IFN-gamma Induction Research Articles

An absolute and restricted requirement for IL-12 in natural killer cell IFN-gamma production and antiviral defense. Studies of natural killer and T cell responses in contrasting viral infections.

IL-12 is a potent induce of IFN-gamma and is associated with a wide variety of immunoregulatory activities. Using murine CMV (MCMV) and lymphocytic choriomeningitis virus (LCMV) models of acute viral infection, we show here that IL-12 is integrally involved in certain aspects of antiviral immunity. Experiments evaluating the production and function of IL-12 demonstrated the induction of biologically active factor early during Smith strain MCMV, but not Armstrong strain LCMV, infection. The MCMV-induced IL-12 was responsible for early NK cell IFN-gamma production and viral control, as in vivo IL-12 neutralization by Ab treatment blocked both of these. In contrast, NK cell IFN-gamma production could not be detected during LCMV infection, and IL-12 neutralization had no effect on early LCMV replication. In both infections, treatments neutralizing IL-12 failed to alter either early NK cell cytotoxicity or later T cell responses, including IFN-gamma production and viral clearance on days 7 and 9 after infection. These data demonstrate the induction of IL-12 production during certain viral infections and the function of IL-12 for eliciting NK cell IFN-gamma production and antiviral defense. Furthermore, they suggest the existence of IL-12-independent mechanisms for IFN-gamma induction and viral control by T cells.

Immunoregulatory properties of ISG15, an interferon-induced cytokine.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

Opposite effects of interleukin-4 on memory T helper cell development depend on interleukin-2

We previously reported that cyclosporin A (CSA) promotes the generation of T helper memory cells during antigenic priming of murine spleen cells in vitro. More recently, we have demonstrated that interleukin-2 (IL2) has a downmodulating effect on T helper memory cell generation. The present data address the role of the other T cell growth factor, IL4, upon induction of these cells. The data presented here show that IL4 can interfere with this process: addition of rIL4 to immunosuppressed priming cultures leads to a considerable decrease in the helper activity of the recovered cells. However, in standard cultures, in which IL2 is normally produced, no effect of IL4 on T helper memory cell generation was found. Addition of IL4 has important consequences for cytokines produced upon antigenic restimulation. In standard cultures, IL4 primes for cells expressing high levels of IL2 and IL4 mRNA. Strikingly, in immunosuppressed priming cultures, IL4 counterbalances the CSA-induced blockade of the IFN gamma gene. Taken together, our results suggest that the unique role of IL4 is to drive T helper memory precursors into an IL4 production differentiation pathway. However, IL4 has a downmodulating effect on memory T helper cell induction when IL2 is not produced. These results confirm that synergy between IL2 and IL4 is mandatory for the directive role of IL4 upon IL4-producing cells. Furthermore, the finding that IL4 promotes the induction of IFN gamma in a CSA-resistant pathway represents a new tool for analysis of regulation of the IFN gamma gene.

Molecular mechanisms underlying IFN-gamma-mediated tumor growth inhibition induced during tumor immunotherapy with rIL-12.

