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Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

STUDY DESIGN We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. OBJECTIVE To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. SUMMARY OF BACKGROUND DATA Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. METHODS Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. RESULTS Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% +/- 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 +/- 507.1 microm, velocity: 0.09 +/- 0.01 microm/min); giant cells (area: 12678 +/- 1637.0 microm, velocity: 0.08 +/- 0.01 microm/min) which grew rapidly without cell division; polygonal cells (area: 3053 +/- 751.2 microm, 0.14 +/- 0.01 microm/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 +/- 235.6 microm, 0.19 +/- 0.01 microm/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. CONCLUSION These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

STUDY DESIGN Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1 beta or TNF-alpha, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. OBJECTIVE To determine the role of signaling through p38 MAPK in nucleus pulposus cell's response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. SUMMARY OF BACKGROUND DATA Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1 beta and tumor necrosis factor-alpha are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. MATERIALS AND METHODS Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-alpha, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. RESULTS Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-alpha induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-alpha. CONCLUSION Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

STUDY DESIGN Cells from degenerated intervertebral discs were transduced with an adenoviral vector delivering cDNA of the catabolic inhibitor, TIMP-1, and alterations in the measured proteoglycan were assessed. OBJECTIVES To assess the potential of TIMP-1 to favorably modify the proteoglycan content of degenerated intervertebral disc cells. SUMMARY OF BACKGROUND DATA Gene therapy with anabolic factors has resulted in increased proteoglycan synthesis in intervertebral disc cells. Biochemical analysis of degenerated discs has revealed elevated levels of the catabolic enzymes, matrix metalloproteinase, suggesting an intimate role of these factors in the degenerative process. The use of TIMP-1, an endogenous inhibitor of matrix metalloproteinase, via gene therapy may provide an additional method to alter the degenerative processes occurring in the intervertebral disc. MATERIALS AND METHODS Degenerated intervertebral disc were isolated from eight patients undergoing elective surgical procedures. Cells were cultured in monolayer and transduced with different concentrations of either an adenoviral-tissue inhibitor of metalloproteinase-1 (Ad-TIMP-1) or adenoviral-bone morphogenic protein-2 (Ad-BMP-2) construct. Cells were cultured in a three-dimensional pellet and proteoglycan synthesis was assessed via 35S-sulfur incorporation. RESULTS Gene delivery of TIMP-1 and BMP-2 increased measured proteoglycan synthesis at each concentration assessed. IVD cells treated with Ad-TIMP-1 demonstrated an optimal response at a multiplicity of infection (MOI) of 100. Cells treated with Ad-BMP-2 demonstrated a progressive increase in proteoglycan synthesis with increasing viral concentrations. CONCLUSIONS Successful delivery of the anticatabolic gene, TIMP-1, results in increased measured proteoglycan in cultured degenerated disc cells. This finding supports catabolic inhibition as a promising avenue of research for the treatment of degenerative disc disease via gene therapy.

Insulin-like growth factor (IGF-1) is critical for normal development and maintenance of cartilage, however arthritic cartilage responds poorly to IGF-1; part of this insensitivity is mediated by nitric oxide (NO). These studies test if cGMP is responsible for NO dependent insensitivity to IGF-1 in chondrocytes in situ in organ culture and in monolayer culture. Lapine cartilage and chondrocytes in monolayer culture and cartilage from osteoarthritic human knees were used. Tissues were exposed to NO from iNOS induced by IL-1, and proteoglycan synthesis in response to IGF-1 was evaluated in the presence and absence of cGMP dependent protein kinase (PKG) inhibitors. PKG activators inhibited IGF-1 responses in cartilage but not chondrocytes in monolayer. IL-1 stimulated cGMP synthesis in both monolayer and organ cultures. However, PKG inhibitors in cartilage slices but not in monolayer cultures restored response to IGF-1. PKG activity was detected in both fresh and monolayer chondrocytes, confirming this part of the cGMP signal cascade is intact in both of the preparations evaluated. Arthritic cartilage response to IGF-1 was restored by both N(G)-monomethyl-L-arginine inhibition of NO synthesis and PKG inhibitors. The data suggests that cGMP mediated effects are critical to NO actions on chondrocytes in situ in the cartilage matrix and supports a role for cGMP in the pathophysiologic effects of NO in osteoarthritis.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso- N-acetylpenicillamine (SNAP) or ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of 35SO4. IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N G-monomethyl-l-arginine(l-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

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.

Einleitung: Interleukin-1 ist ein wichtiger Faktor in der Pathophysiologie der Osteoporose und moglicherweise der periartikularen Knochenresorption bei rheumatoider Arthritis. Die Nutzung von therapeutischen Proteinen, wie Zytokinen und Wachstumsfaktoren als neue therapeutische Modalitat beinhaltet ein groses medikamentoses Potential. Die konventionelle Applikation rekombinanter Proteine ist jedoch fur eine klinischen Anwendung aufgrund kurzer Halbwertzeiten ungeeignet.Fragestellung: Es sollte erstens untersucht werden, ob humane osteoblastare Zellen in vitro retroviral transduziert werden konnen. Zweitens wurde getestet, ob sich die retrovirale Transduktionsrate in vitro optimieren last. Methode: Die untersuchten Zellpopulationen wurden aus humanen Spongiosaproben isoliert und in vitro kultiviert. Das β-Galactosidase-Gen (LacZ) und die cDNA des humanen Interleukin-I-rezeptorantagonistischen Proteins (IL-1Ra) wurden durch retroviral vermittelten Gentransfer auf die Zellkulturen ubertragen. Die LacZ Aktivitat wurde durch Xgal-Farbung bestimmt, IL-1Ra wurde mittels ELISA quantitativ gemessen. Ergebnisse: Die IL-1Ra-Expression erreichte Werte von 8614pg IRAP/50000 Zellen/48h bis 10089pg IRAP/50000 Zellen/48h. Unter Optimierung der Transduktionstechnik lies sich bei der X-gal-Farbung nach retroviralem Transfer des LacZ Gens eine Transduktionsrate von 60% nachweisen. Schlusfolgerungen: Unsere Ergebnisse demonstrieren, das eine retrovirale Transduktion von humanen osteoblastaren Zellen in vitro moglich ist und zu einer reproduzierbaren Produktion der transgenen Produkte fuhrt.

