitive antagonist. CP-105696 inhibited human neutrophil chemotaxis mediated by LTB4 (5 nM) in a noncompetitive manner with an IC50 value of 5.0 +/- 2.0 nM. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on neutrophils indicated that CP-105696 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b upregulation on human neutrophils was competitively inhibited by CP-105696 (pA2 = 8.03 +/- 0.19). CP-105696 at 10 microM did not inhibit either human neutrophil chemotaxis or CD11b upregulation mediated through alternate (i.e., C5a, IL-8, PAF) G-protein coupled chemotactic factor receptors. In isolated human monocytes, LTB4 (5 nM)-mediated Ca++ mobilization was inhibited by CP-105696 with an IC50 value of 940 +/- 70 nM. In vivo, after oral administration, CP-105696 blocked neutrophil and eosinophil infiltration in cavine dermis mediated by either LTB4 or arachidonic acid with ED50 values of 0.3 +/- 0.1 mg/kg. 12(R)-Hydroxyeicosatetraenoic acid-mediated neutrophil infiltration was blocked by 76.4 +/- 14.8% at 3 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukotriene B4 plays a critical role in the progression of collagen-induced arthritis.

Leukotriene B4 (LTB4) is a product of the 5-lipoxygenase pathway of arachidonic acid metabolism. LTB4 is a potent chemotactic factor for neutrophils and has been postulated to play an important role in a variety of pathological conditions including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. The role of LTB4 in such diseases has not yet been defined but in this paper we provide direct evidence that LTB4 plays a critical role in a murine model of RA. CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)- 4-hydroxychroman-7-yl]cyclopentane carboxylic acid, is an LTB4 receptor antagonist that inhibits LTB4 binding to human neutrophil membranes with an IC50 of 3.7 nM and inhibits LTB4-induced chemotaxis of these cells with an IC50 of 5.2 nM. CP-105,696 inhibits LTB4-induced neutrophil influx in mouse skin when administered orally with an ED50 of 4.2 mg/kg. CP-105,696 had a dramatic effect on both the clinical symptoms and histological changes of murine collagen-induced arthritis when administered at doses of 0.3-10 mg/kg. Inhibition was not associated with suppression of the humoral immune response to collagen and was equally effective if drug treatment was commenced just prior to the onset of arthritis or throughout the experiment. These results suggest that LTB4 receptor antagonists may be effective therapeutic agents for the treatment of RA.

Effect of in vivo desensitization to leukotriene B4 on eosinophil infiltration in response to C5a in guinea-pig skin.

1. The effect of in vivo desensitization to leukotriene B4 (LTB4) on eosinophil infiltration in response to recombinant C5a was examined in guinea-pig skin. 2. LTB4 (10-300 ng) and C5a (1-10 micrograms) caused a dose-dependent increase in the levels of eosinophil peroxidase activity (a measure of eosinophil infiltration) 4 h after injection into guinea-pig skin. Leukotriene B4 and C5a were approximately equipotent on a molar basis. Platelet activating factor (0.01-10 micrograms) also caused eosinophil accumulation but was much less active than LTB4 or C5a. 3. 20-Hydroxy-LTB4 caused a dose-dependent desensitization of eosinophil responses to LTB4 (ED50 = 1.6 micrograms kg-1, s.c.) and partially reduced responses to C5a. At a dose of 20-hydroxy-LTB4 (10 micrograms) which inhibited responses to LTB4 completely, responses to C5a were reduced by 56.5 +/- 1.8% (n = 5). The structurally related metabolite of 20-hydroxy-LTB4, 20-carboxy-LTB4, which does not cause desensitization to the effects of LTB4, did not inhibit eosinophil infiltration in response to C5a. 4. The LTB4 receptor antagonist, SC-41,930 (10 mg kg-1, p.o.), also inhibited eosinophil accumulation in response to C5a by 63.0 +/- 3.9% (n = 5) at a dose which inhibited responses to LTB4 by 86.5 +/- 1.9% (n = 5). 5. These data indicate that eosinophil infiltration in response to C5a may, in part, be mediated by the generation of secondary chemotactic factors such as LTB4.

Cyclooxygenase inhibitors enhance tumour necrosis factor production and mortality in murine endotoxic shock.

Intraperitoneal administration of E. coli lipopolysaccharide (5-15 mg/kg) produced a dose-related increase in mortality which was maximal 72 h after induction of shock. Using a suboptimal dose of LPS (10 mg/kg i.p.), pretreatment with indomethacin (0.1-10 mg/kg p.o) or ibuprofen (1-100 mg/kg p.o) 30 min prior to induction of shock led to a significant enhancement of mortality. This enhancement was associated with a 2-3 fold increase in the peak circulating levels of tumour necrosis factor (TNF-alpha) in the serum of animals treated with indomethacin or ibuprofen compared to vehicle-treated control animals. These results indicate that TNF-alpha production is modulated by endogenous prostaglandins in vivo and that enhanced production of TNF-alpha by cyclooxygenase inhibitors may lead to exacerbation of some inflammatory processes.

Specific inhibition of leukotriene B4 (LTB4)-induced neutrophil emigration by 20-hydroxy LTB4: implications for the regulation of inflammatory responses.

