Martine Damonneville

The neuropeptide substance P stimulates the effector functions of platelets

Sensory neuropeptides, such as substance P. appear as potent mediators of various immunological reactions, and inhibit or stimulate a wide range of functions of immune inflammatory cells. Platelets were recently shown to participate as effector cells in an IgE or lymphokine‐dependent killing of parasites. Substance P and its carboxy‐terminal fragment SP (4–11) induce the cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, respectively, by 90% and 40%, whereas the modified C terminal SP, the SP‐free acid, exhibits no effect on the platelets. The neuropeptide effects occur at low doses (10‐8 M), are specific as shown by inhibition studies with a substance P antagonist, theD‐SP. Binding data obtained after flow cytofluorometry with FITC‐SP lead to the conclusion that SP binds specifically to about 20% of the homogenous population of platelets. Moreover, IgE could modulate the SP‐dependent functions of platelets since the pre‐incubation with myeloma human IgE or with AP2 monoclonal antibodies—known to inhibit the IgE‐dependent killing of these cells leads to a dramatic decrease of the SP dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses.

Antigenic properties of Schistosoma mansoni aminopeptidases: Evolution during the development in mammalian hosts

The involvement in the immunity to schistosomiasis of an aminopeptidase activity of schistosomula, 20-day-old and adult worms of Schistosoma mansoni has been studied. This activity, hydrolysing leucine-p-nitroanilide and alanine-p-nitroanilide at pH 7.4 was immunoprecipitated by various infected rat and human sera. These antigenic enzymes were expressed at day 28 after infection in the rat and seemed to be specific for the Schistosoma species. Several aminopeptidase activities were found after analysis by isoelectric focusing of the adult extract but only the pI 8.3 peak was antigenic. Three antigenic peaks were demonstrated after AcA 34 Ultrogel filtration. The biological relevance of these antigenic enzymes in schistosomiasis is discussed.

Role of serine proteases of Schistosoma mansoni in the regulation of IgE synthesis.

The regulation of the IgE response by schistosomula‐released products (SRP) was studied either in vitro with rat and human cell cultures or in vivo by injection into rats of SRP with an unrelated allergen at primary or secondary immunisation. The results obtained in vitro showed that non‐dialysable factors present in SRP potentiate the IgE synthesis by rat and human cells. This enhancing effect was supported by molecules with serine protease activities. On the other hand, the inhibition or depletion of SRP in serine proteases induced a weak synthesis of IgM by rat cells in vitro. The injection of SRP into rats on day 0 with an unrelated allergen led to a potentiation of total IgE production, but an inhibition of specific IgE response. In contrast, a marked elevation or specific IgE response was obtained when SRP was injected upon secondary immunization. Serine proteases of SRP were partly responsible for this potentiative effect.

Induction of a Protective Immune IgE Response in Rats by Injection of Defined Antigens of Schistosomulum-Released Products: Immunochemical Properties of the Target Antigens

Brown Norway rats were injected without adjuvant with the soluble products liberated in a 16-hour culture by schistosomula (schistosomula-released products, SRP-A). A strong cytotoxic and protective IgE response was elicited, mainly directed against 22- and 26-kilodalton (kDa) SRP-A molecules. In the present study, we have attempted to characterize further those molecules. Metaperiodate denaturing treatment of the SRP-A glycans before injection into rats did not modify the immunogenicity of the SRP-A antigens. Results obtained by lectin affinity suggested that the 22- and 26-kDa molecules were glycoconjugates binding to ConA. Preparative sodium dodecylsulfate electrophoresis has allowed the separation of enriched fractions of 22- and 26-kDa molecules which have been injected separately into rats. The corresponding sera were tested in antibody-dependent cell cytotoxicity and displayed a significant cytotoxic IgE response (65 and 53%, respectively) towards the larvae. These results lend further support to the view that the 22- and 26-kDa antigens are the major targets of the protective IgE response and thus appear as potentially protective antigens.

