Jean-Pierre Bouvet

Bacterial Immunoglobulin Superantigen Proteins A and L Activate Human Heart Mast Cells by Interacting with Immunoglobulin E

ABSTRACT Human heart mast cells (HHMC) have been identified in heart tissue, perivascularly, and in the intima of coronary arteries. In vitro activation of isolated HHMC induces the release of vasoactive and proinflammatory mediators (histamine, tryptase, and cysteinyl leukotriene C4 [LTC4]). We investigated the effects of several bacterial proteins on HHMC activation in vitro. HHMC released histamine, tryptase, and LTC4 in response toStaphylococcus aureus Cowan 1 and the immunoglobulin (Ig)-binding protein A, but not to S. aureus Wood 46, which does not synthesize protein A. The effect of protein A was inhibited by preincubation with monoclonal IgM VH3+. Some strains of Peptostreptococcus magnus express an Ig light chain-binding surface protein called protein L. Such bacteria and soluble protein L stimulated the release of preformed and newly synthesized mediators from HHMC. Preincubation of HHMC with either protein A or protein L resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus or anti-IgE. Monoclonal IgE (κ chains) blocked protein L-induced release, whereas IgE (λ chains) had no effect. Streptococcal protein G, formyl-containing tripeptide, and pepstatin A did not activate HHMC. Bacterial products protein A and protein L and intact bacteria (S. aureus and P. magnus) activate HHMC by acting as Ig superantigens.

SYSTEMIC AND SECRETORY HUMORAL IMMUNITY IN THE NORMAL HUMAN VAGINAL TRACT

The molecular status of Abs in the vaginal fluid is reconsidered as a basis for immunization strategies for womens vaccination against HIV. Analysis of separated immunogiobulins (Igs) shows a large proportion of uncleaved IgG, whereas the low amount of IgA includes SIgA, monomers and fragments. SIgM is at a very low level, while free SC molecules are abundant. In addition to the already documented local synthesis, vaginal IgG contains serum‐derived tetanus antitoxins. The IgG could reach the lumen by diffusion, and/or be transported by an Fc receptor‐associated mechanism as suggested by the subclass imbalance in favour of the IgGl isotype. Vaginal SIgA contains very low levels of antibodies to the cell‐wall carbohydrates from a dental caries‐associated streptococcus confirming the participation of the secretory immune system. In addition, the low percentage of IgA2 suggests that a proportion of vaginal SIgA can also derive from actively transported serum polymers. In agreement with our previous studies showing induction of vaginal tetanus antitoxins by intramuscular immunization, these results are in favour of classical parenteral vaccinations to induce protection of the human vagina.

A modified gel filtration technique producing an unusual exclusion volume of IgM: a simple way of preparing monoclonal IgM

The vast majority of monoclonal IgM proteins is eluted just before the total volume of the column when filtered through G200 Sephadex or S200 Sephacryl gels equilibrated in a 0.005 M phosphate buffer but eluted with 0.05 M phosphate buffer containing 1.7 M NaCl. This unusual behaviour in low-ionic buffer is probably due to the poor solubility of IgM in diluted buffers. It allows a 1-step purification procedure under mild conditions and is suitable for both large and small scale preparations.

Delineation Between T and B Suppressive Molecules From Human Seminal Plasma: II. Spermine is the Major Suppressor of T‐Lymphocytes in Vitro

ABSTRACT: The nature of the human semen T‐sup‐pressor was investigated in vitro on human lymphocyte proliferations induced by phytohemagglutinin (PHA) or by alloantigens. Purification by ion‐exchange chromatography, followed by butanol extraction, showed this factor to be present only in the polyamine‐containing fractions. The purified product, obtained by preparative thin‐layer electrophoresis, contained almost exclusively spermine and exhibited the same suppressive activity as this polyamine. Human T‐lymphocyte suppression occurred in the presence of fetal calf serum, but it did not occur in a serum‐free medium. No suppression was observed after preincubation of the fetal calf serum with hydroxylamine, a spermine oxidase inhibitor, whereas a nondialyzable fraction, from normal human serum, decreased the suppression. The semen factor did not act by direct cytotoxicity, as there was no effect of preincubation and suppression could be induced only within the first 6 hr of mitogen activation. These data demonstrate that the in vitro T‐suppressive activity of semen can be assigned mainly to spermine and show that in vivo this suppression must require locally the presence of a spermine oxidase or related enzyme.

Human amniotic IgA inhibits natural IgG autoantibodies of maternal or unrelated origin.

We show that the natural autoantibody activity of amniotic IgG dramatically increases after purification, and that the IgG‐depleted fraction can suppress the activity of IgG natural antibodies from amniotic fluid or from the maternal serum. This suppression is also observed towards serum IgG from unrelated adults but does not impair the tetanus antitoxin activity of serum‐derived IgG. Absorption experiments and immunoglobulin separation by gel permeation demonstrate that this suppression is due to monomeric immunoglobulins of the IgA isotype. The inhibition is associated with an anti‐F(ab ′ )2 activity of the amniotic IgA, involving hypervariable regions of the IgG as demonstrated by different reactivities towards monoclonal IgG sharing the same family of VH and Vκ domains. These results indicate that the inhibition of natural autoantibodies not only occurs with fetal and adult serum IgM, as reported by other groups, but also with amniotic IgA, suggesting a general and important phenomenon. In the case of the amniotic fluid, IgA could protect the fetus against maternal IgG autoantibodies without interfering with simultaneously translocated antigen‐induced IgG antibodies to pathogens.

NON-IMMUNE VH-BINDING SPECIFICITY OF HUMAN PROTEIN FV

The specificity of human F(ab)‐binding Protein Fv (previously called Protein F), a sialoprotein released into the digestive tract mainly during hepatitis, was investigated with fragments or chains of monoclonal immunoglobulins. Protein Fv bound an unreduced H‐chain dimer of a monoclonal human IgA2m(I) molecule but neither the corresponding L‐chain dimer, nor several Bence‐Jones molecules. Using enzymatic subfragments of F(ab)μ(, or F(ab′)2γ, a significant binding was observed with Fv fragments (VH + VL), while Fb fragments (CHI+CL) were inactive. Taken altogether, these results prove that the structure recognized by Protein Fv is located in the VH domain. This structure probably involves discontinuous epitopes linked by a disulphide bond, which are destroyed by combined reduction and dissociation. Protein Fv does not interfere with the antigen‐binding site, since there was no reciprocal inhibition with the antigen‐antibody reaction.

IgM reassociation in the absence of J-chain

Human monoclonal IgM pentamers with different biophysical properties (euglobulin, pseudoglobulin or cryoglobulin) were reduced and reassociated in the absence of J-chain. Reassembly occurred for 50-82% of the monomers. The reassociated molecules consisted of covalent oligomers and pentamers. The deficiency of J-chain (estimated to be less than 0.17% of normal) was shown by alkaline-urea overloaded gel electrophoresis followed by silver staining. The addition of exogenous J-chain, from polymeric IgA or IgM, did not significantly modify the reassembly ratio. Thus J-chain does not seem to be an absolute requirement for IgM polymerization.

HIGH AFFINITY SERUM-DERIVED FAB FRAGMENTS AS ANOTHER SOURCE OF ANTIBODIES IN THE GUT LUMEN OF BOTH NEONATES AND ADULTS

The authors have investigated the presence of serum‐derived immunoglobulin G (IgG) fragments in the human intestine at various ages, these fragments possibly representing another source of antibodies in addition to secretory IgA (SIgA). Fab fragments of the γ isotype were found to be the major molecular form of immunoglobulins in the meconium (median value: 3.7 mg/g of stools), as compared with Fabα (75 μg/g) and IgM (2.6 μg/g). These fragments provided by molecules of the maternal serum displayed a strong antibody activity to the tetanus toxoid and were also found in the stools of 1‐week‐old babies fed with formula milk. The release of serum antibodies into the digestive lumen occurs largely via hepatobiliary secretions, as suggested by the presence of IgG antitoxins in the bile of children operated on extrahepatic biliary atresia. In adults, the Fab antitoxins were also detected in most stool extracts. Affinity of these molecules was found to be similar to that of their serum counterpart with a Ka of 2.1×1010 M1 (median value). These mucosal antibody fragments, associated with the normal pathway of serum IgG catabolism, could provide additional immune defences against pathogens, and be of importance to supplement an immature or deficient secretory immune system.

Non-Immune Binding of Human Protein Fv to Immunoglobulins from Various Mammalian and Non-Mammalian Species

Reactivity of the secretory protein Fv with immunoglobulins (Ig) from various species of vertebrates was investigated. Binding was observed throughout all taxonomic classes: mammalian, avian, reptilian, amphibian and fish. Contrasting with tins wide spectrum, no significant binding was found with most mammalian ungulates, such as horse (Perissodactl), cow, sheep and goat (Artiodactyls), Nevertheless disruption of’the hydrogen bonds of Ig allowed these non‐reactive molecules lo bind. Such a conserved reactivity dining evolution, and our previous data on the effect of the cleavage of the intra‐chain disulphide bonds, suggest that protein Fv recognizes a discontinuous framework structure involving both the FRI and FR3 regions in the variable domain of the heavy chain of Ig.

Immunoglobulin Fab fragment-binding proteins

Five molecules are known to bind the Fab fragments of human immunoglobulins (Ig). Microbial protein A and protein G are primarily Fc-binding molecules but can also bind other structures of the heavy chain, which are located in the variable domain of the third subgroup (VH3) and in the first constant domain of IgG (CH1 gamma), respectively. In contrast, the two other microbial receptors have a sole Ig-binding site, directed to chi chains (protein L) or to Ig polymers (protein P). Protein Fv is synthesized by human liver cells and released in the digestive lumen, where it forms large complexes with secretory Ig after binding to the VH domains. These five molecules, in the main, bind cleaved Ig and most of them recognize all classes of antibodies. Bacterial molecules are, or can be, used as reagents to purify and detect Ig and fragments. Furthermore, a possible use in human therapy or vaccination is envisaged, and the human protein Fv is a key-factor in immune protection against intraluminal pathogens of the gut.