Inflammatory cytokines in small intestinal mucosa of patients with potential coeliac disease.

T helper cell type 1 (Th1) response to gluten has been implicated in the pathogenesis of coeliac disease (CD). To characterize immunological activation and mild inflammations leading to overt CD in potential coeliac patients, jejunal biopsies were obtained from family members of patients with CD or dermatitis herpetiformis (DH). Nine family members and one latent CD, eight CD patients and eight normal controls furnished jejunal biopsy specimens. Immunohistochemical staining of sections for interleukin-1alpha (IL-1alpha), IL-2, IL-4, interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), CD3, gammadelta-T cell receptor (gammadelta-TCR), and alphabeta-TCR was carried out with monoclonal antibodies. Further, expression of IL-4 and IFN-gamma messenger RNA was detected by radioactive in situ hybridization in these same samples. In lamina propria, CD patients and potential CD patients had higher densities of IL-2 (P = 0.028, P = 0.043), IL-4 (P = 0.021, P = 0.034) and IFN-gamma positive cells (P = 0.000, P = 0.009) than did controls. Moreover, CD patients showed a higher density of TNF-alpha positive cells (P = 0.012, P = 0.001) than the other two groups, and expression of IFN-gamma mRNA (P = 0.035) was higher in them than in the other two study groups. Additionally, higher densities of TNF-alpha and IFN-gamma positive cells occurred in potential CD patients with high gammadelta-TCR+ intraepithelial lymphocytes (IELs). Our findings support the hypothesis that lamina propria T cells and macrophages, through their secretion of cytokines, play a central role in the pathogenesis of coeliac disease. The inflammatory cytokines found in potential CD specimens strongly suggest that these inflammatory markers can be identified long before visible villous changes have occurred.

Regulated expression of prostaglandin E(2) receptors EP2 and EP4 in human ovarian granulosa-luteal cells.

Prostaglandins (PGs) have been implicated in regulation of ovarian function. We have previously shown that the expression of cyclooxygenase-2 and the receptor for PGF(2 alpha) are expressed in periovulatory human granulosa cells and upregulated by gonadotropins and cytokines in cultured human ovarian granulosa-luteal (GL) cells. We now show that transcripts for PGE(2) receptor subtypes EP2 and EP4 are expressed in freshly isolated human granulosa cells and in mouse ovaries as detected by Northern blot analysis. However, EP2 and EP4 receptor mRNA levels were low or nondetectable in cultured human GL cells suggesting that these transcripts may be under hormonal and/or cytokine regulation in the ovaries in vivo. Indeed, the proinflammatory cytokine interleukin-1 beta (IL-1 beta) stimulated expression of EP2 and EP4 transcripts in concentration- and time-dependent manner in the GL cells. Furthermore, the transcript for EP2 receptor was localized in the corpus luteum of the mouse ovary by in situ hybridization, and EP2 protein was expressed in human corpus luteum as detected by immunohistochemistry. Our data suggest that IL-1 beta induces expression of EP2 and EP4 receptors in human GL cells, and that EP2 receptor is expressed in both human and murine luteal glands.

Expression of transcription factor GATA-4 during human testicular development and disease.

GATA-4 is a highly conserved transcription factor that plays a critical role in regulating embryonic morphogenesis and cellular differentiation. Given the emerging role of GATA-4 in the development and function of murine gonads, we have now studied its role in human testis. We find that GATA-4 is expressed from early human fetal testicular development to adulthood. This transcription factor is evident in Sertoli cells through fetal and postnatal development. Expression of GATA-4 in Sertoli cells peaks at 19-22 weeks gestation at the time of high circulating fetal FSH. In Leydig cells, GATA-4 is expressed during fetal period and after puberty, coinciding with the periods of active androgen synthesis in the testis; this suggests a link between GATA-4 and steroidogenesis. Also, fetal germ cells and prepubertal spermatogonia express GATA-4, and it is down-regulated in these cells after puberty. As hormonal regulation of GATA-4 in human testis was not possible to study directly, we used testicular samples from patients who had endocrine abnormalities or were hormonally treated. Testicular expression of GATA-4 in hCG-treated cryptorchidism does not differ from that in controls. In androgen resistance, GATA-4 expression in Sertoli and germ cells is weak or totally absent. GATA-4 protein is abundantly present in Sertoli and Leydig cell tumors, suggesting a relationship to tumorigenesis or tumor progression in somatic cell-derived testicular neoplasms.

Transcription factors GATA-4 and GATA-6 and a GATA family cofactor, FOG-2, are expressed in human ovary and sex cord-derived ovarian tumors.

Previous studies have implicated transcription factors GATA-4 and GATA-6 in the regulation of murine ovarian development and function. In rodents, GATA-4 is expressed in granulosa cells of primary and early antral follicles, whereas GATA-6 is expressed in granulosa cells of late antral follicles and luteal glands. Both transcription factors can be detected in lesser amounts in theca cells and interstitial cells. We have now examined the expression of GATA-4 and GATA-6 in human ovaries, human granulosa-luteal (GL) cells and sex cord-derived tumors. We show by in situ hybridization and immunohistochemistry that GATA-4 and GATA-6 messenger RNA (mRNA) and GATA-4 protein are present in granulosa and theca cells in both preantral and antral follicles. Both human ovarian tissue samples and freshly isolated GL cells derived from preovulatory follicles of gonadotropin-treated women express GATA-4, GATA-6, and FOG-2 transcripts, and GATA-6 mRNA expression in GL cell cultures is stimulated by human CG and 8-bromo-cAMP. The vast majority of granulosa and theca cell tumors examined expressed GATA-4 and GATA-6. We also found that mRNA for FOG-2, a recently discovered regulator of GATA-4, is coexpressed with GATA-4 in human ovary samples, normal granulosa cells, and in sex cord-derived tumors. Our results demonstrate that GATA-4, GATA-6, and FOG-2 are expressed in human ovary and in granulosa and theca cell tumors. Our findings support a role for GATA-binding proteins in human ovarian folliculogenesis. Moreover, these data suggest that GATA factors may contribute to the phenotypes of sex cord-derived ovarian tumors.

Expression and regulation of transcription factors GATA-4 and GATA-6 in developing mouse testis.

Previous studies have shown that transcription factors GATA-4 and GATA-6 are expressed in granulosa and thecal cells of the mouse ovary and that GATA-4 expression in ovarian tissue is regulated by gonadotropins. Given the emerging role of GATA-4 and GATA-6 in gonadal cells, we have now studied the expression and regulation of these factors in the mouse testis and testicular cell lines. In situ hybridization demonstrated GATA-4 messenger RNA (mRNA) in the fetal testis at 13.5 days postcoitum. Both GATA-4 and GATA-6 transcripts were observed in late fetal, neonatal, juvenile, and adult Sertoli cells. In addition, GATA-4 mRNA was detected in interstitial cells throughout development. Immunohistochemistry demonstrated GATA-4 protein in both Sertoli and Leydig cells in postnatal animals. The regulation of GATA-4 and GATA-6 expression was explored using established testicular cell lines. Treatment of Leydig tumor cell lines with hCG resulted in a modest, but statistically significant, increase in the steady state level of GATA-4 mRNA, comparable to the previously described effect of FSH on GATA-4 expression in Sertoli cell lines. Gonadotropin or androgen action was not, however, a prerequisite for the basal expression of GATA-4 or GATA-6 in the testis, as their presence in Sertoli and Leydig cells was demonstrated in genetically hypogonadal hpg mice, in rats treated with GnRH receptor antagonist, and in Sertoli cells after chemical abolition of Leydig cells. Cotransfection studies using a GATA-4 expression plasmid and an inhibin alpha promoter/reporter gene construct in Leydig and granulosa tumor cell lines revealed that the inhibin alpha promoter harboring essential GATA-binding sites can be trans-activated by GATA-4. In light of these results, we propose that transcription factors GATA-4 and GATA-6 play differing roles in the maturation and function of testicular somatic cells.

A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis.

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.