Allergens of Schistosoma mansoni. II: Fractionation and characterization of S. mansoni egg allergens

The interaction of Schistosoma mansoni crude soluble egg antigen (SEA) with IgE antibodies in sera from S. mansoni-infected mice, rats and humans has been studied by the radioallergosorbent test (RAST) and the Prausnitz-Küstner (PK) technique. IgE antibodies recognizing egg antigens were present as early as day 21 after the infection in the mouse sera and day 28 in rat sera. IgE in sera of infected humans reacted with antigenic components in the Mr range 70,000-150,000 and focusing as a broad peak in the pH range 4.5-6.5 as measured by RAST. SDS-PAGE followed by western blotting showed the presence of major components at molecular weights of 117,000 and 35,000-43,000. In the PK test, using mouse sera, components focusing in the alkaline pH range also gave a positive reaction. Most of the allergenic activity was bound by concanavalin A-Sepharose and by wheat germ agglutinin-Ultrogel. IgE in serum from an infected non-permissive host (the Fischer rat) apparently recognized egg-stage-specific allergen as indicated by differences in the time course of the IgE response to egg allergens compared to the adult material. When analyzed by SDS-PAGE and western blotting with day 45-infected rat serum, SEA showed some qualitative and quantitative differences to adult worm antigen. Molecules at molecular weights between 25,000 and 30,000 and at about 43,000 in SEA reacted with rat serum IgE and were absent from adult worm antigen. The allergenic similarities between egg and adult worm are discussed.

The effector function of platelets is induced and regulated by T lymphocytes

The demonstration of effector functions of platelets in parasitic diseases raised the question of their possible regulation by T lymphocytes. Normal human platelets, treated with culture supernatants from antigen- or mitogen-stimulated CD4+/CD8- T cells, developed the capacity to kill young larvae of Schistosoma mansoni, in the absence of antibodies. The presence of IFN gamma in the lymphocyte supernatants, the neutralization by monoclonal anti-IFN gamma antibody, and the direct inducer effect of recombinant IFN gamma, clearly identify this lymphokine as one of the stimulator factors. In contrast, ConA- and antigen-stimulated human CD8+/CD4- T-cell supernatants contained a factor able to inhibit the IgE-dependent platelet cytotoxicity toward the same targets. The production of oxygen metabolites by IgE-coated platelets stimulated by anti-IgE was also strongly inhibited by this lymphokine. This platelet activity suppressive lymphokine (PASL) was identified as a polypeptide of 15 to 20 Kd with a pI of 4.6. The in vivo relevance of PASL was demonstrated by the complete abolition, after PASL treatment, of the protection normally conferred following a challenge infection by intravenous passive transfer of platelets from immune to normal rats.

Protection of rats against Schistosoma mansoni infection induced by platelets stimulated with the murine recombinant tumor necrosis factor alpha.

Human recombinant tumor necrosis factor (rTNF alpha) and lymphotoxin (rTNF beta) have been shown to enhance the schistosomicidal activity of human platelets in vitro. In this report, we demonstrated that the murine rTNF was able to induce the in vitro cytotoxic activity of both murine and rat platelets towards the larvae of Schistosoma mansoni, whereas human rTNFs activated only the murine platelets. Passive transfer of platelets stimulated with murine rTNF significantly protected rats up to a 65% reduction of the worm burden. Thus, rTNF may constitute, via its interactions with effector cells, an important regulatory mechanism during the parasitic infection of rats.

IgG response of rats and humans to the released products of schistosomula of Schistosoma mansoni

The participation of products released from Schistosoma mansoni schistosomula (SRP-A) in the IgG antibody response of infected Brown-Norway rats and infected humans has been studied using immunoprecipitation with various antigenic preparations and in in vitro cytotoxicity assays. A large number of SRP-A molecules with a wide range of molecular weights was recognized by infected rat and human sera. Anti-SRP-A antibodies appeared in rat sera from day 28 after infection. In infected humans, a variable pattern of SRP-A recognition was observed between individuals. IgG antibodies obtained by immunization of rats with SRP-A without addition of adjuvants reacted with 3 major schistosomula surface proteins with molecular weights of 38, 32 and 21 kDa. These latter molecules were also revealed strongly by infected rat sera. Moreover, these antibodies were able to kill schistosomula in vitro in the presence of complement or eosinophils.