Noninvasive assessment of hepatobiliary and renal elimination of cysteinyl leukotrienes by positron emission tomography.

N-Acetyl-leukotriene E4 has been identified as an endogenous, biologically less active cysteinyl leukotriene metabolite in rodents and humans. To evaluate the ratio of hepatobiliary to renal elimination of leukotrienes noninvasively by positron emission tomography (PET), we synthesized N-[11C]acetyl-leukotriene E4 by chemical N-acetylation of leukotriene E4. After the intravenous injection of N-[11C]acetyl-leukotriene E4 in normal rats and monkey, uptake by the liver and subsequent excretion into bile were largely responsible for its rapid elimination from blood. In the Cynomolgus monkey, renal excretion of the leukotriene into urine was of additional quantitative importance. Kinetic modeling indicated a mean transit time through the liver of 17 minutes and 34 minutes in rat and monkey, respectively; the corresponding hepatic excretion half-times amounted to 8.5 minutes and 16 minutes. In a mutant rat strain deficient in the hepatobiliary excretion of cysteinyl leukotrienes across the canalicular membrane, the apparent mean liver transit time was 54 minutes, and the hepatic excretion half-time was 29 minutes, indicating prolonged organ storage and metabolism. After transport from the liver back into the circulating blood of omega-oxidized and beta-oxidized metabolites of N-[11C]acetyl-leukotriene E4, renal excretion compensated for the impairment of hepatobiliary elimination in the transport mutant. Metabolite analyses in urine after intravenous injection of N-[3H]acetyl-leukotriene E4 indicated the extensive inactivation of N-acetyl-leukotriene E4 by beta-oxidation from the omega-end in the mutants. A similar shift from hepatobiliary to renal cysteinyl leukotriene elimination was monitored in rats with cholestasis due to bile duct obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of substance P in immediate-type skin reactions induced by staphylococcal enterotoxin B in unsensitized monkeys.

The staphylococcal enterotoxin B (SEB)-induced immediate-type skin reaction in unsensitized monkeys was used as a nonimmunologic mast cell stimulation to search for possible involvement of local neural mechanisms. Evidence is presented that substance P (SP) plays a predominant role in mediating intradermal SEB challenge in unsensitized monkeys. With a rabbit SP antiserum directed against the C-terminal region of SP, a concentration-dependent inhibition of SEB-induced skin reactivity could be demonstrated. Furthermore, a rabbit antiserum directed against the mast cell activating N-terminal part of SP was capable of impeding SEB-induced skin reactions totally. By use of SP antagonists, significant reduction of skin reactions evoked by SEB was found. Finally, capsaicin pretreatment of the skin caused a substantial inhibition of SEB-induced skin reactivity. These data suggest that SEB exerts its effect on cutaneous mast cells via stimulation of primary sensory neurons that contain SP. Moreover, a new in vivo model is described for studies of nerve-mast cell interactions.

Protection against the staphylococcal enterotoxin-induced intestinal disorder in the monkey by anti-idiotypic antibodies.

The staphylococcal enterotoxin serotype B (SEB)-induced enteric intoxication and the immediate-type reaction in the skin of unsensitized monkeys was used to define whether agents competing with SEB for target cell receptors may inhibit pathophysiological effects. For this purpose a duodenal provocation test was developed by use of a pediatric gastroscope, allowing the evaluation of the influence of antagonists on the intestinal disorder upon SEB challenge at the same duodenal site. First, carboxymethylation of histidine residues of SEB caused a complete loss of emetic and skin-sensitizing activity without changing the immunological specificity. However, carboxymethylated SEB is a strong inhibitor of enteric intoxications and immediate-type skin reactions upon SEB challenge. Second, after immunization of BALB/c mice with monoclonal anti-SEB antibodies, monoclonal antiidiotypic antibodies (anti-Id) were obtained by the "hybridoma technique" and purification by idiotype-affinity chromatography. Anti-Id specifically inhibited the binding of horseradish peroxidase-labeled anti-SEB to the ligand, and SEB blocked as well the interaction of these two antibody species, indicating a high degree of binding-site selectivity. Anti-Id completely protected against emetic response and diarrhea upon duodenal provocation with SEB and inhibited immediate-type skin reactions as well. Further, anti-Id acted as an antagonist without triggering biologic functions themselves. This shows that anti-Id constitute a useful tool to protect against a bacterial toxin-induced intestinal disorder.