The present study investigates the molecular mechanisms by which IFN-gamma produced as a result of in vivo IL-12 administration exerts its anti-tumor effects. rIL-12 was administered three or five times into mice bearing CSA1M fibrosarcoma, OV-HM ovarian carcinoma or MCH-1-A1 fibrosarcoma. This regimen induced complete regression of CSA1M and OV-HM tumors but only transient growth inhibition of MCH-1-A1 tumors. The anti-tumor effects of IL-12 were associated with enhanced induction of IFN-gamma because these effects were abrogated by pretreatment of hosts with anti-IFN-gamma antibody. Exposure in vitro of the three types of tumor cells to rRFN-gamma resulted in moderate to potent inhibition of tumor cell growth. IFN-gamma stimulated the expression of mRNAs for an inducible type of NO synthase (iNOS) in CSA1M cells and indoleamine 2,3-dioxygenase (IDO), an enzyme capable of degrading tryptophan, in OH-HM cells, but induced only marginal levels of these mRNAs in MCH-1-A1 cells. In association with iNOS gene expression, IFN-gamma-stimulated CSA1M cells produced a large amount of NO which functioned to inhibit their own growth in vitro. Although OV-HM and MCH-1A1 cells did not produce NO, they also exhibited NO susceptibility. Whereas the tumor masses from IL-12-treated CSA1M-bearing or OV-HM-bearing mice induced higher levels of iNOS (for CSA1M) or IDO and iNOS (for OV-HM) mRNAs, the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNA alone. Moreover, massive infiltration of CD4(+) and CD8(+) T cells and Mac-1(+) cells was seen only in the CSA1M and OV-HM tumors. Thus, these results indicate that IFN-gamma produced after IL-12 treatment induces the expression of various genes with potential to modulate tumor cell growth by acting directly on tumor cells or stimulating tumor-infiltrating lymphoid cells and that the effectiveness of IL-12 therapy is associated with the operation of these mechanisms.

Regulation of interleukin-12 signalling during T helper phenotype development.

The experiments described above have allowed us to define the molecular events in IL-12 signalling. Within minutes after IL-12 treatment of responsive cells, Stat1, Stat3, and Stat4 are tyrosine phosphorylated. These molecules form nuclear DNA-binding complexes consisting of homodimeric Stat1 and heterodimeric Stat3-Stat4 complexes. In a murine in vitro phenotype development model, T cells rapidly and selectively lose their capacity to respond to IL-12 upon acquisition of the Th2 phenotype. This hyporesponsiveness is manifested by the inability of IL-12 to induce IFN gamma production in differentiated Th2 cells, as well as the inability of IL-12 to induce the activation of Stat4. Despite the functional defect of IL-12 signalling in Th2 cells, all known components of the IL-12 signal transduction pathway are present. We speculate that in Th2 cells, the second receptor chain may be absent or one of the other components may be modified. Genetic experiments in Balb/c and B10.D2 strains of mice have demonstrated several differences in T helper differentiation in vitro. Stimulation of T cells under neutral conditions results in a bias of Balb/c T cells toward the Th2 extreme and B10 T cells toward the Th1 extreme of cytokine production. Following stimulation under neutral conditions, B10 T cells retain the ability to respond to IL-12 while Balb/c T cells lose IL-12 responsiveness. Mating experiments have demonstrated that the B10 genetic effect is dominant in F1 mice. Analysis of backcrossed animals has suggested that the ability to respond to IL-12 in the secondary stimulation may be controlled by a single dominant B10 gene. The results we describe may have profound implications for allergy. Since allergic responses are largely due to the activation of the Th2 subset of T lymphocytes, a better understanding of T cell phenotype development may reveal multiple targets for therapeutic intervention. First, a better understanding of Th1 phenotype induction in response to IL-12 may allow prevention of in vivo allergic responses using pharmacological tools which bias allergen-specific responses to the Th1 subset. Second, a molecular explantation of why Th2 cells fail to reverse phenotype in response to IL-12 may allow treatment of atopic individuals to remove the disease-promoting T lymphocyte compartment. Finally, a better understanding of the basis for genetic differences in murine T helper cell differentiation may allow us to identify a causative genetic element in humans, yielding better diagnostic and therapeutic methods.

Potentiation by Thyroxine of Interferon-γ-Induced HLA-DR Expression Is Protein Kinase A- and C-Dependent

