Local effects of granulomatous inflammation on functional activation of T cells in athymic mice.

We investigated the effect of granulomatous inflammation in skin on lymphocyte maturation in athymic (nu/nu) mice. Hepatic egg granulomas developed in euthymic (nu/+) mice with schistosomiasis were transplanted into skin of nu/nu mice. During skin granuloma development the rate of DNA synthesis and interleukin 2 activity of lymphocytes from lymph nodes, with and without concanavalin A stimulation, showed that the nu/nu cells were activated to levels of untreated nu/+ lymph node cells. Activation of splenic lymphocytes was not detected in the grafted nu/nu mice. Also, immunohistochemical staining demonstrated an increase in cells expressing Thy 1.2, Lyt-1 or L3T4 surface markers in the skin and lymph nodes, but not in spleen. The findings indicate that a granulomatous reaction in nu/nu mouse skin induces local, but not systemic, proliferation and differentiation of lymphocytes, to a low degree compatible with resting nu/+ mice.

Skin granuloma formation in mice immunosuppressed by cyclosporine.

Granulomatous tissue reactions appear in athymic mouse skin, indicating that initiation of granuloma formation may be T-cell independent. To further evaluate the relationships between granuloma formation and T-cell function, we treated euthymic BALB/c mice with cyclosporine (Cs), a potent immunosuppressive drug, injected intramuscularly (150 mg/kg/day) 5 times a week. Hepatic granulomas were isolated from mice with schistosomiasis and transplanted into the skin of mice treated with Cs for 2 weeks. Cyclosporine injection was continued for 3 additional weeks. Blood levels of the drug increased during treatment (489 ng/ml at 2 weeks and 822 ng/ml at 5 weeks). Morphologically identical granulomas developed in both treated and untreated mice. Examination for T-cell functions showed that by the end of 2 weeks treatment, concanavalin A, phytohemagglutinin responses, and IL-2 activity were markedly depressed, and IL-2 receptor expression was not detected in either lymph nodes or spleen of the Cs-treated mice; however, after hepatic granuloma graft, T-cell functions in regional lymph nodes, but not in spleen, as well as peripheral blood eosinophilia were stimulated in Cs-treated mice. These data strongly suggest that intact T-cell activity is not essential for the initiation of granuloma formation. In addition, granuloma grafts appear to stimulate Cs-resistant T-cell activation locally, which amplifies and organizes the granulomatous response.

A novel synthetic vitamin A-like compound (a polyprenoic acid derivative, E-5166) inhibits the release of arachidonic acid stimulated by epidermal growth factor.

Little is known about the mechanisms of anti-inflammatory activity of retinoids. A new synthetic vitamin A-like compound (polyprenoic acid derivative, E-5166) has a strong in vitro binding affinity to intracellular binding proteins for acidic retinoids. In order to elucidate the anti-inflammatory activity of E-5166, we studied the effect of E-5166 on the epidermal growth factor (EGF)-stimulated arachidonic acid (AA) release of pig epidermis. E-5166 significantly inhibited the EGF-stimulated AA release and this inhibitory effect of E-5166 required a longer incubation than hydrocortisone did. Furthermore, E-5166 inhibited the EGF-stimulated phosphatidylinositol (PI) turnover of pig epidermis. These results indicate that E-5166 inhibited the EGF-stimulated AA release through the inhibition of the EGF-stimulated PI turnover.

Transfer of granulomatous inflammation with nonviable preparations of schistosome granulomas in naive mice.

Hepatic granulomas of euthymic (nu/+) mice infected with Schistosoma mansoni were freeze-dried or freeze-thawed 3 times and transplanted subcutaneously into naive nu/+ and athymic (nu/nu) mice. The grafted sites, studied histologically, showed formation of organized granulomas in nu/+ mice similar to donor granulomas as observed after grafting of freshly isolated granulomas. On the other hand, in nu/nu mice, the nonviable transplants elicited small and disorganized granulomas, like hepatic granulomas in nu/nu mice with schistosomiasis, but different from fresh nu/+ transplants in nu/nu skin. The findings indicate viable cells are not required for transfer of granulomatous reactions, but T cells are needed for full expression.

T-cell independent transfer of organized granuloma formation.

A direct role for delayed hypersensitivity in organized granuloma formation has been emphasized in the literature, but some clinical and experimental studies have suggested a more complicated relationship. In order to further evaluate T-cell function in cutaneous granulomatous inflammation, hepatic granulomas isolated from athymic mice with schistosomiasis were grafted into the skin of athymic and euthymic mice. Granulomas developed in athymic mice, as seen previously after transplantation of euthymic hepatic granulomas, indicating that granulomas develop in the skin without T-cell function. However, the granulomas in athymic mice skin were smaller and lacked eosinophils and mast cells, whereas the athymic mouse granulomas grafted in euthymic mice skin were larger, better organized, and associated with tissue eosinophilia. These findings suggest that granuloma initiation itself is T-cell independent, but that T-cell participation enhances the granulomatous reaction.

