Basophil histamine release induced by a substance from stimulated human platelets

Platelet activation may occur during immunoglobulin E antibody (IgE)-mediated reactions. In these studies, we confirm that platelet-derived supernatants (PDS) induce histamine release from human mixed leukocytes containing basophils, one of the initial target cells in IgE-mediated reactions. In extending this observation, we have shown that this PDS-induced histamine release is both temperature- and calcium-dependent. Kinetic studies of release induced by PDS indicate that release is more rapid than that associated with IgE-dependent mechanisms. This platelet-derived, histamine-releasing activity is produced by platelet stimulation with collagen (5 micrograms/ml) and acetylglyceryl ether phosphorylcholine (10(-7)), as well as thrombin (1 U/ml). Initial characterization has shown that it is stable to acid and to freeze-thawing but not to boiling for 10 min. In addition, although this histamine-releasing activity is nondialyzable (i.e., greater than 3500 m.w.), it cannot be attributed to platelet factor 4. Thus, platelets, once activated, can produce a soluble substance or substances which can initiate basophil-mediated reactions, further suggesting that platelet activation can enhance allergic and inflammatory reactions.     

The effect of a substance extracted from the urine of prepubertal children on circulating rat gonadotropins]

In the male rat, castration causes an increase of the LH level measured by radioimmunoassay. The application of a compound extracted from a impuber child urine and having anti-gonadotropic effects revealed biologically cannot stop this increase that is even put up by the treatment.     

An endogenous digitalis-like compound extracted from human urine: biochemical and chemical studies

Plasma and urine levels of an endogenous digitalis-like compound (EDLC) are increased in low renin Na+-dependent experimental hypertension, in some normotensive offspring of hypertensive patients and in some essential hypertensive patients. Urine-drived EDLC was purified from 550 L of urine from essential hypertensive patients (n = 8) and from normotensive subjects with a family history of hypertension (n = 27), using flash chromatography on C18 reversed-phase, anion exchange chromatography and various reversed-phase high performance liquid chromatographies. The mechanism of Na+-K+ ATPase inhibition and the related effects of semipurified urine-derived EDLC were studied and compared with those of ouabain. Its action was similar to that of ouabain in 8 out of 10 of the tests applied. The main effects of such a compound were the depression of Na+-K+ pump activity of human erythrocytes, the inhibition of 5-hydroxytryptamine reuptake by human platelets, and the induction of natriuresis in urethanized rats. Therefore, EDLC may be considered as one of the natriuretic hormones whose mechanism of action closely resembles that of ouabain.     

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.     

Helper activity for antibody synthesis encoded by mRNA extracted from human peripheral blood mononuclear cells

Translation products in Xenopus laevis of mRNA from human peripheral blood mononuclear cells were tested for their capacity to replace T cells in the anti-SRBC response of nude spleen cells. When the starting material came from PHA or FCS-stimulated lymphocytes, the translated lymphokines, displaying such a B cell helper activity were found to be encoded by mRNA sedimenting at 6-7S and 13S on a sucrose density gradient. 6-7S mRNA from control, non-stimulated lymphocytes was also able to code for B cell helper activity. Thorough T-cell depletion of mouse responder cell populations left unchanged the activity of 6-7S mRNA products while preventing that of 13S products. The latter were found to contain IL-2, which suggests that their action on B cells was indirect, mediated by T-cell stimulation.     

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper–mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity–mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.     

Analysis of the mechanism of histamine release induced by substance P

Substance P causes release of histamine from rat peritoneal mast cells; the structure-activity relationship shows that N-terminal residue is essential and the hydrophobic region of C-terminal plays an important role. Electrical conductivity of black lipid membrane containing phosphatidic acid was augmented by substance P. In this case, N-terminal residues and C-terminal hydrophobicity were also unavoidable. The partitioning of substance P into the organic phase increased in the presence of phosphatidic acid. The CD spectrum of substance P was changed from the unordered form to beta-form by coexistence of phosphatidic acid/PC liposomes in the medium. The addition of calcium or magnesium in the test solution is effective to prevent either of these phenomena. These findings indicate that substance P probably binds to negatively charged sites of membrane lipids, and subsequent penetration of C-terminal into the hydrophobic core of lipid bilayer may induce an increase of membrane permeability and the following histamine release.     

The mechanism of mediator release from human basophils induced by platelet-activating factor.

Platelet-activating factor (PAF) is a lipid mediator able to induce a variety of inflammatory processes in human peripheral blood cells. We have investigated the effect of PAF on the release of chemical mediators from human basophils of allergic and normal donors. PAF (10 nM to 1 microM) caused a concentration-dependent, noncytotoxic histamine release (greater than or equal to 10% of total) in 27 of 44 subjects tested (24 atopic and 20 nonatopic donors). The release process was either very rapid (t1/2 approximately equal to 10 s) or quite slow (t 1/2 approximately equal to 10 min), temperature- and Ca2(+)-dependent (optimal at 37 degrees C and 5 mM Ca2+). Coincubation of PAF with cytochalasin B (5 micrograms/ml) enhanced the release of histamine induced by PAF and activated the release process in most donors (42 of 44). Atopics did not release significantly more histamine than normal subjects, and the percentage of PAF responders (greater than or equal to 10% of total) was nearly the same in the two groups. Histamine release was accompanied by the synthesis and release of leukotriene C4, although this lagged 1 to 2 min behind histamine secretion. Lyso-PAF (100 nM to 10 microM), alone or together with cytochalasin B, did not release significant amounts of histamine. The release of histamine activated by PAF was inhibited by the specific PAF receptor antagonist, L-652,731, with an IC50 of 0.4 microM. There was a partial desensitization to PAF when the cells were preincubated with PAF (100 nM to 1 microM) for 2 min in the absence of Ca2+, whereas the cells remained responsive to anti-IgE (0.1 micrograms/ml). If neutrophils were removed from the basophil preparation by a Percoll gradient or a countercurrent elutriation technique, there was a significant decrease in PAF-induced histamine release. PAF (1 microM) was able to induce a very rapid, transient rise (peak less than 10 s) in [Ca2+]i in purified basophils analyzed by digital video microscopy. Finally, among human histamine-containing cells, the basophils are unique in degranulating following a PAF challenge. Mast cells from human lung, skin, or uterus failed to respond to PAF (10 nM to 1 microM) regardless of the presence or absence of cytochalasin B (5 micrograms/ml). Our results demonstrate that PAF is able to induce the release of inflammatory mediators from human basophils, and that neutrophils can influence this response. It is suggested that PAF-induced basophil activation can play a role in the pathogenesis of allergic disorders.     

Alterations of gene expression in Novikoff hepatoma cells induced by a factor in human urine

Proteins and protein subunits from Novikoff hepatoma cells have been mapped by two-dimensional polyacrylamide gel electrophoresis utilizing the BASO-DALT system to resolve the basic proteins. Utilizing this technique, it has been demonstrated that human urine contains proteins that retain biological activity and can stimulate synthesis of several new proteins in neoplastic cells. This stimulatory activity has been detected in urine from cancer patients and normal individuals.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.