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) potentiate the antiviral state induced by interferon-gamma(IFN-gamma) in homologous cells by a mechanism that is dependent upon calcium/phospholipid-dependent protein kinase (PKC). L-T4 and T3 also potentiate induction by IFN-gamma of MHC class II HLA-DR antigen expression in HeLa cells. In the present studies of HLA-DR expression, the PKC inhibitor staurosporine (0.1-1 nM) enhanced the expression of HLA-DR when the inhibitor was added simultaneously with IFN-gamma, 100 IU/ml. In the presence of IFN-gamma and 10(-7) M T4, the same concentrations of staurosporine inhibited potentiation of HLA-DR expression by thyroid hormone. A more specific PKC inhibitor, CGP41251 (0.5-5 nM), similarly enhanced HLA-DR expression in the presence of IFN-gamma but inhibited thyroid hormone potentiation of antigen expression. Both actions of CGP41251 were suppressed when cells were also treated with phorbol 12-myristate 13-acetate (PMA). A phospholipase C inhibitor, U73122 (1-1000 nM), did not alter the potentiating ability of T4, although it inhibited in a concentration-dependent manner the expression of HLA-DR induced by IFN-gamma. The potentiating effect of T4 was much more sensitive to a cyclic AMP-dependent protein kinase (PKA) inhibitor,KT5720 (1-1000nM), than was the induction of HLA-DR by IFN-gamma. The inhibitory effects of KT5720 were reversed by concurrent 8-bromo-cAMP treatment. The calmodulin antagonist W-7 (5-50 microM) did not alter IFN-gamma induction of HLA-DR in either the presence or absence of T4. HLA-DR expression in HeLa cells appears to be under PKC-associated inhibition; IFN-gamma reverses this inhibition to promote the appearance of the DR antigen. In contrast, potentiation by T4 of induction of HLA-DR by IFN-gamma requires activation of PKC. PKA is involved both in DR induction by IFN-gamma and in potentiation of the latter by T4. Thus, PKA and PKC have discrete roles in IFN-gamma-induced MHC class II antigen expression and its modulation by thyroid hormone.

Cyclosporine inhibition of leukocyte calcineurin is much less in whole blood than in culture medium.

Recent reports have shown that cyclosporine (CsA)-treated patients have abundant calcineurin phosphatase (CN) activity in vivo despite CsA blood concentrations that would be completely inhibitory in vitro. We sought to determine whether this disparity was a result of altered distribution of CsA in culture medium (CM) compared with whole blood (WB). CN activity was measured in peripheral blood leukocytes (PBL) that had been exposed in vitro to CsA in either WB or CM. Cells from both groups were also stimulated with OKT3 to determine the effect of CsA on the induction of IFN-gamma synthesis. CsA accumulation in PBL was determined by liquid scintillation counting of PBL exposed to 3H-CsA. The IC50 for CsA inhibition of CN activity was significantly lower for PBL in CM (2 micrograms/L) compared with PBL in WB (102 micrograms/L, P < or = 0.005). Likewise, for CsA inhibition of IFN-gamma induction, the IC50 was 18 micrograms/L for PBL in CM compared with 690 micrograms/L for PBL in WB (P < or = 0.005). The shift in IC50 was accompanied by a 10-fold increase in 3H-CsA in PBL in CM compared with PBL in WB. We conclude that PBL exposed to CsA in CM accumulate significantly more CsA than PBL in WB. The result is that CsA inhibition of CN activity and cytokine induction appears at least an order of magnitude greater than its true effect in biologic fluids. This disparity is due to partitioning of CsA to irrelevant CsA binding sites, which are abundant in WB and in vivo, but absent in culture medium.

Augmentation or inhibition of IFN-gamma-induced MHC class II expression by lipopolysaccharides. The roles of TNF-alpha and nitric oxide, and the importance of the sequence of signaling.

MHC class II expression on macrophages is one determinant of Ag presentation and the vigor of CD4+ T cell immunity. We show that LPS may either inhibit or augment IFN-gamma-induced MHC class II on macrophages depending on the sequence of the IFN-gamma and LPS signals. LPS inhibited MHC class II when added simultaneously with IFN-gamma, but augmented class II expression when added after IFN-gamma. Inhibition was due to nitric oxide (NO), which was only produced if LPS was given simultaneously with IFN-gamma. However, even when NO production was inhibited, LPS given simultaneously with IFN-gamma did not augment MHC class II expression. This suggests that LPS delivers different signals when given simultaneously vs after IFN-gamma. LPS augmentation of class II expression was functionally important because it correlated with increased Ag presentation. Augmentation by LPS of IFN-gamma-induced class II expression by macrophages has not been previously reported. We found that TNF-alpha, like LPS, inhibited IFN-gamma-induced class II expression if NO was produced, but augmented it in the absence of NO formation. Studies with a neutralizing anti-TNF-alpha Ab, however, indicate that LPS augmentation of MHC class II did not require TNF-alpha. LPS augmentation involved a different mechanism than IFN-gamma induction of MHC class II. LPS augmentation occurred at a post-transcriptional level, whereas IFN-gamma-induction occurred at the level of gene transcription. LPS augmentation was apparent after 2 h of stimulation by LPS, while IFN-gamma induction of class II expression required more than 8 h.

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.

Differential utilization of IFN-gamma-responsive elements in two maturationally distinct macrophage cell lines.

We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.

IFN-gamma up-regulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines.

On human mature monocytes the immunomodulator IFN-gamma has been shown to down-regulate the receptor for the anaphylatoxic peptide C5a (CD88, C5aR). In this study, we show that in immature myelo-/monoblastic U937, HL60, and MonoMac6 cells, IFN-gamma induces C5aR-ligand binding activity. In U937 cells, this induction cannot be blocked by the protein kinase C inhibitor staurosporine. An increase in free cytosolic Ca2+ upon ligand binding indicates functional coupling of this receptor in U937 and HL-60 cells. G-Proteins involved in this C5a responsiveness after IFN-gamma induction are completely pertussis toxin sensitive. Our data suggest that an additional pertussis toxin-resistant pathway exists in U937 cells after induction by dibutyryl cAMP. However, this is not due to changes in the mRNA level of the pertussis toxin-insensitive G-protein subunit G alpha 16. Induction by dibutyryl cAMP, but not that by IFN-gamma, resulted in C5a-dependent release of N-acetyl-beta-D-glucosaminidase, further highlighting functional differences in the effects of the inducers. Our data show an IFN-gamma-dependent increase in C5aR expression and suggest a maturation-related change in signaling of the C5aR, presumably at the level of receptor coupling.

Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.

A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.

Deoxyspergualin inhibits kappa light chain expression in 70Z/3 pre-B cells by blocking lipopolysaccharide-induced NF-kappa B activation.

Deoxyspergualin (DSG) is a potent immunosuppressive agent that is currently undergoing clinical trials for treatment of transplant rejection, preventive of human anti-mouse Ab response, and blocking autoimmune disease progression. The mechanism of action of DSG appears to be novel, with in vivo activity attributable to the suppression of both humoral and cell-mediated immunity. In this study we investigated the effect of DSG on the induction of lg expression in the 70Z/3 murine pre-B cell line. Treatment of 70Z/3 cells with DSG for 24, 48, or 72 h before LPS or IFN-gamma induction resulted in a time-dependent inhibition of surface lgM expression, with greater than 80% inhibition observed after 72 h of pretreatment. Inhibition of surface expression was specific for lgM, as neither MHC class I nor CD45 (B220) surface expression was affected by DSG pretreatment. Cyclosporin A was ineffective at suppressing surface igM induction. DSG pretreatment results in a 10-fold reduction in LPS- or IFN-gamma-induced kappa L chain protein and mRNA expression. No change was observed in either mu or beta-actin mRNA levels. Analysis of nuclear and cytoplasmic NF-kappa B expression using electrophoretic mobility shift analysis and Western analysis, revealed that DSG blocked LPS-induced NF-kappa B nuclear translocation, but had no effect on cytoplasmic NF-kappa B levels. We conclude that DSG may act to suppress humoral immune responses by blocking the transcriptional activation of kappa L chain expression during certain stages of B cell development.

Dissection of strain difference in acquired protective immunity against Mycobacterium bovis Calmette-Guérin bacillus (BCG). Macrophages regulate the susceptibility through cytokine network and the induction of nitric oxide synthase.

Protection against infection with intracellular pathogens operates in two stages, early innate resistance and late acquired protective immunity (API), in inbred mouse strains. Although both C57BL/10 (B10) and BALB/c mice bear the susceptible phenotype of innate resistance, Calmette-Guérin bacillus (BCG) vaccination generated efficient API in B10 but not in BALB/c mice. Employing a specific nitric oxide (NO) synthase inhibitor, we revealed that NO production plays a pivotal role in the API of B10 mice. Consistent with this, expressions of the inducible isoform of NO synthase (iNOS) protein and mRNA were significantly higher in the spleen of B10 mice than in that of BALB/c mice. Furthermore, IFN-gamma, a potent inducer of iNOS, and mRNAs for IL-12 (p40); an inducer of IFN-gamma and IL-2 were also vigorously expressed in the spleen of B10 mice compared with that of BALB/c mice. In an attempt to clarify the mechanism by which the different capacities for API are generated, we analyzed the cytokine network between T cells and macrophages in both B10 and BALB/c mice. We found that multiple functions of macrophages, which include capacities to express IL-12 (p40) mRNA in response to BCG and to express mRNAs for iNOS and IL-12 (p40) in response to IFN-gamma, were impaired in BALB/c mice as compared with B10 mice. However, T cells appeared to express comparable level of IFN-gamma mRNA in both strains when stimulated with IL-12. Taken together, these results indicate that the macrophage functions play a pivotal role in both the induction and effector phases of API to determine the susceptibility of mice to BCG infection.

RB and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal IFN-gamma induction of the class IL transactivator CIITA in class II non-inducible RB-defective tumor lines.

The major histocompatibility (MHC) class II genes encode cell surface proteins that bind antigenic peptide for presentation to T-cells. The class II proteins are expressed constitutively on B-cells and EBV-transformed B-cells, and are inducible by IFN-gamma on a wide variety of cell types. Retinoblastoma protein (RB) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. RB-defective tumor lines are non-inducible for MHC class II by IFN-gamma, or very weakly inducible, but transfection of 2 different lines with RB expression vectors re-establishes or substantially enhances class II inducibility. Therefore, we examined the RB status of a series of B-cell mutants that are defective in class II expression, generated either in vitro or derived from Bare Lymphocyte Syndrome (BLS) patients. Nuclear matrix-bound RB was detectable in all cases, indicating that loss of RB is not responsible for decreased class II expression in these lines. A second E2F-I binding protein, most likely DP-I, was also apparently normal in both class II-positive and -negative B-cell lines. We also examined the IFN-gamma induction of CIITA in RB-defective lines. CIITA is a class II gene transactivator known to be defective in one form of BLS and to be required for the induction of MHC class II by IFN-gamma. CIITA mRNA is normally inducible by IFN-gamma in class II non-inducible, RB-defective lines, and in one line, re-expression of RB has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in RB-defective cells is not due to a block in CIITA inducibility.

Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression.

The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.

IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-gamma.

