Inhibitory effects of recombinant human interleukin 10 on disease manifestations in a P-->F1 model of acute graft versus host disease.

Interleukin 10 (IL-10) is a cytokine with both antiinflammatory and immunosuppressive properties. In the present study, we have examined the effects of recombinant human IL-10 (rHuIL-10) on the development of acute graft-vs.-host disease (GVHD) in unirradiated (C57B1/6JxA/J) F1 recipients of parental A/J lymphocytes. rHuIL-10 (2.5 to 100 micrograms/mouse administered subcutaneously) caused a significant reduction in splenomegaly in GVH mice. GVH splenocytes exhibited an augmented capacity to produce IFN-gamma when stimulated in culture with Con A or LPS. The IFN gamma produced in response to LPS stimulation was found to be derived from CD4+ and CD8+ T cells with little or no contribution from the NK1.1+ subpopulation of the GVH spleen. Treatment with IL-10 in vivo was found to diminish the capacity of splenocytes to produce IFN gamma when stimulated with LPS but not with Con A. IL-10 did not protect GVH mice from a lethal dose of LPS but caused a marked reduction in the serum TNF alpha response triggered by the LPS challenge. We conclude that IL-10 may be useful in controlling those clinical manifestations of acute GVHD that arise as a result of the activities of proinflammatory cytokines such as IFN gamma and TNF alpha.

The cooperative effects of TNF-alpha and IFN-gamma are determining factors in the ability of IL-10 to protect mice from lethal endotoxemia.

Recent studies have demonstrated that interleukin-10 (IL-10) has the capacity to protect mice from the lethal effects of endotoxin. In this investigation, we have examined the ability of IL-10 to protect both normal mice and Corynebacterium parvum-primed mice against endotoxin lethality. In the overwhelming majority of experiments, recombinant murine IL-10 (rMuIL-10) and recombinant human IL-10 (rHuIL-10) did not protect normal BALB/cJ mice from lipopolysaccharide (LPS)-induced lethality at doses up to 10 micrograms/mouse. Despite their inability to protect, both IL-10 preparations were highly effective in preventing the increase in serum tumor necrosis factor alpha (TNF-alpha) that occurred in response to the lethal dose of LPS. Moreover, a neutralizing antibody against TNF-alpha gave only partial protection when administered alone to BALB/cJ mice. Treatment with a combination of neutralizing antibodies against TNF-alpha and interferon-gamma (IFN-gamma) resulted in complete protection. In contrast to BALB/cJ mice, normal BDF1 mice were protected from lethal endotoxemia by treatment with both rMuIL-10 and rHuIL-10. However, IL-10 did not protect C. parvum-primed BDF1 against LPS lethality even though it caused a reduction in the LPS-induced serum TNF-alpha response in C. parvum-primed mice as well as in normal BDF1 mice. Neutralizing antibodies against TNF-alpha and IFN-gamma were protective when administered alone to normal BDF1 mice, as previously demonstrated in C. parvum-primed mice. These findings suggest that lethal endotoxemia is a result of the cooperative activities of TNF-alpha and IFN-gamma in normal mice of the BALB/cJ and BDF1 strains as well as in C. parvum-primed BDF1 mice. IL-10 appears to be less effective in protecting mice from lethal endotoxemia when cooperation between IFN-gamma and TNF-alpha is facilitated by high-level production of the cytokines as in C. parvum-primed mice or when there is evidence of strong synergy between them as in normal BALB/cJ mice.

Lipopolysaccharide-induced cytokine production and mortality in mice treated with Corynebacterium parvum.

Tumor necrosis factor alpha (TNF-alpha) has been shown to be an important mediator of the lethal effects of endotoxin in several experimental models of septic shock. However, studies with a recombinant human interleukin-1 (IL-1) receptor antagonist protein (IL-1ra) suggest a role for IL-1 as a mediator of septic shock as well. In the present study, we show that mice treated in vivo with Corynebacterium parvum are primed for the production of interferon-gamma (IFN-gamma) and exhibit an enhanced capacity to produce serum IL-1 alpha, TNF-alpha, and IL-6 when challenged intravenously with lipopolysaccharide (LPS). The majority of C. parvum-treated mice die within 24 h of an LPS challenge. Pretreatment with a rat antimouse TNF-alpha monoclonal antibody (mAb) protected 90% of the animals against the lethal endotoxin challenge, while an anti-IFN-gamma mAb gave approximately 75% protection. The anti-IFN-gamma mAb also caused a reduction in LPS-induced serum TNF-alpha and IL-1 alpha. Anti-IL-1 alpha, anti-IL-1 beta, and anti-IL-6 neutralizing mAb did not protect against lethality when administered to mice prior to the LPS challenge. These results indicate that TNF-alpha and IFN-gamma are major mediators of endotoxin shock in C. parvum-treated mice. The results further suggest that the IFN-gamma produced by C. parvum-primed mice in response to an LPS challenge serves as a stimulus for enhanced production of TNF-alpha and IL-1 alpha. These findings are consistent with an increasing body of evidence suggesting a major role for IFN-gamma in lethal endotoxemia.

A study of cytokine production in acute graft-vs-host disease.

The role of cytokines in the development of acute graft-vs-host disease (GVHD) was investigated in B6AF1 mice that were injected with parental A/J lymphocytes. Splenocytes from GVH mice exhibited an increased capacity to produce interleukin (IL)-1, IL-6, and TNF-a when stimulated in culture with lipopolysaccharide (LPS). This enhanced capacity was diminished following in vivo treatment with immunosuppressive drugs. Concanavalin A-stimulated GVH spleen cells produced significantly lower levels of IL-2 but higher levels of interferon-gamma (IFN-gamma) than did syngeneic spleen cells. Immunosuppressive therapy in vivo increased the capacity of GVH spleen cells to produce IL-2. However, immunosuppressants differed in their effects on IFN-gamma production. Sch 24937 (6-bromo-5-chloro-2-[1-(methylsulfonyl)acetyl] 3-(2-pyridyl)indole) enhanced or had no effect while cyclosporin A consistently decreased the capacity of splenocytes to produce this lymphokine. These results indicate that the capacity of GVH splenocytes for cytokine production can be differentially affected by the actions of some pharmacological agents. The data also indicate that there may be differential regulation of the production of IL-2 and IFN-gamma by the Th1 subset in the GVH spleen.

Inhibition of collagen II-induced arthritis in mice--a comparison of the effects of Sch 24937, immunosuppressants and nonsteroidal antiinflammatory drugs on the clinical expression of disease.

Previous studies from this laboratory have described Sch 24937 as a potent immunosuppressive agent that is particularly effective in suppressing humoral immune responses in mice. These findings prompted an evaluation of the effects of Sch 24937 in type II collagen-induced arthritis in mice where disease manifestations include the development of a strong humoral response to the collagen antigen. Sch 24937 reduced the incidence and severity of arthritis in collagen sensitized mice which appeared to be directly related to the immunosuppressive properties of the drug. However in contrast to the steroid betamethasone which also exhibited immunosuppressive activity, Sch 24937 did not prevent the changes occurring in the lymphocyte population of the draining lymph nodes of mice immunized with type II collagen. While the exact mechanism of the immunosuppressive activity of Sch 24937 remains to be elucidated, its mode of action in suppressing arthritis differs at least to some extent from that of a steroid.