A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta(1) in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

TGF-beta and CD23 are involved in nitric oxide production by pulmonary macrophages activated by beta-glucan from Paracoccidioides brasiliensis

Pulmonary macrophages (PM), which are CD11b/CD18(+) and CD23(+), may be involved in the onset of inflammatory events caused by Paracoccidioides brasiliensis in the lungs. In the present study, we measured the nitric oxide (NO) and interleukin in PM production after intratracheal (i.t.) inoculation of an enriched beta-glucan cell wall fraction from P. brasiliensis (Fraction F1). BALB/c and C57/BL6 (B6) mice were i.t. treated with Fraction F1, and their PM were restimulated in vitro with LPS and interferon-gamma up to 14 days after treatment. Macrophages BALB/c mice produced less NO than PM from B6 mice. The lower NO production was caused by higher production of TGF-beta by pulmonary macrophages of BALB/c and was abrogated by anti-TGF-beta MoAb in vitro and in vivo. Other interleukins such as IL-10, IL-4 and a combination of IL-1, TNF-alpha and IL-6 were not involved in NO production induced by Fraction F1. Expression of CD11b increases and expression of CD23 decreases on PM of BALB/c mice after in vivo treatment whereas PM of B6 mice do not show a variation of their phenotype. Moreover, the ability of pulmonary macrophages to induce lymphocyte proliferation was reduced in mixed cultures of CD11b(+) or CD23(+) macrophages but was restored when lymphocytes were cultivated in the presence of NO inhibitor (L-NMMA). Thus, the results presented herein indicate that in BALB/c but not in B6 mice TGF- is strongly induced by Fraction 1 in PM in vivo and suppresses NO production. Low NO production by PM is associated with a change in CD11b/CD23 expression and with a high lymphocyte proliferative response. Thus, CD11b(+)/CD23(+) PM modulate NO and TGF-beta production in the pulmonary microenvironment.

The choroid plexus transcriptome reveals changes in type I and II interferon responses in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a marked decline in cognition and memory function. Increasing evidence highlights the essential role of neuroinflammatory and immune-related molecules, including those produced at the brain barriers, on brain immune surveillance, cellular dysfunction and amyloid beta (Aß) pathology in AD. Therefore, understanding the response at the brain barriers may unravel novel pathways of relevance for the pathophysiology of AD. Herein, we focused on the study of the choroid plexus (CP), which constitutes the blood-cerebrospinal fluid barrier, in aging and in AD. Specifically, we used the PDGFB-APPSwInd (J20) transgenic mouse model of AD, which presents early memory decline and progressive Aß accumulation, and littermate age-matched wild-type (WT) mice, to characterize the CP transcriptome at 3, 5-6 and 11-12months of age. The most striking observation was that the CP of J20 mice displayed an overall overexpression of type I interferon (IFN) response genes at all ages. Moreover, J20 mice presented a high expression of type II IFN genes in the CP at 3months, which became lower than WT at 5-6 and 11-12months. Importantly, along with a marked memory impairment and increased glial activation, J20 mice also presented a similar overexpression of type I IFN genes in the dorsal hippocampus at 3months. Altogether, these findings provide new insights on a possible interplay between type I and II IFN responses in AD and point to IFNs as targets for modulation in cognitive decline.

Some biological properties of the human amniotic membrane interferon

Human amniotic interferon was investigated to define the species specificity of its antiviral action and compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cell in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Decrease in beta-Cell Proliferation Precedes Apoptosis during Diabetes Development in Bio-Breeding/Worcester Rat: Beneficial Role of Exendin-4

In autoimmune type 1 diabetes mellitus, proinflammatory cytokine-mediated apoptosis of beta-cells has been considered to be the first event directly responsible for beta-cell mass reduction. In the Bio-Breeding (BB) rat, an in vivo model used in the study of autoimmune diabetes, beta-cell apoptosis is observed from 9 wk of age and takes place after an insulitis period that begins at an earlier age. Previous studies by our group have shown an antiproliferative effect of proinflammatory cytokines on cultured beta-cells in Wistar rats, an effect that was partially reversed by Exendin-4, an analogue of glucagon-like peptide-1. In the current study, the changes in beta-cell apoptosis and proliferation during insulitis stage were also determined in pancreatic tissue sections in normal and thymectomized BB rats, as well as in Wistar rats of 5, 7, 9, and 11 wk of age. Although stable beta-cell proliferation in Wistar and thymectomized BB rats was observed along the course of the study, a decrease in beta-cell proliferation and beta-cell mass from the age of 5 wk, and prior to the commencement of apoptosis, was noted in BB rats. Exendin-4, in combination with anti-interferon-gamma antibody, induced a near-total recovery of beta-cell proliferation during the initial stages of insulitis. This highlights the importance of early intervention and, as well, the possibilities of new therapeutic approaches in preventing autoimmune diabetes by acting, initially, in the insulitis stage and, subsequently, on beta-cell regeneration and on beta-cell apoptosis.

The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic beta cells.

