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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.

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.

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.

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.

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.

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.

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.

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 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.

We have isolated a continuous cell line from soft tissue lining the knee joints of rabbits. Designated HIG-82, this line was produced by spontaneous establishment of an aging, late-passage culture of primary cells. Like unpassaged, primary cells, HIG-82 cells can be activated by a number of stimuli, including phorbol myristate acetate (PMA), interleukin-1 (IL-1), and the endocytosis of latex beads. Activated cells secrete collagenase, gelatinase, caseinase (stromelysin), and prostaglandin E2 (PGE2) into their culture medium. Pseudodiploid, HIG-82 cells combine a high plating efficiency with a doubling time of approximately 24 h. As primary tissue of this origin is difficult to obtain in large quantities and shows cellular heterogeneity, the HIG-82 cell line should facilitate research into the biology and biochemistry of the fibroblastic cells that line the diarthrodial joints of mammals. Such cells are likely to be important in the pathophysiology of various arthritides.