Pathogenetic difference between collagen arthritis and adjuvant arthritis.

Daily treatment with cyclosporin at a dose of 25 mg/kg for 14 d gave complete suppression of the development of collagen arthritis and adjuvant arthritis in Sprague-Dawley rats during an observation period of 45 d. To study whether the immunologic unresponsiveness produced by cyclosporin is antigen specific, we rechallenged the cyclosporin-protected rats with either type II collagen or complete Freund's adjuvant (CFA) after discontinuation of cyclosporin treatment. Type II collagen-immunized, cyclosporin-protected rats did not develop arthritis in response to reimmunization with type II collagen, but, they did develop arthritis in response to a subsequent injection of CFA. Similarly, CFA-injected, cyclosporin-protected rats showed a suppressed arthritogenic reaction in response to reinjection of CFA, whereas their response to a subsequent immunization with type II collagen was unaffected. On the other hand, the rats that were treated with cyclosporin without any prior antigenic challenge could develop arthritis in response to a subsequent injection of CFA or type II collagen after cessation of cyclosporin treatment. These results indicate that specific immunologic unresponsiveness can be induced by cyclosporin in the two experimental models of polyarthritis, collagen arthritis and adjuvant arthritis, and that there is no cross-reactivity between type II collagen and the mycobacterial cell wall components. The results further indicate that immunity to type II collagen plays a critical role in the pathogenesis of collagen arthritis but that its pathogenetic role in adjuvant arthritis is insignificant.

Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival.

We have tested the effects of FK506 (FK), a new immunosuppressive agent, on a rat limb allograft model. Histoincompatible BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 2 mg/kg, 10 mg/kg, or 50 mg/kg of FK on the day of limb transplantation (day 0) significantly prolonged graft survival in a dose-dependent manner--i.e., mean limb survival times (MST) based on gross signs of skin rejection were 16 +/- 3 days, 51 +/- 6 days, or 104 +/- 17 days, respectively (P less than 0.01). Delayed treatment with a single injection of 10 mg/kg of FK at when early signs of rejection were visible (day 7 or day 10) reversed the ongoing rejection. The MSTs in these groups were comparable to that of those treated with the same dosage of FK on day 0. The FK-induced unresponsiveness toward limb allografts was donor-specific because limb-allografted. FK-protected rats could not accept the skin grafts from a third-party donor. In the next set of experiments, rats were given a single administration of 10 mg/kg of FK on the day of limb allograft, followed by intermittent injections of 3 mg/kg of FK once a week. This regimen produced complete graft survival for more than 200 days, though Pneumocystis carinii pneumonia occurred in most of the recipients. These results represent the unique effects of FK in preventing or reversing the graft rejection and in inducing indefinite survival in this animal model of composite tissue allografts.

Collagen arthritis in rats: the importance of humoral immunity in the initiation of the disease and perpetuation of the disease by suppressor T cells.

Arthritis could be passively transferred with a serum concentrate from collagen arthritic rats to nude rats and cyclosporin-treated, type II collagen-tolerant rats. These findings suggest that collagen arthritis could be inducible by humoral immunity alone in the absence of cellular immunity to type II collagen or functional T cells. In addition, passive arthritis induced by anticollagen antibody is a mild, transient disease from which the animals normally recover and the rats that have recovered from passive arthritis are resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, when a serum concentrate was transferred while cyclosporin was administered continuously, transferred arthritis in cyclosporin-treated, type II collagen-tolerant rats lasted as long as cyclosporin treatment and arthritis was significantly enhanced compared to those of naive recipients. Further, enhancement and prolongation of passively transferred arthritis in nude rats was observed. Furthermore, treatment with cyclophosphamide reversed acquired resistance to collagen arthritis subsequent to recovery from passive arthritis. These findings suggest that suppressor T cells might, at least in part, affect the clinical course of collagen arthritis and reverse acquired resistance to arthritis.

Prolonged limb allograft survival with short-term treatment with FK-506 in rats.

The effect of the new immunosuppressive agent FK-506 on rat limb allograft was investigated across the BN-to-F344 histocompatibility barrier. A 14-day course of FK-treatment at doses of 1 mg/kg per day or more, begun on the day of operation, significantly increased the period of graft survival. Additional studies demonstrated that single treatment with FK only on the day of operation prolonged the graft survival in a dose-dependent manner. These results stress the profound immunosuppressive effect of FK and suggest the possible future application of composite tissue allograft in humans.

Limb allografts in rats immunosuppressed with cyclosporine: as a whole-joint allograft.

We performed limb allografts in three inbred rat strains immunosuppressed with cyclosporine. As controls, 10 autografts and 10 isografts exhibited an excellent result. In minor-mismatched allografts (Lewis to Fischer, n = 45), with the use of cyclosporine, the grafted limbs survived and the articular cartilage retained normal architecture and cell viability 52 weeks after grafting, but without cyclosporine treatment severe degeneration and destruction of articular cartilage were shown by 16 weeks after operation. In major-mismatched allografts (Brown Norway to Fischer, n = 35), the articular cartilage of cyclosporine-treated animals maintained normal architecture and cell viability 52 weeks after operation despite the gross appearance of skin rejection, while that of non-cyclosporine-treated animals was degenerated and destroyed by 6 weeks. These results suggest the possibility of whole-joint allografts in humans with the use of cyclosporine, as well as other organ transplantations.

Treatment with cyclophosphamide reverses acquired resistance to collagen arthritis subsequent to recovery from passive arthritis.

Passive arthritis induced by anticollagen antibody was a mild, transient disease from which the animals normally recovered and the rats that had recovered from passive arthritis were resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, treatment of the recovered rats with cyclophosphamide (CY) shortly before a second administration of a serum concentrate induced a second episode of acute polyarthritis in 82% of the rats. In addition, CY treatment without further administration of a serum concentrate induced a recurrence of arthritis in 25% of the recovered rats. Similarly, treatment of the recovered rats with CY shortly before the challenge with type II collagen induced a second episode of arthritis in 83% of the rats. However, treatment with CY shortly before the challenge was unable to restore the suppressed humoral response to type II collagen. These results provide evidence that the resistant state is mediated, at least in part, by CY-sensitive events.

Inhibition by FK506 of established lesions of collagen-induced arthritis in rats.

We investigated the superior potency of the immunosuppressive agent FK506 on collagen-induced arthritis in rats. In our initial studies, we demonstrated that only one shot administration of FK506 at a dose of 10 mg/kg on the same day as type II collagen immunization suppressed the incidence of arthritis completely as well as humoral and delayed-type hypersensitivity (DTH) skin test responses to type II collagen. Yet no major side effects were observed in the rats treated with such a high dose of FK506. Additional studies demonstrated that pretreatment with FK506 on day -7 or day -3 was effective in suppressing the severity of arthritis and immune responses to type II collagen. The immunosuppressive effect of a single high-dose administration of FK506 continued for at least 1 week in this animal model of arthritis. A single administration of FK506 at a dose of 10 mg/kg on day 12 or 15, after the clinical onset of arthritis, was also effective in suppressing the severity of arthritis and immune response to type II collagen. We conclude that FK506, in this model, possesses an important, curative action when applied therapeutically. The outlook of FK506 treatment in clinical autoimmunity is promising at present.

Reversal of antigen-induced resistance to collagen arthritis by cyclophosphamide.

Treatment of rats with intravenous injection of 1 mg of soluble native type II collagen induced resistance against the subsequent induction of active arthritis by type II collagen immunization. This resistant state was accompanied by suppressed antibody response and delayed-type hypersensitivity (DTH) skin reaction to type II collagen. However, pretreatment of rats with 20 mg/kg of cyclophosphamide (CY), an agent reputed to damage suppressor T-cell function, 2 days before intravenous injection of soluble type II collagen abrogated the antigen-induced resistance against the subsequent induction of active arthritis. The DTH skin reaction to type II collagen was completely restored and the antibody response to type II collagen was significantly though not completely restored by CY pretreatment. These results provide evidence that antigen-induced resistance to collagen arthritis is mediated, at least in part, under the control of CY-sensitive events.

Effect of cyclophosphamide and its analogs on collagen arthritis in rats.

The effects of cyclophosphamide (CY) and its structurally related analogs, ifosfamide (Ifo), sufosfamide (Sufo), and mafosfamide (Mafo), on collagen arthritis in Sprague-Dawley rats were examined. Prophylactic treatment with 7.5-10 mg/kg/day of CY. 15 mg/kg/day of Ifo, and 10-15 mg/kg/day of Sufo for the first 10 days starting on the same day as the type II collagen immunization suppressed arthritis induction as well as humoral immune response to type II collagen. Prophylactic treatment with Mafo at doses ranging from 10 to 40 mg/kg/day for 10 days was ineffective in suppressing the disease development. When drug treatment was started only during the immediate preclinical phase of arthritis, the development of arthritis was suppressed in the animals treated with 10 mg/kg/day of CY and 15 mg/kg/day of Ifo from Day 5 to Day 14. Additional studies demonstrated that treatment with 10 mg/kg/day of CY and 15 mg/kg/day of Ifo started at the time of disease onset significantly suppressed the severity of arthritis compared with the control group. These results show the effectiveness of Ifo and CY on this animal model of polyarthritis and suggest the possibility of clinical use of Ifo for the treatment of human arthritides similar to CY.

Enhancing effects of tilorone on collagen arthritis and humoral immune response to type II collagen.

The effect of tilorone, which is known to suppress adjuvant arthritis, on the induction of collagen arthritis in rats was investigated. Combined data of the present experiments show that all of the tilorone-treated rats except one in the lowest dosage group developed arthritis but that the incidence of arthritis in the tilorone-treated groups was not significantly different from that of the control group. The results also show that the two higher dosages (12.5 and 25 mg/kg/day) of tilorone caused a significant increase in the severity of collagen arthritis. Humoral immune response to type II collagen was significantly augmented in these two higher dosage groups; however, delayed-type hypersensitivity response to type II collagen was suppressed while tilorone was administered continuously. In addition, treatment with tilorone caused a significant increase in the concentration of anticollagen IgG extractable from the joint tissue. Anticollagen IgG subclass analysis revealed that the major subclass was IgG2a in both the serum and paw extract, with minor amounts of IgG2b, IgG2c, and IgG1. The response of all these subclasses was almost equally activated by tilorone treatment.

Serum transfer of collagen arthritis in congenitally athymic nude rats.

The role of cellular immunity in collagen arthritis was investigated with congenitally athymic nude rats (rnu/rnu) and their heterozygous littermates (rnu/+). Immunization with type II collagen induced polyarthritis and definite immunity to type II collagen in rnu/+ rats, whereas rnu/rnu rats did not develop arthritis or immunity to collagen. An additional study demonstrated that collagen arthritis could be passively transferred with a serum concentrate from arthritic Sprague-Dawley rats to naive rnu/rnu rats as well as to rnu/+ rats. Histopathologically, the passively transferred arthritis in rnu/rnu rats resembled that in rnu/+ rats. Despite no difference in clearance of anti-type II collagen antibody after transfer between them, the passively transferred arthritis in rnu/rnu rats was significantly enhanced and prolonged in comparison with that in rnu/+ rats. These results indicate that arthritis may be inducible by humoral immunity in the absence of functional T cells and also suggest that anti-type II collagen-antibody is not the sole regulatory factor and that the suppressor cell system might regulate the clinical course of the disease.

Serum transfer of collagen arthritis to cyclosporin-treated, type II collagen-tolerant rats.

Collagen arthritis has been passively transferred with a serum concentrate from immunized donors to immunologically naive recipients as well as cyclosporin-treated, type II collagen-tolerant rats. These findings point to an important role for anticollagen antibody and appear to rule out a role for cellular immunity to type II collagen in the initiation of this disease. The passively transferred arthritis was a transient lesion in the majority of naive recipients and in the cyclosporin-treated, type II collagen-tolerant rats as well when a serum concentrate was transferred after the cessation of cyclosporin treatment. When cyclosporin-treated, type II collagen-tolerant rats received transfer concentrate while cyclosporin was administered continuously, arthritis was significantly enhanced, and lasted as long as cyclosporin was administered and in the majority of rats up to 2 weeks after the cessation of cyclosporin treatment. These results, together with a rapid clearance of anticollagen antibody from the serum, suggest that anticollagen antibody is not the sole regulatory factor and that a cellular suppressor system, sensitive to cyclosporin, might participate in the regulation of this disease process.

Effects of cyclosporin on collagen induced arthritis in mice.

We have studied the effect of the immunosuppressive agent cyclosporin on collagen induced arthritis in mice. Cyclosporin, when given prophylactically, was capable of suppressing the development of collagen induced arthritis and the immunological response to native type II collagen in a dose dependent manner. Furthermore, treatment with cyclosporin, started on the same day as the booster injection with type II collagen, also resulted in inhibition of development of arthritis and of immunity to collagen. These findings suggest that the time of a booster injection, three weeks after the initial immunisation, might be still within the induction phase of arthritis since reinoculation is required to produce a high incidence of arthritis in mice. In addition, therapeutic treatment with cyclosporin did not affect the clinical course of the disease or the immune response to collagen.

Comparative studies of the effects of FK506 and cyclosporin A on passively transferred collagen-induced arthritis in rats.

We investigated the effect of a novel immunosuppressive agent, FK506, in comparison with cyclosporin A (CsA) on the development of passive arthritis induced by anti-type II collagen (CII) antisera in rats. FK506 pretreatment shortly before serum transfer markedly suppressed the incidence and the severity of passive arthritis, while CsA pretreatment had no observable effects on this disease when used in doses sufficient to suppress the development of active arthritis induced by CII immunization. In an additional study, we examined whether these agents affect antibody-mediated tolerance induction. CII-specific immunological tolerance was induced by serum transfer, but was unaffected by either FK506 or CsA pretreatment in our regimen. While its precise mechanism of the immunosuppressive activity remains to be elucidated, FK506 can act on the antibody-mediated effector phase of arthritis and may offer new insights into the possible role of potential therapeutic utility in human autoimmune diseases.

High-density proteoglycan induces specific suppression of adjuvant-induced arthritis in rats.

In vitro data support the view that T cells in adjuvant-induced arthritis (AIA) respond to the proteoglycan (PG) component of articular cartilage; however, an in vivo role for PG in AIA has yet to be shown. To do so, we examined the effects of pretreatment with bovine cartilage high density PG (HDPG) on AIA induced by heat-killed Mycobacterium butyricum in Lewis rats. Purified bovine cartilage HDPG emulsified in Freund's incomplete adjuvant (FIA) was injected intradermally into rats 7 days before challenge with Myco. butyricum. The severity of arthritis was significantly suppressed in rats pretreated with as little as 0.75 mg of HDPG, and the arthritis was completely suppressed in rats pretreated with 3.0 mg of HDPG. This suppression was specific, as the same treatment did not protect against type II collagen-induced arthritis. Suppression of AIA is primarily a property of the HDPG, as suppression of the arthritis was significantly less with pretreatment with 3.0 mg of middle density fractions of PG, and no suppression was observed with pretreatment with the lowest density fraction of PG. Thus we report that pretreatment with cartilage HDPG, but not lower density PG, can induce specific suppression of AIA. These in vivo results support the view that immunity to cartilage HDPG plays a major role in the pathogenesis of AIA, and can induce specific tolerance to this type of arthritis.

Autologous blood transfusion with recombinant erythropoietin treatment. 22 arthroplasties for rheumatoid arthritis.

12 anemic and 10 non-anemic patients with rheumatoid arthritis were treated with recombinant human erythropoietin (rHuEPO) before arthroplasty. The patients received 400-800 units/kg of rHuEPO subcutaneously once a week. Autologous blood was collected after the hemoglobin concentration was increased by 5 percent or more. All but one of the patients responded to the treatment. They were given 1-3 units of autologous blood, and underwent the operation without homologous blood transfusion. The mean duration of the treatment was 1 month. In 1 patient with severe anemia, additional transfusion with 2 units of blood was necessary during the operation. In all patients, there was a tendency for the hemoglobin response ratio to rHuEPO to correlate negatively with the initial CRP levels. The treatment did not affect the patients' clinical rheumatologic condition and there were no adverse effects. These results demonstrated that the treatment with subcutaneous rHuEPO is both effective and non-toxic and can therefore eliminate the need for homologous blood transfusion in anemic patients undergoing arthroplasty for rheumatoid arthritis.

The effects of recombinant human granulocyte colony-stimulating factor on passive collagen-induced arthritis transferred with anti-type II collagen antibody.

Type II collagen-induced arthritis (CIA) is a pathologic process mediated, in part, by humoral immune mechanisms. Because many antibody-mediated reactions are neutrophil-dependent, the role of this cell population was examined in passive CIA transferred with anti-type II collagen (CII) antibody. In cyclophosphamide (CY)-induced leukocytopenic rats, swelling and inflammation associated with the arthritic response were significantly reduced. Concomitant administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to leukocytopenic rats for 7 consecutive days from the day of CY injection resulted in the recovery of peripheral blood neutrophils count and the abrogation of the suppression of arthritis with an optimal dose of anti-CII antibody. Further study demonstrated that a prior administration of rhG-CSF to naive rats for five consecutive days resulted in the significant and specific increase of peripheral blood neutrophils count and enhancement of passive arthritis with a suboptimal dose of anti-CII antibody. It was suggested that neutrophils played an important role in the development of passive CIA.