Cysteinyl leukotrienes as mediators of staphylococcal enterotoxin B in the monkey.

The role of cysteinyl leukotrienes (LTs) in the action of staphylococcal enterotoxin B (SEB) was investigated in unsensitized monkeys using inhibitors of prostanoid synthesis and LT action and by measuring generation of LT in vivo. LY 171883, a selective LTD4/LTE4 receptor antagonist, proved highly efficient in inhibiting immediate-type hypersensitivity reactions in the skin and protecting against the emetic response provoked by SEB in a concentration-dependent manner. Inhibition of prostanoid formation by pretreatment of monkeys with indomethacin or aspirin did not influence SEB responses. Based on chromatographic and radioimmunologic analysis, the generation of endogenous cysteinyl LTs was demonstrated in vivo. The concentration of LTE4, the major biliary cysteinyl LT detected, increased ten-fold and a novel cysteinyl LT metabolite in urine indicated strongly enhanced LT generation upon challenge with SEB. Cysteinyl LTs are important mediators in the pathophysiology of SEB-induced enteric intoxication. Therefore, cysteinyl LT antagonists may be of therapeutic value in the treatment of this intestinal disorder.

Staphylococcal enterotoxin B as a nonimmunological mast cell stimulus in primates: the role of endogenous cysteinyl leukotrienes.

The immediate-type skin reaction and the emetic response in unsensitized monkeys on challenge with staphylococcal enterotoxin B (SEB) were studied to define the role of cysteinyl leukotrienes (LTs) in the action of the toxin. LY 171883, a selective LTD4/LTE4 receptor inhibitor, antagonized SEB-induced skin reactions and emetic responses completely. Inhibition of prostanoid formation by indomethacin, however, and pretreatment with BW 755C, a dual lipoxygenase and cyclooxygenase inhibitor, did not influence these reactions. The generation of endogenous cysteinyl LTs upon intragastric SEB administration was established in vivo. There was a tenfold increase in LTE4, the major biliary cysteinyl LT, and a novel cysteinyl LT metabolite in urine occurred, indicating strongly enhanced LT generation on SEB challenge. These results provide the first evidence that cysteinyl LTs may be important mediators in the pathophysiology of SEB-induced effects, as a model for pseudo-allergic reactions.

Anti-idiotypic antibodies as staphylococcal enterotoxin receptor probes on monkey mast cells.

The immediate-type skin reaction in unsensitized monkeys upon challenge with staphylococcal enterotoxin B (SEB) was studied to define the role of mast cell receptors in the action of the toxin. For this purpose anti-idiotypic antibodies (anti-Id) were raised in BALB/c mice against monoclonal anti-SEB antibodies and purified by idiotype affinity chromatography. Anti-Id completely abolished skin reactions upon challenge with SEB without having biological functions itself. The data are compatible with the view that receptors for staphylococcal enterotoxin actually exist on the mast cell membrane of primates and anti-Id may be of potential value to influence the course of staphylococcal enterotoxin-mediated effects.

Metabolism and analysis of cysteinyl leukotrienes in the monkey.

Predominant hepatobiliary elimination from blood and subsequent enterohepatic circulation of cysteinyl leukotrienes is demonstrated in the monkey Macaca fascicularis. From intravenous [3H]leukotriene C4, about 40% were recovered as metabolites in bile and about 20% in urine within 5 h. [3H]Leukotriene E4 was a predominant metabolite of defined structure in blood plasma, bile, and urine. From intraduodenal [3H]leukotriene C4, about 5% were recovered as metabolites in bile and about 8% in urine within 8 h. Endogenous cysteinyl leukotrienes generated in vivo were measured after implantation of a subcutaneously looped biliary bypass. Tapping of the loop allowed access to bile and prevented interference by leukotrienes produced by surgical trauma (Denzlinger, C., Rapp, S., Hagmann, W., and Keppler, D. (1985) Science 230, 330-332). Endogenous cysteinyl leukotrienes were analyzed in bile, urine, and blood plasma by the sequential use of high-performance liquid chromatography and a radioimmunoassay that was optimized for leukotriene E4 as a predominant metabolite detected in the tracer studies. Biliary leukotriene E4 rose from less than 0.2 to 9 nmol/liter, when leukotriene synthesis was elicited in anesthesized monkeys by staphylococcal enterotoxin B administered intragastrically. This study provides an approach to the analysis of cysteinyl leukotrienes in primates and serves to define the role of these mediators under pathophysiological as well as physiological conditions in vivo.

Anti-idiotypic antibodies that inhibit immediate-type skin reactions in unsensitized monkeys on challenge with staphylococcal enterotoxin.

The staphylococcal enterotoxin B (SEB)-induced immediate-type skin reaction in unsensitized monkeys was used as a nonimmunological mast cell stimulus to examine whether the toxin exerts its effect via specific receptors on the target cell membrane. Anti-idiotypic antibodies (anti-Id) were raised in BALB/c mice against monoclonal anti-SEB antibodies (anti-SEB) and purified by idiotype affinity chromatography. The anti-Id nature of the antibody was demonstrated by its ability to inhibit the binding of 125I-labeled anti-SEB to the ligand in a concentration-dependent manner. Moreover, binding of anti-SEB to anti-Id was antagonized by the SEB ligand in a competitive way. These antibodies completely abolished skin reactions in unsensitized monkeys on challenge with SEB and impeded those provoked by staphylococcal enterotoxins A and C1 but did not have the biological activity of the toxin. These data are compatible with the view that receptors for staphylococcal enterotoxins may exist on the membrane of mast cells in the skin of unsensitized monkeys. The data suggest an experimental approach for producing anti-cell receptor antibodies that are of potential value to influence the course of staphylococcal enterotoxin-mediated effects.

Skin reactivity of unsensitized monkeys upon challenge with staphylococcal enterotoxin B: a new approach for investigating the site of toxin action.

The correlation between skin tests and emetic responses in unsensitized monkeys was used to elucidate the cellular site of action of staphylococcal enterotoxin B (SEB). Evidence is presented that SEB administered intradermally provoked immediate-type skin reactions associated with mild degranulation of cutaneous mast cells. The cytoplasma showed signs of synthetic and metabolic activity, with formation of vesicles and increased prominence of mitochondria. Carboxymethylation of histidine residues of SEB altered the molecule (cSEB) from more alkaline components to more acidic species with increased microheterogeneity. This modification caused a loss in toxicity and completely abrogated the skin-sensitizing activity without changing the immunological specificity. cSEB, however, could compete with SEB for binding sites on the target cell surface. Previously, compound 48/80-treated skin sites behaved refractively to challenge with SEB, indicating that mediators from cutaneous mast cells are required for SEB-induced skin reactions. Skin reactions as well as emetic responses challenged with SEB were completely inhibited by H2 receptor antagonists and calcium channel blockers but not by H1 antihistamine or competitive antagonists of serotonin. This new approach provides a model for investigating the mechanisms of SEB action.

Direct skin test in highly sensitized guinea pigs for rapid and sensitive determination of staphylococcal enterotoxin B.

The direct skin test in highly sensitized guinea pigs was developed as a rapid and extremely sensitive assay for detection of staphylococcal enterotoxin B (SEB) in foods. This report details the experimental conditions required to elicit optimal sensitization of guinea pigs to SEB. An intense and persistent immunoglobulin E (IgE) anti-SEB response was established in strain 13 guinea pigs pretreated with cyclophosphamide followed by four sensitizing doses of 10 micrograms of SEB 1 month apart. The conditions, however, optimal for eliciting IgE responses led to a sustained failure to produce antibody of the IgG1 subclass. With the use of highly sensitized guinea pigs, one can achieve a sensitivity ranging from 0.1 to 1.0 pg of purified SEB by the direct skin test for at least 7 months after the last challenge. For analysis of SEB in food extracts, the entire assay can be accomplished within 20 min with a sensitivity of 10 to 100 pg SEB per ml of prepared food samples, and the recovery of enterotoxin from spiked food products ranged between 75 and 89% of the amount added.