Pancreatic NOD beta cells express MHC class II protein and the frequency of I-A(g7) mRNA-expressing beta cells strongly increases during progression to autoimmune diabetes.

AIMS/HYPOTHESIS: In the NOD mouse model, attempts to show MHC class II expression by pancreatic beta cells were unsuccessful so far. We readdressed this question by analysing I-A(g7) expression in single pancreatic beta cells. METHODS: Single-cell multiplex RT PCR and single-cell immunofluorescence were used to study MHC class II expression in NOD and NOD/SCID beta cells. RESULTS: Pancreatic beta cells from NOD mice express the I-A(g7) protein as well as the corresponding mRNA. The frequency of MHC class II mRNA-expressing beta cells is drastically increased during the progression to overt diabetes. MHC class II protein is accumulated intracellularly, and invariant chain is co-expressed. Beta cells from 9- to 10-week-old NOD/SCID mice express MHC class II at the same low frequency as beta cells from 3-week-old NOD mice. CONCLUSION/INTERPRETATION: NOD beta cells express I-A(g7) and could be a direct target of autoreactive CD4+ T cells. This MHC class II expression is triggered by infiltrating lymphocytes.

CXCR4/CXCL12 expression and signalling in kidney cancer.

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12alpha/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12alpha, A-498 cells were found to express CXCR4. More importantly, real-time RT-PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12alpha and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12alpha was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12alpha. Comparing gene expression profiles in rhCXCL12alpha stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention.

Fingerprints of anergic T cells.

Peripheral T cell tolerance may result from activation-induced cell death [1], anergy [1], and/or immune response modulation by regulatory T cells [2]. In mice that express a transgenic receptor specific for peptide 111-119 of influenza hemagglutinin presented by E(d) class II MHC molecules as well as hemagglutinin under control of the immunoglobulin-kappa promoter, we have found that anergic T cells [3] can also have immunoregulatory function and secrete IL-10 [4]. In order to obtain information on molecular mechanisms involved in anergy and immunoregulation, we have compared expression levels of 1176 genes in anergic, naive, and recently activated CD4+ T cells of the same specificity by gene array analysis. The results provide a plausible explanation for the anergic phenotype in terms of proliferation, provide new information on the surface phenotype of in vivo-generated anergic CD4+ T cells, and yield clues with regard to new candidate genes that may be responsible for the restricted cytokine production of in vivo-anergized CD4+ T cells. The molecular fingerprints of such T cells should enable the tracking of this small population in the normal organism and the study of their role in immunoregulation.

Glucocorticoid production in the chicken bursa and thymus.

Glucocorticoid (GC) hormones play an important role in thymic T cell selection and in the development of autoimmune diseases. Previous studies have shown that the mammalian thymus itself is able to produce GC. In order to assess the importance of these findings in terms of the evolutionary development of the immune system, we investigated the functional presence of steroidogenic enzymes in primary lymphoid organs of chickens, which represent one of the best studied non-mammalian species. To this end, we attempted to demonstrate enzyme activities of the whole set of steroidogenic enzymes for the synthesis of GC in the bursa of Fabricius and the thymus. We isolated steroidogenic organelles from primary lymphoid tissues, incubated these with radioactive (precursor) steroids in vitro and visualized the resulting products by thin-layer chromatography. Our results show that the chicken bursa as well as the chicken thymus possesses all enzymes and cofactors required for GC production. The observation of GC production in an organ responsible for B cell selection and maturation is a further step in uncovering the yet ill-defined mechanism of B cell selection. These results provide the biochemical basis for the in situ hormonal effects, and underline the general importance of GC hormones on T and B lymphocyte development and selection.

Altered circadian rhythms of the stress hormone and melatonin response in lupus-prone MRL/MP-fas(Ipr) mice.

