An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.     

Hydroxyl radical-induced hydrogen/deuterium exchange in amino acid carbon-hydrogen bonds

BB rats are used as models of autoimmune human IDDM. Genetic control of IDDM in both species is complex, including both major histocompatibility complex (MHC)-linked and non-MHC-linked genes. DP-BB rats develop IDDM spontaneously. Expression of disease in these animals requires homozygosity at the lyp locus, which causes lymphopenia. All genetic analyses of BB rat diabetes to date have backcrossed to the DP-BB strain or used (DP-BB x non-BB)F2 animals to ensure that a fraction of progeny are homozygous for lyp. Here we report the analysis of a backcross of the DP-BB rat to the histocompatible WF rat. Neither WF nor (WF x DP-BB)F1 animals develop spontaneous IDDM. However, 95% of (WF x DP-BB)F1 rats and a fraction of (WF x DP-BB) x WF backcross animals readily develop IDDM after treatment with polyinosinic:polycytidylic acid and a cytotoxic anti-RT6.1 monoclonal antibody. Using simple sequence length polymorphism analysis, we have mapped loci on chromosomes 4 and 13 that show significant linkage to IDDM expression and insulitis. The susceptibility locus on chromosome 4 is linked to, but not identical to, lyp. We propose a disease model for the BB rat that requires 1) the RT1u MHC haplotype for disease susceptibility, 2) a new locus on chromosome 4 for disease initiation (as measured by insulitis), 3) a new locus on chromosome 13 for disease progression in response to environmental perturbation, and 4) lyp for spontaneous expression of disease.     

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.     

A novel subset of adult gamma delta thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of gamma delta thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5% and 30% of total gamma delta thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1dull gamma delta thymocytes from DBA/2 mice with anti-gamma delta monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-gamma (IFN-gamma), IL-4, IL-10, and IL-3. In contrast, only IFN-gamma was detected in parallel cultures of Thy-1bright gamma delta thymocytes. Virtually all Thy-1dull gamma delta thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1dull gamma delta thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1dull gamma delta thymocytes from DBA/2 mice express TCR encoded by the V gamma 1 gene and a novel V delta 6 gene named V delta 6.4. Sequence analysis of these functionally rearranged gamma and delta genes revealed highly restricted V delta-D delta-J delta junctions, and somewhat more diverse V gamma-J gamma junctions. We conclude that Thy-1dull gamma delta thymocytes exhibit properties that are equivalent to those of natural killer TCR alpha beta T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Antigen activation rescues recent thymic emigrants from programmed cell death in the BB rat

One of the diabetes susceptibility genes of the BB rat is a mutation at the lyp locus that decreases the thymic output of T cells and the life span of most recent thymic emigrants (RTE). Consequently, there is a 10-fold reduction in the number of CD4+ and CD8+ T cells in secondary lymphoid organs. Results presented in this work demonstrate that the BB rat lyp mutation is associated with an accelerated apoptotic death in vitro of mature CD4+ 8- and CD4- 8+ thymocytes and peripheral T cells. The stability of the pool of recirculating T cells (PRL) of BB rats over time results from a > 10-fold increase in the mitotic activity of T cells as assessed in vivo by bromodeoxyuridine incorporation. This increased mitotic activity is not observed when BB T cells develop in the context of a normal sized PRL. MHC haploidentical WF and BB rats differ at minor histocompatibility loci. Intravenous injection of (WF x BB)F1 T cells into euthymic BB rats led to the rejection of donor T cells within 3 wk by unprimed recipients and within 1 wk by primed recipients. This secondary immune response was unaffected by postpriming thymectomy. F1 T cells were not rejected, but rather expanded after their injection into thymectomized BB rats that had been primed as early as 48 h after thymectomy. These results strongly suggest that the BB rat PRL is devoid of long-lived naive T cells and that rescue of recent thymic emigrants from programmed cell death is initiated by Ags, exclusively.     