In insulin-secreting cells, cytokines activate the c-Jun N-terminal kinase (JNK), which contributes to a cell signaling towards apoptosis. The JNK activation requires the presence of the murine scaffold protein JNK-interacting protein 1 (JIP-1) or human Islet-brain 1(IB1), which organizes MLK3, MKK7 and JNK for proper signaling specificity. Here, we used adenovirus-mediated gene transfer to modulate IB1/JIP-1 cellular content in order to investigate the contribution of IB1/JIP-1 to beta-cell survival. Exposure of the insulin-producing cell line INS-1 or isolated rat pancreatic islets to cytokines (interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta) induced a marked reduction of IB1/JIP-1 content and a concomitant increase in JNK activity and apoptosis rate. This JNK-induced pro-apoptotic program was prevented in INS-1 cells by overproducing IB1/JIP-1 and this effect was associated with inhibition of caspase-3 cleavage. Conversely, reducing IB1/JIP-1 content in INS-1 cells and isolated pancreatic islets induced a robust increase in basal and cytokine-stimulated apoptosis. In heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene, the reduction in IB1/JIP-1 content in happloinsufficient isolated pancreatic islets was associated with an increased JNK activity and basal apoptosis. These data demonstrate that modulation of the IB1-JIP-1 content in beta cells is a crucial regulator of JNK signaling pathway and of cytokine-induced apoptosis.

Selective expression of the V beta 14 T cell receptor on Leishmania guyanensis--specific CD8+ T cells during human infection.

Peripheral blood mononuclear cells from subjects never exposed to Leishmania were stimulated with Leishmania guyanensis. We demonstrated that L. guyanensis-stimulated CD8(+) T cells produced interferon (IFN)- gamma and preferentially expressed the V beta 14 T cell receptor (TCR) gene family. In addition, these cells expressed cutaneous lymphocyte antigen and CCR4 surface molecules, suggesting that they could migrate to the skin. Results obtained from the lesions of patients with localized cutaneous leishmaniaisis (LCL) showed that V beta 14 TCR expression was increased in most lesions (63.5%) and that expression of only a small number of V beta gene families (V beta 1, V beta 6, V beta 9, V beta 14, and V beta 24) was increased. The presence of V beta 14 T cells in tissue confirmed the migration of these cells to the lesion site. Thus, we propose the following sequence of events during infection with L. guyanensis. After initial exposure to L. guyanensis, CD8(+) T cells preferentially expressing the V beta 14 TCR and secreting IFN- gamma develop and circulate in the periphery. During the infection, these cells migrate to the skin at the site of the parasitic infection. The role of these V beta 14 CD8(+) T cells in resistance to infection remains to be determined conclusively.

Interferon-gamma impacts at multiple points during the progression of autoimmune diabetes.

The role of interferon-gamma in autoimmune diabetes was assessed by breeding a null mutation of the interferon-gamma receptor alpha chain into the nonobese diabetic mouse strain, as well as into a simplified T cell receptor transgenic model of diabetes. In contrast to a previous report on abrogation of the interferon-gamma gene, mutation of the gene encoding its receptor led to drastic effects on disease in both mouse lines. Nonobese diabetic mice showed a marked inhibition of insulitis-both the kinetics and penetrance-and no signs of diabetes; the transgenic model exhibited near-normal insulitis, but this never evolved into diabetes, either spontaneously or after experimental provocation. This failure could not be explained by perturbations in the ratio of T helper cell phenotypes; rather, it reflected a defect in antigen-presenting cells or in the islet beta cell targets.

Variations in IB1/JIP1 expression regulate susceptibility of beta-cells to cytokine-induced apoptosis irrespective of C-Jun NH2-terminal kinase signaling.

We previously reported that interleukin-1beta (IL-1beta) alone does not cause apoptosis of beta-cells, whereas when combined with gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), it exerts a distinct apoptotic effect. Studies in beta-cell lines indicated that IL-1beta reduced expression of islet brain (IB)-1/JNK interacting protein (JIP)-1, a JNK scaffold protein with antiapoptotic action. We examined whether variations in IB1/JIP-1 expression in purified primary beta-cells affect their susceptibility to cytokine-induced apoptosis. Exposure to IL-1beta for 24 h decreased cellular IB1/JIP-1 content by 66 +/- 17%; this IL-1beta effect was maintained in the presence of TNF-alpha + IFN-gamma, which did not influence IB1/JIP-1 levels by themselves. Addition of IL-1beta to TNF-alpha + IFN-gamma increased apoptosis from 20 +/- 2% to 59 +/- 5%. A similar increase in TNF-alpha + IFN-gamma-induced apoptosis was produced by adenoviral expression of antisense IB1/JIP-1 and was not further enhanced by addition of IL-1beta, indicating that IL-1beta-mediated suppression of IB1/JIP-1 in beta-cells increases their susceptibility to cytokine-induced apoptosis. However, adenovirally mediated overexpression of IB1/JIP-1 also potentiated TNF-alpha + IFN-gamma-induced apoptosis, suggesting that the antiapoptotic effect of IB1/JIP-1 depends on well-defined cellular levels. We conclude that the IB1/JIP-1 level in beta-cells can control their susceptibility to apoptosis independent of JNK signaling.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia.