Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.     

Cutting edge: antigen-dependent regulation of telomerase activity in murine T cells

Telomeres, structures on the ends of linear chromosomes, function to maintain chromosomal integrity. Telomere shortening occurs with cell division and provides a mechanism for limiting the replicative potential of normal human somatic cells. Telomerase, a ribonucleoprotein enzyme, synthesizes telomeric repeats on chromosomal termini, potentially extending the capacity for cell division. The present study demonstrates that resting T cells express little/no activity, and optimal Ag-specific induction of telomerase activity in vitro requires both TCR and CD28-B7 costimulatory signals. Regulation of telomerase in T cells during in vivo Ag-dependent activation was also assessed by adoptive transfer of TCR transgenic T cells and subsequent Ag challenge. Under these conditions, telomerase was induced in transgenic T cells coincident with a phase of extensive clonal expansion. These findings suggest that telomerase may represent an adoptive response that functions to preserve replicative potential in Ag-reactive lymphocytes.     

Induction of circulating IL-1 receptor antagonist by IFN treatment

This study was undertaken to determine whether IFN induce IL-1 receptor antagonist (IL-1Ra), a specific inhibitor of IL-1. Plasma samples were obtained from healthy volunteers (n = 5) and patients with chronic hepatitis C (n = 5) treated with IFN-alpha, and from patients with renal cell carcinoma (n = 6) treated with IFN-gamma and assayed for IL-1Ra by a specific radioimmunoassay. Both types of IFN were administered subcutaneously. In vitro studies were carried out with PBMC from healthy volunteers. A single, low and nontoxic dose (1 x 10(6) U) of IFN-alpha induced circulating IL-1Ra, which reached peak levels within 12 h. This effect was dose-dependent and more pronounced with a higher dose (5 x 10(6) U). Peak IL-1Ra levels 12 h after 5 x 10(6) U IFN-alpha were 4.16 +/- 0.35 ng/ml in healthy volunteers and 5.7 +/- 0.73 ng/ml in patients with chronic hepatitis C (difference not significant). Thereafter levels declined but remained elevated for 24 h. IFN-gamma treatment led only to a modest increase of circulating IL-1Ra even at a dose of 400 micrograms; this dose, however, was associated with side effects similar to those seen after injection of 5 x 10(6) U IFN-alpha. PBMC stimulated with IFN-alpha or IFN-gamma produced IL-1Ra in vitro. The induction of IL-1Ra may contribute to the antiviral, anti-inflammatory, and antiproliferative effects of IFN.     

Cutting edge: protective effects of notch-1 on TCR-induced apoptosis

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1 beta

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1 beta (IL-1 beta). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1 beta, IL-6 and IL-8. IL-1 beta activation of GF cells showed that IL-1 beta non only induces the expression of IL-6, IL-8 and TNF-alpha, but also acts in an autocrine manner of GF cells and induces IL-1 beta expression. Furthermore, the continuous presence of IL-1 beta in GF cell cultures did not down regulate the response of GF cells to IL-1 beta. Pretreatment of GF cells with IL-1 beta resulted in the enhanced synthesis of TNF-alpha in response to additional IL-1 beta. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation

We have recently developed approaches for the generation of encephalitogenic T cell clones from mouse strains considered resistant to experimental allergic encephalomyelitis (EAE). By allowing for the direct use of knockout and mutant strains of mice, such clones allow for the efficient characterization of the relevance of specific gene products in the effector phase of EAE. Recent studies have suggested that Fas/FasL-mediated cell death may play a role in the pathogenesis of MS. To assess the role of Fas/FasL in EAE, we have tested the ability of wild-type C57BL/6-derived, encephalitogenic T cell clones to mediate adoptively transferred EAE in Fas-deficient C57BL/6-lpr mice. We now report that mice with the lpr mutation are fully susceptible to the adoptive transfer of EAE. Our results suggest that Fas/FasL-mediated cell death in the central nervous system does not play an integral role in the effector phase of acute EAE.     

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia

This brief history of the dental diamond bur is intended to provide both a historical perspective and an evaluation of the current state of bur technology. An understanding of the origins of dental diamonds and the issues facing manufacturers transforms the dentist from a simple user into an informed consumer. The author contends that this can improve dental care and enable the dentist to collaborate with manufacturers in developing improved dental burs.     

Kinetics of intraocular cytokines in the suppression of experimental autoimmune uveoretinitis by type I IFN

The systemic administration of IFN-alpha/beta was previously found to suppress inflammation in rats with experimental autoimmune uveoretinitis (EAU); however, an effect on the systemic immune response was not identified. In order to investigate an immunological basis for suppression at the intraocular level, rats immunized with interphotoreceptor retinoid-binding protein (IRBP) were administered daily intramuscular injections of 10(5) IU IFN-alpha/beta and cytokines were measured by ELISA in intraocular extracts prepared by ultrasonification at various timepoints throughout the course of EAU. In control EAU, intraocular concentrations of IFN-gamma were found to be non-detectable on day 8 before the onset of inflammation, significantly elevated on day 12 at peak inflammation (182+/-106 pg/ml), then non-detectable again on day 16 after inflammation had begun to subside. In contrast, intraocular IFN-gamma in IFN-alpha/beta-treated rats remained non-detectable or low at all timepoints. Measurement of intraocular IL-2 revealed no difference between the two groups of rats. Intraocular IL-4 concentrations were elevated in rats treated with IFN-alpha/beta, although this cytokine was also detected in the same range in controls as well as normal rats. Finally, intraocular IL-10 was non-detectable on day 8, significantly elevated at peak inflammation on day 12 (588+/-139 pg/ml), then decreased to low levels on day 16 in control EAU rats, while remaining non-detectable or low in IFN-alpha/beta-treated rats. These results suggest that acute inflammation in IRBP-induced EAU in rats involves both IFN-gamma and IL-10 at the local intraocular level, and that systemic administration of IFN-alpha/beta inhibits EAU via a mechanism that involves suppression of both cytokines.     

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.     

Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor

Insulin and insulin-like growth factor-1 (IGF-1) treatment of cells overexpressing the insulin receptor or the IGF-1 receptor promotes phosphorylation and activation of Janus kinases JAK-1 and JAK-2 but not of TYK-2. With insulin, we observed maximal phosphorylation of JAK-1 within 2 min (5.2 +/- 0.6-fold) and maximal phosphorylation of JAK-2 within 10 min (2.4 +/- 0.6-fold). In cells incubated with IGF-1, we found maximal phosphorylation of JAK-2 within 2 min (1.9 +/- 0.2-fold) and of JAK-1 within 5 min (4.5 +/- 0.4-fold). The JAKs from insulin- or IGF-1-stimulated cells were activated, as shown by their autophosphorylation in vitro. Moreover, they were able to phosphorylate in vitro native insulin receptor substrate (IRS)-1 and a fragment of IRS-2 (GST-IRS-2591-786). Comparison of 32P-peptide maps of IRS-1 phosphorylated in vitro by the insulin receptor vs. JAK-1 showed the occurrence of different phosphopeptides, suggesting that different sites are likely to be phosphorylated by the two kinases. Finally, coprecipitation of receptors and JAK-1 was seen, and phosphorylation of both receptors was found to be necessary for receptor binding to JAK-1. Two domains of JAK- 1 are involved in the formation of the complex between receptor and JAK-1, i.e. the N-terminal portion containing JH7 and JH6 domains, and the C-terminal kinase domain (JH1 domain). Taking our data together, we conclude that: 1) insulin and IGF-1 lead to phosphorylation and activation of JAK-1 and JAK-2 in intact cells; 2) phosphorylation of IRS-I by JAK-1 seems to occur on sites different from those phosphorylated by the insulin receptor; 3) JAK-1 interacts directly with phosphorylated insulin and IGF-1 receptors; and 4) the JH7-JH6 and JH1 domains of JAK-1 are responsible for the interaction with insulin and IGF-1 receptors.     

Engineering the C-region of human insulin-like growth factor-1: implications for receptor binding

Recombinant wild-type human IGF-1 and a C-region mutant in which residues 28-37 have been replaced by a 4-glycine bridge (4-Gly IGF-1) were secreted and purified from yeast. An IGF-1 analogue in which residues 29-41 of the C-region have been deleted (mini IGF-1) was created by site-directed mutagenesis and also expressed. All three proteins adopted the insulin-fold as determined by circular dichroism. The significantly raised expression levels of mini IGF-1 allowed the recording of two-dimensional NMR spectra. The affinity of 4-Gly IGF-1 for the IGF-1 receptor was approximately 100-fold lower than that of wild-type IGF-1 and the affinity for the insulin receptor was approximately 10-fold lower. Mini IGF-1 showed no affinity for either receptor. Not only does the C-region of IGF-1 contribute directly to the free energy of binding to the IGF-1 receptor, but also the absence of flexibility in this region eliminates binding altogether. As postulated for the binding of insulin to its own receptor, it is proposed that binding of IGF-1 to the IGF-1 receptor also involves a conformational change in which the C-terminal B-region residues detach from the body of the molecule to expose the underlying A-region residues.