1. The interaction between leukotriene B4 (LTB4) and its metabolite, 20-hydroxy LTB4 in the control of neutrophil emigration was examined in guinea-pig skin. 2. Leukotriene B4 (10-300 ng) elicited a dose-dependent increase in neutrophil infiltration (as measured by myeloperoxidase activity) 4 h after injection into guinea-pig skin. In contrast, 20-hydroxy LTB4 (30-1000 ng) displayed only weak inflammatory activity in this assay. 3. Although 20-hydroxy LTB4 had low agonist activity, this metabolite caused a potent dose-dependent inhibition of responses to LTB4 (100 ng), when administered systemically (ED50 = 1.3 micrograms kg-1, s.c.) without significantly affecting neutrophil infiltration in response to C5a (2 micrograms). Systemic administration of 20-carboxy LTB4 (10 micrograms) did not affect neutrophil accumulation in response to LTB4 or C5a. In addition, neither 15(S)-hydroxy 5(S)-HPETE(10 micrograms) nor lipoxin A4 (10 micrograms) inhibited responses to LTB4. 4. Addition of 20-hydroxy LTB4 (10(-11)-10(-8) M) to human blood prior to isolation of the neutrophils led to concentration-dependent decrease in the number of LTB4 receptors and decreased chemotactic responsiveness to LTB4 without affecting responses to C5a. Incubation of blood with 20-carboxy LTB4 (10(-8) M) did not reduce LTB4 receptor number of chemotactic responsiveness to LTB4. 5. These data indicate that although 20-hydroxy LTB4 is a weak agonist at LTB4 receptors, it can desensitize neutrophils to the effects of LTB4 via down-regulation of the high affinity receptor and thus provides evidence for a mechanism whereby inflammatory responses may be regulated.

Co-operation between interleukin-1 and the fibrinolytic system in the degradation of collagen by articular chondrocytes.

1. The interaction between interleukin 1 (IL-1) and the fibrinolytic system in the control of collagen degradation by rabbit chondrocytes has been investigated in a tissue-culture system where cells are grown on a 14C-labelled collagen matrix. 2. Culture of rabbit chondrocytes in the presence of human recombinant IL-1 beta at a concentration of 57pM for 48 h led to the presence of procollagenase but not active collagenase in the medium. The latent collagenase could be activated by incubation with an organomercurial, aminophenylmercuric acetate (APMA). 3. Addition of IL-1 beta to chondrocytes grown on a 14C-labelled collagen matrix did not increase the degradation of the matrix compared to control over a 48 h period. However, in the presence of plasmin (200 micrograms ml-1) or plasminogen (100 micrograms ml-1), IL-1 beta (57 pM) caused almost complete degradation of the collagen matrix. Plasmin or plasminogen alone caused only slight degradation of the collagen matrix. 4. Tissue inhibitor of metalloproteinases (TIMP) or the selective metalloproteinase inhibitor, SC44463, inhibited the degradation induced by IL-1 beta and plasminogen in a concentration-related manner and at concentrations that were correlated with inhibition of collagenase. 5. When concentrations of IL-1 beta which caused only minimal degradation of the matrix in the presence of plasminogen were combined with fibrin (1 microgram ml-1), there was almost total degradation of the matrix by 48 h. 6. These results indicate there is a synergistic interaction between IL-1 and the fibrinolytic system in the degradation of collagen by rabbit chondrocytes in culture.

Metalloproteinases and cartilage proteoglycan depletion in chronic arthritis. Comparison of antigen-induced and polycation-induced arthritis.

Chronic monarticular arthritis can be induced in ovalbumin-sensitized rabbits by intraarticular injection of ovalbumin (antigen-induced arthritis) or in naive rabbits by injecting hyaluronic acid mixed with the polycation poly-D-lysine (polycation-induced arthritis). Both models show some points of similarity, including joint swelling, the presence of inflammatory leukocytes and the inflammatory mediator prostaglandin E2, and the kinetics of cartilage proteoglycan loss. However, the assessment of the capacity of synovial lining and articular cartilage to synthesize and secrete neutral metalloproteinases reveals a difference between these models. We found that articular cartilage from the inflamed joints of rabbits with antigen-induced arthritis did not synthesize neutral metalloproteinases, although the synovial lining did. In contrast, both the synovial lining and the articular cartilage from the inflamed joints of rabbits with polycation-induced arthritis synthesized neutral metalloproteinases. These findings suggest that in inflammatory synovitis, different mechanisms can operate to produce damage to the matrix of articular cartilage.

Effect of indomethacin on swelling, lymphocyte influx, and cartilage proteoglycan depletion in experimental arthritis.

The effects of indomethacin on antigen induced arthritis in rabbits have been investigated. Arthritis was induced in the knee joints of sensitised rabbits by intra-articular injection of antigen. Swelling of the joints was measured for 14 days after antigen challenge, and groups of animals were killed on days 1, 7, or 14 for collection of synovial fluids and tissues. Indomethacin (1 mg/kg, three times daily) reduced joint swelling and the prostaglandin E2 concentrations in synovial fluid. In addition, indomethacin increased the loss of proteoglycan from articular cartilage and the numbers of lymphocytes in the inflamed synovial lining. These findings suggest that the symptomatic benefits of indomethacin and related drugs in inflammatory arthritis may be achieved at the expense of significant adverse effects on joint tissues.

Cartilage proteoglycan depletion in acute and chronic antigen-induced arthritis.

We examined the kinetics of proteoglycan (PG) depletion in rabbits with antigen-induced arthritis. There was a rapid loss of PG from arthritic cartilage, reaching 35-40% at day 7. Thereafter, the rate of PG depletion declined, and by day 42, the maximum loss was 55-60%. The initial loss of PG was accompanied by the appearance of large amounts of sulfated glycosaminoglycans (GAGs) in the joint fluid (measured as total sulfated GAGs by dye binding and as keratan sulfate by radioimmunoassay). However, by day 14, the levels of sulfated GAGs in arthritic joint fluid declined to control levels, even though the cartilage demonstrated a sustained depletion of PG. The cartilage PG degradation observed in antigen-induced arthritis could also be produced in normal animals by a single intraarticular injection of recombinant interleukin-1. The acute loss of cartilage PG occurred independently of neutrophil accumulation, both in the case of antigen-induced arthritis and after injection of interleukin-1.