Effects of cytochrome P-450 inhibitors on the in vivo metabolism of all-trans-retinoic acid in rats.

This study examines the effects of ketoconazole, R 75 251 and some other cytochrome P-450 inhibitors on the in vivo metabolism of all-trans-retinoic acid (RA) in normal rats. Oral treatment with ketoconazole or R 75 251 (40 mg/kg, -1 hr) reduced the elimination rate of i.v. injected RA from plasma: the half-life of RA increased from 27 min in control-treated animals to 43 min and 76 min after dosing with ketoconazole and R 75 251, respectively. However, neither drug had an effect on the distribution volume of the retinoid. Two hours after i.v. injection of RA, residual plasma levels of the retinoid were 11.2 ng/ml in ketoconazole and 22.7 ng/ml in R 75 251-treated rats. The other P-450 inhibitors, aminoglutethimide, cimetidine, itraconazole, metyrapone and saperconazole, showed no sparing effect on RA elimination: plasma levels of the acid were below 1 ng/ml, as in control-treated animals. Administration of ketoconazole or R 75 251 (40 mg/kg, -2 hr) to rats also enhanced endogenous plasma concentrations of RA. Levels of the retinoid were raised from mostly undetectable values (less than 0.5 ng/ml) to 1.3 +/- 0.1 and 2.5 0.1 ng/ml after treatment with ketoconazole and R 75 251, respectively. These data are indicative of the important contribution of the cytochrome P-450 enzyme system to the in vivo metabolic process of RA. In vivo inhibition of the P-450 pathway not only increased the biological half-life of exogenously administered RA, but also enhanced the endogenous plasma level of this vitamin A derivative.

Arabinogalactan- and dextran-induced ear inflammation in mice: differential inhibition by H1-antihistamines, 5-HT-serotonin antagonists and lipoxygenase blockers.

Intravenous injection of arabinogalactan or dextran together with pontamine sky-blue dye into mice increased vascular permeability and led to marked blueing of the ears. Arabinogalactan caused a rapidly progressing ear blueing (maximal coloration 20-30 min after injection). This response was suppressed by pretreating the animals with the histamine H1-antihistamines levocabastine and loratadine. In contrast, dextran induced a slowly evolving ear inflammation (maximal coloration 60-90 min after injection), which was blocked by the 5-HT-serotonin antagonists cinanserin, metergoline and ritanserin. Furthermore, the dextran reaction was inhibited by the lipoxygenase (LO)/cyclooxygenase (CO) inhibitors BW540C, BW755C and phenidone and by the specific 5-LO inhibitor AA-861. Both arabinogalactan and dextran responses were inhibited by aprotinin, a kallikrein inhibitor, and the mixed H1/5-HT antagonists astemizole and azatadine. The inflammogenic activity of the polysaccharides was not affected by administration of the CO inhibitors indomethacin and suprofen, the thromboxane synthetase inhibitor dazoxiben, the H2-antihistamines cimetidine and ranitidine, the anticholinergics isopropamide or the PAF-antagonist L-652, 731. These data indicate the existence of distinctive endogenous molecules that mediate the pinnal extravasation reaction to both polysaccharides: histamine for arabinogalactan, serotonin and lipoxygenase-derived arachidonic acid metabolites for dextran.

Ketoconazole inhibits the in vitro and in vivo metabolism of all-trans-retinoic acid.

Ketoconazole, an antifungal agent and inhibitor of certain mammalian cytochrome P-450-dependent enzymes, was studied for its effects on the in vitro and in vivo metabolism of all-trans-retinoic acid (RA). In vitro, ketoconazole (Ki = 0.75 microM) inhibited, in an apparently competitive manner, the cytochrome P-450-mediated metabolism to 4-hydroxy- and 4-keto-retinoic acids by hamster liver microsomes. In vivo, ketoconazole suppressed the formation of polar RA metabolites by normal rats dosed intrajugularly with 200 ng of [3H]RA. After p.o. treatment with ketoconazole (2.5-40 mg/kg) given 1 hr before the [3H]RA injection, the radioactivity extracted from the liver consisted of 25 to 50% polar metabolites (control 66 +/- 1%) and 50 to 75% undegraded RA (control 34 +/- 1%) as evidenced by reverse-phase high-performance liquid chromatography. Time course experiments showed that ketoconazole's inhibitory effects lasted for 3 hr. Our data indicate the quantitative importance of the cytochrome P-450 enzymatic pathway in the biotransformation of RA. They also suggest that ketoconazole is capable of prolonging the biological half-life of RA and of improving the tissue levels of this compound.

