Spontaneous and prostatic steroid binding protein peptide-induced autoimmune prostatitis in the nonobese diabetic mouse.

Chronic nonbacterial prostatitis is a poorly defined syndrome of putative autoimmune origin. To further understand its pathogenesis, we have analyzed autoimmune prostatitis in the NOD mouse, a strain genetically prone to develop different organ-specific autoimmune diseases. Spontaneous development of autoimmune prostatitis in the NOD male, defined by lymphomonuclear cell infiltration in the prostate gland, is well-established by approximately 20 wk of age and is stably maintained afterward. Disease development is indistinguishable in NOD and NOR mice, but is markedly delayed in IFN-gamma-deficient NOD mice. A T cell response to the prostate-specific autoantigen prostatic steroid-binding protein (PSBP) can be detected in NOD males before development of prostate infiltration, indicating lack of tolerance to this self Ag. The intraprostatic inflammatory infiltrate is characterized by Th1-type CD4(+) T cells, which are able to transfer autoimmune prostatitis into NOD.SCID recipients. We characterize here experimental autoimmune prostatitis, detected by intraprostatic infiltrate and PSBP-specific T cell responses, induced in 6- to 8-wk-old NOD males by immunization with synthetic peptides corresponding to the C1 subunit of PSBP. Three PSBP peptides induce in NOD mice vigorous T and B cell responses, paralleled by a marked lymphomononuclear cell infiltration in the prostate. Two of these peptides, PSBP(21-40) and PSBP(61-80), correspond to immunodominant self epitopes naturally processed in NOD mice after immunization with PSBP, whereas peptide PSBP(91-111) represents a cryptic epitope. These model systems address pathogenetic mechanisms in autoimmune prostatitis and will facilitate testing and mechanistic analysis of therapeutic approaches in this condition.

Prostatein or steroid binding protein (PSBP) induces experimental autoimmune prostatitis (EAP) in NOD mice.

In a previous study, we showed that nonobese diabetic (NOD) mice, a strain that present an inherited predisposition to develop both spontaneous and induced autoimmune lesions, are susceptible to the induction of experimental autoimmune prostatitis (EAP), developing a severe inflammatory reaction in the prostate, accompanied by humoral and T-cell-mediated responses. In this study we asked whether the protein steroid binding protein (PSBP) or prostatein (a major autoantigen in the rat model of EAP) is a potential autoantigen in the NOD mouse model and examined the ability of purified PSBP to induce EAP in this strain. Our results indicate clearly that NOD male mice react immunologically to PSBP by developing lymphocytic inflammatory lesions in prostatic tissue and producing both a cellular- and humoral-specific autoimmune response. But our results suggest also the existence of other prostatic autoantigens present only in total prostate extract. Such additional antigens could enhance the autoimmune response and result in more severe forms of inflammation. We also analyzed the respective contributions of MHC antigens and CD4/CD8 T-cell subsets in NOD mice lacking expression of beta 2-microglobulin (NOD.beta2m degrees/degrees) or MHC class II beta chain (NOD.Abeta degrees/degrees) and demonstrate an essential role for CD4(+) T cells in the development of EAP in the NOD model. In conclusion, we demonstrate that PSBP is an autoantigen recognized by the NOD immune system, capable of generating humoral and cellular autoimmune responses and of inducing EAP. Moreover, using selected knock-out NOD mice we demonstrate an essential role for CD4(+) T cells in the development of EAP.

Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor.

Prostatic steroid binding protein (PSBP) is the major protein produced ( approximately 20% of the total cytosolic protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.

Prostatein (or rat prostatic steroid binding protein) is a major autoantigen in experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is a disease that could be considered an experimental model of human non-bacterial prostatitis. In this experimental model, male rats are intradermally immunized with a saline extract of male sex accessory glands (RAG) in an adequate adjuvant. The prostatitis observed in the immunized animals develops as a consequence of the immune response against RAG antigens, and the histological lesion is strikingly similar to the pattern of prostatic inflammation observed in the human disease. In this study, we purified one of the prostatic autoantigens recognized by the autoantibodies in our model. Amino acid sequence analysis identified the purified protein as prostatein or rat prostatic steroid binding protein, a member of the uteroglobin superfamily. Prostatein was recognized not only by the humoral autoimmune response, but also by the cellular autoimmune response. Certainly, the DTH response and lymph node cell proliferative assays against prostatein in immunized animals yielded positive results. Prostatein is not only the target of the autoimmune response in animals immunized with the whole extract, but also an inducing antigen of the disease. Purified prostatein, when incorporated to an adequate adjuvant, elicited cellular and humoral autoimmune response and lesion in the prostate gland. The identification of one of the target antigens in autoimmune prostatitis has provided a further refinement and characterization of our model, which could serve for a better understanding of the aetiology, pathogenesis and pathophysiology of non-bacterial prostatitis.

