Experimental studies on the bridging hypothesis of anaphylaxis: haptenic determinants required to elicit immediate-type reactions in calf skin by separate or concurrent sensitization with reagins of different specificity.

The present study has established, that cows suitably immunized with either DNP-edestin (DNP-Ed), di-DNP-gramicidin-J [(DNP)(2)-Gram], respectively, or p-azobenzenearsonate-Ed (ABA-Ed) synthesized and secreted reaginic antibodies (IgE) into colostrum. Whereas ABA-Ed failed to elicit more than a low response, there was however a persistent and increased antibody synthesis between 10 and 56 days after priming with DNP-Ed. Bivalent and multivalent DNP haptens differing in molecular size, degree of substitution, and rigidity were compared for their effectiveness in eliciting Prausnitz-Küstner (P-K) reactions in either newborn colostrum-deprived calves or in those 4 wk of age. The sensitization with reaginic anti-DNP antibody has been accomplished either by feeding colostrum of the immunized dam or by intradermal injection of reaginic serum or colostral whey. It could be demonstrated that equimolar doses of the bivalent alpha,N-(epsilon,N-DNP-aminocaproyl-)-epsilon,N-DNP-L-lysine and the multivalent dinitrophenylated bovine serum albumin were equally effective in eliciting reactions in skin sites provided that a high affinity antibody was used for sensitization. By contrast, the comparatively rigid, bivalent hapten, (DNP)(2)-Gram consistently failed to induce comparable reactions. Furthermore, it was clearly shown that optimal distances of determinant groups on the haptenic molecule are a prerequisite for positive P-K reactions, since alpha,epsilon,N-bis-DNP-lysine failed to induce comparable reactions. Concurrent sensitization of skin sites with reaginic anti-DNP and anti-ABA antibodies provides the final proof that cross-linking of two adjacent reaginic molecules on the mast cell surface by a bivalent hapten is required for effective elicitation of immediate-type reactions. This has been accomplished by utilizing the bivalent epsilon,N-DNP-alpha,N-[(4-hydroxy-3-azobenzenearsonic acid)-phenacetyl]-L-lysine (DNP-ABA) carrying noncross-reactive haptenic groups, which was consistently effective in eliciting P-K reactions in doubly but never in singly sensitized skin sites. It is apparent from the results that equimolar doses of monovalent haptens could completely inhibit the response to DNP-ABA. The present studies finally establish that mast cells of newborn colostrum-deprived calves lack IgE molecules on their surface. Thus, mast cells of newborn calves may be unique, to investigate the molecular mechanisms involved in immediate-type reactions more precisely.

Effect of platelet-activating factor on human polymorphonuclear leukocyte enhancement of chemiluminescence and antibody-dependent cellular cytotoxicity.

The effect of platelet-activating factor on human granulocytes was determined by luminol-dependent chemiluminescence (CL), antibody-dependent cellular cytotoxicity (ADCC), and rosette assay. An activation of the respiratory burst could be measured by CL and be shown to depend on the presence of extracellular calcium. This direct stimulation of PMN was proved to be inhibited by oxygen radical scavengers as well as by the calcium channel blocker diltiazem. Furthermore, the presence of PAF enhanced the activation of PMN via Fc- or C3b-receptors, as demonstrated by CL and ADCC. On the other hand, no influence on the rosette-forming capacity of PMN could be detected. The results support the concept of the import role of PAF in inflammatory processes.

The immune system in chickens.

