Carol Terminelli

The anti-inflammatory activities of cannabinoid receptor ligands in mouse peritonitis models

In this report, we describe experiments in which cannabinoid receptor ligands were evaluated for effects on the development of a peritoneal inflammation when elicited in mice with thioglycollate broth or staphylococcus enterotoxin A. The cannabinoid receptor agonists [(-)-11-hydoxy-Delta(8) tetrahydrocannabinol-dimethylheptyl] (HU-210) and [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl[pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl) methanone] (WIN 55212-2) blocked the migration of neutrophils into the peritoneal cavity in response to these inflammatory stimuli. This effect was caused by a delay in the production of the neutrophil chemoattractants, KC and macrophage inflammatory protein-2. HU-210 and WIN 55212-2 blocked neutrophil chemokines and neutrophil migration whether administered subcutaneously (s.c.) or intracerebroventricularly (i.c.v.). Their modulatory effects on the inflammation were antagonized by centrally administered [N-(piperdin-1-yl)-5-(4-chloropheny)-1-(2,4-dichloropheny)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] (SR141716A), a selective cannabinoid CB(1) receptor antagonist. This latter observation, and the ability of the cannabinoid receptor agonists to suppress the peritoneal inflammation at relatively low doses when administered i.c.v., indicated a role for central cannabinoid CB(1) receptors in the anti-inflammatory activities of HU-210 and WIN 55212-2. The cannabinoid receptor agonists had no effect on monocyte migration elicited by thioglycollate, despite their ability to suppress monocyte chemotactic protein-1 levels in lavage fluids. The cannabinoid CB(2) receptor antagonist, [N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-choro-3 methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide] (SR144528) inhibited the peritoneal inflammation in a manner analogous to that of HU-210 and WIN 55212-2 when administered i.c.v., but it did not appear to act through central cannabinoid CB(1) receptors. The present results add to the body of literature indicating that cannabinoid receptor ligands have diverse anti-inflammatory properties.

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

Tumor necrosis factor a (TNF‐α) 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‐lra) 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‐γ (IFN‐γ) and exhibit an enhanced capacity to produce serum IL‐Ια, TNF‐a, 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‐α monoclonal antibody (mAb) protected 90% of the animals against the lethal endotoxin challenge, while an anti‐IFN‐γ mAb gave approximately 75% protection. The anti‐IFN‐7fgamma; mAb also caused a reduction in LPS‐ induced serum TNF‐α and IL‐Ια. Anti‐IL‐la, anti‐ IL‐1β, 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‐α and IFN‐γ are major mediators of endotoxin shock in C. parvum‐treated mice. The results further suggest that the IFN‐γ produced by C. parvum‐primed mice in response to an LPS challenge serves as a stimulus for enhanced production of TNF‐α and IL‐Ια. These findings are consistent with an increasing body of evidence suggesting a major role for IFN‐γ in lethal endotoxemia.

The cooperative effects of TNF-α and IFN-γ 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 μg/mouse. Despite their inability to protect, both IL‐10 preparations were highly effective in preventing the increase in serum tumor necrosis factor α (TNF‐α) that occurred in response to the lethal dose of LPS. Moreover, a neutralizing antibody against TNF‐α gave only partial protection when administered alone to BALB/cJ mice. Treatment with a combination of neutralizing antibodies against TNF‐α and interferon‐7 (IFN‐γ) 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‐α response in C. parvum‐primed mice as well as in normal BDF1 mice. Neutralizing antibodies against TNF‐α and IFN‐γ 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‐α and IFN‐γ 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‐γ and TNF‐α 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. J. Leukoc. Biol. 55: 711–718; 1994.

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.

Modulation of cytokine responses in Corynebacterium parvum-primed endotoxemic mice by centrally administered cannabinoid ligands.

The cannabinoid receptor agonists [(-)-11-hydoxy-Delta(8)tetrahydrocannabinol-dimethylheptyl] (HU-210) and [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl[pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl) methanone] (WIN 55212-2) were previously shown to downregulate inflammatory cytokines (tumor necrosis factor alpha and interleukin-12) and to upregulate antiinflammatory interleukin-10 when administered intraperitoneally (i.p.) to mice before an endotoxin challenge. Cytokine modulation coincided with the onset of behavioral changes that are associated with cannabinoid agonist activated central cannabinoid CB(1) receptors. Both effects were antagonized by [N-(piperdin-1-yl)-5-(4-chloropheny)-1-(2,4-dichloropheny)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] (SR141716A) a selective cannabinoid CB(1) receptor antagonist. In the present study, we have investigated further the apparent role of central CB(1) cannabinoid receptors in cytokine modulation by HU-210 and WIN 55212-2. When administered intracerebroventricularly (i.c.v.), the drugs modulated cytokine responses at doses that were threefold to fourfold lower than those found effective by the i.p. route. SR141716A blocked cytokine modulation when coadministered centrally with the agonists, while a selective cannabinoid CB(2) receptor antagonist, (N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-choro-3 methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide) (SR144528) had no effect. Surprisingly, SR144528 was found to modulate cytokines itself when injected i.c.v.

Synthesis and structure-activity relationships of heteroaryl substituted-3,4-diamino-3-cyclobut-3-ene-1,2-dione CXCR2/CXCR1 receptor antagonists.

Comprehensive SAR studies were undertaken in the 3,4-diaminocyclobut-3-ene-1,2-dione class of CXCR2/CXCR1 receptor antagonists to explore the role of the heterocycle on chemokine receptor binding affinities, functional activity, as well as oral exposure in rat. The nature of the heterocycle as well as the requisite substitution pattern around the heterocycle was shown to have a dramatic effect on the overall biological profile of this class of compounds. The furyl class, particularly the 4-halo adducts, was found to possess superior binding affinities for both the CXCR2 and CXCR1 receptors, functional activity, as well as oral exposure in rat versus other heterocyclic derivatives.

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.

Comparative Effects of Azathioprine, Cyclophosphamide and Frentizole on Humoral Immunity in Mice

Data is presented comparing the activities of three immunosuppressive agents, cyclosphosphamide, frentizole and azathioprine in models of humoral immunity in mice. Cyclophosphamide and frentizole suppressed the primary and secondary plaque forming cell responses to sheep erythrocytes at lower doses than did azathioprine. Prolonged suppression of serum antibody titers occurred following short-term therapy with cyclophosphamide or frentizole, but not azathioprine. Azathioprine was also the least effective agent in suppressing a primary response to the T-independent antigen, trinitrophenylated lipopolysaccharide. All three agents were found to inhibit the induction and activity of suppressor cells at immunosuppressive doses.

Elevated levels of NO in both unchallenged and LPS-challenged C. parvum-primed mice are attributable to the activity of a cytokine-inducible isoform of iNOS.

Elevated levels of nitric oxide (NO2 ‐/NO3 ‐) were detected in the serum of mice 3–7 days after priming with Corynebacterium parvum (Propiombacterium acnes). The serum NO2 ‐/NO3 ‐ response was completely inhibited when C. parvum‐primed (C. parvum) mice were treated with NG‐monomethyl‐l‐arginine (l‐NMMA) or aminoguanidine (AG) on days 6 and 7 post priming. The response was also inhibited when the mice were treated with interleukin‐10 (IL‐10) and the cytokine was most effective when given in multiple doses beginning on the day of priming. In contrast to l‐NMMA and AG, IL‐10 had no effect on the serum NO2 ‐/NO3 ‐ response when administered to the mice on days 6 and 7 post priming. The inducible isoform of NOS (iNOS) appeared to be responsible for the elevated NO2 ‐/NO3 ‐ response in C. parvum mice because iNOS transcripts were readily detected in their livers. Moreover, these transcripts as well as the circulating levels of NO2 ‐/NO3 ‐ were dramatically reduced when the mice were treated with anti‐tumor necrosis factor α (anti‐TNF‐α) or antiinterferon‐γ (anti‐IFN‐γ) monoclonal antibodies (mAbs) during the priming interval. There was a modest increase (less than twofold) in the serum NO2 ‐/NO3 ‐response following a lipopolysaccharide (LPS) challenge to C. parvum mice (C. parvum/LPS mice). LPS had a more dramatic stimulatory effect if the levels of NO2 ‐/NO3 ‐ preexisting in C. parvum/LPS mice were reduced by treatment with l‐NMMA, AG, or IL‐10 before the challenge. Thus the levels of NO2 ‐/NO3 ‐that preexisted in C. parvum/LPS mice appeared to influence their ability to mount a NO2 ‐/NO3 ‐ response subsequent to the LPS challenge. The NO2 ‐/ NO3 ‐ response did not contribute to lethality in C. parvum/LPS mice because anti‐TNF‐α and anti‐IFN‐γ mAbs were protective but had no effect on serum NO2 ‐/NO3 ‐ levels when administered to mice 24 h before the LPS challenge. J. Leukoc. Biol. 61:24–32; 1997.

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.