Shift from anti- to proinflammatory cytokine profiles in microglia through LPS- or IFN-gamma-mediated pathways

In this study, cytokine mRNA profiles in microglia from newborn rats were detected by in situ hybridization. Under natural culture conditions, microglia expressed the immunosuppressive transforming growth factor-beta 1 (TGF-beta 1) and interleukin (IL) 10 to a greater degree than the pro-inflammatory cytokines IL-1 beta, IL-6, IL-12, interferon-gamma (IFN-gamma) and TNF-alpha. High TGF-beta 1 and IL-10 levels could reflect one mechanism for immune privilege within the CNS under physiological conditions. Stimulation of microglia with LPS or IFN gamma resulted in strong up-regulation of proinflammatory cytokines, while TGF-beta 1 and IL-10 were down-regulated. These effects of LPS or IFN-gamma are anticipated to reflect immunopathogenic processes within the CNS.     

Delayed cytokine expression in rat brain following experimental contusion

Proinflammatory cytokines mediate brain injury in experimental studies. This study was undertaken to analyze the production of proinflammatory cytokines in experimental contusion. A brain contusion causing delayed edema was mimicked experimentally in rats using a weight-drop model. Intracerebral expression of the cytokines interleukin (IL)-1 beta, tumor necrosis factor-alpha (TNF alpha), IL-6, and interferon-gamma (IFN gamma) was studied by in situ hybridization and immunohistochemistry. The animals were killed at 6 hours or 1, 2, 4, 6, 8, or 16 days postinjury. In the injured area, no messenger (m)RNA expression was seen during the first 2 days after the trauma. On Days 4 to 6 posttrauma, however, strong IL-1 beta, TNF alpha, and IL-6 mRNA expression was detected in mononuclear cells surrounding the contusion. Expression of IFN gamma was not detected. Immunohistochemical double labeling confirmed the in situ hybridization results and demonstrated that mononuclear phagocytes and astrocytes produced IL-1 beta and that mainly astrocytes produced TNF alpha. The findings showed, somewhat unexpectedly, a late peak of intracerebral cytokine production in the injured area and in the contralateral corpus callosum, allowing for both local and global effects on the brain. An unexpected difference in the cellular sources of TNF alpha and IL-1 beta was detected. The cytokine pattern differs from that seen in other central nervous system inflammatory diseases and trauma models, suggesting that the intracerebral immune response is not a uniform event. The dominance of late cytokine production indicates that many cytokine effects are late events in an experimental contusion: Different pathogenic mechanisms may thus be operative at different times after brain injury.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

The effects of oophorectomy and female sex steroids on glucose kinetics in the rat

In the present study, we have analyzed the appearance and maturation of gamma/delta T cells, recognized with a new mAb V65, in the central and peripheral lymphoid organs of fetal, neonatal, and adult Wistar rats. Cytofluorometrical analysis demonstrated the first V65+ gamma/delta T cells in the thymus of 16-17-day embryonic rats, although by immunohistology, they were identified only in 19-day rat embryos in both the cortico-medullary border and thymic medulla. Phenotypically, gamma/delta thymocytes from fetal and neonatal thymus expressed CD3, CD2, and CD5, but only 60-80% were CD8+ and approximately 40-50% expressed the alpha chain (p55) of the IL-2R. In the periphery, the immunohistological study identified for the first time gamma/delta T cells in the splenic white pulp and the gut of 21-day fetal rats, where they occurred within the epithelium as well as in the lamina propria. After birth, gamma/delta lymphocytes appeared in the skin, where they were present as dendritic epidermal T cells in increasing numbers during postnatal life. Whereas these gamma/delta T cells formed the predominant T-cell population in the rat skin, gamma/delta T cells in peripheral lymphoid organs, BALT, or the gut only represented a minor T-cell population. These results are discussed in comparison to gamma/delta T cells of other vertebrate species.     

Modulation of IL-12 by transforming growth factor-beta (TGF-beta) in Mycobacterium tuberculosis-infected mononuclear phagocytes and in patients with active tuberculosis

In humans, tuberculosis is associated with suppression of T-cell responses to antigens of Mycobacterium tuberculosis. Recently, the macrophage product, transforming growth factor-beta (TGF-beta) has been implicated in suppression of T-cell proliferation and cytokine production during tuberculosis. We studied the effect of TGF-beta on production of IL-12, and on the augmentation of M. tuberculosis-induced IFN gamma production by IL-12, in patients with pulmonary tuberculosis and by M. tuberculosis. Induction of IL-12 p35, but not IL-12 p40, by M. tuberculosis in monocytes was dependent on prior priming of the cells with IFN gamma. Expression of both IL-12 p40 and p35, however, was suppressed by TGF-beta. Further, TGF-beta interfered with the bioactivity of IL-12 in the enhancement of M. tuberculosis-induced IFN gamma mRNA expression and cytokine production. However, in mononuclear cells from patients with tuberculosis the main effect of TGF-beta on IL-12 appeared to be counter action to IL-12 induced IFN gamma production in response to M. tuberculosis.     

