Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Retrovirus mediated in vivo gene transfer to synovium in bacterial cell wall-induced arthritis in rats.

Gene therapy may provide an effective alternative to conventional approaches for treating rheumatoid arthritis. Direct in vivo gene delivery to synovium has a distinct advantage with respect to clinical use. To date, retroviral vectors are the best studied constructs for gene delivery, and almost all approved gene therapy trials in humans rely on retroviral vectors. However, the applicability of retroviral transduction is limited by requirement for cell division, and attempts to transduce normal synovium in situ using retroviral vectors are reported to fail. The present study was undertaken in order to investigate susceptibility of inflamed synovium to retroviral infection in vivo. Using an experimental model of bacterial cell wall (BCW)-induced arthritis in rats, we attempted two approaches for delivery of retroviral vectors to synovium. In the first approach, recombinant retroviral vectors carrying reporter genes lacZ and neo were directly injected into inflamed rat ankle joints. Alternatively, inflamed joints were inoculated with gamma-irradiated murine retroviral vector-producing packaging cells. We found that about 1% of cells in explants from joints inoculated with packaging cells were lacZ-neo-positive. The lacZ+ neo+ cells in joint explants proliferated in culture and were of rat origin as determined using species-specific polymerase chain reaction (PCR) analysis. There was no evidence of transduction in explants from joints directly injected with retroviral vectors or from contralateral, control joints. These findings show that arthritic joints have a population of cells susceptible to retroviral infection in situ and demonstrate the possibility of using retroviral vectors for direct gene delivery to inflamed synovium.

Control of glucan-induced systemic granulomatosis by cyclosporine A.

This study has shown that cyclosporine A (CyA), under certain conditions, is a powerful inhibitor of intravascular and extravascular monocyte/macrophage accumulation. Experiments were carried out in Lewis rats in which intravenous injection of particulate glucan calls forth a striking granulomatous response in lung, liver, and spleen and produces a marked stimulation of splenic erythro- and myelopoiesis. In agreement with the results of others, there was also a considerable elevation in monocyte/macrophage chemoattractant levels in the bronchoalveolar lavage fluid, which is held to be a key reaction in the pathogenesis of the histologic lesions. Treatment of the animals with subcutaneous injections of CyA prevented the rise in the chemoattractant activity and suppressed the granulomatous organ infiltration as well as the splenic hemopoiesis. The findings supply new insights into the activities of CyA and would support its clinical use in macrophage-dominated diseases.

Superantigen can reactivate bacterial cell wall-induced arthritis.

Intravenous injection of toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus, can reactivate arthritis in a rat ankle joint that has been previously inflamed by injection of peptidoglycanpolysaccharide polymers isolated from the cell walls of group A streptococci. The severity and chronicity of this renewed arthritis is dose dependent and at higher doses (125 micrograms/kg) a prolonged joint inflammation with pannus formation and marginal erosion of cartilage and bone is induced after a single injection of TSST-1. Only modest synovial hyperplasia is induced in control ankle joints by systemic injection of TSST-1. Another superantigen, streptococcal pyrogenic exotoxin induces a much weaker, acute reactivation of arthritis that resolves by 2 days. Repeated injections of TSST-1 at 7-day intervals give the same undiminished pattern of joint response, but the joint swelling persists at a higher level with each succeeding injection. Cyclosporin A suppresses all phases of the recurrent arthritis, indicating that TSST-1 could be functioning through its property of a superantigen activating T lymphocytes. II-1 receptor antagonist and anti-TNF-alpha neutralizing antibody, which reduce reactivation of arthritis by peptidoglycan-polysaccharide polymers, have no effect on reactivation by TSST-1. This experimental model provides a means to examine in vivo the possible role of superantigens in rheumatoid arthritis and related diseases, and to analyze the cellular and molecular pathways induced by this family of microbial products.

Suppression of local and systemic responses in streptococcal cell wall-induced acute inflammation of the air pouch by cyclosporine A. Comparison with the effects of two anti-inflammatory bis-benzimidazoles.

Injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide into a subcutaneous air pouch causes local outpouring of neutrophils and macrophages and distant hemopoietic proliferation in spleen and bone marrow. Cyclosporine A (CyA) suppressed neutrophil accumulation and all cell lines of hemopoiesis. trans-1,2-Bis(5-amidino-2-benzimidazolyl)ethene (BBE) also interfered with neutrophil exudation, yet reduced only the erythroid component of the hemopoietic process. The ethane analogue of BBE, on the other hand, did not prevent neutrophil emigration, but held down splenic erythropoiesis and myelopoiesis. All three compounds stimulated streptococcus group A cell wall-derived peptidoglycan polysaccharide uptake by pouch macrophages. CyA being the least active, BBE and its ethane analogue also produced a shift of wear-and-tear pigment from large numbers of small splenic macro-phages into small numbers of large macrophages. The pouch model is very useful in the study of anti-inflammatory compounds and has furnished the first evidence of CyA interference with massive neutrophilic infiltration and with hemopoietic signals.

Pro- and anti-inflammatory roles of interleukin-1 in recurrence of bacterial cell wall-induced arthritis in rats.

A specific interleukin-1 (IL-1) receptor antagonist (IL-1ra) was used to examine the roles of IL-1 in an experimental model designed to analyze the reactivation phase of erosive arthritis, induced in rats with peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptococci. Monoarticular arthritis was initiated by injection of a small dose of PG-APS into an ankle joint, and reactivation was induced by intravenous injection of PG-APS 20 days later. Human recombinant IL-1ra given at a dose of 2 to 3 mg/kg at the time of reactivation of arthritis and at 6-h intervals inhibits the increase in joint swelling by at least 60%. Joint swelling is suppressed 30 to 50% when the initial treatment with IL-1ra is delayed until 6 h after reactivation. IL-1ra is not effective when the initial injection is delayed 12 or 24 h. With an injection schedule of IL-1ra given at the time of reactivation and every 6 h, treatment can be stopped at 24 h and the suppression of swelling is no different from that in rats for which injections are continued for 4 days. The results indicate that IL-1 has a prominent, although not exclusive, role in initiating inflammation in this model and is involved in the amplifying processes in progressive inflammation and chronic erosive disease. An anti-inflammatory function of IL-1 is also indicated from data showing that IL-1ra treatment limited to 6 h or less after the induction of reactivation enhances joint swelling, whereas intravenous injection of human recombinant IL-1 beta 24 h before reactivation suppresses the reactivation of arthritis.

Streptococcal cell wall-induced systemic disease. Beneficial effects of trans-bis(5-amidino-2-benzimidazolyl)ethene, a novel, macrophage-directed anti-inflammatory agent.

Previously bis(5-amidino-2-benzimidazolyl)methane (BABIM) was identified as a strong inhibitor of the multisystem inflammatory disease induced in Lewis rats by injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide (PG-APS). A BABIM derivative, trans-bis(5-amidino-2-benzimidazolyl)ethene (BBE), has attracted attention because of striking qualitative and quantitative differences in its activities when compared with the parent compound. BBE could control destructive tibial osteitis and necrotizing granulomatous splenitis and hepatitis, regardless if given in a preventive or curative mode. The compound had little effect on synovitis, however. BABIM, on the other hand, was active against synovitis and osteitis, but not against splenic granuloma formation. To be effective, it needed to be applied in a preventive mode. BBE caused a characteristic enlargement of PG-APS-laden splenic and hepatic macrophages suggesting that those cells represent targets of the inhibitor. BBE may be a powerful tool for the study of granulomatous lesions.

Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.

Streptococcal antigen-induced dislocation and dysplasia of the hip in newborn rats. Radiologic and histologic evaluation of a model of congenital dislocation of the hip.

Dislocation of the hip developed in 62% of newborn rats with streptococcal antigen-induced synovitis. Age at the time of the induction of synovitis is critical since dislocation is not observed in older rats. Synovitis with distention and laxity of the joint capsule is most likely responsible for the hip dislocation. Although congenital dislocation of the hip in children is not mediated by an inflammatory process, the current model of dislocation of the hip in rats is similar in being critically age-dependent, and associated with ligamentous laxity. Our model may be helpful in studying this important clinical entity.

Protracted anemia associated with chronic, relapsing systemic inflammation induced by arthropathic peptidoglycan-polysaccharide polymers in rats.

Mild hypoproliferative anemia with abnormal iron metabolism frequently accompanies chronic inflammation and infection in humans. To determine whether anemia is associated with chronic relapsing arthritis induced by bacterial cell wall polymers, serial determinations of the hematocrit were measured in rats injected intraperitoneally with sonicated peptidoglycan-polysaccharide fragments from group A streptococci. Acute anemia peaked 5 to 10 days after injection, and chronic, spontaneously relapsing anemia persisted for 309 days. 51Cr labeling demonstrated decreased erythrocyte survival, i.e., a half-life of 8.4 days in cell wall-injected rats versus 11.8 days in controls. Erythrocytes were mildly microcytic, and leukocyte counts were elevated during early spontaneous reactivation of arthritis, 15 days after injection of peptidoglycan-polysaccharide. Bone marrow myeloid/erythroid precursor ratios were elevated in arthritic rats (P less than 0.0001). Purified peptidoglycan produced an acute anemia lasting 10 days, while injection of group A streptococcal polysaccharide and mutanolysin-digested cell wall did not affect the hematocrit. The minimal effective dose of peptidoglycan-polysaccharide was 5 micrograms of rhamnose per g (body weight). Serum iron and transferrin levels were decreased in cell wall-injected rats (P less than 0.005) and were closely correlated with hematocrit values and joint inflammatory scores. Stainable iron was increased in the liver, spleen, and mesenteric lymph nodes and unchanged in the bone marrow of cell wall-injected rats. Anemia accompanying chronic, relapsing systemic inflammation induced by peptidoglycan-polysaccharide polymers appears to be an excellent animal model of the anemia of chronic disease.

Suppression of streptococcal cell wall-induced arthritis by a potent protease inhibitor, bis(5-amidino-2-benzimidazolyl)methane.

Bis(5-amidino-2-benzimidazolyl)methane, a powerful synthetic trypsin inhibitor, proved to be highly effective in suppressing the arthritis induced by streptococcal cell wall fragments in Lewis rats. It reduced not only the degree of synovitis, osteitis, and hematopoietic hyperplasia in the distal extremities, but also the degree of associated granulomatous inflammation in the liver. The results suggest that trypsin-like proteases play an important role in this arthritis model and that inhibitors may be useful in the treatment of similar arthritic conditions in humans.

Mast cell activation by group A streptococcal polysaccharide in the rat and its role in experimental arthritis.

Acute edematous responses were induced in Sprague-Dawley rats by the intravenous injection of group-specific polysaccharide (PS) isolated from group A streptococci. Thirty minutes after the intravenous injection of PS there was marked degranulation of subcutaneous and periarticular mast cells in all 4 feet, carbon particle labeling of adjacent venules, and an 8-fold increase in Evans blue dye content of the extremities. This acute reaction to PS was completely blocked by pretreatment with compound 48/80, but the polyarticular relapsing arthritis following the systemic injection of an arthropathic dose of streptococcal cell wall fragments containing large, covalently bound peptidoglycan-polysaccharide (PG-PS) was not blocked.

Localized gut-associated lymphoid tissue hemorrhage induced by intravenous peptidoglycan-polysaccharide polymers.

A hemorrhage into gut-associated lymphoid tissue developed as early as 3 min after the intravenous injection of group A streptococcal peptidoglycan-polysaccharide polymers into rats. Extravasated erythrocytes were specifically located in the lamina propria and organized lymphoid follicles of the intestines and mesenteric lymph nodes and did not occur in the lungs, kidneys, liver, spleen, adrenal glands, or submandibular and popliteal lymph nodes, as determined by gross and histologic observations and measurement of radiolabeled erythrocytes. Petechial hemorrhage was preferentially located within the intestine to the distal ileum, Peyer's patches, and lymphoid aggregates of the colon. The hemorrhage was transient and occurred in a dose-dependent fashion. It was maximal 5 min after injection and resolved completely by 3 days. A unique feature of this altered vascular permeability was the absence of polymorphonuclear leukocytic infiltration, edema, vasculitis, and tissue necrosis.

Comparison of inflammatory reactions induced by intraarticular injection of bacterial cell wall polymers.

Cell wall polymers isolated from group A streptococci, as well as lipopolysaccharide from Salmonella typhimurium and synthetic muramyl dipeptide, were injected into the ankle joints of rats. The inflammatory responses were assessed by gross and histologic examination, and edema was measured by accumulation of radiolabeled albumin in the limbs. The isolated group-specific polysaccharide induced extensive edema of the articular and periarticular tissue immediately after injection, and this resolved in 24 hours. The peptidoglycan moiety did not produce early edema, but induced an acute exudative reaction followed by a proliferative synovitis which resolved after 5 days. Reactions induced by covalently bound complexes of peptidoglycan and the group-specific polysaccharide (PG-APS) varied, depending on the size of the complex. Small fragments, derived from mutanolysin digestion, caused both an acute edematous reaction and transient arthritis. Larger fragments did not cause the immediate edematous reaction, but induced an acute arthritis that appeared within 24 hours and evolved into a chronic process. Episodes of recurrent inflammation, a distinctive feature of joint inflammation induced by systemic injection of PG-APS polymers, were not observed following intraarticular injection of any of the cell wall polymers. The relative susceptibility of different rat strains to arthritis induced by intraarticular injection paralleled the responses to systemic injection of PG-APS. These results demonstrate that variations in arthropathogenicity are due, in part, to inherent differences in the phlogistic activities of different cell wall polymers, and that the genetic control of susceptibility involves regulation of the inflammatory responses rather than the quantity of cell wall distributed to the joint.

Arthropathic properties of cell wall polymers from normal flora bacteria.

Peptidoglycan-polysaccharide (PG-PS) fragments were purified from cell walls of group D streptococci (Streptococcus faecium, strains ATCC 9790 and F-24) with a protocol which minimizes autolytic activity and tested for ability to induce arthritis in rats. PG-PS fragments from cell walls of other normal flora bacteria (Peptostreptococcus productus, and Propionibacterium acnes), group A streptococci, and pseudomurein-PS fragments from cell walls of Methanobacterium formicicum, were similarly purified and tested. Upon intraarticular injection into rat ankles, all PG-PS polymers induced acute inflammation; pseudomurein-PS fragments were approximately five times less active than the PG-PS preparations. After intraperitoneal injection, P. acnes PG-PS induced a minimal acute arthritis, Peptostreptococcus productus PG-PS induced a moderately severe acute joint inflammation followed by a mild chronic arthritis, and both group A and group D streptococcal PG-PS induced severe acute arthritis which evolved into chronic, erosive joint disease; pseudomurein-PS fragments were without effect, consistent with a crucial role for the PG moiety of PG-PS. Chronic arthritis induced by group D streptococcal PG-PS subsided after 60 days, whereas that induced by group A streptococcal PG-PS was still active after 128 days. The arthropathic properties of this modest number of common normal flora bacteria suggest that different PG-PS structures derived from the normal flora have the potential to induce a wide range of responses, from transient acute to chronic erosive joint disease.

Measurement of streptococcal cell wall in tissues of rats resistant or susceptible to cell wall-induced chronic erosive arthritis.

The quantity of streptococcal cell wall localized in the joints of rats of strains which are either susceptible (Sprague-Dawley, LEW/N, M520/N) or resistant (Buffalo, WKY/N, F344/N) to cell wall-induced chronic erosive arthritis was measured after intraperitoneal injection of group A streptococcal cell wall fragments. Susceptibility or resistance was not associated with a difference in the amount of cell wall localized in limbs or other tissues. It is concluded that although localization of cell wall in joint tissue is essential for development of arthritis, the relative resistance of certain rat strains reflects genetic regulation of inflammatory response rather than a quantitative difference in localization of cell wall in joints.

Measurement of bacterial cell wall in tissues by solid-phase radioimmunoassay: correlation of distribution and persistence with experimental arthritis in rats.

We have developed sensitive and specific solid-phase radioimmunoassays to quantitate the distribution and persistence of bacterial antigen in rats developing arthritis in response to a single injection of streptococcal cell wall material. Three separate assays were specific for either the A polysaccharide (N-acetyl-D-glucosamine), A-variant polysaccharide (polyrhamnose), or peptidoglycan (D-ala-D-ala) moieties of the streptococcal cell wall. Antigen was detected in all tissues surveyed, although the greatest amount was in the liver and spleen. By using three fractions of cell wall separated by size, we have shown that the development of arthritis correlates with the degree of cell wall deposited and persisting in the joints. Further statistical analyses suggested differences in metabolism by different tissues and differential metabolism of different antigenic epitopes in some cases.

Relationship of complement to experimental arthritis induced in rats with streptococcal cell walls.

Experimental arthritis developed in rats injected intraperitoneally with aqueous suspensions of peptidoglycan-polysaccharide complexes (PG-APS) isolated from group A streptococcal cell walls. Reduction of serum complement by pretreatment with cobra venom factor (COV) reduced acute joint inflammation over the first 3 days following injection of PG-APs. Thereafter, the course of the disease was not different in the COV-treated rats. The serum levels of complement were depressed below detectable levels by 24 hr in rats injected only with cell walls, but rebounded to normal levels or above 3 days after injection. In rats injected with COV before cell walls, the complement levels also increased 3 days after injection of cell walls, in contrast to sustained depressed levels in rat injected only with COV. The correlation between severity of joint inflammation and serum complement levels at day 3 was positive in COV-treated rats. The quantity of cell wall per joint at day 3 correlated with the severity of joint disease. However, COV treatment did not alter the amount of cell wall localized in joint tissue. Therefore, although complement does appear to have a role in early joint inflammation, its effect is not upon the transport of cell wall into joint tissue.

Arthropathic properties related to the molecular weight of peptidoglycan-polysaccharide polymers of streptococcal cell walls.

The covalently bound polymers of peptidoglycan and group-specific polysaccharide (PG-APS) were isolated from the cell walls of group A streptococci. Arthritis was induced in rats with a single intraperitoneal injection of an aqueous suspension of PG-APS fragments derived by sonication. The joint lesions induced with this polydisperse suspension followed a bimodal pattern consisting of an acute phase, which reached a peak 5 days after injection and then receded, followed by a chronic, remittent, erosive arthritis lasting several months. The relative severities of the acute and chronic phases could be manipulated by selection of the size of PG-APS fragments. The fragments of PG-APS obtained by sonic treatment were resolved on the basis of size into three major populations by sucrose gradient or differential centrifugation. Based upon light scattering and gel filtration, the average molecular weight of the largest family of fragments was estimated to be about 500 x 10(6), the intermediate fragments were 50 x 10(6) daltons, and the predominant size in the smallest population was 5.3 x 10(6) daltons. The larger fragments induced negligible acute inflammation, but chronic disease became apparent 5 to 9 weeks after injection. The smallest fragments induced the most severe acute inflammation, with relatively little late, chronic joint disease. The particles of intermediate size induced moderate acute inflammation and the most severe chronic, erosive joint lesions. A single injection of fragments of the isolated peptidoglycan moiety of the PG-APS induced only a moderate acute inflammation of joints, with no apparent capacity to maintain the injury and induce chronic disease.

Cell-mediated immune response during experimental arthritis induced in rats with streptococcal cell walls.

Chronic, remittent, erosive arthritis was produced in rats by a single intraperitoneal injection of an aqueous suspension of cell wall fragments isolated from group A streptococci. Arthritis could be induced in rats which had been immunologically compromised by neonatal thymectomy. Delayed hypersensitivity to cell wall peptidoglycan could not be elicited in these rats, although progressive joint disease was obvious by clinical and radiological measurements. A delayed skin test was elicited with peptidoglycan in non-thymectomized rats at 6 to 14 days after injection of low doses of cell wall fragments. Between 2 to 4 weeks after cell wall injection the skin test could not be elicited and these rats could not be sensitized again with peptidoglycan. After a high dose of cell wall the skin test could not be elicited at any time. These non-thymectomized rats which had been injected with cell walls remained hyporesponsive to peptidoglycan for at least 3 months. Lymphocytes from non-thymectomized cell wall-injected rats also showed a non-specific depression of lymphocyte response to phytohaemagglutinin in vitro, but this function was recovered between 2 to 4 weeks after cell wall injection. We conclude that cell-mediated immunity against bacterial cell wall antigens is not a pathogenetic factor in this experimental model of arthritis.