The most sophisticated feature of the immune system expressed in vertebrates is recognition of foreign molecules by distinct types of immunocompetent cells. Birds are the first vertebrates in which a clear dichotomy of the lymphoid system has been established: 1. Thymus-derived (T) lymphocytes, the effector cells in cell-mediated (immunity and 2. Bursa-derived (B) lymphocytes, the precursor cells of the antibody-synthesizing plasma cell. Lymphocyte differentiation begins with the migration of haemopoietic stem cells from yolk sac and liver into bursa and thymus early in embryonic life followed by clonal expansion within the central lymphoid tissues. The second stage of differentiation is considered to begin with the migration of lymphocytes to the peripheral lymphoid tissue (spleen and lymph nodes). Based on genetic information, B cells are capable of recognizing foreign antigens by specific immunoglobulin molecules whereas T cell receptors are presumed to be products of the immune response genes. Surface differences provide the basis for analyzing the population dynamics of T and B cells and the pathway of immunologic diseases as well. There is compelling evidence that antigen entering the body stimulates a conventional type of systemic immune response. Antigen which remains in the mucosa, however is apt to induce a local type of response. The responses include B cells, which remain in the peripheral lymphoid tissues for the most part, and secrete immunoglobulins of different classes (IgM, IgG, IgA). T cells, however, are represented by the circulating pool of lymphocytes, and do not synthesize antibodies but instead release various mediators upon interaction with the antigen, which play a role in cell-mediated immunity. The antibody response to most antigens requires cooperation between T and B cells and macrophages as well, but in some instances T cells can also suppress B cell function. There is some evidence that the immune response of chickens is genetically controlled, which is particularly pertinent to susceptibility and resistance to diseases. Since humoral and cell-mediated immunity can separately be affected by removal of central lymphoid organs, chickens may serve as a useful model to elucidate the function of the immune system in health and disease.

Appearance and immunohistochemical localization of UDP-glucose: coniferyl alcohol glucosyltransferase in spruce (Picea abies (L.) Karst.) seedlings.

UDP-glucose: coniferyl alcohol glucosyltransferase was detected in hypocotyls of spruce (Picea abies (L.) Karst.) seedlings. Enzyme activity rose after germination to maximal activity on about the 10th day and then rapidly declined. An antiserum against the glucosyltransferase isolated from cambial sap of spruce (Schmid and Grisebach, 1982, Eur. J. Biochem. 123, 363-370) was employed for localization of the enzyme in cross sections of hypocotyls from 10-d-old spruce seedlings, using the immunofluorescent technique. The results show that the transferase is located predominantly in the epidermal and subepidermal layers and in the vascular bundles. Intracellularly, the enzyme is located in the parietal cytoplasmic layer. The results corroborate the assumption that coniferin (coniferyl alcohol ß-D-glucoside) participates in lignification of spruce.

Relationship between enterotoxic- and T lymphocyte-stimulating activity of staphylococcal enterotoxin B.

The enterotoxins produced by Staphylococcus aureus cause a gastrointestinal intoxication probably via their action on intramucosal neuronal cells. Staphylococcal enterotoxins are also the most powerful mitogens known, activating CD3+ T lymphocytes of several species in a clonally variable and MHC class II-dependent fashion. We examined a possible relationship between enterotoxic and mitogenic activity of staphylococcal enterotoxin serotype B (SEB). We used a monoclonal anti-Id directed against the combining site of an anti-SEB mAb. This anti-Id failed to elicit an enteric response by itself but could block the enteric response in monkeys to a 6000-fold excess of SEB. The anti-Id was mitogenic, however, for human and monkey T cells, triggering a fraction of CD4+ and CD8+ T cells. Not all SEB-reactive T cells were activated by the anti-Id. The anti-Id bound to T cells with a similarly low affinity as did SEB. Additional evidence for a separation of enterotoxic and mitogenic activity comes from studies with carboxymethylated SEB. Although this modified SEB had lost its enterotoxic activity, it was as mitogenic as the unmodified molecule. These results support the notion that the enteric reaction to SEB is not mediated via its effect on T lymphocytes. We conclude that SEB and anti-Id might bind to a common structure of different receptors on T cells and target cells in the intestinal mucosa, probably peripheral sensory neurons.

Antibody-dependent cellular cytotoxicity and chemiluminescence as a tool for studying the mechanism of anti-glomerular basement membrane nephritis. The role of the cytotoxic potential of polymorphonuclear granulocytes and monocytes.

An in vitro system was elaborated to study the mechanisms inducing tissue injury in anti-glomerular basement membrane (GBM) nephritis. Collagenase-digested GBM (CGBM) was covalently attached to Fab' specific for chicken red blood cells (CRBC). The preparation of the CGBM-Fab' conjugate was effected by using iodoacetyl chloride coupling in analogy to a procedure described by Chiang & Koshland (1979). This conjugate was used for coating CRBC (CGBM-CRBC). In this system the antibody-dependent cellular cytotoxicity (ADCC) and the chemiluminescence mediated by purified bovine polymorphonuclear (PMN) and mononuclear cells (MNC) as well as rabbit MNC against CGBM-CRBC were compared in the presence of sheep anti-GBM IgG. All three cell populations were potent effectors in ADCC and chemiluminescence and evidence was obtained that the cytotoxic potential of MNC has to be attributed to monocytes. If compared at low effector target cell ratios in a 2 hr assay bovine PMN, however, were significantly more efficient than bovine MNC. The extent of both ADCC and chemiluminescence was directly related to the amount of anti-GBM IgG present in the system. Based on the inhibition experiments with oxygen intermediate scavengers, both ADCC and chemiluminescence by bovine PMN is dependent on generation of reactive oxygen species indicating that such radicals could play a role in vascular (endothelial) injury as documented in the loss of structural integrity of GBM.

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.

Appearence and localization of a ß-glucosidase hydrolyzing coniferin in spruce (Picea abies) seedlings.

ß-Glucosidase activity for coniferin (coniferyl alcohol ß-D-glucoside) is not present in spruce (Picea abies L. Karst.) seeds but appears in the young seedlings. Lignification starts at ca. day 9 of germination in the vascular bundles. An antiserum against glucosidase 1 isolated from spruce seedlings (Marcinowski and Grisebach, Eur J. Biochem. 1978) was employed for the localization of the enzyme in cross sections of hypocotyls using immunofluorescent technique. The results indicate that at this stage of development the glucosidase is localized at the inner layer of the secondary cell wall. Glucosidase activity was present in all cells of the investigated hypocotyl tissue.

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.

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.

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.

Stimulation and suppression of rat mast cell functions by alloantibodies.

The stimulatory as well as the inhibitory capacity of alloantisera has been investigated with respect to rat mast cell functions. Alloantibody against alloantigens coded for by the major histocompatibility (H-1) gene region promoted histamine release from purified LEW mast cells. This process was found to be complement-independent but demonstrated an absolute requirement for calcium. Pretreatment of mast cells with anti-H-1 antisera in the absence of calcium markedly suppressed the IgE-dependent histamine release challenged either by antigen or by anti-IgE antibody. The alloantisera, however, did not interfere with the ability of compound 48/80-associated histamine liberation. Additionally, antibodies specific for H-1 antigens were highly effective in inhibiting the binding of IgE to the mast cell surface. Alloantisera absorbed with erythrocytes lost their capacity to block mast cell functions. Based on these data the possible ralationship between H-1 alloantigens and the IgE receptor on the mast cell surface is discussed.

In vitro studies on the selective binding of IgG from different species to tissue sections of the bovine mammary gland.

Tissue sections of the mammary gland of cattle provide a sensitive and reproducible model for studying the initial events of the selective transport of bovine IgGs by the acinar epithelium (AE). Mammary tissue was reacted with purified antibody to horseradish peroxidase (HRP) and exposed to HRP. The sites of peroxidase activity were revealed cytochemically. The highly selective binding of IgGs by AE of the colostrum-forming gland but never of the lactating gland was most notable, whereas IgG1 and IgG2 subclasses and IgM and IgA lacked any binding capacity. An inhibition assay showed that the inhibitory capacity of IgGs was abolished by either alanylation or acetylation, whereas binding of IgGs was blocked by both Fab/c and isolated H-chains, but not by F(ab')2, Fab and Fc. The inhibitory pattern suggests that the region on the IgGs molecule involved in binding to the AE receptor may be located within the CH2 domain. In addition, IgG preparations of human, sheep and rabbit origin were found to be equally efficient in inhibiting the binding of IgGs to AE, whereas no inhibition was obtained with nonmammalian (turtle and chicken) IgG. Similarly, human myeloma proteins of the subclasses IgG1 and IgG3 caused complete inhibition, while IgG2 and IgG4 were inefficient. It is suggested that molecular evolution of IgG within mammalian species is accompanied by the conservation of structures fitting to receptors on the cell surface of different species.

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.