Ionophore A23187 stimulates arachidonic acid release and cyclic GMP accumulation in pig epidermis.

We studied the effect of Ionophore A23187 (A23187) on phospholipid degradation and the accumulation of cyclic GMP in pig epidermis. A23187 stimulated the release of AA, probably partially through the activation of phospholipase A2. A23187 also stimulated the accumulation of epidermal cyclic GMP, but the activity of cyclic GMP-dependent phosphodiesterases was not altered. Mepacrine, an inhibitor of phospholipase A2, inhibited the A23187-stimulated accumulation of cyclic GMP. The results suggest that A23187 stimulates the accumulation of epidermal cyclic GMP by a calcium-dependent process which also requires products of phospholipid degradation.

Effects of 12-o-tetradecanoylphorbol-13-acetate and polyprenoic acid derivative on calcium-activated phospholipid-dependent protein kinase of pig skin.

12-o-Tetradecanoylphorbol-13-acetate (TPA) activates calcium-activated, phospholipid-dependent protein kinase (protein kinase C) partially purified in an ion exchange column from pig epidermis. Protein kinase C was activated by TPA in a concentration-dependent manner with simultaneous addition of Ca2+ and phospholipid. Polyprenoic acid derivative (E5166) which is a newly synthesized retinoic acid derivative, inhibited the TPA activation of protein kinase C. This inhibition may explain the mechanisms by which retinoids inhibit TPA-induced tumor promotion.

Epidermal growth factor stimulates release of arachidonic acid in pig epidermis.

Epidermal growth factor (EGF) at physiologic concentrations (0.001-0.1 microgram/ml) stimulated the release of [14C] arachidonic acid [14C-AA] from pig epidermis. Although EGF stimulated the release of AA in the absence of exogenously added calcium to some extent, the addition of calcium (0.3-1.2 mM) significantly potentiated the release of AA stimulated by EGF. Ionophore A23187, which is known to stimulate phospholipase A2 activity by opening the calcium gates, potentiated the EGF-stimulated release of AA. The stimulatory effect of EGF was partially inhibited by the addition of mepacrine (70% inhibition at 10 microM) and by the pretreatment of hydrocortisone (60% inhibition at 1.0 microM). The loss of 14C-labeled phospholipids in pig epidermis was mainly due to the degradation of 14C-labeled phosphatidylcholine. Present results and recent reports by other workers suggest that EGF stimulates phospholipase A2 activity and result in the increased release of AA.

Epidermal growth factor stimulates tyrosine phosphorylation of pig epidermal fibrous keratin.

Epidermal growth factor (EGF) stimulated phosphorylation of pig epidermal proteins, one of which was pig epidermal keratin. In order to further characterize phosphorylated proteins and specify the EGF-dependent protein phosphorylation, we attempted to identify phosphorylated keratin proteins and to analyze phosphorylated phosphoamino acids of keratin proteins stimulated by EGF. Four major polypeptide bands of pig epidermal keratin were immunoprecipitated by antihuman callus keratin antibody which reacted with fine networks of fibrous keratin of pig epidermal cells grown in vitro. Four major polypeptide bands were greatly phosphorylated by EGF in a dose-dependent manner. The analysis of phosphorylated phosphoamino acids revealed that EGF stimulated tyrosine phosphorylation of pig epidermal fibrous keratin.

Insulin-like activity of photochemically obtained monovalent monomeric concanavalin A in the presence of anti-concanavalin A antibodies: dependence on multivalency for stimulation of glucose oxidation of rat fat cells.

Insulin-like action of monovalent monomeric concanavalin A (m-Con A) was examined in rat adipocytes in the presence of anti-m-Con A antiserum. The antisera from rabbits injected with m-Con A reacted with not only monovalent monomeric but also tetravalent tetrameric concanavalin A (alpha-Con A) in Ouchterlony double diffusion analysis. m-Con A alone did not show any appreciable effect on glucose oxidation of adipocytes while it slightly inhibited glycerol release stimulated by epinephrine. In contrast, exposure of adipocytes to m-Con A in the presence of antibodies to m-Con A resulted in stimulation of glucose oxidation and inhibition of epinephrine-stimulated lipolysis. The stimulation and the inhibition with m-Con A in the presence of the antibodies were of the same degree as those with alpha-Con A. Both alpha- and m-Con A were slightly active in inhibiting 125I-labeled insulin binding. These results demonstrate that the ability of anti-m-Con A antiserum to aggregate m-Con A bound to receptors on the isolated-adipocyte plasma membrane allowed m-Con A to mimic the biological activity of insulin and that the aggregation of receptors for ligands other than insulin can induce insulin-like action in rat fat cells.