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This study compared the ability of rabbit medial collateral ligament, posterior cruciate ligament, and anterior cruciate ligament tissue to synthesize nitric oxide, and determined its effects on matrix synthesis, an important component of ligament repair. It is not known whether ligament cells can produce nitric oxide and, if so, whether it influences healing of ligament injuries. The anterior cruciate and posterior cruciate ligament tissue produced large amounts of nitric oxide in response to the inflammatory cytokine interleukin-1. Medial collateral ligament, in contrast, produced only modest amounts of nitric oxide. Furthermore, anterior cruciate ligament and, to some degree, posterior cruciate ligament synthesized nitric oxide spontaneously in culture, whereas medial collateral ligament never did so. When nitric oxide was supplied to these tissues, it strongly inhibited collagen synthesis by the two cruciate ligaments, but had little effect on collagen synthesis by the medial collateral ligament. Endogenously synthesized nitric oxide was also able to inhibit collagen synthesis as well as proteoglycan synthesis by the two cruciate ligaments, but had little effect on matrix synthesis by the medial collateral ligament. We propose a novel hypothesis, based on nitric oxide production and matrix synthesis, that may help explain why the two cruciate ligaments have such limited healing capacity compared with the medial collateral ligament.

Objective: Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1beta (IL-1beta). If NO production is blocked, much of the IL-1beta inhibition of proteoglycan synthesis is prevented. We tested the hypothesis that this inhibitory effect of NO on proteoglycan synthesis is secondary to changes in chondrocyte transforming growth factor beta (TGFbeta)., Methods: Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFbeta bioactivity in chondrocyte- and cartilage-conditioned medium (CM) was measured with the mink lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35S-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns., Results: IL-1beta increased active TGFbeta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFbeta were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFbeta without affecting the early TGFbeta activation. IL-1beta stimulated a NO-independent, transient increase in TGFbeta3 at 24 hours; however, TGFbeta1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1beta increased CM concentrations of TGFbeta1 from 24-72 hours of culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGFbeta1. Anti-TGFbeta1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1beta + L-NMA, confirming that NO inhibition of TGFbeta1 in IL-1beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFbeta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillamide. Similar results were seen with cartilage slices in organ culture. The autocrine increase in CM TGFbeta1 levels following prior exposure to TGFbeta1 was also blocked by NO., Conclusion: NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFbeta1 by chondrocytes exposed to IL-1beta. It prevents autocrine-stimulated increases in TGFbeta1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.

Slices of lapine meniscus produced large amounts of nitric oxide after stimulation with interleukin-1, tumor necrosis factor alpha, or a mixture of lapine synovial cytokines known as chondrocyte-activating factors. Monolayer cultures of meniscal cells produced from the proteolysis of meniscal tissue contained a mixed population of chondrocytic and fibroblastic cells. These cultures also produced large amounts of nitric oxide in response to cytokines. Monolayer cultures of meniscal cells produced by the explant method, in contrast, were uniformly fibroblastic and did not produce nitric oxide in response to cytokines. We conclude that menisci contain two populations of cells, one fibroblastic and the other chondrocytic. The chondrocytic cells are responsible for generating most of the nitric oxide in response to cytokines. Endogenously generated nitric oxide suppressed the synthesis of collagen and proteoglycan by menisci but protected proteoglycan from the catabolic effects of interleukin-1. The inhibitory effect of nitric oxide on collagen synthesis occurred without greatly altering the abundance of mRNAs encoding the various collagen alpha chains. During further investigation, arginine was unexpectedly found to stimulate the synthesis of collagen and, to a lesser degree, of noncollagenous proteins but not of proteoglycans. Fragments of meniscus, but not meniscal cells in monolayer culture, increased their production of matrix metalloproteinases, lactate, and, especially, prostaglandin E2 in response to interleukin-1. Inhibition of nitric oxide production with NG-monomethyl-L-arginine enhanced production of matrix metalloproteinases but had little effect on the synthesis of lactate or prostaglandin E2.

The addition of human recombinant interleukin-1beta (IL-1beta) to cultures of lapine articular chondrocytes provoked the synthesis of large amounts of NO and reduced the production of type-II collagen. NG-Monomethyl-l-arginine (L-NMA), an inhibitor of NO synthase, strongly suppressed the production of NO and partially relieved the inhibition of collagen synthesis in response to IL-1beta. The NO donor S-nitrosoacetylpenicillamine (SNAP), on the other hand, inhibited collagen production. IL-1 lowered the abundance of Col2A1 mRNA in an NO-independent manner. Collectively, these data indicate that IL-1 suppresses collagen synthesis at two levels: a pretranslational level which is NO-independent, and a translational or post-translational level which is NO-mediated. These effects are presumably specific as L-NMA and SNAP had no effect on total protein synthesis or on the distribution of newly synthesized proteins between the cellular and extracellular compartments. Prolyl hydroxylase is an important enzyme in the post-translational processing of collagen, and its regulation and cofactor requirements suggest possible sensitivity to NO. Extracts of cells treated with IL-1 or SNAP had lower prolyl hydroxylase activity, and L-NMA was partially able to reverse the effects of IL-1. These data suggest that prolyl hydroxylase might indeed be a target for NO. Because underhydroxylated collagen monomers fail to anneal into stable triple helices, they are degraded intracellularly. Inhibition of prolyl hydroxylase by NO might thus account for the suppressive effect of this radical on collagen synthesis.

Study Design: Normal and herniated human intervertebral disc specimens were cultured to study the effects of interleukin-1 beta on the production of nitric oxide, interleukin-6, prostaglandin E2, and matrix metalloproteinases. The effects of endogenously produced nitric oxide on the synthesis of other mediators also were studied., Objectives: To test the hypothesis that the cells of the intervertebral disc are metabolically active and are capable of responding to biochemical stimuli such as interleukin-1 beta in a manner that could engender degenerative changes. As part of this study, the authors also investigated some of the possible autocrine regulatory mechanisms that may operate during the biochemical responses of disc cells., Summary of Background Data: The authors previously showed, for the first time, that herniated cervical and lumbar disc specimens spontaneously produce increased amounts of nitric oxide, interleukin-6, prostaglandin E2, and certain matrix metalloproteinases. These results suggest that these biochemical agents are in some manner involved with degenerative processes in the intervertebral disc. This novel hypothesis merits further evaluation; the current communication reports the results of experiments designed to do so., Methods: Fourteen normal, nondegenerated discs (control group) were obtained from seven patients undergoing anterior spinal surgery for trauma or lumbar scoliosis. Thirty-six herniated discs (18 lumbar and 18 cervical) were obtained from 30 patients undergoing surgery for persistent radiculopathy. The specimens were placed into tissue culture and incubated for 72 hours in the presence or absence of interleukin-1 beta and NG-monomethyl-L-arginine, and inhibitor of nitric oxide synthases, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 were performed., Results: Normal, control disc specimens significantly increased their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Herniated lumbar and cervical discs, which were spontaneously releasing increased levels of these biochemical agents, further increased their production of nitric oxide, interleukin-6, and prostaglandin E2 in response to interleukin-1 beta. Blocking the biosynthesis of nitric oxide in interleukin-1 beta-stimulated disc cells provoked a large increase in the production of interleukin-6., Conclusions: Cells of the intervertebral discs are biologically responsive and increase their production of matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 when stimulated by interleukin-1 beta. The effect is more dramatic in normal, nondegenerated discs where spontaneous synthesis of these mediators is low. Nevertheless, cells of the herniated degenerated discs where spontaneous production was high were still capable of further increasing their synthesis of several of these biochemical agents in response to interleukin-1 beta. Endogenously produced nitric oxide appears to have a strong inhibitory effect on the production of interleukin-6, which suggests that autocrine mechanisms play an important role in the regulation of disc cell metabolism.

Study Design: Herniated lumbar disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced., Objectives: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in intervertebral disc degeneration and in the pathophysiology of radiculopathy., Summary of Background Data: Low back pain with or without radiculopathy is a significant clinical problem, but the etiology of low back pain and the exact pathophysiology of radiculopathy remain elusive. The biochemical events that occur with intervertebral disc degeneration and, in particular, the role of biochemical mediators of inflammation and tissue degradation have received sparse attention in the literature. There is some preliminary evidence that inflammatory mediators may have an important role in the pathophysiology of radiculopathy., Methods: Eighteen herniated lumbar discs were obtained from 15 patients undergoing disc surgery. The specimens were cultured and incubated for 72 hours, and the media were collected subsequently for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, eight lumbar disc specimens were obtained from four patients undergoing anterior surgery for scoliosis and traumatic burst fractures, and similar biochemical analyses were performed., Results: The culture media from the herniated lumbar discs showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated discs compared with the control discs. Interleukin 1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of either the herniated or control discs., Conclusions: Herniated lumbar discs were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These products may be involved intimately in the biochemistry of disc degeneration and the pathophysiology of radiculopathy. Their exact roles certainly need further investigation, but their mere presence implicates biochemical processes in intervertebral disc degeneration.

Objective: To determine whether rabbit synovial fibroblasts can synthesize nitric oxide (NO) and, if so, how production is regulated by cytokines., Methods: Primary cultures of synovial fibroblasts (type B synoviocytes) were established from synovia excised from the knee joints of New Zealand white rabbits. Synthesis of NO was measured as nitrite accumulation in conditioned media in the presence or absence of various cytokines and other activators., Results: Resting cultures of synoviocytes normally produced little or no NO. However, production of this free radical was induced by interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha) or the phagocytosis of latex beads; in some cultures, the synthesis of NO occurred spontaneously. In each case, NO synthesis began approximately 9 h after the addition of cytokines, suggesting the involvement of an inducible form of NO synthase. Antagonists of this phenomenon included interferon gamma (IFN-gamma), which weakly inhibited NO production, and transforming growth factor beta (TGF-beta), a very strong inhibitor. Platelet derived growth factor (PDGF) inhibited NO synthesis by cells stimulated with IL-1, but not by cells stimulated with TNF-alpha. Synovial autocrine factors (CAF) modestly induced NO synthesis, but inhibited synthesis by IL-1; TGF-beta was identified as an inhibitory component of CAF. Phorbol myristate acetate (PMA) had only a small inductive effect, and inhibited induction by IL-1. However, the protein kinase inhibitor staurosporin was a strong inducer. Modulators of cyclic nucleotides, in contrast, had relatively modest effects on NO synthesis. Inhibition of NO biosynthesis by NG-monomethyl-L-arginine (NMA) had no effect upon the increase in the production of prostaglandin E2 (PGE2), matrix metalloproteinases (MMP) or lactate by synoviocytes responding to IL-1. The rabbit synoviocyte cell line, HIG-82, did not synthesize detectable NO under any of the culture conditions tested., Conclusion: Synoviocytes are a potential source of NO in arthritic joints.

Addition of human, recombinant interleukin-1 beta (hrIL-1 beta) to cultures of lapine articular chondrocytes provoked a delayed increase in the production of both nitric oxide (NO) and lactate. These two phenomena followed a similar time course and shared a parallel dose-response sensitivity to hrIL-1 beta. A causal relationship is suggested by the ability of N-monomethyl-L-arginine (NMA), an inhibitor of NO synthase, to blunt the glycolytic response to hrIL-1 beta. Furthermore, addition of S-nitroso-N-acetylpenicillamine (SNAP), which spontaneously generates NO in culture, increased lactate production to the same degree as IL-1. However, 8-Br-cGMP and isobutylmethylxanthine (IBMX) had no effect either in the presence or absence of IL-1. Even under standard, aerobic, cell culture conditions, chondrocytes consumed little oxygen, either in the presence or absence of IL-1 or NMA. Furthermore, cyanide at concentrations up to 100 microM had no effect upon NO synthesis or lactate production. Thus, the increases in glycolysis under study were not secondary to reduced mitochondrial activity. Although cells treated with IL-1 had increased rates of glycolysis, their concentrations of ATP fell below those of untreated chondrocytes in a time-dependent, but NMA-independent, manner. Transforming growth factor-beta (TGF-beta) and synovial cytokines (CAF) also increased lactate production. However, TGF-beta failed to induce NO, and its effect on glycolysis was independent of NMA. Furthermore, cells treated with TGF-beta were not depleted in ATP. These data are consistent with hypotheses that rates of proteoglycan synthesis are, in part, regulated by the intracellular concentration of ATP or by changes in pericellular pH. These two possibilities are not mutually exclusive.

Although IL-1 is an important modulator of chondrocyte metabolism, the postreceptor events triggered by IL-1 remain obscure. The present study shows that IL-1 induces the biosynthesis of nitric oxide (.N = O) by articular chondrocytes. Synthesis of .N = O is also induced by LPS. Other inflammatory mediators such as IFN-gamma, fibroblast growth factor, and TNF-alpha fail to provoke the production of .N = O, but they increase the potency of IL-1. A combination of IL-1, LPS, and TNF-alpha was shown to induce maximal production of 355 +/- 51 nmol/10(6) cells/72 h of nitrite (NO2-), which was measured as a stable end-product of .N = O generation. The biosynthesis of .N = O requires an induction period of approximately 6 h and continues for at least 72 h. Inhibition of .N = O production with the competitive inhibitor NG-monomethyl-L-arginine (NMA) leads to a suppression of gelatinase and PGE2 synthesis by chondrocytes activated with IL-1 alone. In contrast, NMA enhances the synthesis of both gelatinase and PGE2 after activation with a combination of IL-1, LPS, and TNF-alpha. An increase of PGE2 synthesis from 42.0 +/- 21.0 to 174.0 +/- 33.5 ng/10(6) cells/72 h resulted from the addition of NMA when these stimulatory agents were combined. Exposure of IL-1 and fibroblast growth factor-stimulated chondrocytes to authentic, exogenous .N = O led to an increase of PGE2 synthesis from 5.6 +/- 1.7 of untreated cells to 15.8 +/- 6.8 ng/10(6) of .N = O treated cells within the 1st h. This was followed by a suppression of PGE2 synthesis within the next 2 h.