The immune system interacts with the hypothalamo-pituitary-adrenal axis via so-called glucocorticoid increasing factors, which are produced by the immune system during immune reactions, causing an elevation of systemic glucocorticoid levels that contribute to preservation of the immune reactions specificities. Previous results from our laboratory had already shown an altered immuno-neuroendocrine dialogue via the hypothalamo-pituitary-adrenal axis in autoimmune disease-prone chicken and mouse strains. In the present study, we further investigated the altered glucocorticoid response via the hypothalamo-pituitary-adrenal axis in murine lupus. We established the circadian rhythms of corticosterone, dehydroepiandrosterone-sulfate, adrenocorticotropic hormone and melatonin, as well as the time response curves after injection of interleukin-1 of the first three parameters in normal SWISS and lupus-prone MRL/MP-fas(Ipr) mice. The results show that lupus-prone MRL/ MP-fas(Ipr) mice do not react appropriately to changes of the light/dark cycle, circadian melatonin rhythms seem to uncouple from the light/dark cycle, and plasma corticosterone levels are elevated during the resting phase. Diurnal changes of dehydroepiandrosterone-sulfate and adrenocorticotropic hormone were normal compared to healthy controls. These data indicate that MRL/ MP-fas(Ipr) mice not only show an altered glucocorticoid response mediated via the hypothalamo pituitary adrenal axis to IL-1, but are also affected by disturbances of corticosterone and melatonin circadian rhythms. Our findings may have implications for intrathymic T cell development and the emergence of autoimmune disease.

Monitoring gene expression of TNFR family members by beta-cells during development of autoimmune diabetes.

Autoimmune diabetes results from destruction of pancreatic beta-cells by islet-infiltrating leukocytes. Different molecular mechanisms seem to be involved in this destruction but the results from many studies have not provided a clear picture so far. Therefore, we have developed a multiplex single-cell reverse transcription polymerase chain reaction to analyze the expression of genes of the tumor necrosis factor receptor (TNFR) family in pancreatic beta-cells during the development of autoimmune diabetes in a TCR-HA x INS-HA double transgenic as well as a non-obese diabetic (NOD) animal model. To this end we have followed the expression of cell surface receptors of the TNFR family in NOD mice as well as in double transgenic mice that express in their T cells class II MHC-restricted TCR specific for peptide 111 - 119 from influenza hemagglutinin (TCR-HA) as well as hemagglutinin under the control of the rat insulin promoter (INS-HA). Both types of mice develop insulitis and diabetes spontaneously. The data show a significant increase in the expression of Fas and TNFR2 (p75) during the development of insulitis, whereas TNFR1 (p55) is already expressed in beta-cells before the onset of insulitis. As ligands for these receptors are already expressed at high levels during the phase of insulitis, it is possible that beta-cell death is regulated by intracellular inhibitors of apoptosis pathways.

Glucocorticoid production in the murine thymus.

Glucocorticoid hormones are known to act as important modulatory factors in the development of autoimmune diseases, and to play an important role in thymic T-cell selection. There seems to be a finely balanced equilibrium between the apoptosis-inducing effects of glucocorticoid and T cell receptor ligand binding. Here we are investigating whether glucocorticoid-induced T cell apoptosis is mainly dependent on circulating glucocorticoid levels or if the thymus itself is able to produce glucocorticoids. To this end, we attempted to demonstrate enzyme activities of the whole set of steroidogenic enzymes for the synthesis of glucocorticoids in murine thymic tissue. We isolated steroidogenic organelles from thymic tissue, incubated these with radioactive (precursor) steroids in vitro, and visualized the resulting products by thin-layer chromatography. Our results show that the thymus possesses all enzymes and cofactors required for glucocorticoid production. However, an intact thymic architecture is necessary for glucocorticoid production, since 11beta-hydroxylase was not detected in irradiated thymi or in a thymic epithelial cell line. The results of these experiments show that the whole glucocorticoid metabolism takes place within the thymus. This finding provides the biochemical basis for the in situ effects of glucocorticoid hormones on thymocyte development and selection.

Autoimmune insulitis and diabetes in the absence of antigen-specific contact between T cells and islet beta-cells.

Autoimmune diabetes develops following recognition of organ-specific antigens by T cells. The disease begins with peri-islet infiltration by mononuclear cells, proceeds with insulitis and becomes manifest with destruction of insulin-producing islet beta-cells. T cells are necessary to induce insulitis and diabetes, but it is not clear by what mechanisms they can do so, i. e. whether the T cells need to make antigen-specific contact with the beta-cell or whether other interactions are sufficient to induce beta-cell death. In the present study we have constructed chimeric mice in which the bone marrow-derived antigen-presenting cells, but not the islet beta-cells, are capable of presenting antigen to monospecific T cells. We show that both insulitis as well as beta-cell destruction can proceed in the absence of islet beta-cell surface antigen recognition by T cells. Our results support the notion that diabetes can be caused by distinct effector mechanisms.

Hypothalamic beta-endorphin concentrations are decreased in animals models of autoimmune disease.