Histoplasma capsulatum is a pathogenic fungus found in discrete geographic locations throughout the world. The fungus invades the reticuloendothelial organs such as the spleen and liver of immunocompetent hosts where it is usually controlled. However, in individuals with immune deficiency, histoplasmosis is a severe and potentially fatal disease. Resistance to this infection is due primarily to a cellular immune response mediated by T cells and macrophages. Moreover, IFN-gamma is critical in activating macrophages to kill the organism. Herein we study the regulation of cytokine induction in mice infected with H. capsulatum and the effects of IL-12 in the course of infection. Mice infected with H. capsulatum and treated with neutralizing Abs to IFN-gamma, TNF-alpha, or IL-12 experienced accelerated mortality, indicating that endogenous production of these cytokines plays an important role in response to infection. In contrast, mice treated with IL-12 or a neutralizing Ab to IL-4 at the initiation of infection had substantially diminished mortality. Moreover, mice infected and treated with IL-12 show a two- to threefold increase in the amount of IFN-gamma following in vitro stimulation with specific H. capsulatum Ag compared with the control infected mice. The protective effect of IL-12 could be abrogated if a neutralizing Ab to IFN-gamma was given at the same time, demonstrating that the role of IL-12 in protection was mediated by IFN-gamma. Additionally, infected mice treated with IL-12 had a severalfold decrease in the colony counts of H. capsulatum in spleen cells after 5 days of infection as compared with control animals. Lastly, spleen cells from infected animals treated with IL-12 showed a striking decrease in their proliferative response to mitogen or H. capsulatum Ag. Responses could be restored by adding inhibitors of IFN-gamma or of nitric oxide to the in vitro cultures. The above observations suggest that IL-12 may be useful in immunologic intervention against this opportunistic pathogen.

Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses.

Genes containing the interferon-stimulated response element (ISRE) enhancer have been characterized as transcriptionally responsive primarily to type I interferons (IFN alpha/beta). Induction is due to activation of a multimeric transcription factor, interferon-stimulated gene factor 3 (ISGF3), which is activated by IFN alpha/beta but not by IFN gamma. We found that ISRE-containing genes were induced by IFN gamma as well as by IFN alpha in Vero cells. The IFN gamma response was dependent on the ISRE and was accentuated by preexposure of cells to IFN alpha, a treatment that increases the abundance of ISGF3 components. Overexpression of ISGF3 polypeptides showed that the IFN gamma response depended on the DNA-binding protein ISGF3 gamma (p48) as well as on the 91-kDa protein STAT91 (Stat1 alpha). The transcriptional response to IFN alpha required the 113-kDa protein STAT113 (Stat2) in addition to STAT91 and p48. Mutant fibrosarcoma cells deficient in each component of ISGF3 were used to confirm that IFN gamma induction of an ISRE reporter required p48 and STAT91, but not STAT113. A complex containing p48 and phosphorylated STAT91 but lacking STAT113 bound the ISRE in vitro. IFN gamma-induced activation of this complex, preferentially formed at high concentrations of p48 and STAT91, may explain some of the overlapping responses to IFN alpha and IFN gamma.

Down-regulating effects of IL-4 and IL-10 on the IFN-gamma response in atopic dermatitis.

Atopic dermatitis (AD) is a chronic allergic disease associated with toxin (superantigen)-producing Staphylococcus aureus skin infections, impaired delayed hypersensitivity responses, and the expansion of IL-4-secreting Th2 cells, as well as diminished IFN-gamma synthesis. IL-12 is known to induce IFN-gamma synthesis and to augment Th1 responses. In this study, therefore, we examined the potential role of IL-12 in the immunopathogenesis of AD. We show that, after stimulation with staphylococcal toxic shock syndrome toxin-1 (TSST-1) or IL-12, PBMC from patients with AD are deficient in their ability to produce IFN-gamma. PBMC from AD patients, however, produced normal quantities of IL-12 and expressed normal levels of IL-12R. Induction of IFN-gamma by TSST-1 was decreased by neutralizing anti-IL-12 Ab in normal donors, but not in AD patients. The latter observation is consistent with a defective response to IL-12 in AD PBMC. Because AD is associated with increased production of IL-4 and IL-10, we examined the effect of IL-4 on IL-12- or TSST-1-induced IFN-gamma production in normal donors. IL-4 inhibited IL-12-induced IFN-gamma production. Furthermore, Ab neutralization of IL-4 caused increased production of IFN-gamma in AD PBMC. However, neutralization of IL-10 activity caused an even greater augmentation of IFN-gamma production. Our data suggest that despite normal levels of IL-12 production and IL-12R expression, PBMC from AD patients are unable to generate normal IL-12-induced IFN-gamma responses. This defective response may be due to the excess production of IL-4 and IL-10 in this common allergic condition.