Cytoprotective effects of disodium cromoglycate on rat stomach mucosa.

The cytoprotective effects of the anti-asthmatic drug, disodium cromoglycate (DSCG), on gastric mucosal necrosis induced by ethanol in rats were studied. Subcutaneous, but not oral, DSCG prevented the formation of gastric lesions and this effect was dose-dependent between 1.25 and 40 mg kg-1, with an ED50 value of 6.8 mg kg-1. Maximal cytoprotection occurred 15-30 min after DSCG treatment. Histological examination revealed that DSCG effectively protected the gastric mucosa against ethanol-induced vascular congestion, haemorrhage, epithelial desquamation and mucosal oedema. Enhanced production of endogenous prostaglandins, which are known cytoprotective compounds, could not explain the mucosal protection. At a dose of 40 mg kg-1, DSCG did not change prostaglandin E2 or 6-keto-prostaglandin F1 alpha concentrations in gastric mucosal tissue, although its cytoprotective activity was partially inhibited by prior treatment of the animals with indomethacin.

CD3 modulation inhibits pokeweed mitogen-induced T-cell help for immunoglobulin production.

The CD3 molecule is considered to be a signal transducer in the process of T-cell activation. Modulation of the CD3 molecule of peripheral blood T cells can be accomplished by incubation at 37 degrees C with UCHT-1, a mouse IgG1 anti-CD3 monoclonal antibody, under experimental conditions avoiding T-cell activation. We have examined the effect of CD3 modulation on T-cell-dependent polyclonal immunoglobulin (Ig) production induced by pokeweed mitogen (PWM) in cultures of peripheral blood lymphocytes. CD3 modulation strongly inhibited (greater than 80%) IgG and IgM production. This was due to inhibition of the production of soluble helper factors by the T cells, and not to induction of suppressor cells. These data support the concept that the CD3 molecule is an essential signal transducer in the process of PWM-induced helper T-cell activity, and that CD3 can function as a receptor transmitting negative signals to helper T cells.

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation.

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex.

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function.

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells.

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.

Human T lymphocyte activation by monoclonal antibodies; OKT3, but not UCHT1, triggers mitogenesis via an interleukin 2-dependent mechanism.

OKT3 and UCHT1 monoclonal antibodies, which recognize the same human T cell surface antigen, induce proliferation in T lymphocytes. In this report, we compared the mechanism by which these antibodies trigger DNA synthesis in human peripheral blood mononuclear cell (PBMC) cultures. Whereas PBMC from all donors tested were mitogenically inducible by OKT3, cells from only 25 of 40 donors were responsive to UCHT1 . UCHT1 treatment of PBMC from responders, but not from nonresponders, resulted in the expression by T cells of membrane binding sites reactive with anti-Tac monoclonal antibody, which specifies the human interleukin 2 (IL 2) receptor. UCHT1 -induced PBMC supernatants from nonresponders, but unexpectedly, also from responders, contained no measurable IL 2 activity. In keeping with this finding, anti-Tac monoclonal antibody failed to suppress UCHT1 -triggered [3H]thymidine incorporation into PBMC from responsive donors. By contrast, OKT3 treatment of PBMC from all donors led to the emergence of IL 2 receptors, and substantial IL 2 production, and the resultant DNA synthesis was inhibitable by anti-Tac antibody. These data indicate that the interaction of OKT3 and UCHT1 monoclonal antibodies with the same T cell structure leads to the induction of proliferation via two different mechanisms: one dependent on the availability of IL 2 (OKT3) and one independent on the production and processing of this lymphokine ( UCHT1 ). PBMC unresponsiveness to UCHT1 could therefore not be related to a dysfunction in IL 2 synthesis or IL 2 receptor display.

The mitogenic activity of OKT3 and anti-Leu 4 monoclonal antibodies: a comparative study.

The mitogenic properties of OKT3 (IgG2a) and anti-Leu 4 (IgG1), two monoclonal antibodies directed at the same surface antigen of human T cells, have been studied. Both antibodies, at subnanomolar concentrations, induced thymidine incorporation of comparable magnitude into human peripheral blood mononuclear cell cultures. Their mitogenicity reached peak values 3 days after stimulation, was totally inhibitable by monoclonal OKT11A antibody, and was strictly dependent on the presence of monocytes as auxiliary cells. Whereas mononuclear cells from all blood donors tested were proliferative to OKT3, cells from 12 of 30 donors (40%) were unresponsive to anti-Leu 4. The incidence of this unresponsiveness was significantly higher for cells from female than male donors, showing 60 and 20% nonresponders, respectively. Anti-Leu 4 unresponsiveness was found to be due to the inability of autologous monocytes to exert their auxiliary function; thus, lymphocytes from anti-Leu 4 nonresponders became mitogenically inducible by anti-Leu 4 after addition of monocytes from anti-Leu 4 responders, whereas lymphocytes from anti-Leu 4 responders failed to respond to anti-Leu 4 after supplementation of monocytes from anti-Leu 4 nonresponders. Considering the different immunoglobulin subtype of OKT3 (IgG2a) and anti-Leu 4 (IgG1), our results indirectly suggest that the auxiliary function of monocytes is mediated by their Fc receptors. Unresponsiveness to anti-Leu 4 could then be explained by the absence, paucity, or functional deficiency of monocytic Fc receptors for IgG1.

OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties.

OKT3, a monoclonal anti-human T cell antibody (IgG2), was found to induce DNA synthesis in human peripheral lymphocyte cultures. OKT3 induced maximal mitogenesis at a concentration of 10 to 20 ng/ml and was about 20-fold more potent than PHA as a mitogen. No high-dose inhibition of thymidine incorporation was noticed at concentrations up to 2.5 microgram OKT3/ml. The monovalent Fab fragment of OKT3 was also mitogenic but about 100 times less potent than the parent IgG. OKT3 appeared to be a T lymphocyte mitogen as only sheep red blood cell rosetting lymphocytes were responsive. Quantitative studies on the binding of 125I-labeled Fab fragment of OKT3 to human lymphocytes showed an average of 5.1 x 10(4) receptor sites/cell with an association of about 10(8) M-1 at 37 degrees C, with no heterogeneity of the cell binding sites. These data suggest a strong interaction of the monoclonal OKT3 with a limited number of identical T cell membrane receptors. As this interaction can trigger mitogenesis, the cell membrane determinant recognized by OKT3 could be described as a "T cell stimulation receptor." The mitogenecity of the lymphocytes is not solely dependent on cross-linking of these receptors.

Carbohydrate binding specificity of the lectin from the pea (Pisum sativum).

Hapten inhibition measurements on the precipitin reaction between Pisum sativum lectin and Pichia pinus phosphomannan showed the lectin to bind D-mannose, D-glucose, D-fructose and L-sorbose. Unmodified hydroxyl groups at the C-4 and the C-6 positions of the D-glucopyranose ring were essential for binding to the protein. Modification of the C-2 hydroxyl group was allowed in the D-glucopyranose ring but not in the D-mannopyranose configuration. Substitution of the hydroxyl hydrogen atom at the C-3 position of D-glucose increased the binding efficiency. With the exception of gentiobiose, the beta-linked glycobioses tested were not bound to the lectin, whereas the alpha-linked glycobioses were potent inhibitorsmin general, the P. sativum lectin was found to be less sensitive to structural variation of inhibiting carbohydrates than concanavalin A, the lectin from Canavalia ensiformis.