Distribution of immunoreactive androgen-binding protein/sex hormone-binding globulin in tissues of the fetal rat.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein that binds androgens and estrogens with high affinity. In the adult, ABP/SHBG is thought to function in the male reproductive system and the general circulation in both sexes to modulate the actions of sex steroids. The ABP/SHBG gene is also expressed in the embryonic rat liver, where SHBG is secreted into the fetal blood of male and female rats. The embryo also expresses an alternative SHBG with a unique N-terminal sequence. In this study, the distribution of immunoreactive SHBG in the 17-day-old male fetal rat was determined with six antisera. In general, all of the antisera reacted with the same structures. Specific tissue immunoreactivity was mostly cytoplasmic and/or extracellular. By far the most prominent immunoreactive structures were the mesoderm-derived tissues: connective tissue, striated and cardiac muscle, cartilage, and the liver hematopoietic system. In addition, all regions of the fetal brain contained immunoreactive neurons. In the developing male reproductive system, there was minor reactivity in the testicular cords, whereas the connective tissue in the differentiating Wolffian duct stained with all of the antisera. The Wolffian duct epithelium and epithelia in other developing organs contained small amounts of immunoreactive SHBG, except for the lung, which stained in the epithelial extracellular matrix. An antibody raised against a unique N-terminal peptide specific for the alternative SHBG protein revealed that it was also present in many tissues. These data suggest that SHBG is important for the differentiation of mesodermal tissues. SHBG may modulate the action of androgens in embryonic stroma, thereby regulating development of the epithelium in hormone-dependent tissues.

Identification of probasin-related antigen as cystathionine gamma-lyase by molecular cloning.

We reported previously that a monoclonal antibody against probasin (rat prostatic secretory protein) recognizes a 40-kDa protein localized in rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissues. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression library using an affinity-purified polyclonal antibody. The amino acid sequence deduced from the determined cDNA sequence included sequences identical with those of proteolytic fragments of PRB-RA, which covered about 70% of the deduced sequence. Northern blot hybridization of poly(A)+ RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in the liver and kidney. A sequence homology search revealed that PRB-RA is almost completely identical to rat cystathionine gamma-lyase (cystathionase) and that it does not show overall homology with probasin. Three candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, neutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally related to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in liver parenchymal cells.

Localization of secretory, membrane-associated and cytoskeletal proteins in rat testis using an improved immunocytochemical protocol that employs polyester wax.

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38 degrees C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule.(ABSTRACT TRUNCATED AT 400 WORDS)

An anti-probasin monoclonal antibody recognizes a novel 40-kDa protein localized in rat liver and a specific region of kidney urinary tubule.

Immunoblotting with a monoclonal antibody against probasin (rat prostatic secretory protein) showed that a 40-kDa protein antigenically related to probasin was localized in rat liver and kidney. The contents of probasin in these organs were negligible. Immunostaining revealed that the 40-kDa protein (probasin-related antigen: PRB-RA) was expressed in the liver parenchymal cells and the kidney urinary tubular epithelial cells in outer stripe. The content of PRB-RA in the kidney was low during 0 to 2 weeks of age, then rapidly increased about 10-fold from 2 to 8 weeks of age. The content in the liver increased about 2-fold during the period, reaching a value of 10-12 ng/micrograms protein, which was ten times higher than that in the kidney. PRB-RA was purified from rat liver by ion-exchange chromatography, gel filtration and fast protein liquid chromatography on a hydroxyapatite column. The purified protein formed insoluble aggregates in the absence of a detergent, and it had a blocked amino terminal. The amino acid sequence of a peptide generated by tryptic digestion of alkylated PRB-RA was determined. Computer analysis showed that there was no protein having a significant homology with the peptide. These results indicate that a novel 40-kDa protein with a structural similarity to probasin is localized in rat liver and kidney, and might bear a function specific to these organs.

The 56 kDa androgen-binding protein in human genital skin fibroblasts: its relation to the human androgen receptor.

We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.

Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver.

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.

Monoclonal antibodies to rat androgen-binding protein recognize both of its subunits and cross-react with rabbit and human testosterone-binding globulin.

A series of four monoclonal antibodies to rat androgen-binding protein (ABP) has been developed. These antibodies recognize both the heavy (48,400-dalton) and light (43,000-dalton) subunits of the native ABP molecule. In addition, they recognize the subunits from which Asn-linked oligosaccharides have been removed by treatment with N-glycanase, indicating that these moieties do not form the immunological determinants recognized by the antibodies. Two of these antibodies are capable of recognizing both nondenatured ABP, as assessed by dot blot analysis, and denatured ABP, as determined by Western blot analysis of ABP after electrophoresis under denaturing conditions. The immunoreactivity of denatured ABP is decreased with two of the antibodies, suggesting that they more readily recognize the antigenic epitopes when the protein is in its native configuration. The antibodies were capable of immunoprecipitating covalently labeled ABP from solution. All four monoclonal antibodies produced were determined to be immunoglobulins M by both enzyme-linked immunosorbent assay and Ouchterlony immunodiffusion even though the initial serum response of the immunized animals indicated the presence of immunoglobulins G. All of the monoclonal antibodies raised against rat ABP cross-reacted with rabbit and human testosterone-binding globulin (TeBG). They were able to detect two subunits when Western blots of intact rabbit [mol wt (Mr, 43,000 and 40,500] or human (Mr, 47,600 and 44,500) TeBG were probed, but only a single subunit (Mr, 39,300) when deglycosylated samples of rabbit TeBG were analyzed.

Production of an antibody to mouse salivary androgen binding protein (ABP) and its use in identifying a prostate protein produced by a gene distinct from Abp.

We previously demonstrated polymorphism of a mouse salivary protein which, because of its ability to bind androgen, we designated androgen binding protein (ABP) and its structural gene, Androgen binding protein (Abp). This report describes the purification of salivary ABP and presents the amino acid composition of its subunits. Using an antibody raised against the purified protein, we demonstrated the presence of cross-reactive material (CRM) in mouse parotid, submaxillary, sublingual, and prostate glands by double immunodiffusion. Immunohistochemical detection of proteins on electroblots of polyacrylamide electrophoresis and isoelectric focusing gels, however shows that the prostate CRM is a protein with electrophoretic and isoelectric focusing behavior distinct from that of salivary ABP isoproteins. Because the DBA/2J strain has a variant of salivary ABP, that strain was analyzed to determine if a prostate ABP-CRM variant was present. The approach was hampered by an inability to detect CRM in the prostates of DBA/2J mice. Prostate CRM was detected, however, in some progeny from repeated backcrosses of DBA/2J X C3H/St hybrids to the C3H/St and DBA/2J parental strains. The prostate CRM detected in samples from animals heterozygous for salivary Abp appears to be identical to C3H/St prostate CRM, suggesting that the gene controlling prostate ABP-CRM is related to, but distinct from, Abp. The reason for reduced or absent CRM in the prostates of DBA/2J males is unknown but this finding suggests that there are strain-related differences in the expression of this prostate protein.

Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties.

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.

Decreased fertility and motility of spermatozoa from rats immunized with a prealbumin epididymal-specific glycoprotein.

This study investigated the effects on the progressive motility, zona-binding capacity, and fertility of spermatozoa from the cauda epididymidis of adult male rats that were actively immunized against an acidic glycoprotein secreted by the epididymis. The percentage of motile spermatozoa was less than 5% in nine of ten rats that received the epididymal antigen, and 40 to 50% in eight of the 10 control rats. In animals immunized against the antigen, there was a dramatic decrease, but not a complete suppression, in the capacity of epididymal spermatozoa to bind the zona pellucida as compared with the nonimmunized controls. Fertility was decreased two weeks after the end of the treatment, but partial restoration of fertility was observed 6 months later.

Human testicular androgen-binding protein shares immunodeterminants with serum testosterone-estradiol-binding globulin.

In the human, there are two glycoproteins, testosterone-estradiol-binding globulin (hTeBG) and androgen-binding protein (hABP), which bind testosterone. Although these two proteins have similar physicochemical properties, they can be distinguished on the basis of origin and lectin binding. hTeBG is made in the liver and exhibits high affinity for Concanavalin A (Con A), while hABP from the testes is only partially bound to this lectin. That is, when testicular extracts were applied to Con A-Sepharose columns, a portion of the testosterone-binding material showed no interaction with the lectin and eluted in the void volume (peak I), while the remainder interacted strongly and could be eluted with alpha-methyl-D-glucoside (peak II). These observations are consistent with the proposal that peak I contains only hABP, whereas peak II contains hTeBG and/or hABP with carbohydrate units that permit binding to Con A. To further study the properties of these binding proteins, a hTeBG RIA using a monospecific antiserum was employed to compare the proteins in testes and serum. The results indicated that the testosterone-binding activities in peaks I and II of testicular extracts could not be distinguished immunologically from hTeBG in sera of normal women. These findings suggested that hTeBG and hABP share common epitopes. We next determined whether hABP was secreted into the blood or amniotic fluid by fractionating these fluids in Con A-Sepharose columns. Unlike testicular extracts, male serum and amniotic fluid contained single immunoreactive and steroid-binding species which bound specifically to Con A. We conclude from these observations that hABP (peak I), peak II activity, and hTeBG have common immunodeterminants and that if hABP is secreted into the blood of men, then its carbohydrate chains bind to Con A, making it indistinguishable from hTeBG under these conditions.