Heinz Gulle

Anti-inflammatory properties of human serum IgA : induction of IL-1 receptor antagonist and FcαR (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-α) and IL-6 in human monocytes

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL‐1 and TNF‐α accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL‐1 receptor antagonist (IL‐1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL‐1 activity. The present study shows that human serum IgA induces significant IL‐1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL‐1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL‐1β release, and even inhibited LPS‐induced IL‐1β release. Induction of IL‐1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of FcαR with MoAb MY‐43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA‐induced IL‐1Ra production, suggesting that FcαR‐mediated induction of tyrosine phosphorylation is required for the IgA‐induced stimulation of IL‐1Ra release. In addition, triggering of FcαR with MoAb specifically down‐regulated TNF‐α and IL‐6 release in human monocytes activated with Hib. By the induction of IL‐1Ra and down‐regulation of the release of inflammatory cytokines such as IL‐1β, TNF‐α and IL‐6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.

Direct blotting with viable cells of protein mixtures separated by two-dimensional gel electrophoresis

A procedure is described which combines the high resolution power of two-dimensional (2D) gel electrophoresis with the advantage of direct probing with viable cells. This device permits the transfer by electroelution of 480 distinct fractions from a 2D gel into soluble phase. Transferred fractions are virtually nontoxic, thus allowing direct probing with viable cells. Using this procedure it was shown that T cells from normal healthy individuals recognized a multitude of Mycobacterium tuberculosis antigens and that the fine antigen recognition pattern of T cells changed after short-term culture in vitro. The application of this procedure to the verification of antigen purity at the T cell level and to the identification of antigens within crude bacterial lysates which are recognized by cloned T cells is described. This approach should be applicable to the rapid identification and characterization of any interesting T cell antigen, for example from important pathogens against which a subunit vaccine is desirable. Moreover, it could be helpful for the analysis of interactions between soluble ligands and their target cells.

Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome

Up to now there is no treatment for staphylococcal toxic shock syndrome, a disease mainly induced by toxic shock syndrome toxin-1(TSST-1). There is great demand in finding means to control the disease, one of them is the development of an effective and safe vaccine against TSST-1. In this study we constructed a series of vaccine candidates and investigated their biological activity, toxicity, and potential to invoke an immune response. TSST-1 was isolated from Stahylococcus aureus supernatants and recombinantly expressed as a N-terminal 6x histidine-tagged protein in Escherichia coli. In order to obtain molecules with minimal toxicity we constructed single mutants (G31R and H135A) and one double mutant (G31R/H135A) with both residues exchanged. We also detoxified native TSST-1 isolated from S. aureus, and recombinantly expressed TSST-1 by treatment with formaldehyde. Functional activity of native and recombinant TSST-1 and grade of inocuity of mutants and toxoids was determined by investigating mitogenity, T-cell activation, and cytokine release upon stimulation of human mononuclear cells with the vaccine candidates. All substances were tested in a rabbit immunization study. After primary immunization and three additional boosts all vaccinated animals developed antibody titers against TSST-1 and were protected against challenge with a lethal doses of superantigen potentiated with lipopolysaccharide.

Epitope mapping of neutralizing TSST-1 specific antibodies induced by immunization with toxin or toxoids

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a potent stimulator of the immune system. T-cells are activated by crosslinking of MHC class II molecules on antigen presenting cells with T-cell receptors (TCR). TSST-1 is associated with the majority of the cases of menstrual staphylococcal toxic shock, a severe and life-threatening multisystem disorder. Even though antibody mediated protection has been studied, information on antibody specificity directed to individual antigenic determinants of the protein is incomplete. To obtain immunogens with low toxicity, we generated a double-site mutant (dmTSST-1), modified at solvent-exposed residues predicted to be important for both MHC class II and TCR binding, and detoxified recombinantly expressed TSST-1 (rTSST-1) as well as native TSST-1 (nTSST-1) isolated from Staphylococcus aureus by treatment with formaldehyde. Rabbits were immunized with rTSST-1, nTSST-1, dmTSST-1, and formaldehyde inactivated toxoids. The sera obtained were used to map the antigen-reactive regions of the molecule and to identify specificities of antibodies induced by immunization with the different antigens. To detect linear antigenic epitopes of TSST-1 the reactivity of the sera with 11-meric peptides having an overhang of four residues, covering the entire molecule of TSST-1, have been studied. We found that sera of TSST-1 immunized rabbits predominantly reacted with N-terminal residues 1-15, while sera generated with formaldehyde inactivated toxoid recognized a total of 7 regions located at the N- and C-terminus and internal sites of TSST-1. Despite different specificities all sera were able to inhibit TSST-1 induced proliferation of human mononuclear cells.

Analysis of Primary T Cell Responses to Intact and Fractionated Microbial Pathogens

Freshly isolated human T lymphocytes were tested for their response to mycobacteria, mycobacterial lysates, 2 dimensional (2D) PAGE separated mycobacterial lysates, leishmania and defined leishmanial antigen preparations. While gamma delta T cells proliferated vigorously in the presence of mycobacteria and mycobacteria derived lysates, a significant stimulation from 2 D gel separated lysates was not detected. In addition gamma delta T cells failed to respond towards leishmania or leishmanial components. In the alpha beta T cell compartment some donors, presumably according to their state of immunity against mycobacteria, responded to mycobacteria, mycobacterial lysates and 2 D gel separated mycobacterial lysates. Neither freshly isolated gamma delta T cells nor alpha beta T cells from naive donors did mount a significant immune response against leishmania.

Tuberculosis and Leprosy: Attempts to Identify T-Cell Antigens of Potential Value for Vaccine Design

Tuberculosis and leprosy are chronic bacterial infectious diseases which represent major health problems worldwide. It is generally accepted that, on the one hand, effective vaccination strategies are required for satisfactory control of these diseases and, on the other hand, that currently available vaccination measures are insufficient for this purpose. Ideally, a subunit vaccine should be designed which is composed of one or a few protective antigens. In this brief treatise our approach towards the identification of antigens with potential value for vaccine design is described. It comprises high resolution fractionation by two‐dimensional gel electrophoresis, transfer of separated fractions by electroelution and testing of separated fractions with viable T cells and accessory cells. Using this approach we find: (1) multiple antigens are recognized by T cells from leprosy and tuberculosis patients as well as healthy contacts; (2) apparently, suppressive antigens exist in leprosy; (3) an antigen cluster which is apparently indicative for immunity against M. tuberculosis is present among secreted proteins. We hope that further improvement of this methodology will help in the rational design of subunit vaccines against tuberculosis and leprosy.

Nitrocellulose particles adsorbed to immunoglobulins are a new and effective approach to induce cell activation dependent on receptor aggregation

Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.

A rapid immunological spot test for the identification of proteins in covalently linked protein-nucleic acid complexes

A method is described for the rapid immunological identification of proteins in studies of multicomponent systems. In this case the system is the E. coli ribosome, and the ribosomal proteins to be identified are covalently attached to fragments of labelled ribosomal RNA as a result of chemical cross-linking procedures. Antisera raised against the individual ribosomal proteins are spotted onto a nitrocellulose sheet, and an aliquot of the covalent complex under test is added to each antibody spot. After suitable washing procedures, a positive reaction with one or other of the antisera is visualized by autoradiography of the labelled RNA moiety attached to the antibody via the ribosomal protein. Amounts of protein as low as 10 pg can readily be detected.