The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin 0, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins. The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor. The toxins also induce the release of leukotriene-metabolizing enzymes such as y-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4. Dipeptidase activity is more pronounced than they -glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan.
The effect of serum factors on Ig synthesis (IgE, IgG) in vitro was analyzed. Spleen and mesenteric lymph node cells were obtained from Nippostrongylus brasiliensis-infected and non-infected mice. Sera and ammonium sulphate precipitated serum fractions from mice of different genetic origin (Balb/c - H-2d, A.CA - H-2f, B10.G - H-2q) suppressed in vitro IgE synthesis whereas a pronounced enhancement of IgG antibody synthesis was obtained in several experiments. Our results obtained with sera from both high and low IgE responder strains demonstrated that no strain specificity exists as to the inhibitory efficacy of mouse sera for total IgE synthesis in vitro. The suppressive activity of the mouse sera was concentrated in a fraction precipitated with 20%-50% saturated ammonium sulphate. Amicon XM50 ultrafiltration suggested that this fraction had an apparent molecular weight greater than 50,000 daltons. Suppressive activity was removed by immunoadsorption of the 20-50% fraction with anti-IgE Sepharose. After exogenous addition of monoclonal IgE to an inactive fraction in vitro neither the fraction enriched in IgE nor monoclonal IgE alone were able to suppress IgE synthesis in the culture. Our results suggest that one or more serum factors in the presence of IgE are responsible for the suppression of total IgE synthesis in vitro.
We investigated the IgE-antibody response in 36 inbred mouse strains during infection with Nippostrongylus brasiliensis. With regard to the N. brasiliensis-specific IgE-antibody activity responder and nonresponder mice were obtained. Mice with the H-2-f haplotype (A.CA, B10.M, A.TFR 5) are high specific responded. It is suggested that one Ir gene for the N. brasiliensis-induced IgE-antibody response is localized within the K to J region of the mouse major histocompatibility complex. To obtain a medium IgE-antibody response it appears that a complementation between two Ir genes is necessary. An additional genetic control beyond the H-2 complex cannot be excluded. As to the total nonspecific serum IgE levels the parasitic infection leads to a 20- to 40-fold increase in high and low specific responder strains as well. Pretreatment of high responder mice (A.TFR 5, B10.M) with sera that were obtained from normal or complete Freund’s adjuvant treated low (B10.G) and nonresponder (A.TFR 1) mice led to significant inhibition or enhancement of the N. brasiliensis-specific IgE-antibody response.
In vitro IgE synthesis by lymphoid cells was studied during the course of infection of mice with Nippostrongylus brasiliensis. The studies involved inbred strains of mice which had been shown to be high IgE responders (A.CA, B10.M), or non-responders (Balb/c, B10.D2) to parasite antigen. In addition, F1 hybrids of low and high responders and irradiated non-responders were studied. Infection with N. brasiliensis led to an increase in IgE synthesis in vitro which was most pronounced during reinfection of mice. Addition of mitogens e.g. pokeweed mitogen (PWM), lipopolysaccharide (LPS), concanavalin A (ConA) to the cultures induced enhancement, suppression or had no effect on IgE synthesis. Addition of N. brasiliensis homogenate or worm culture supernatant led to a fluctuating pattern of IgE synthesis. No correlation was found between lymphocyte proliferative response to mitogen and worm antigens and IgE synthesis. Our data suggest, that PWM is more likely to enhance IgE synthesis in vitro than LPS or ConA. An enhancement is more easily observed with the cells of non-infected animals or during the early phase of infection or reinfection. The mitogen-induced increase in IgE synthesis did not exceed the values obtained during infection or reinfection.
An eosinophil chemotactic factor (ECF) can be released from human polymorphonuclear neutrophils (PMN), rat mononuclear and rat mast cells by the calcium ionophore (A23187), during phagocytosis, by arachidonic acid and phospholipase A2. It has been suggested that stimuli such as the ionophore and the phagocytic event lead to phospholipid turnover with the generation of arachidonic acid which is subsequently transformed by a lipoxygenase-like enzyme into ECF. Addition of phospholipids such as phosphatidylethanolamine and phosphatidvlinositol during ionophore stimulation of various cells increased the ECF release significantly. ECF activity is also enhanced in the presence of indomethacin at concentrations which inhibit prostaglandin synthesis. With bromphenylacylbromide and eicosatetraynoic acid, ECF generation as well as the chemotaxis of eosinophils is inhibited suggesting that the phospholipase A2-arachidonic acid pathway represents a common link for ECF release as well as for the chemotaxis of eosinophils. From the cytosol of human PMN an ECF-containing enzyme was obtained. Incubation of phospholipase A2 and phospholipids with the ECF-converting enzyme led to potent ECF indicating that addition of phospholipids provides the soluble ECF-generating system with an additional source of arachidonic acid. The data represent a molecular approach to analyze the mechanisms of ECF release from soluble components after immunological triggering of the cells.
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