Leptin normalizes the impaired response of proinsulin mRNA to long chain fatty acids in heterozygous Zucker diabetic fatty rats

To determine if underleptinization of islets of Zucker diabetic fatty (ZDF) rats is the proximal cause of their inability to compensate for obesity, we compared the proinsulin/beta-actin mRNA ratio in heterozygous (fa/+) ZDF rats with that of wild-type (+/+) and homozygous (fa/fa) ZDF rats. In +/+ islets cultured with 2 mM free fatty acids (FFA) the proinsulin mRNA ratio rose 2.4-fold at 12 h. In fa/+ islets, the ratio rose only 65% above normal. There was no change in fa/fa islets. The presence of leptin (20 ng/ml) in the culture medium increased the FFA-induced response of proinsulin mRNA of fa/+ islets to that of +/+ islets while reducing FFA incorporation into triglycerides. The leptin-induced improvement in the proinsulin mRNA response was independent of any changes in glucose usage. These findings support a causal relationship between diminished leptin action on islets and the impaired beta-cell response to FFA in ZDF rats.     

A role for T-helper type 1 and type 2 cytokines in the pathogenesis of various human diseases

Mosmann first proposed the existence of subsets of CD4+ T cells that produce distinct types of cytokines. Native T lymphocytes (Thp cells) differentiates into either CD4+ Th1 cells that produce IL-2, IFN gamma, and lymphotoxin which promote cell-mediated immunity, or into Th2 cells that produce IL-4, IL-5, IL-6, IL-10 and IL-13, which promote antibody production and humoral immunity. These T cell subsets reciprocally regulate one another since one of the Th1 products, IFN gamma, inhibits the proliferation and functions of Th2 cells, whereas the Th2 products, IL-4 and IL-10, suppress cytokine production by Th1 cells. A distinct Th1/Th2 divergence determine resistance versus susceptibility to diseases such as leishmaniasis and toxoplasmosis in mice. In allergic diseases such as atopic dermatitis and allergic asthma, allergen-specific T cells acquired the Th2 phenotype. These Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. These cytokines induce eosinophilia and an Ig class switch to IgG4 and IgE. These Th2 cells are responsible for the enhanced production of IgE antibodies. These findings indicate that Th2 cytokines play an important role in the development of allergic diseases. The importance of cell-mediated immunity, particularly donor-anti-host CTL, in mediating acute GVHD suggests that Th1 cytokines may be important in the induction of acute GVHD. To further characterize the roles of Th1 and Th2 cytokines in the development of acute GVHD, analysis of IL-2, IFN gamma, IL-4 and IL-10 cytokine genes was performed by RT-PCR on biopsied skin specimen. An increase in mRNA expression for IL-2 and IFN gamma was observed, whereas there was no significant increase in IL-4 and IL-10 mRNA. These data suggest that Th1 cytokines may be essential for the development of acute GVHD. It is apparent that Th1 cytokines are generally harmful to the maintenance of pregnancy. We have shown that Th2 cytokines are produced by maternal T lymphocytes at the maternal-fetal surface (retroplacental blood lymphocytes). This finding strengthens the hypothesis of a significant contribution of Th2 cytokines to a successful pregnancy.     

Anti-TNF antibody modulates cytokine and MHC expression in cardiac allografts

Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine evoked in response to alloantigen stimulation and may be involved in lymphocyte activation, adhesion molecule expression, and regulation of MHC class II antigens. Anti-TNF treatment prolongs cardiac allograft survival. We investigated the role of anti-TNF in the regulation of MHC class II antigens and cytokine mRNA expression of TNF, interferon-gamma (IFN), IL-2, IL-4, and IL-10 in cardiac allografts to elucidate its immunological mechanism. These in vivo studies were conducted using a rat MHC mismatch Brown-Norway to Lewis (BN to LEW) heterotopic cardiac transplant model. In control untreated rats, allografts were rejected at 6.8 +/- 0.6 days. Allograft survival was significantly prolonged to 12.7 +/- 1.4 days with anti-TNF treatment. MHC class II expression, analyzed by indirect immunofluorescence cytometry, demonstrated that MHC class II-positive cells increased by 25% in spleens of untreated allografted rats compared to naive rats, while anti-TNF-treated allografted rats had a similar percentage of MHC II cells as naives. Further, naive, untransplanted rats and both anti-TNF and untreated, transplanted rats had heart and spleens harvested on Day 5 post-transplant. Cytokine mRNA expression was determined by semiquantitative RT-PCR. In heart and spleen cells from naives, TNF mRNA expression was undetectable or very weak. However, in rejecting allografts and spleen cells from untreated recipients, TNF expression was remarkably increased, while anti-TNF attenuated this TNF expression in both heart graft and spleen cells. Furthermore, IL-2, IL-10, and IFN expression were absent in naive hearts. However, in untreated allografts IL-2, IL-10, and IFN were strongly expressed, which was markedly decreased after anti-TNF treatment. Finally, IL-4 expression was found equally in naive hearts, untreated allografts, and anti-TNF-treated allografts. These results suggest that anti-TNF antibody treatment may not only neutralize TNF activity but also play a role in altering cytokine mRNA expression and MHC class II expression.     

Promoter analysis of the human mismatch repair gene hMSH2

On the basis of both the interleukin-2-receptor (IL-2R) alpha-chain expression on 16-day-old fetal rat thymocytes and the occurrence of interleukin-2 (IL-2) mRNA-containing cells early during rat thymus ontogeny, we have investigated the possible role of IL-2/IL-2R complex in rat T-cell maturation. For this purpose, we analyzed the effects of the addition of either recombinant rat IL-2 or anti-CD25 (OX-39)-blocking monoclonal antibodies to fetal thymus organ cultures (FTOC), established from 16-day-old rat embryos. IL-2 stimulated the growth of thymocytes and, as a result, induced T-cell differentiation, whereas OX-39 mAb blocked the maturation of thymic-cell progenitors. Accordingly, these results support the involvement of IL-2/IL-2R complex in rat T-cell development.     

Tumour-derived, endocrine, exogenous and therapeutic factors differentially modulate cytokine secretion in whole blood cell culture

Following our previous results which showed that TGF-beta 1 suppressed the secretion of certain cytokines, we investigated the effects of different endogenous and exogenous factors on cytokine secretion in whole blood cell culture by using an enzyme-linked immunosorbent assay (ELISA) for measurement of cytokine concentrations. Several molecules including dexamethasone, noradrenaline (NA) and ethanol differentially inhibited mitogen-induced cytokine secretion. Dexamethasone and noradrenaline suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha, IL-1 alpha and IL-1 beta. beta-Endorphin and Leu-Enkephalin had no significant influence on cytokine secretion. Suppression of cytokine secretion by TGF-beta 1 was further intensified significantly and dose dependently by addition of noradrenaline. GM-CSF stimulated the secretion of IL-1 alpha, IL-1 beta and TNF gamma, but had no influence on the secretion of IL-2, IFN alpha and IFN gamma. G-CSF, IL-3 and SCF did not significantly influence secretion of all cytokines tested. Thus, endogenous and exogenous factors differentially influence cytokine secretion by immunocompetent cells.     

Antimutagenic activity of extracts from Japanese eggplant

There is increasing evidence that Schwann cells play an important role in the pathogenesis of autoimmune inflammatory peripheral nerve disease. Schwann cells have been reported to express major histocompatibility complex class I and II (MHC I and II) and intercellular adhesion molecule-1 (ICAM-1), and to produce interleukin-1 (IL-1), prostaglandin E2 and thromboxane A2. In this study we investigated freshly dissociated neonatal Lewis rat Schwann cells and a SV40 transfected neonatal rat Schwann cell line (Schwann cell line) for production of mRNA for the immunomodulatory cytokines IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN gamma), and tumor necrosis factor-alpha (TNF alpha) employing RT-PCR. Primary Schwann cells and Schwann cell line were examined following IFN gamma stimulation and were found to express TNF alpha and IL-6 mRNA. These results further support a role for Schwann cell participation in inflammatory responses within the peripheral nervous system (PNS).