Objective Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1β (IL-1β). If NO production is blocked, much of the IL-1β 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 β (TGFβ). Methods Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFβ 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-1β increased active TGFβ in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFβ were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFβ without affecting the early TGFβ activation. IL-1β stimulated a NO-independent, transient increase in TGFβ3 at 24 hours; however, TGFβ1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1β increased CM concentrations of TGFβ1 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 TGFβ1. Anti-TGFβ1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1β + L-NMA, confirming that NO inhibition of TGFβ1 in IL-1β–treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFβ 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 TGFβ1 levels following prior exposure to TGFβ1 was also blocked by NO. Conclusion NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFβ1 by chondrocytes exposed to IL-1β. It prevents autocrine-stimulated increases in TGFβ1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.

Gene therapy is a technique that may offer advantages over current methods of cytokine delivery to ligaments. To determine if implanted genes could be expressed in normal and injured knee ligaments, the medial collateral ligament and anterior cruciate ligament were studied in 18 rabbits. A retroviral ex vivo technique using allograft medial collateral ligament and anterior cruciate ligament fibroblasts and an adenoviral in vivo technique were compared as methods for delivering the LacZ marker gene to knee ligaments. Bilateral knee surgeries were performed, and the rabbits were equally divided into three groups. Group 1 received the retrovirus and the medial collateral ligament was ruptured, Group 2 received the adenovirus and the medial collateral ligament was ruptured, and Group 3 received the adenovirus and the medial collateral ligament was not injured. The anterior cruciate ligament was not injured in any group. The medial collateral and anterior cruciate ligaments of the right knees received 10(6) allografted, transduced ligament fibroblasts or 10(9) adenovirus particles, whereas the ligaments of the left knee received a similar volume of saline solution only. Equal numbers of rabbits were killed at 10 days, 3 weeks, and 6 weeks following the procedure. Ligament samples were stained with X-gal to detect the expression of the LacZ gene product, beta-galactosidase. LacZ gene expression was evident in ruptured and uninjured medial collateral ligaments as well as in the anterior cruciate ligament. The expression lasted between 10 days and 3 weeks in the medial collateral and anterior cruciate ligaments with use of the retrovirus and between 3 and 6 weeks in the medial collateral ligament and at least 6 weeks in the anterior cruciate ligament with the adenovirus. The length of gene expression in the ruptured and uninjured medial collateral ligaments did not differ. These preliminary studies indicate that gene transfer to normal and injured knee ligaments is possible.

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.

Although it has been reported that several growth factors modulate soft-tissue healing, the specific effects of growth factors on protein synthesis during ligament healing have not been widely investigated. In this study, we examined the effects of basic and acidic fibroblast growth factors, transforming growth factor beta 1, and epidermal growth factor on collagen and noncollagenous protein synthesis by cultured fibroblasts from medial collateral ligament and anterior cruciate ligament in vitro. Uptake of tritiated proline was used to measure synthesis of collagen and noncollagenous protein, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the type of collagens synthesized. Our data showed that transforming growth factor beta 1 increased both collagen and noncollagenous protein synthesis by medial collateral and anterior cruciate ligament fibroblasts on a dose-dependent basis. Collagen synthesis by cultured fibroblasts from the medial collateral and anterior cruciate ligaments was increased by treatment with transforming growth factor beta 1 by as much as approximately 1.5 times that of untreated controls. Although the response to transforming growth factor beta 1 by anterior cruciate ligament fibroblasts was equal to that by medial collateral ligament fibroblasts, the amounts of matrix proteins synthesized by anterior cruciate ligament fibroblasts were approximately half of that by medial collateral ligament fibroblasts. The increase was mostly in type-I collagen. Treatment of anterior cruciate ligament fibroblasts with epidermal growth factor increased collagen synthesis by approximately 25% but had little effect on medial collateral ligament fibroblasts. Neither basic nor acidic fibroblast growth factor increased either collagen or noncollagenous protein synthesis. These findings suggest that topical application of transforming growth factor beta 1, alone or in combination with epidermal growth factor, may have the potential to strengthen the ligament by increasing matrix synthesis during its remodeling and healing processes.

Growth factors have been shown to stimulate fibroblast proliferation during wound and ligament healing. In this study, we analyzed individual effects of eight growth factors in vitro on the proliferation of fibroblasts from the medial collateral (MCL) and anterior cruciate (ACL) ligaments of skeletally mature rabbits. We compared the proliferative response of growth factor-treated and nontreated fibroblasts of both ligaments. The growth-factor treated fibroblasts of the MCL and ACL were also compared. We found that the fibroblasts exposed to epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-BB proliferated significantly more than untreated fibroblasts. Acidic fibroblast growth factor at a dose of 1.0 ng/ml caused significant increases in fibroblast proliferation only in the MCL. Transforming growth factor-beta 1, insulin-like growth factor-1, platelet-derived growth factor-AA, and interleukin-1 alpha did not significantly stimulate fibroblast proliferation. MCL fibroblasts generally did not proliferate significantly more than ACL fibroblasts with the exception of MCL fibroblasts exposed to the highest doses of basic fibroblast growth factor, acidic fibroblast growth factor and platelet-derived growth factor-BB. The data were also compared with those obtained earlier using fibroblasts from skeletally immature rabbits (Schmidt et al., JOR 1995). The proliferative response of both the MCL and the ACL fibroblasts was found to decrease with skeletal maturation. Thus, our findings suggest that animal age and fibroblast origin are important factors in determining the proliferative response to growth factors.

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.

Study Design. In vitro and in vivo testing of a gene expression control system. Objective. The purpose of this study is to establish the ability of controlling gene expression using an adeno-associated viral vector containing a novel control system (AAV-RheoSwitch GFP [Intrexon Corp., Blacksburg, VA]) in intervertebral disc cells for potential use in gene therapy trials. Summary of Background Data. Gene therapy for disc degeneration shows promise; however, concern remains regarding safety. Careful control of gene expression is needed to facilitate translation into clinical trials. Methods. Rabbit nucleus pulposus cells were treated in vitro with increasing multiplicities of infection of AAV-RheoSwitch GFP, followed by increasing concentrations of Intrexon's activator ligand, and examined for fluorescence during and after removal of ligand. New Zealand white rabbits were injected with AAV-RheoSwitch GFP and killed either before or after 5 days of daily ligand injection. Tissues were analyzed for the presence of green fluorescent protein (GFP) with fluorescence microscopy and immunohistochemical staining. Results. In vitro, GFP expression was noted to be dose and time dependent, decreased 24 hours after removal of ligand, and was minimally detectable in cells after 48 hours. In vivo, increasing GFP expression was seen in animals treated with viral vector and ligand. No GFP expression was evident in tissues from rabbits that received only virus, or activator ligand alone. In addition, no GFP expression was evident in the adjacent discs, spinal cord, dura, bone, liver, or brain of any animals. Conclusion. These data demonstrate that in vitro ligand-induced gene expression can be stimulated and effectively turned off by removal of the ligand. In addition, we demonstrated the in vivo utility of this system through showing up-regulation of GFP without nonspecific gene expression or expression in adjacent tissues. This system, therefore, has the potential to increase the safety of gene therapy in the treatment of intervertebral disc degeneration.

Lapine synovial fibroblasts produce prostaglandin E2 (PGE2) and neutral metalloproteinases in response to phorbol 12-myristate 13-acetate (PMA), human recombinant interleukin-1 (hrIL-1) and, in an autocrine fashion, in response to partially purified preparations of their own cytokines known as cell-activating factors (CAF). Here we have examined the possible role of protein kinase C (PKC) in these responses. Whereas the 80-kDa substrate for PKC could not be detected in synovial fibroblasts, these cells contained a 35-kDa protein which fulfilled the criteria for qualifying as a specific substrate of PKC. Translocation assays based upon phosphorylation of the 35-kDa protein and Western blotting techniques allowed the movement of PKC from the cytosolic to the particulate fraction in response to PMA and CAF to be detected but not in response to 4 alpha-PMA or hrIL-1. Inhibitors of PKC suppressed synovial activation by PMA, partially blocked activation by CAF but had no effect on activation by hrIL-1. There thus appear to be PKC-dependent and PKC-independent routes to synovial cell activation. Our data suggest that IL-1 uses the latter, while CAF contains cytokines which utilize both routes.

Joints are difficult organs to target therapeutically. Intravenous, intramuscular, and oral routes of drug delivery provide poor access to the joint, and expose the body systemically to the therapeutic agent. Although intraarticular injection provides direct access to the joint, most injected materials have a short intraarticular half-life. We propose to circumvent these problems by introducing into the synovium gene(s) coding for proteins with antiarthritic properties. Two methods of gene delivery to synovium are under development. In the direct approach, in situ transduction of synoviocytes follows the injection of suitable vectors into the joint. In the indirect approach, synovium is removed from the joint, its synoviocytes are isolated, and the cells transduced in vitro. Genetically modified cells are subsequently transplanted back into the synovium. Using retroviral vectors, we have been able to express the lacZ and neo genes in lapine synovial fibroblasts in vitro. Following neoselection, all cells became LacZ+. Neo-selected cells carrying the lacZ marker gene were transplanted back into the knees of recipient rabbits to examine the persistence and expression of these genes in vivo. Islands of LacZ+, transplanted cells persisted in the recipient joints for at least 3 months. Furthermore, Neo+ cells could be grown from synovia recovered from these joints. Initial attempts to use retroviruses for the direct, in situ transduction of synovium have failed, probably because synoviocytes in the normal synovium are mitotically inactive. Present efforts are directed towards further development of our techniques for transferring genes to joints, and using these techniques to antagonize the intraarticular actions of interleukin-1.(ABSTRACT TRUNCATED AT 250 WORDS)

In view of the possible link between collagenase and the formation of aortic aneurysms we have determined whether cells within the aorta are able to synthesize this enzyme. Explanted cells obtained from fragments of lapine abdominal aorta secreted little or no collagenase. Two related metalloproteinases, gelatinase and stromelysin, were also produced at very low levels. Treatment with purified human monocyte interleukin-1 beta, partially purified lapine, synovial IL-1 or phorbol myristate acetate strongly induced the synthesis of all these enzymes. These activators also increased synthesis of prostaglandin E2. The identity of collagenase was confirmed by detection of the characteristic TCA and TCB breakdown fragments of collagen and by demonstration of collagenase mRNA within activated aortic cells. Unactivated aortic cells contained no detectable collagenase mRNA, suggesting a pretranslational level of regulation. Aortic cells thus possess the ability to express several neutral metalloproteinases and, if a sufficient inflammatory stimulus was present, they might do so in arteries undergoing aneurysmal degeneration.

Synoviocytes secrete factors which induce the synthesis of neutral metalloproteinases (NMP) and prostaglandin E2 (PGE2) by chondrocytes in a response called "chondrocyte activation". We analyzed synovial chondrocyte activating factors (CAF) for the presence of cytokines which modulated the NMP production by articular chondrocytes. These studies suggested the presence of several other cytokines in addition to interleukin-1 (IL-1). Both resting and activated synoviocytes contained mRNA for basic fibroblast growth factor (bFGF) which is a synergist for IL-1 induced NMP production, and secreted bFGF into their culture media. They also expressed mRNA for transforming growth factor beta (TGF beta) which inhibits IL-1 induced NMP production. These cells also produce tumor necrosis factor alpha (TNF alpha) and trace amounts of interleukin-6 (IL-6). In addition to these there is evidence for a synovial activator of chondrocytes which is distinct from IL-1. Since a number of recombinant cytokines including TNF alpha, IL-6 and bFGF failed to activate chondrocytes, this could be a novel cytokine.

STUDY DESIGN: A cohort of young, healthy New Zealand White rabbits was followed longitudinally with serial magnetic resonance imaging (MRI) analysis and terminal analysis of histologic changes and gene expression. OBJECTIVE: To examine the changes observed during normal aging in the intervertebral disc. SUMMARY OF BACKGROUND DATA: Although there is a correlation between aging and the onset of intervertebral disc degeneration (IDD), evidence suggests that distinct pathways are involved in these processes. Our group has characterized a reproducible rabbit model of IDD by MRI, radiograph, histology, and mRNA expression. However, no similar analysis has been performed longitudinally for intervertebral disc aging to allow comparison of these 2 important processes. METHODS: Four skeletally mature female NZW rabbits were housed for 122 weeks, and lumbar spine MRIs were characterized serially. Histologic and quantitative gene expression analysis of the nucleus pulposus of these aging animals was performed, and compared with adult and young rabbits. RESULTS: Mean MRI index decreased by

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.

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor- β 1 (TGF- β 1 ) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48–51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF- β 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TGF- β 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increases in collagen and total protein synthesis due to TGF- β 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.

OBJECTIVE This study explored the therapeutic effect of interleukin-1 receptor antagonist (IL-1Ra), administered by gene transfer, on the progression of osteoarthritic (OA) lesions in an experimental dog model. METHODS Seventeen mature mongrel dogs were divided into 3 groups. Group 1 (n = 7) had an anterior cruciate ligament (ACL) section of the right knee through a stab wound incision. Groups 2 and 3 (n = 5 per group), had an ACL section of the right knee and partial synovectomy of the left knee. Each dog's synovium was subjected to enzymatic digestion, and the synovial fibroblasts were propagated in monolayer culture. Synovial cells from each dog were transduced in vitro using the retrovirus MFG with either the Escherichia coli beta-galactosidase (lac Z) gene (group 2) or the human IL-1Ra gene (group 3). Two days after surgery, the dogs received intraarticular injections as follows: group 1 phosphate buffered saline (PBS) (2 ml); group 2 autologous cells (60 x 10(6) cells/2 ml of PBS) transduced with the lac Z gene; group 3 autologous cells transduced with the IL-1Ra gene. Synovial fluid was aspirated at 2 weeks and 4 weeks. All dogs were euthanized at 4 weeks postsurgery. The right knees were dissected, and lesions were scored for macroscopic and microscopic changes. Synovial explants were dissected and representative specimens were used for histology or were cultured for 48 hours. The levels of IL-1Ra in synovial fluid and synovial explant conditioned medium were measured by specific enzyme-linked immunosorbent assay. RESULTS The level of IL-1Ra in synovial fluid of group 3 was 202.8 +/- 131.5 ng/ml (mean +/- SEM) at 2 weeks and 2.8 +/- 2.2 ng/ml at 4 weeks after surgery. Membrane explants isolated from dogs that received synovial cells transduced with the IL-1Ra gene (group 3) actively produced IL-1Ra (4.0 +/- 2.0 ng/gm of tissue wet weight). The severity of OA cartilage lesions was similar in groups 1 and 2. In contrast, group 3 dogs had a marked reduction in macroscopic lesion severity on the tibial plateaus (P < 0.01 for grade; P < 0.04 for size) and femoral condyles. Moreover, the histologic lesion severity was decreased on both plateaus (P < 0.06) and condyles. CONCLUSION This study showed that a local increase in IL-1Ra production in OA knee joints by intraarticular injection of transduced synovial cells can reduce the progression of experimentally induced lesions.

The addition of human recombinant interleukin-1β (IL-1β) 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-1β. 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.

Objective. To assess the abilities of various vectors to transfer genes to the synovial lining of joints. Methods. Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5′ long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). Results. Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. Conclusion. None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.

Induction of stromelysin and collagenase mRNAs in response to phorbol myristate acetate (PMA) and autocrine factors (CAF) was compared in primary cultures of lapine synovial fibroblasts and an immortalized line of these cells known as HIG-82. In both cell types, message induction was quicker for CAF than for PMA. Appearance of both stromelysin and collagenase mRNAs occurred earlier in HIG-82 cells and, unlike primary cells, HIG-82 cells partially resisted inhibition by cycloheximide. To determine whether differences in AP-1 activity could account for these observations, the induction of c-fos and c-jun mRNAs was studied in conjunction with gel shift assays for AP-1 binding. Both inducers increased the abundance of c-fos mRNA, although the response was weaker in HIG-82 cells. However, the increase in c-jun mRNA was more marked in HIG-82 cells; furthermore, this increase was sustained for over 6 h. Gel shift assays confirmed that in both types of cells PMA and CAF increased AP-1 binding activity. In primary cells, this activity was sensitive to cycloheximide, but in HIG-82 cells, there was only partial sensitivity to cycloheximide. The gel shift analyses and data from experiments using an AP-1-CAT reporter construct revealed, in many cultures, constitutive AP-1 activity in the absence of stromelysin and collagenase expression, suggesting that AP-1 alone is insufficient for matrix metalloproteinase induction. Antisense oligonucleotides to c-fos and c-jun strongly inhibited the induction of stromelysin mRNA in primary cells treated with PMA, but was only weakly active against message induction in HIG-82 cells. In neither primary cells nor HIG-82 cells did antisense oligonucleotides strongly inhibit stromelysin induction in response to CAF. These data suggest there may exist an AP-1-independent route to message induction or that factors other than c-FOS and c-JUN may be used in certain circumstances. Western blot analyses detected no marked difference between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in their resting levels of c-FOS and c-JUN. Thus the differences reported here between HIG-82 cells and primary cells in the kinetics and cycloheximide sensitivity of MMP induction may reside in their abilities to modify posttranslationally the relevant transcription factors.

Study Design. Herniated cervical 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 cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy. Summary of Background Data. Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain. We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens. Methods. Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E 2 , and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed. Results. The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E 2 , and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs. Interleukin-1a, interleukin-1β, tumor necrosis factor-α, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs. Conclusions. Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E 2 , and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.

Growth factors have been shown to stimulate fibroblast division and thus may influence ligament healing. We analyzed the effects of individual growth factors on the proliferation of fibroblasts from the medial collateral and anterior cruciate ligaments of the rabbit in vitro in order to identify growth factors that might enhance proliferation of fibroblasts and to compare the responses of the fibroblasts from the two ligaments to these growth factors. Through measurement of the uptake of [3H]-thymidine into DNA, fibroblasts from these ligaments that had been treated with epidermal growth factor and basic fibroblast growth factor were found to proliferate nearly eight times more than control fibroblasts. Additionally, the fibroblasts of both ligaments proliferated at similar rates when exposed to platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-1-alpha. However, epidermal growth factor and transforming growth factor-beta caused the fibroblasts from the medial collateral ligament to proliferate at a rate 1.3-1.4 times greater than that of fibroblasts from the anterior cruciate ligament. The reverse was true with acidic fibroblast growth factor, which stimulated the fibroblasts from the anterior cruciate ligament to proliferate at a rate 1.3-1.6 times greater than that of fibroblasts from the medial collateral ligament. This study demonstrated that growth factors can stimulate cell division in ligaments and may be effective in enhancing ligament healing but that these differences were not great enough to explain fully the clinical differences observed between healing of the medial collateral and anterior cruciate ligaments.

Slices of rabbit articular cartilage synthesized large quantities of nitric oxide (NO) following exposure to human recombinant interleukin-1 beta (hrIL-1 beta) or rabbit synovial cytokines (CAF). Each of these stimuli also strongly suppressed the biosynthetic incorporation of 35SO4(2-) into the glycosaminoglycans (GAGs) of cartilage proteoglycans. Treatment of cartilage fragments with L-NG-monomethylarginine (L-NMA), a competitive inhibitor of NO synthase, both inhibited NO synthesis in response to IL-1 and CAF and restored proteoglycan synthesis. D-NMA was inactive in this regard, and L-arginine reversed the effects of L-NMA. S-nitrosylacetylpenicillamine (SNAP), an organic donor of NO, reversibly mimicked the effect of IL-1 and CAF on 35SO4(2-) incorporation. These data suggest that endogenously synthesized NO is the mediator which reduces cartilage proteoglycan synthesis in response to cytokines such as IL-1 and CAF. Antagonists of NO production may promote cartilage matrix synthesis and thus have potential as chondroprotective or chondroreparative agents.

In an effort to understand the mechanism of matrix metalloproteinase (MMP) induction, lapine synoviocytes were isolated and incubated with phorbol myristate acetate (PMA) and autocrine “cell-activating factors” (CAF), agents which significantly increase MMP mRNA abundance. AP-1 complexes, formed by c-fos and c-jun which bind to 5′ residues of the MMP genes, seem causally related to MMP gene expression in response to PMA. However, although AP-1 DNA binding activity is strongly induced following exposure of synoviocytes to CAF, MMP gene expression in response to CAF does not correlate well with AP-1 activity and is not inhibited by antisense DNA to fos and jun. We hypothesize that there is a CAF-response factor involved in MMP gene expression and that this factor competes with the binding of the AP-1 complex to its target response element.

Summary There have been recent reports of adverse clinical results with freeze-dried ethylene oxide-treated bone-patellar tendon-bone (FD-ETO-BPTB) allografts used in anterior cruciate ligament (ACL) reconstruction. Ethylene oxide and its residues were implicated as the cause of many of the failures. Wear particles generated from both freeze-dried ethylene oxidetreated and deep frozen bone-patellar tendon-bone (DF-BPTB) allografts were placed in culture with lapine synoviocytes. The resulting synovialconditioned media were then assayed for interleukin-1 (IL-1) content. IL-1 is a potent mediator of tissue inflammation. FD-ETO-BPTB wear particles generated statistically significant levels of IL-1 when compared with both a negative control and DF-BPTB wear particles.

Preparations of lapine synovial 'chondrocyte activating factors' (CAF) were analyzed for the presence of individual cytokines which modulate the production of neutral metalloproteinases (NMPs) and prostaglandin E2 (PGE2) by articular chondrocytes. A combination of different biochemical analyses suggested that synovial fibroblasts secrete IL-1 alpha, which activated chondrocytes directly, bFGF, which potentiated the activity of IL-1, and TGF-beta 1, which produced a bivalent response. TGF-beta 1 suppressed NMP synthesis by chondrocytes, but enhanced PGE2 synthesis. The IL-1 receptor antagonist protein (IRAP) eliminated chondrocyte activation by IL-1, but only partially inhibited activation by CAF. Thus, CAF may contain a cytokine in addition to IL-1 which activates chondrocytes. This putative additional factor was more thermosensitive than IL-1, and had an apparent molecular weight of approx. 20,000 when estimated by size exclusion chromatography. Of a variety of purified cytokines tested for their ability to induce NMPs in chondrocytes, only IL-1 was active. This favours the possibility that the activity which resists suppression by IRAP reflects the presence of a novel cytokine.

The lapine synovial cell line HIG-82 secretes factors that activate cultures of articular chondrocytes. We showed that these "chondrocyte-activating factors" (CAF) also activate quiescent cultures of HIG-82 cells in an autocrine fashion. After exposure to partially purified preparations of CAF, HIG-82 cells increased their synthesis of prostaglandin E2 (PGE2) and the neutral proteinases collagenase, gelatinase, and stromelysin. CAF also induced their own synthesis. Both PGE2 synthesis and endogenous production of CAF started to increase between 1 and 3 h after treatment of cells with exogenous CAF, but the neutral proteolytic activity of the conditioned medium took approximately 12 h to increase. Induction of neutral proteinases by CAF was inversely related to the degree of cell confluency, whereas their induction by phorbol myristate acetate (PMA) was independent of this parameter. Both CAF and PMA provoked morphologic changes in subconfluent cultures of HIG-82 cells. Although the intracellular concentration of free Ca2+ increased rapidly in response to CAF, the results of experiments with calcium channel blockers and ionophores failed to support a role for Ca2+ fluxes in induction of neutral proteinases. In similar types of experiments, no evidence could be found to implicate fluxes in cyclic AMP or cyclic GMP in the induction of collagenase, gelatinase, or stromelysin. Because PMA is such a strong inducer of these enzymes, protein kinase C may be involved in signal transduction, but further work is needed to determine whether this is so.

We have been examining the role of protein kinase C (PKC) in synovial cell activation in response to interleukin-1 (IL-1). Attempts to measure PKC in soluble extracts of synovial fibroblasts by standard techniques failed. Western blotting with anti-PKC antibodies detected only a low level of PKC in synovial cells compared to rat basophilic leukemia cells and crude brain extracts. However, synovial PKC could be detected by measuring the Ca(2+)- and phospholipid-dependent phosphorylation of endogenous substrates. In this way, a 35 kDa protein was identified as the major endogenous cytosolic substrate for PKC. Treatment of synoviocytes with phorbol myristate acetate (PMA) strongly induced the synthesis of neutral metalloproteinases (NPs) and prostaglandin E2 (PGE2). Both Western blotting and assays based upon phosphorylation of the 35 kDa protein confirmed translocation of PKC from the cytosol in response to PMA. Although IL-1 induced the NPs and PGE2, it did so without detectable translocation of PKC. There thus appear to be PKC-dependent and PKC-independent routes of synovial cell activation. Our data suggest that IL-1 uses the latter.

After exposure to human recombinant interleukin-1 (hrIL-1), chondrocytes increase their synthesis of prostaglandin E2 (PGE2) and neutral metalloproteinases (NMPs). This response, known as chondrocyte activation, is also elicited by partially purified preparations of rabbit synovial IL-1, known as ‘chondrocyte activating factors’ (CAF). CAF activates chondrocytes more strongly than does hrIL-1, probably because it contains additional growth factors. Basic fibroblast growth factor (bFGF) is one such factor, although CAF also contains others which modulate the activation of chondrocytes. Chondrocyte activation by hrIL-1 is strongly potentiated by bFGF and phorbol myristate acetate (PMA). A series of experiments was conducted to examine the possible role of protein kinase C (PKC) in mediating these effects. Inhibitors of PKC partially blocked the induction of NMPs by CAF and completely suppressed the potentiating effect of PMA. However, induction by 10 units of hrIL-1/ml and potentiation by bFGF were not affected by these inhibitors. Furthermore, cells whose PKC had been down-regulated by prolonged exposure to PMA remained responsive to IL-1. Surprisingly, inhibitors of PKC greatly increased the amounts of NMPs produced in response to a low dose (1 unit/ml) of hrIL-1. These inhibitors also enhanced the synthesis of PGE2 by cells responding to 1 and 10 units of hrIL-1/ml. Phosphorylation of the 80 kDa substrate for PKC was augmented by PMA and CAF, but not by hrIL-1 or bFGF. Moreover, Western-blotting techniques, which confirmed translocation of PKC in response to PMA and CAF, did not detect translocation in cells treated with hrIL-1 or bFGF. Western blotting also demonstrated the presence of PKC isoenzyme type III (alpha), but not types I (gamma) or II (beta). These data argue that PKC does not mediate the effects of hrIL-1 or bFGF in chondrocytes. However, CAF contains additional substances which activate this enzyme and whose effects may in part be mediated by PKC.

As first shown by Stadler et al. [1], articular chondrocytes synthesize copious amounts of free radical nitric oxide (NO) following activation by interleukin-1 (IL-1). Present research is directed towards identifying a role for NO in the physiology or pathophysiology of chondrocytes and cartilage. Suppression of proteoglycan synthesis is one of the major metabolic responses of chondrocytes to IL-1 [2]. By inhibiting proteoglycan synthesis, IL-1 interferes with one of the main processes involved in maintaining the integrity of articular cartilage. In so doing, it may potentiate cartilage loss in both osteoand rheumatoid arthritis. The present paper reports data implicating NO in the process through which IL-1 inhibits proteoglycan synthesis by articular chondrocytes.

This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/106 cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 108 infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 × 105 infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.

Objective To determine whether articular chondrocytes express growth factor genes delivered by adenoviral vectors and whether expression of these genes influences matrix synthesis in the presence and absence of interleukin-1 (IL-1). Methods Monolayer cultures of rabbit articular chondrocytes were infected with recombinant adenovirus carrying genes encoding the following growth factors: insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGFβ1), and bone morphogenetic protein 2 (BMP-2). As a control, cells were transduced with the lac Z gene. Cultures were also treated with each growth factor supplied as a protein. Levels of gene expression were noted, and the synthesis of proteoglycan, collagen, and noncollagenous proteins was measured by radiolabeling. Collagen was typed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography. The effects of growth factor gene transfer on proteoglycan synthesis in the presence of IL-1 were also measured. Results The expression of all transgenes was high following adenoviral transduction. Proteoglycan synthesis was stimulated ∼8-fold by the BMP-2 gene and 2–3-fold by the IGF-1 gene. The effects of BMP-2 and IGF-1 genes were additive upon cotransduction. Synthesis of collagen and noncollagenous proteins, in contrast, was most strongly stimulated by the IGF-1 gene. In each case, collagen typing confirmed the synthesis of type II collagen. IL-1 suppressed proteoglycan synthesis by 50–60%. IGF-1 and TGFβ genes restored proteoglycan synthesis to control levels in the presence of IL-1. The BMP-2 gene, in contrast, elevated proteoglycan synthesis beyond control levels in the presence of IL-1. Conclusion Transfer of growth factor genes to articular chondrocytes can greatly increase matrix synthesis in vitro, even in the presence of the inflammatory cytokine IL-1. This result encourages the further development of gene therapy for the repair of damaged cartilage.

Addition of CoCl2 solutions to the culture media of confluent monolayers of lapine or human synoviocytes stimulated their production of the neutral proteinases collagenase, gelatinase, and caseinase. With lapine cells, maximum stimulation occurred at 10(-7) M CoCl2, while human cells required 10(-4)-10(-5) M CoCl2 to achieve a maximum stimulation. Production of prostaglandin E2 by lapine cells was enhanced some 30-40% by concentrations of CoCl2 that maximally stimulated synthesis of the neutral proteinases, whereas all concentrations of CoCl2 slightly depressed the production of prostaglandin E2 by human cells. Lapine synovial cells that had been stimulated by CoCl2 also produced a substance, or substances, that provoked the synthesis of collagenase, gelatinase, caseinase, and prostaglandin E2 by monolayers of articular chondrocytes. Chondrocytes themselves, however, resisted activation by CoCl2. These findings may be relevant to the aseptic loosening of joint prostheses.

Cultures of lapine articular chondrocytes were exposed to purified, human, recombinant interleukin-1 α or partially purified preparations of lapine, synovial, cytokines in the presence of [32P]orthophosphate. After 30 min incubation, phosphoproteins were extracted from the cells, separated by two-dimensional gel electrophoresis and visualized autoradiographically. Analysis of the autoradiograms revealed that interleukin-1 and the synovial factors produced marked changes in the pattern of protein phosphorylation. The synovial cytokines induced many of the same changes as interleukin-1, as well as a number of unique changes. This finding is consistent with the notion that, in addition to interleukin-1, synoviocytes secrete other cytokines which modulate the metabolism of chondrocytes. These data support the idea that signal transduction in chondrocytes responding to interleukin-1 involves the activation of one or more protein kinases.

The cDNA probe H-9, originally constructed to recognize a portion of the mRNA for lapine synovial collagenase, also hybridized with a RNA of the same size (approximately 2.0 kb) isolated from activated lapine articular chondrocytes. Primary, monolayer cultures of lapine articular chondrocytes did not contain detectable amounts of this RNA, nor did they secrete measurable amounts of collagenase into their culture media. Following exposure to synovial factors, the chondrocytes contained high levels of collagenase mRNA, while their conditioned media had considerable collagenolytic activity. Collagenase mRNA started to appear in chondrocytes 3-5 h after treatment with the synovial material. Maximum levels occurred after 12-24 h. Recombinant human interleukin-1 also induced the appearance of this mRNA. We conclude that chondrocyte collagenase is likely to be the same gene product as synovial collagenase, and that its regulation by lapine articular chondrocytes probably occurs at a pretranslational level.

Growing evidence suggests that biochemical mecha nisms play a role in the pathogenesis of arthritis. Car tilaginous wear particles have been shown to induce destructive enzymes and cytokines. To assess the biocompatibility of artificial ACL replacements, the ef fects of wear particles from the following ligaments were analyzed biochemically and histologically: GORE- TEX, Stryker Dacron Ligament Prosthesis, Versigraft carbon, Kennedy LAD, Xenograft, Leeds-Keio, and hu man patellar tendon allograft. Ligaments were frozen and ground to produce wear particles similar to those seen clinically and were added to lapine synovial cell cultures. The resulting conditioned medium was ana lyzed for collagenase, gelatinase, and chondrocyte ac tivating factor (CAF) production. All of the ligaments induced significantly elevated enzyme and CAF production by the synoviocytes, with Xenograft and carbon inducing significantly higher en zyme levels than those of the other five ligaments. Five milligrams of wear particles were injected into the knees of 4 kg to 5 kg rabbits that were analyzed histologically after 14 weeks. Wear particles accumu lated in the periarticular synovial folds and induced modest to severe macrophage infiltration in the syno vium. A hypothetical model explaining the role of artifi cial ligament wear particles in the pathogenesis of arthritis is presented.

In the experiments described here, we have sought to determine whether primary cultures of cells derived from articular cartilage will, upon subsequent subculture, undergo in vitro senescence in a manner analogous to that described for several other types of diploid cell. Using cells from the articular cartilage of rabbits, dogs and man, we have established that the population doubling capacity of cultures of these cells is directly related to the specific lifespan of the donor organism. Furthermore, the doubling capacity of the initial cultures of lapine articular chondrocytes is inversely related to the age of the donor rabbit. By these criteria, serially passaged primary cultures of cells derived from articular cartilage appear, a priori , to be a valid system for studies of cellular ageing. Monolayer cultures of lapine chondrocytes appear to “dedifferentiate” after several passages. However, the same cells can be grown as clones, under which conditions they appear to retain better their differentiated properties. Even under these circumstances, lapine articular chondrocytes have a limited capacity for growth, which can be calculated to approximate to the same average number of cell divisions as undergone by monolayer cultures. Lapine chondrocytes frequently transform into established lines of fibroblastic cells. Transformation of canine chondrocytes was more rare, while human chondrocytes have not been observed to transform. This suggests that resistance to transformation is somehow related to lifespan. In addition to furthering our understanding of cellular ageing, studies of the senescence of articular chondrocytes could provide new insights into the aetiology of primary osteoarthritis.

The lapine, synovial cell line, HIG-82, secretes 'chondrocyte activating factors' (CAF) which induce the synthesis of collagenase (EC 3.4.24.7), gelatinase, caseinase and prostaglandin E2 (PGE2) by confluent, monolayer cultures of lapine, articular chondrocytes. Partially purified CAF increased the production of PGE2 by chondrocytes within 3 h; in certain cultures this occurred in as little as 1 h. Increased levels of the three neutral metalloproteinases, in contrast, were only measurable in the conditioned medium after a delay of 9-18 h. After removal of the CAF, the synthesis of PGE2 reverted to basal levels within 1-4 h, but synthesis of the three proteinases remained high for an additional 4 days. Indomethacin, at concentrations which completely inhibited PGE2 synthesis, had no effect upon the coordinate induction of collagenase, gelatinase and caseinase. However, cycloheximide, alpha-amanitin and 5,6-dichlororibosylbenzimidazole (DRB) suppressed induction of these proteinases suggesting that CAF derepressed the genes coding for these enzymes. Once the chondrocytes had been activated by CAF, the inhibitors of transcription had a much weaker effect on the production of the neutral proteinases, indicating that their mRNAs may be relatively stable. In the presence of CAF, inhibition under these conditions was weaker still, possibly due to stabilisation of these mRNA molecules. Experiments with a number of compounds which modulate cellular Ca2+, cAMP or cGMP failed to support a straightforward role for these mediators in the induction of neutral metalloproteinases in chondrocytes. High concentrations of phorbol myristate acetate (PMA) provoked only a slight synthesis of these enzymes.