IFN-gamma production of adult rat astrocytes triggered by TNF-alpha

IFN-gamma plays an important role in modulating inflammatory responses within the CNS. The cell type responsible for IFN-gamma production within the CNS is less well defined. We examined the production and regulation of IFN-gamma by adult rat astrocytes. IFN-gamma was hardly detectable in cultured astrocytes, while addition of TNF-alpha dose-dependently induced IFN-gamma production by astrocytes. No IFN-gamma production by astrocytes could be induced by LPS, IL-10 or TGF-beta 1. TNF-alpha-induced IFN-gamma production by astrocytes was inhibited by treatment of astrocytes with TGF-beta 1, but not IL-10. TNF-alpha induced IFN-gamma production by astrocytes was confirmed by using immunocytochemical staining. The data suggest that astrocyte-derived IFN-gamma induced by TNF-alpha may participate in local immune reactions of the brain in an autocrine and paracrine fashion.     

Cytokine regulation of human microglial cell IL-8 production

IL-8 involvement in neutrophil activation and chemotaxis may be important in inflammatory responses within the central nervous system, secondary to meningitis, encephalitis, and traumatic injury. The source of IL-8 within the brain during these inflammatory processes, however, is unknown. To explore the role of microglia in the production of IL-8, human fetal microglia, which are the resident macrophages of the brain, were treated with LPS and pro- and anti-inflammatory cytokines to determine their effects on IL-8 production. We found that IL-8 protein levels increased in response to LPS or IL-1 beta, or to TNF-alpha, which also corresponded to elevated IL-8 mRNA levels by RT-PCR. Pretreatment with IL-4, IL-10, or TGF-beta 1 potently inhibited the stimulatory effects of these proinflammatory agents. These findings indicate that human microglia synthesize IL-8 in response to proinflammatory stimuli, and that anti-inflammatory cytokines down-regulate the production of this chemokine. These results may have important therapeutic implications for certain central nervous system insults involving inflammation.     

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.     

In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony-stimulating factor

LPS tolerance is characterized by a diminished monocytic synthesis of TNF-alpha and, interestingly, IL-10 after LPS restimulation. We wondered whether granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and IFN-gamma can prevent or reverse this down-regulation of TNF-alpha and IL-10 production. The LPS-induced TNF-alpha amounts in desensitized PBMC treated with GM-CSF, IFN-gamma, or IL-12 and in naive, non-cytokine-primed cultures were similar, while much more TNF-alpha was induced in cytokine-primed naive cells. The effect of IL-12 was dependent on the presence of nonmonocytic cells and could be completely blocked with an IFN-gamma antiserum. Treatment of LPS-desensitized pure monocytes with IFN-gamma or GM-CSF resulted in a very high TNF-alpha expression and no difference to cytokine-primed naive monocytes was evident any longer. While IFN-gamma and IL-12 decreased IL-10 expression in naive PBMC, it was increased by both and by GM-CSF in LPS-tolerant cultures. Again, only IL-12 was dependent on the presence of nonmonocytic cells. For prevention of LPS tolerance, similar results were obtained. Recently, we have shown that IL-10 and TGF-beta mediate LPS desensitization in vitro and can be used to establish LPS hyporesponsiveness in the absence of LPS. IFN-gamma and GM-CSF prevented and reversed down-regulation of TNF-alpha and IL-10 synthesis also in the model of IL-10/TGF-beta1-induced LPS hyporesponsiveness, while IL-12 was ineffective because of its obvious inability to induce IFN-gamma. In summary, after LPS desensitization/hyporesponsiveness, IFN-gamma and GM-CSF tended to normalize pro- and anti-inflammatory monocytic behavior. Our results suggest that during LPS desensitization/hyporesponsiveness, monocytes acquire a hitherto unknown functional state with an altered reaction to biologic response modifiers.     

Factors involved in the differentiation of TGF-beta-producing cells from naive CD4+ T cells: IL-4 and IFN-gamma have opposing effects, while TGF-beta positively regulates its own production

TGF-beta has been shown to play a central role in regulating inflammatory responses; thus, understanding the factors involved in the generation of TGF-beta-producing cells could lead to interventions that are useful in effecting disease progression. In initial studies, the capacity of naive CD4+ T cells from TCR transgenic (Tg) mice to produce TGF-beta following primary and secondary stimulation was assessed. TGF-beta, IL-4, or IFN-gamma production could not be detected from highly purified naive CD4+/lymphocyte endothelial cell adhesion molecule (LECAM)-1high cells following primary stimulation for 36 h with plate-bound anti-CD3, anti-CD28, and IL-2. This population was subsequently used to study the differentiation of TGF-beta-producing CD4+ T cells. In further studies, naive CD4+/LECAM-1high cells from TCR transgenic mice of both the BALB/c and B10.A backgrounds were stimulated with T-depleted spleen cells (TDS) and specific peptide in the presence of various cytokines and/or cytokine antagonists for 5 days, restimulated, and TGF-beta, IL-4, and IFN-gamma production were measured. Priming conditions favoring high IL-4 production and/or low IFN-gamma production greatly enhanced TGF-beta production in secondary cultures. Furthermore, the presence of IL-10 in cultures was associated with an increase in TGF-beta production following restimulation. The importance of IL-4 and IFN-gamma in regulating TGF-beta production was confirmed in studies showing that cells from IFN-gamma(-/-) mice produced more TGF-beta, while cells from IL-4(-/-) mice produced less TGF-beta compared with wild-type controls. Finally, the addition of exogenous TGF-beta to priming cultures significantly enhanced the production of TGF-beta upon restimulation, demonstrating that TGF-beta has a role in self-regulating its own production.     

The importance of TGF-beta in murine visceral leishmaniasis

IFN-gamma is critical for the cure of leishmaniasis in humans and mice. BALB/c mice are genetically susceptible to infection with the visceralizing species of Leishmania, L. chagasi. We have evidence that a soluble factor(s) inhibits IFN-gamma production by cultured liver granuloma cells from BALB/c mice during L. chagasi infection. In contrast, liver granulomas from C3H.HeJ mice, which are genetically resistant to L. chagasi infection, produce abundant IFN-gamma. According to ELISAs and neutralization studies, there was not evidence that the Th2-type cytokines IL-10 or IL-4 contributed to IFN-gamma suppression. However, both Ab neutralization and immunohistochemistry showed that granuloma-derived TGF-beta was, at least in part, responsible for inhibiting IFN-gamma release by CD4+ cells in BALB/c liver granuloma cultures. Consistently, TGF-beta levels were high in liver granulomas from susceptible BALB/c mice but low in resistant C3H mice or in BALB/c mice that were immunized against L. chagasi disease. Administration of recombinant adenovirus expressing TGF-beta (AdV-TGFbeta) but not IL-10 (AdV-IL10) caused genetically resistant C3H mice to become significantly more susceptible to L. chagasi infection. In contrast, either AdV-TGFbeta or AdV-IL10 could abrogate the protective immune response achieved by immunization of BALB/c mice. We conclude that locally secreted TGF-beta inhibits Th1-associated cure of murine visceral leishmaniasis caused by L. chagasi, independently of Th2-type cytokines.     

Alterations in cytokine production following intraocular injection of soluble protein antigen: impairment in IFN-gamma and induction of TGF-beta and IL-4 production

Immune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-gamma production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-gamma production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-gamma production required IL-4. Interestingly, significant levels of TGF-beta were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-beta was produced primarily by the splenic CD4 and non-T cells and was of the TGF-beta1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-beta at several different (including the effector) phases during the response.     

