Description and comparison of three in vitro methods for the detection of guinea pig MHC antigens.

Two methods (an immunoenzymatic and a cell dot immunobinding assay) are described and compared with a reference protein A rosetting assay, for the detection of guinea pig class I and class II histocompatibility antigens. The main requirement of these three assays for obtaining good results is the use of whole fresh target cells since all tested experimental conditions for fixing cellular antigens (glutaraldehyde, formaldehyde, paraformaldehyde), as well as for obtaining cell monolayers (adherence, desiccation, poly-L-lysine) presented several drawbacks and were unsuccessful in our hands. The comparison between the three methods shows that the immunoenzymatic assay gives good results but that it is less reproducible than the other two. The cell dot immunobinding is highly sensitive, reproducible and economical. It can be used for genetic typing and alloantigen analysis of guinea-pig lines, instead of the rosetting method, which is highly reliable but time consuming.

A rapid and reliable in vivo method for anti-GPLA (class I and class II antigens) antibody titration and GPLA typing.

A reverse Arthus reaction (RAR) may be successfully used in guinea pigs to detect histocompatibility alloantigens of the GPLA type. Epidermal cells carry class I GPLA antigens, and Langerhans cells also bear class II alloantigens. It is therefore possible to elicit an RAR by intradermal injection of relevant alloimmune sera in the skin of guinea pigs of known GPLA haplotype. RAR is detected by increased vascular permeability due to IgG1 antibody and hemorrhage due to IgG2 antibody. Compared with an in vitro protein A-rosetting method RAR is easier and quicker. It proved more sensitive for class II antigens in which Langerhans cells are the target for anti-class II antibody and rather less sensitive for class I antigens. RAR is a convenient method for following the course of GPLA alloimmunization, allowing titration of antibodies against both classes of antigen. It may also be used to type guinea pigs of unknown GPLA haplotype.

Alloantibody bipolar bridging; a new mechanism of cell surface activation.

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found l(1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (Fc gamma R) were specific for IgG2 subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG1, IgG2 and F(ab')2 anti-DNP-DNP25 BSA); and (3) that specific alloantibodies of IgG2 subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O2-) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG1 nor F(ab')2 portion were able to trigger O2- release. This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.

17 beta estradiol affects the expression of guinea pig blood leukocyte MHC antigens.

Sex hormones have an effect on various immune responses but the mechanisms of action are unknown. One of these mechanisms might be a modification of expression of major histocompatibility complex (MHC) antigens in blood leucocytes. Estradiol-induced variations of the expression of guinea pig blood leukocytes MHC antigens (GPL-A) was studied. Class I and class II MHC antigens were detected by a sensitive rosetting method using specific alloimmune sera (AIS) and staphylococcal protein A-coated sheep red blood cells (SPA-SRBC) and evaluated by counting the number of bound SPA-SRBC per 100 cells. MHC antigens decreased after estrogen treatment. Estradiol modifies the expression of GPL-A antigens on the mononuclear cells including the Kurloff cells, which are involved in immunity or in a natural killer effect, but did not affect the expression of polymorphonuclear cells, ones which are not involved in immunity.

Studies on human spermatozoa autoantigens. I. Fractionation of sperm membrane antigens: evidence of three antigenic systems.

In order to identify human spermatozoal surface autoantigens, suspensions of previously frozen washed sperm were ground and ultracentrifuged (170,000 g for 60 min). The antigenicity of the fast supernatant (FS) and the fast pellet (FP) were defined by specific inhibition of spermotoxic and various sperm-agglutinating activities of autoimmune human sera (WHO Reference Bank). The FS and the urea-soluble extract of FP were fractionated on Sephadex G-200 columns, and the antigenicity of these fractions was similarly defined. Both FS and FP inhibited, to variable extents, the anti-sperm activities. Inhibition of head-to-head (H-H) agglutination by FS was twice as strong as by FP. The reverse was observed with tail-to-tail (T-T) agglutination. Ten times more FS than FP was necessary to inhibit the spermotoxicity of all tested sera. Four fractions were collected after FS filtration on Sephadex G-200. F1, a homogeneous protein, inhibited spermotoxicity and H-H agglutination F2 inhibited all activities (including T-T agglutination). F3, a low molecular weight fraction, selectively inhibited H-H agglutination. F4 was inactive. Treatment by 8 M urea allowed a partial solubilization of FP antigenicity. Urea-soluble fractions inhibited spermotoxicity and H-H but not T-T agglutination. The antigen(s) involved in T-T agglutination is (are) destroyed by urea since the urea-treated FP was no longer able significantly to decrease T-T agglutination. These results suggest that at least three different autoantigens are responsible for H-H sperm agglutination, T-T sperm agglutination and spermotoxicity respectively.