THERAPEUTIC EFFECT OF PROLONGED HYPERBARIC OXYGEN IN ADJUVANT ARTHRITIS OF THE RAT
JOEL WARREN, MIRIAM R. SACKSTEDER, and CLAIRE A. THUNING
Development of adjuvant disease in the rat is suppressed if hyperbaric oxygen is administered within one day after the inoculation of an oil suspension of Mycobacterium tuberculosis and continued for 16 to 17 days. Although 10 to 40% of the rodents developed mild arthritis after discontinuance of therapy, most remained symptom-free for at least 50 days. Oxygen administered after arthritis is advanced still exerted a significant curative effect. Possible mechanisms underlying the therapeutic action of oxygen are discussed. Prolonged exposure of rodents to hyperbaric oxygen (HBO) will suppress experimental autoimmune disease and block the delayed hypersensitivity reaction (DHR) to several antigens. Adjuvant disease in the rat (AD), a systemic, immune disorder induced by exogenous antigen and a useful model for the development of anti-arthritic drugs, is also modified by HBO. Fischer 344 rats were exposed to oxygen at 2 atmospheres absolute (15 psi) for 6 hours daily beginning at various times after their sensitization with Mycobacterium tuberculosis From the Leo Goodwin Institute for Cancer Research, Nova University, Fort Lauderdale, Florida. Joel Warren: PhD; Miriam R. Sacksteder: AB; Claire A. Thuning: MS. Supported by a grant from The Mellen Foundation. Address reprint requests to Dr. Joel Warren, Leo Godwin Institute for Cancer Research, Nova University, 3301 College Avenue, Ft. Lauderdale, Florida 333 14. Submitted for publication June 19, 1978; accepted December 5 , 1978.
Arthritis and Rheumatism, Vol. 22, No. 4 (April 1979)
in oil. When treatment was initiated one d later nd continued for 16 to 17 days, the number of arthritic rodents was significantly reduced as was the severity of their symptoms. Four to 14 days after the cessation of HBO therapy, characteristic arthritis appeared in a portion of the rats but was invariably a milder disease with minimal systemic involvement. When oxygen therapy was delayed until the tenth or eighteenth day, a time when joint inflammation was well advanced, it was still capable of reducing the incidence and severity of joint disease. Decreased histopathologic changes, and particularly the suppression of periosteal granulomata, corroborated the clinical effectiveness of oxygen therapy. We believe that continued daily HBO selectively and reversibly depletes or alters one or more immunocyte populations in rodents and thereby directly or indirectly influences several compartments of the immune response. The effects of prolonged exposure to oxygen on the immune response of animals or human beings have apparently not been investigated despite the repeated observations that several functions of the leukocyte and macrophage are inhibited when these cells are maintained in oxygen-rich atmospheres in vitro (1,2). It has recently been found that exposure of rodents to tolerable pressures of hyperbaric oxygen (HBO) markedly alters a diverse set of inflammatory, cell-mediated disease processes. These include the suppression of allograft rejection (3) and the delayed hypersensitivity response to tuberculin and myelin protein (4,5). In
OXYGEN FOR ADJUVANT ARTHRITIS
experimental allergic encephalomyelitis (EAE) there is a reduction of the inflammatory granulomatous response to complete Freund adjuvant inoculated into the footpads of guinea pigs or rats. Subsequent mononuclear cell infiltration into the site of antigen deposition and the accumulation of myelin activated-macrophages in the central nervous system is blocked, resulting in complete clinical and histologic suppression of the disease in guinea pigs ( 5 ) . These immunosuppressive phenomena require prolonged exposure for approximately 6 hours to 99% oxygen at 1.5-2.0 atmospheres absolute (ATA) or 7.5-15 psi. The effects of hyperoxygenemia were reversible and immunoresponsiveness to myelin antigen or tuberculin was regained within 6 to 10 days after the animal was returned to air breathing (5). In the following we amplify our preliminary report of the ability of hyperbaric oxygen to modify another cell-mediated syndrome, adjuvant disease (AD) in the Fisher 344 rat. This arthritis and periostitis, first described by Pearson in 1956, induced by the subcutaneous inoculation of mycobacterial lipopolysaccharide, has become a useful model in the study of autoimmune joint disease (6). Recent reports suggest that at least two subpopulations of thymocytes are involved in the causation of AD; a T lymphocyte resistant to anti-thymocyte serum and probably long-lived (7), and a second group with shorter lifetimes, which are sensitive to hydrocortisone and alkylating agents, and which may act as suppressor cells (8). The adjuvant arthritic rat is a reliable pharmacologic assay system because of the parallelism between its response to antiarthritic and antiinflammatory compounds and their efficacy in rheumatoid arthritis in human beings (9). Thus, chemical immunosuppressants, corticosteroids, radiation, gold salts, and salicylates all demonstrate comparable degrees of dose/responsiveness in man and the rat (10).
MATERIALS AND METHODS Induction and grading of arthritis. Fischer 344 rats (obtained from Sprague-Dawley) weighing 150-200 gm were inoculated subcutaneously at the distal end of the tail with 5 mg of heat-killed M tuberculosis (Difco) suspended in 0. I ml of sterile, heavy mineral oil (Squibb). Twenty to 40 rats of equal sex distribution were used in each experiment. The extent and severity of the ensuing arthritis were graded by a system similar to that described by Koga and Pearson (1 1). One point was given for each involved wrist or ankle and 3 points were scored for the degree of swelling of any one joint. Thus, a clinical index of 16 would be assigned to an animal with se-
vere inflammation and swelling of all four extremities. The mean daily indices for the arthritic animals were plotted for periods of 50 to 100 days beginning 10 days after sensitization. The individuals evaluating the degree of clinical involvement were not aware of the treatment received by a particular group. Treatment with oxygen (HBO). Steel pressure chambers were fitted with observation windows, loading ports, a floor plate, and tray for holding silica gel as the moisture absorbant.* Commercial oxygen tanks, set to deliver 100 psi, fed purge gas into the chambers at 5 psi. After at least 5 minutes of flushing, the exhaust valve was closed and the flow adjusted so that the rate of compression to the preset limit of 15 psi never exceeded 1-2 psi/minute. At the end of treatment the tanks were decompressed at a slightly slower (0.5-1 .O psi/min) rate. Animals, caged in groups of 6 to 10, were kept in the tanks for 6 hours daily measured from the time the pressure reached 2 ATA. Food and water were withheld from both control and HBO groups during periods of treatment. Histologic examination. At varying times after sensitization, rats from each group were sacrificed and knee and wrist joints and major viscera were removed and fixed in 10% formalin. Sections through the mid-joint were stained with hemotoxylin-eosin and, in some cases, the Gomori trichrome stains.
RESULTS The course of adjuvant arthritis in untreated Fischer 344 rats. The distribution and intensity of arthritic involvement in this strain of rats have not been reported but were found to be similar to that described for the Lewis and Sprague-Dawley strains by Swingle et a1 (12). Median time to first appearance of joint inflammation was 12.5 f 2.6 days in 65 untreated animals. Al-. though the incidence of illness was similar in both sexes (28 of 28 males, 35 of 37 females), the local, acute, inflammatory phase was frequently more severe in the females. Maximum swelling of the joints was present between the eighteenth and twenty-fifth days and was associated with weight loss, and less frequently, a purulent conjunctivitis, nasal discharge, scaling of the nostrils and balanitis, as described by Pearson and Wood (13). The rats had difficulty in locomotion and whimpered when disturbed, as if they were in considerable pain. These signs stabilized or subsided by the fiftieth day, leaving articular deformities in 70 to 100% of the untreated rodents, depending upon the experiment. The variability in response in identical, repeated experiments noted by Swingle et a1 was also observed in our studies (12). *Additional information on the design of these chambers and their controls is available on request.
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Inhibitory effect of hyperbaric oxygen on the development of adjuvant arthritis. The course of arthritis was markedly altered in animals receiving daily hyperbaric oxygen. The earlier treatment was begun after sensitization, the more complete was the therapeutic response. When oxygen was started one day after inoculation of antigen and continued for 16 to 17 days, there was a marked reduction in the severity of arthritis and in the number of afflicted rodents. Clinical arthritis never developed during the period of early HBO therapy, and when it appeared there was an interval of 4 to 14 days after discontinuance of 0,. Delayed AD was usually a milder disease with considerably less impairment of motion. The clinical course shown in Figure 1 summarizes data from three experiments in which oxygen was administered for 16 days. A total of 12 of 26 rats (46%) developed only mild to moderate arthritic swelling and inflammation after the discontinuance of oxygen. In contrast, the incidence of severe clinical arthritis was 37 of 38 (99%) in the controls (P>0.001). It is noteworthy that systemic signs such as penile induration, balanitis, and inflammation of the nose and eyes were not observed in those rats that developed adjuvant disease after the discontinuance of HBO therapy. Therapeutic effects of hyperbaric oxygen in adjuvant arthritis. Oxygen therapy was still capable of reducing the incidence and severity of joint disease if delayed until joint inflammation was well advanced, but before cartilage destruction was present. The cumulative clinical responses to HBO in two experiments when it was administered continuously are illustrated in Figure 2. Three separate groups of rats received HBO beginning 1, 10, or 18 days after the inoculation of antigen and continuing for 16 days thereafter. Each of 26 untreated animals had developed severe inflammation of 3 to 4 limbs by the twenty-first day (mean clinical index = 13.4). The least joint involvement and the mildest disease occurred in the group in which treatment was begun one day after sensitization. Twelve of 2 1 animals on this schedule had not developed clinical signs of illness by the fiftieth day of observation. If oxygen was not initiated until the tenth or eighteenth day, when 18 of 20 rats exhibited joint disease of varying intensity, it was still capable of significant amelioration of arthritis during the 16-day exposure, i.e., days 10 to 26 or days 18 to 34. Some increase in the clinical indices of arthritis of varying degree appeared after the discontinuance of oxygen. However, by 50 days, at the termination of the experiment, the overall severity of the disease in the groups treated on day 1 or day 10 was less than that in
the untreated controls. In 14 rats treated day 1 to 16 and observed for 132 days, arthritis failed to develop after hyperbaric oxygen was discontinued. Comparative efficacy of continuing daily and alternating hyperbaric oxygen in adjuvant disease. In earlier studies of allergic encephalomyelitis it was found that the suppressive effects of oxygen could be prolonged in guinea pigs if treatment was administered on alternate days (15). A comparable suppression of adjuvant arthritis in the rat was observed if oxygen treatment was started one day after sensitization and then administered on alternate days but the amount of time of administration was more than doubled. Figure 3 il-
Control Oxygen -treated
No. Rats with A.A. No. Rats at Risk
Days After Antigen Inoculation Figure 1. Suppressive effect of HBO on adjuvant disease in the Fischer Rat. Animals were inoculated into the distal tail with a mineral oil suspension of 5 mg of heat-killed M tuberculosis on day 0. The severity of clinical arthritis was graded for each paw on a scale of &4 (maximum score = 16 for each rat). The mean daily indices for each group of rodents were plotted for the duration of the observation. The difference in the mean severity of arthritis was subjected to Student's r distribution. The plots for the HBO treated rats in this figure were considered significant (P< 0.05).
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Schedule of Oxygen Treatment Control 0 Q.D. I - 16 A Q.D. 10-26 0 Q.D. 18-34
rats sacrificed on day 30, or 14 days after the discontinuance of oxygen. In untreated rats sacrificed later in the disease (fiftieth day) the granulomatous ligamental tissues were still heavily infiltrated with mononuclear histiocytes, plasma cells, and neutrophils. In contrast, in oxygentreated animals, after an interval of 33 days of air breathing, there was a marked reduction in joint size and the granulation tissue contained a relatively small number of mononuclear cells embedded in active fibroblasts. Between the tenth and eighteenth day of illness, inflammation of the fusiform phalangeal, the metatarsal, and metacarpophalangeal joints was at its peak. However, within one week after the initiation of HBO, the joint swelling had decreased, the livid, tender, acutely inflamed skin of the extremities had changed to a pale, gray-white color and histologically the entire process was slowed in relation to arthritis of the same duration in control rats. Control
o Oxygen : daily, Q.D. I 17
A Oxygen : alternate days,
Days After Antigen Inoculation Figure 2. Relation of initiation and duration of HBO treatment to adjuvant arthritis. Procedure is the same as that in Figure 1 except that HBO was administered for three different periods after sensitization; on days 1-16, days 10-26, days 18-34. Optimal suppression of disease was greatest with early treatment. However, HBO initiated as late as day 18 retarded the development of arthritis.
lustrates a plot of the clinical severity of AD in untreated and treated animals by use of continuous (17 days) or intermittent (34 days) therapy. Although the disease in the control animals was less severe in the experiment, effective suppression was attainable with the well-tolerated, staggered schedules. Histopathologic observations. Examination of sections of wrist and ankle joints from control and treated rodents corroborated the clinical effectiveness of prolonged oxygen therapy. Treatment initiated 24 hours after inoculation of antigen and continued for 16 days completely prevented the initial rapid growth of granulomatous tissue and macrophage infiltration of the tendons, synovial sheaths, and synovia. The articular cartilages and perichondrium showed no or minimal cellular infiltration, and bone destruction was not observed in
Days After Antigen Inoculation Figure 3. Comparative effectiveness of HBO administered daily or on alternate days. Groups of 10 rats received HBO on days 1-17 daily or from days 1-34 on alternate days. Although the arthritis in the controls was of only moderate severity, both oxygen regimens were therapeutically effective. P 5 0.01 was considered as highly significant.
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In both treated and control Fischer rats, there was a gradual return of lost weight and a subsidence of inflammation beginning about the seventh week. At this time, residuals in the form of stiff ankylosis and flexion deformities were observed grossly and microscopically in 8 of 13 controls examined at this time but in only 1 of 9 rats in which treatment was begun on day 10.
DISCUSSION The immunosuppressive effects of hyperbaric oxygen in experimental allergic encephalomyelitis and adjuvant disease have a number of similarities that suggest an underlying common mechanism. Both require an induction period of several days of HBO to become manifest, and the continued administration of oxygen prevented the development of clinical signs for at least 34 days. The return of immunoresponsiveness, as judged by the appearance of clinical signs of arthritis (AD) or paralysis (EAE), required approximately 4 to 10 days. The suppression of granuloma formation resulting from several antigens including M tuberculosis, tuberculin, and myelin proteins was a common and prominent characteristic (5). The ability of tissue hyperoxygenemia to block effector pathways, particularly those which are lymphocyte and/or macrophage mediated, is apparent. This gains support from our earlier finding that oxygen therapy was still capable of modifying the course of EAE and preventing infiltrative monocytosis in the spinal cord even when its administration was delayed for a significant period (6 to 10 days) after antigen ( 5 ) . At this time the mobilization and sensitization of lymphocytes to myelin protein are well advanced, the vascular permeability of the capillaries of the central nervous system is increased, and the passive transfer of EAE by sensitized lymphocytes and the appearance of dermal sensitivity to myelin antigen are demonstrable shortly thereafter (14,15). A block in the effector side of the immune response is suggested by the preliminary observation that passive transfer of myelin-sensitized rat lymphocytes to oxygen-treated recipients failed to induce EAE although it did so in untreated control rats (16). In a similar fashion, the remission of clinical signs of arthritis in rats when HBO was started 10 days after sensitization was associated with a marked reduction in the antigendirected, mononuclear infiltration of the joints, granuloma formation, and osteonecrosis. Each of the foregoing characteristics of oxygen
immunosuppression suggests that it involves interference with lymphocyte mobilization and/or lymphocyte function. Among the nonexclusive mechanisms whereby hyperbaric oxygen could modify cell-mediated responses are: 1) a direct effect of oxygen on one or more cell populations and 2) an indirect result of endogenous steroids induced by the stress of hyperoxygenemia. The detrimental effects of oxygen on phagocytic cells in vitro are well documented (1,17,18). In the animal this may be related to the intracellular reduction of oxygen to superoxide (0,) and H,02,both of which have been suggested as key factors in the bactericidal activity of phagocytes (19). Under study in our laboratory is the possibility that an increase in tissue PO, could induce superoxide levels in the activated macrophage which exceed the capacity of its protective oxidases resulting in the death of this cell. Alteration of the immune response via stress induced elevations of steroid levels has been described by Monjan and Collector (20). Bean and his associates observed that an increased level of adrenocortical activity also results from hyperoxygenemia and that hypophysectomy diminished oxygen toxicity 'in rats (2 1). Adrenocortical hyperplasia and elevated serum levels of corticosterones were observed in rats exposed for 2 to 3 days at 1 atmosphere and their return to air breathing resulted in a drop in steroid levels within a few hours (22). Exogenous corticosteroids are capable of suppressing essentially all delayed hypersensitivity responses including adjuvant disease (23). A partial list of specific immunocomponents reported to be modified by steroids includes the uptake and digestion of particulates, the reduced ability of RES cells to destroy phagocytized bacteria, suppression of generation of sensitized cytotoxic lymphocytes, suppression of macrophage MIF, and the loss of cortical thymocytes (24). In the case of adjuvant disease, such characteristics of oxygen suppression as the reduction of granulomata, the reactivation of inflammation after discontinuance of therapy, and the effectiveness of alternating daily treatment, resemble those of steroid immunosuppression. However, steroid activity alone does not explain the lasting effect of early oxygen treatment in blocking the progression of arthritis long after HBO is discontinued and while there is ample residual oil-antigen histologically persisting in the site of inoculation. In order to clarify the mechanism of oxygen immunosuppression and its possible involvement of ad-
OXYGEN FOR ADJUVANT ARTHRITIS
renocortical stimulation we plan to examine the effects of oxygen on various components of cellular and humoral immunity in parallel with measurement of its effects on adrenocortical activity. The unique and unexpected effects of increased tissue Po, could provide a useful probe for basic research on the humoral and cellular immune systems.
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by M Rosenthale, H Mansmann. New York, Spectrum, 1975, pp 155-171 Koga T, Pearson CM: Immunogenicity and arthritogenicity in the rat of an antigen from M. tuberculosis wax D. J Immunol 1 I1:599-608, 1973 Swingle KF, et al: Differences in the severity of adjuvant arthritis in four strains of rats. Proc SOCExp Biol Med 132:608-612, 1969 Pearson CM, Wood FD: Studies of polyarthritis and other lesions induced in rats by injection of mycobacterial adjuvant. I. General clinical and pathologic characteristics and some modifying factors. Arthritis Rheum 2:440459, 1959 Oldstone MBA, Dixon FJ: Immunohistochemical study of allergic encephalomyelitis. Am J Pathol 52:25 1-263, 1968 Shaw CM, Alvord EC, Kaku J, et al: Correlation of EAE with delayed-type skin sensitivity to specific homologous encephalitogen. Ann NY Acad Sci 122:318-331, 1965 Paterson PY, Thuning CA, Sacksteder MR, et al: Unpublished Weislow OS, Pakman L: Inhibitors of Pseudomonas aeruginosa by HBO interaction with mouse peritoneal exudate cells. Infect Immunol 10546-552, 1974 Allison AC: Role of lysosomes in oxygen toxicity. Nature 4967: 141- 143, 1965 Stossel TB: Phagocytosis. N Engl J Med 290:717-723, 774-780, 883-839, 1974 Monjan AA, Collector MI: Stress induced modulation of the immune response. Science 196:307-308, I977 Bean JW, Smith CW: Hypophyseal and adrenocortical factors in pulmonary damage induced by oxygen at atmospheric pressure. Am J Physiol 172:169-174, 1952 Houlihan RT, et al: Adrenocortical response to simulated high altitude and to oxygen at high pressures. Physiologist 9:206-2 1 I, 1966 Ward JR, Cloud RS: Comparative effect of anti-rheumatic drugs on adjuvant-induced polyarthritis in rats. J Pharmacol Exp Ther 152:1 16- 121, 1966 Fauci AS, et al: Glucosteroid therapy: mechanisms of action and clinical consideration. Ann Int Med 84:304-315, 1976