Interleukin-18 enhances lipopolysaccharide-induced interferon-gamma production in human whole blood cultures

Interleukin-18 (IL-18) is a newly described cytokine, formerly called interferon-gamma (IFN-gamma)-inducing factor. In a simple 24-h human whole blood culture, IFN-gamma was produced by the combination of lipopolysaccharide (LPS) plus IL-18. To liberate cytokines in the leukocyte and red cell compartments, the detergent Triton X-100 was added to the entire blood culture. The combination of low concentrations of LPS plus IL-18 induced a 3- to 5-fold greater production of IFN-gamma than did either stimulant alone. Tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were also produced. The presence of IL-10 completely suppressed the production of IFN-gamma and reduced that of TNF-alpha, IL-6, and IL-8. Thus, IFN-gamma, TNF-alpha, IL-8, and IL-6 are produced in a single whole blood culture, making correlations in the synthesis of a T helper type 1 cytokine and proinflammatory cytokines with disease activity possible in a single culture.     

In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry

CD8+ T cells often differentiate into highly cytotoxic cells, secreting a Th1-like or type 1 cytokine pattern characterized by the production of IFN-gamma. However, cytotoxic, and in some reports, noncytotoxic, type 2 cells that secrete IL-4, IL-5, or IL-10 instead of IFN-gamma, can be generated when CD8+ T cells are primed in the presence of IL-4. Here, we show that IL-4 can also generate typical CD8 type 1 responses. Indeed, while presence of TGF-beta biases the development of CD8 T cells that, then, produce little cytolytic activity and IFN-gamma, addition of IL-4 results in the recovery of cytotoxicity and IFN-gamma production. The cooperative effects of TGF-beta and IL-4 imply dual functions, not only for IL-4, but also for TGF-beta. Indeed, depending on the presence or absence of IL-4, TGF-beta either inhibits or induces the generation of type 1 CD8+ T cells. Physiologically, the ratio of local IL-4/TGF-beta concentration may therefore be a critical element in determining the outcome of T cell responses to pathogen and autoantigens. It allows CD8 T cells to switch from an immunotolerant state in the presence of only TGF-beta or IL-4, to an immunocompetent proinflammatory type 1 state in the absence or presence of both cytokines.     

Expression of interleukin-3 and tumor necrosis factor-beta mRNAs in cultured microglia

The function of interleukin-3 (or multi-CSF) in the hemopoietic system has been studied in great detail. Although its growth promoting activity on brain microglial cells has been confirmed both in vitro and in vivo, its presence in the brain and even in cultured brain cells has repeatedly been questioned. We have shown recently that isolated rat microglia express mRNA(IL-3) and synthesize IL-3 polypeptide. It is shown here by use of the PCR method, that mRNA(IL-3) is found also in C6 glioblastoma, in rat aggregate cultures, and in newborn and adult rat brain. Quantitation of amplified cDNA(IL-3) was achieved by non-competitive RT-PCR using an elongated internal standard. IL-3 messenger RNA was almost undetectable in vivo and low in (serum-free) aggregate cultures. In isolated microglia, mRNA(IL-3) was increased upon treatment with LPS, PHA, with the cytokines IL-1 or TNF-alpha, with retinoic acid, dbcAMP or the phorbol ester TPA. Effects of LPS were inhibited by dexamethasone, while the glucocorticoid by itself had no effect on basal IL-3 expression. LPS increased mRNA(IL-3) in a concentration-dependent manner beginning with 10 pg/ml and reaching plateau levels at 10 ng/ml. LPS also increased mRNAs of TNF-alpha and TNF-beta. TNF-alpha mRNA was already detectable in untreated microglia and LPS-increased levels were sustained for a few days. In contrast, TNF-beta mRNA was observed only between 4 and 16 h of LPS incubation. It was absent in LPS-free microglia, and after 24 h of LPS-treatment or later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects

Steroid hormones have long been known to modulate immune function, and recent studies indicate that one of the means by which they do so involves effects on the secretion of immunoregulatory cytokines. Our laboratory has found recently that estradiol (E2) selectively modifies cytokine secretion in proteolipid protein (PLP)-specific, CD4+ T cell clones isolated from patients with the demyelinating disease, multiple sclerosis, and from normal control subjects. The data suggest that E2 may play a role in regulating the balance between pro- and antiinflammatory conditions, especially at concentrations typical of pregnancy. To determine whether other pregnancy-associated steroid hormones are capable of similar activity, we expanded our testing to include estrone (E1), estriol (E3), progesterone, and dexamethasone. The results indicate that E1 and E3 enhance secretion of Ag- or anti-CD3-stimulated IL-10 and IFN-gamma in dose-dependent fashion, almost identical to that of E2. The effect on IL-10 was more potent than occurred with IFN-gamma. In addition, E1 and E3, like E2, had a biphasic effect on TNF-alphabeta secretion, with low concentrations stimulatory, and high doses inhibitory. None of the estrogens influenced IL-4 or TGF-beta secretion. Progesterone enhanced secretion of IL-4, without affecting any other tested cytokine. Finally, dexamethasone induced TGF-beta secretion, but inhibited IFN-gamma and TNF-alphabeta. This differential effect of steroid hormones on the secretion of cytokines by CD4+ human T cell clones is consistent with the possibility that, collectively, they promote antiinflammatory conditions at high concentrations typical of pregnancy.     

Cuticular proteins from the lobster, Homarus americanus

The interleukin-12 receptor (IL-12R)beta1 chain is an essential component of the functional IL-12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti-CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up-regulation of IL-12Rbeta1 expression and IL-12 binding. Kinetic studies revealed that maximum expression of IL-12Rbeta1 and IL-12 binding occurred on days 3-4. Anti-CD3-induced expression of IL-12Rbeta1 chain and IL-12 binding by PBMC was augmented by anti-CD28 mAb, indicating that the potentiating effect of anti-CD28 on T cell responses to IL-12 could be mediated, at least in part, by the enhancement of IL-12R expression. Among 16 cytokines tested, IL-2, IL-7 and IL-15 markedly induced IL-12Rbeta1 expression and IL-12 binding on resting PBMC, whereas IL-1alpha and tumor necrosis factor-alpha had a minimal enhancing effect. In contrast, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, interferon (IFN)-alpha, IFN-gamma, granulocyte/macrophage colony-stimulating factor and transforming growth factor (TGF)-beta2 had no detectable enhancing effect. Anti-CD3-induced expression of IL-12Rbeta1 and of low-affinity IL-12 binding sites was partially inhibited by TGF-beta2, IL-10 and IL-4; however, TGF-beta2 and IL-10 completely abolished anti-CD3-induced expression of high-affinity IL-12 binding sites. Consistent with the reduction of high affinity IL-12 binding sites, PBMC activated with anti-CD3 mAb in the presence of TGF-beta2 or IL-10 failed to produce IFN-gamma or to proliferate in response to IL-12. These results suggest that Th2 cell-derived cytokines can inhibit IL-12-induced biological functions by inhibiting IL-12R expression and that expression of a second subunit of the IL-12R (IL-12Rbeta2), required for the formation of high-affinity IL-12 binding sites, may be more highly regulated by TGF-beta2 and IL-10 than is expression of IL-12Rbeta1.     

CD40-CD154 interaction and IFN-gamma are required for IL-12 but not prostaglandin E2 secretion by microglia during antigen presentation to Th1 cells

IL-12 and PGE2 promote and inhibit, respectively, the development of Th1 responses. Production of these mediators by APC residing in the central nervous system (CNS) may be involved in the local regulation of the T cell phenotype during infectious and autoimmune CNS diseases. In the present study we have examined IL-12 and PGE2 secretion by cultured microglia and astrocytes from the mouse brain upon Ag-dependent interaction with I-Ad-restricted, OVA323-339 specific TCR transgenic Th1 and Th2 cell lines. We show that microglia, which restimulate efficiently both Th1 and Th2 cells, secrete IL-12 upon Ag-dependent interaction with Th1, but not with Th2 cells. Th1-driven IL-12 production depends on TCR ligation by MHC class II/peptide complexes, CD40 engagement on microglia, and IFN-gamma secretion by activated Th1 cells. Th1 and, to a lesser extent, Th2 cells also stimulate the production of PGE2 by microglia. T cell-mediated induction of PGE2 requires MHC class II/peptide/TCR interactions but does not depend on CD40 engagement or on the presence of IFN-gamma. Astrocytes, which preferentially activate Th2 cells, fail to produce IL-12 and secrete negligible amounts of PGE2 upon interaction with either Th1 or Th2 cells. These results suggest that during CNS infection or immunopathology, IL-12 produced by microglia upon Ag-specific interaction with Th1 cells may further skew the immune response to Th1, whereas the T cell-dependent production of PGE2 by microglia may represent a negative feedback mechanism, limiting the propagation of Th1 responses.     

Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines

Interleukin (IL)-4, IL-10, IL-13 and transforming growth factor beta (TGF-beta) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT-PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) expression in human monocytes. The effects of IL-4 and IL-10 on monocyte chemoattractant protein-1 (MCP-1)-and C-reactive protein (CRP)-induced TF expression were also studied. A direct comparison revealed that IL-4, IL-10 and IL-13 all down-regulated LPS-induced TF expression in a concentration-dependent manner without the need for priming. In contrast, TGF-beta required 4 h of priming to inhibit TF expression induced by LPS. IL-10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL-4 and IL-13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL-4 and IL-10 showed similar concentration-dependent inhibition of MCP-1- and CRP-induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL-10, in particular, may be a possible candidate as a TF-preventing drug.     

Role of transforming growth factor-beta in the preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B

Stimulation of murine CD4+ T cells with staphylococcal enterotoxin B (SEB) results in the preferential development of T helper (Th) 1 cells [i.e. high interferon (IFN)-gamma and low interleukin (IL)-4, IL-5 and IL-10]; whereas in response to plate-bound anti-CD3 or anti-T cell receptor-alpha beta, Th1 as well as Th2 cells develop. In the present study, we examined the mechanism which is responsible for the selective Th1 development in the SEB system. The addition of IL-4 resulted in a strong development of Th2 cells showing that SEB stimulation can result in Th2 differentiation. Co-stimulation with anti-CD28 was insufficient in this regard. Lack of Th2 development in the SEB system was in part due to the inhibitory effect of endogenously produced transforming growth factor-beta (TGF-beta), because anti-TGF-beta allowed the development of Th2 cells. Similarly, TGF-beta inhibited Th2 development and stimulated Th1 development in the anti-CD3 system. This shift was only partially prevented by also including IL-4 in the cultures. The effects of TGF-beta could only partially be explained by stimulation of IFN-gamma or inhibition of IL-4 as intermediatory cytokines: (1) TGF-beta stimulated Th1 development even in the presence of anti-IL-4 and anti-IFN-gamma, and (2) a strong inhibitory effect of anti-TGF-beta on Th1 development was still observed when anti-IL-4 and IFN-gamma were simultaneously added to the cultures. It is concluded that SEB favors Th1 development by stimulation of TGF-beta production. Inhibition of Th2 development by TGF-beta is due, in part, to inhibition of IL-4 and stimulation of IFN-gamma, and, in part, to a direct effect of TGF-beta on the responding T cells.     

Primary adenoid cystic carcinoma of the skin. Histologic and ultrastructural study of two cases localized in the external auditory canal

Multiple sclerosis (MS) is probably caused by multiple factors, but there is evidence that an autoimmunological process is relevant for the pathogenesis. Cytokines can operate in different ways in MS and the animal model "experimental allergic encephalomyelitis (EAE). "Interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), TNF-beta, interleukin-1 (IL-1) and IL-2 are important inflammation mediators within the MS plaque, whereas IFN-alpha, IFN-beta, transforming-growth-factor-beta (TGF-beta) and IL-10 exert mainly immunosuppressive functions. Application of these anti-inflammatory cytokines and the selective block of pro-inflammatory cytokines are promising new therapeutic strategies with fewer side effects than the commonly used cytostatic drugs.