Complex interactions between the neuroendocrine and the immune systems are present in autoimmune diseases. The central opioid peptide beta-endorphin (BE) has been shown to modulate peripheral immune responses in normal animals. In the present study we analyze the hypothalamic concentrations of this peptide in two models of spontaneous autoimmune disease, the MRL [corrected] lpr/lpr mouse, that develops a lupus-like autoimmune disease, and the obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis. In both instances, hypothalamic concentrations of BE are significantly lower than normal controls. In MRL [corrected] lpr/lpr mice, BE is already lower at 1 month of age, when no clinical sign of the disease is yet present. Similarly, low levels of BE are observed in OS chickens before the onset of thyroiditis, i.e., already at the embryonic stage. Moreover, a further decrease of BE is observed in OS chickens in correspondence with the first signs of thyroid mononuclear infiltration. Considering the immunosuppressive effects exerted by central BE, these results are suggestive of the fact that in autoimmune disease prone animals the low hypothalamic concentrations may be one of several factors predisposing for the development of autoimmune disease.

Pleiotropic changes controlled by the pre-T-cell receptor.

The construction of various gene-deficient mice has facilitated the understanding of the role of various receptors and signaling pathways that control the generation of alphabeta lineage cells. A predominant role is occupied by the pre-TCR, which not only generates large numbers of alphabeta lineage cells but also controls TCRbeta allelic exclusion as well as commitment to the gammadelta lineage versus the alphabeta lineage.

Genomic structure of the human pre-T cell receptor alpha chain and expression of two mRNA isoforms.

The pre-TCR, which is minimally composed of the TCRbeta chain, the pre-Talpha chain, and the CD3 complex, regulates early T cell development. The pre-Talpha chain is a 33-kDa type I transmembrane glycoprotein with an extracellular part similar to the constant domain of the immunoglobulin supergene family. We have sequenced (11 kb) the human pTalpha gene, which like the murine pTalpha gene consists of four exons: exon 1 encodes the 5' untranslated region, the leader peptide and the first three amino acids of the mature protein, exon 2 the extracellular immunoglobulin (Ig)-like domain, exon 3 a 15-amino acid peptide including a cysteine required for heterodimerization with TCRbeta, exon 4 the transmembrane region, the cytoplasmic tail and the 3' untranslated sequence. The human pTalpha gene is located on chromosome 6p21.3, close to the HLA-A locus. Reverse transcription-PCR studies with human thymus and leukemic cells showed that alternative splicing produces a shorter pTalpha isoform, which lacks the Ig-like domain but contains the transmembrane elements and the extracytoplasmic cystein and which could thus permit pairing with TCRbeta chain and association with CD3 molecules. The conserved splice sites suggest a yet ill-defined biological function of the short pTalpha protein.

Neuroendocrine-immune disturbances in animal models with spontaneous autoimmune diseases.

According to our concept, the development of autoimmune disease depends on the presence of two sets of essential genes, one coding for an abnormal autoreactivity of the immune system, the other for a primary susceptibility of the target organ/structure for the immune attack. The final outcome of the disease in a given individual is then fine tuned by modulatory factors, such as diet or hormones. With regard to the latter, the immuno-endocrine interaction via the hypothalamo-pituitary-adrenal (HPA) axis has proven to be of special importance. Investigating the so-called Obese strain (OS) of chickens, an animal model with a spontaneously occurring Hashimoto-like autoimmune thyroiditis, we have first shown an impaired surge of glucocorticoid hormones after stimulation of the HPA axis by antigens or certain cytokines (glucocorticoid-increasing factors--GIFs). More recently, we have found a similar behavior in models with systemic autoimmune diseases, that is, murine lupus erythematosus and avian scleroderma. More detailed studies have, however, proven that the mechanisms underlying this altered immuno-endocrine communication via the HPA axis differs in different models. Finally, recent data point to the possibility that the classical pathways of glucocorticoid-T-cell interactions also take place in the thymus itself, which has been shown to be a site of steroid hormone production.

Disturbed immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in murine lupus.

Immune reactions and mitogen stimulation of mammals and chickens lead to an increase of glucocorticoid (GC) plasma levels concomitant with the immune response. Interleukin (IL) 1, one of the most important glucocorticoid increasing factors produced by cells of the immune system, acts via the hypothalamo-pituitary-adrenal (HPA) axis. This pattern of immunoendocrine feedback communication is altered in autoimmune disease (AID) and represents a possible site of action for GC therapy. In the present study we investigated the role and possible underlying mechanisms of a disturbed immunoendocrine communication via the HPA axis in murine lupus. We analyzed the response to recombinant human (rhu) IL-1alpha in AID-prone mice [NZB, NZW, (NZB/NZW)F1, MRL/MP-lpr] in comparison to nonautoimmune, normal control mice (Swiss, C3H/HeJ, MRL/MP-+/+) at different levels of the HPA axis. To this end, we quantified the plasma levels of ACTH, corticosterone, and corticosterone-binding globulin (CBG) and determined various pathology parameters for autoimmunity. AID-prone mice produced nearly the same levels of plasma corticosterone after injection of rhu IL-1alpha as normal mice, but had baseline corticosterone levels consistently higher, thus resulting in significantly lower corticosterone increasing ratios. ACTH levels increased after rhu IL-1alpha injection, but there was no clearcut difference in the increasing ratios of AID-prone and normal strains. CBG levels showed no difference. As expected, there was a correlation of pathology parameters for autoimmunity and the altered immunomodulatory response to rhu IL-1alpha per group. On an individual basis, there was no such correlation. In conclusion, our results confirm the existence of a disturbed immunoendocrine communication in AID-prone mice. This disturbance clearly differs from individual to individual and also among different types of AID.

[Fragments of human chorionic gonadotropin (hCG): diagnosis of pregnancy and tumors]. / Fragmente des humanen Choriongonadotropins (hCG): Schwangerschafts- und Tumordiagnostik.

The pregnancy and tumor marker human chorionic gonadotropin (hCG) belongs to the family of the glycoprotein hormones. Information on epitope forming sequences of hCG and its subunits hCG alpha and hcg beta has significant impact on the examination of intra- and extracellular metabolism and the standardization of diagnostic assay systems. Variants of hCG appear in biological fluids with variable modifications on different parts of the molecule. These changes may influence the binding patterns of monoclonal antibodies (MCA), thereby causing erroneous results in hCG immunoassays. The aim of the present work was to investigate the influence of peptide bond cleavages and the loss of certain segments of the molecule, which were induced by proteases on the expression of the seven hCG alpha-(alpha 1-alpha 7), nine hCG beta- (beta 1-beta 9) and four hCG beta-core-fragment-epitopes (beta 10-beta 13), previously identified by us [1-10]. To this end, we digested hCG alpha and hCG beta with chymotrypsin. Hormone fragments were separated by high performance liquid chromatography (HPLC) and subsequently immunochemically examined by direct binding radioimmunoassay (DB-RIA), competitive RIA and immunoenzymometric assays (IEMA). Fractions containing hCG-like immunoreactivity were sequenced by Edman and carboxypeptidase-Y degradation. It appeared that: (I) Amino acids (AA) alpha 41-47 and the peptide bonds between AA alpha 40/41, alpha 47/48 and alpha 29/30 do not influence the expression of the 7 alpha-epitopes, (II) The absence of the hCG beta N-terminus plays a crucial role for the formation of epitopes beta 10 and beta 13. (III) Neither the presence nor the absence of the C-terminal peptide of hCG beta (hCG beta CTP, AA beta 114-145) has any importance for the expression of epitopes beta 1-beta 7 and beta 10-beta 13 (IV).(ABSTRACT TRUNCATED AT 250 WORDS)

Free alpha subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains.

Immunochemical studies were undertaken to identify surface-orientated epitopes of the free alpha subunit of human chorionic gonadotrophin (hCG-alpha) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-alpha, an enzymatically digested hCG-alpha subunit, and a reduced and alkylated hCG-alpha preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-alpha, seven different epitopes (alpha 1-alpha 7), arranged in four spatially distinct domains, could be distinguished: A, alpha 1,2,4; B, alpha 3,5; C, alpha 6; D, alpha 7. The peptides spanning hCG-alpha(13-18), hCG-alpha(17-22) and hCG-alpha(33-42) appeared to contribute to the formation of epitopes alpha 2, alpha 4 and alpha 6 respectively. Since epitope alpha 6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-alpha(33-42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (alpha 7) are destroyed by reducing and alkylating hCG-alpha. In contrast, chymotryptic digestion of hCG-alpha, leading to release of the heptapeptide hCG-alpha(41-47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-alpha epitopes by radioreceptor assay revealed that the three hCG-alpha peptides corresponding to epitopes alpha 2, alpha 4 and alpha 6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (alpha 1-alpha 5), shared by hCG and hCG-alpha, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains.