Regulation of MHC class II expression and antigen processing in murine and human mesenchymal stromal cells by IFN-gamma, TGF-beta, and cell density

Mesenchymal stromal cells (MSC) possess immunosuppressive properties, yet when treated with IFN-gamma they acquire APC functions. To gain insight into MSC immune plasticity, we explored signaling pathways induced by IFN-gamma required for MHC class II (MHC II)-dependent Ag presentation. IFN-gamma-induced MHC II expression in mouse MSC was enhanced by high cell density or serum deprivation and suppressed by TGF-beta. This process was regulated by the activity of the type IV CIITA promoter independently of STAT1 activation and the induction of the IFN regulatory factor 1-dependent B7H1/PD-L1 encoding gene. The absence of direct correlation with the cell cycle suggested that cellular connectivity modulates IFN-gamma responsiveness for MHC II expression in mouse MSC. TGF-beta signaling in mouse MSC involved ALK5 and ALK1 TGF-beta RI, leading to the phosphorylation of Smad2/Smad3 and Smad1/Smad5/Smad8. An opposite effect was observed in human MSC where IFN-gamma-induced MHC II expression occurred at the highest levels in low-density cultures; however, TGF-beta reduced IFN-gamma-induced MHC II expression and its signaling was similar as in mouse MSC. This suggests that the IFN-gamma-induced APC features of MSC can be modulated by TGF-beta, serum factors, and cell density in vitro, although not in the same way in mouse and human MSC, via their convergent effects on CIITA expression.     

High level class II trans-activator induction does not occur with transient activation of the IFN-gamma signaling pathway

Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-gamma signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-gamma. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-gamma signaling pathway is regulated by the duration of the activation of the primary IFN-gamma-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-gamma is required for MHC class II gene activation despite the fact that the level of the IFN-gamma-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-gamma signaling. The possible implications of this result for tumorigenesis are discussed.     

Inhibition of IFN-gamma-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-gamma induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-gamma-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-gamma-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1(-/-) macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-gamma-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-gamma signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-gamma-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4(+) T cells.     

Suppressors of cytokine signaling (SOCS)-1 and SOCS-3 are induced by CpG-DNA and modulate cytokine responses in APCs

During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-gamma as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-gamma-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.     

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

Attenuation of MHC class II expression in macrophages infected with Mycobacterium bovis bacillus Calmette-Guérin involves class II transactivator and depends on the Nramp1 gene.

The natural resistance associated macrophage protein 1 (Nramp1) gene determines the ability of murine macrophages to control infection with a group of intracellular pathogens, including Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). The expression of the resistant allele of the Nramp1 gene in murine macrophages is associated with a more efficient expression of several macrophage activation-associated genes, including class II MHC loci. In this study, we investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression in three types of macrophages: those expressing a wild-type allele of the Nramp1 gene (B10R and 129/Mphi), those carrying a susceptible form of the Nramp1 gene (B10S), and those derived from 129-Nramp1-knockout mice (129/Nramp1-KO). Previously, we published results showing that Ia protein expression is significantly higher in the IFN-gamma-induced B10R macrophages, compared with its susceptible counterpart. In this paper, we also show that the higher expression of Ia protein in B10R cells is associated with higher I-Abeta mRNA expression, which correlates with a higher level of IFN-gamma-induced phosphorylation of the STAT1-alpha protein and subsequently with elevated expression of class II transactivator (CIITA) mRNA, compared with B10S. Furthermore, we demonstrate that the infection of macrophages with M. bovis BCG results in a down-regulation of CIITA mRNA expression and, consequently, in the inhibition of Ia induction. Therefore, our data explain, at least in part, the molecular mechanism involved in the inhibition of I-Abeta gene expression in M. bovis BCG-infected macrophages activated with IFN-gamma.     

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.     

Regulation of MHC class II antigen expression. Opposing effects of tumor necrosis factor-alpha on IFN-gamma-induced HLA-DR and Ia expression depends on the maturation and differentiation stage of the cell

MHC class II induction by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. TNF-alpha has been found both to enhance and also to inhibit IFN-gamma-induced MHC class II expression. In the present studies, the effect of TNF-alpha on IFN-gamma induced MHC class II expression was tested in various cell lines. On the basis of the data, we propose that, depending on the stage of differentiation and maturation of the cells, TNF-alpha might synergize or antagonize the affects of IFN-gamma on the regulation of MHC class II expression. Thus, in immature cells such as HL-60 or THP-1, TNF-alpha enhances IFN-gamma-induced class II expression. However, when differentiation was induced in these cells by TPA or IFN-gamma, the additive effect of TNF-alpha on the IFN-gamma induced DR expression was eliminated. Furthermore, TNF-alpha down-regulates the IFN-gamma-induced class II expression in differentiated cells such as human skin fibroblasts or activated macrophages. In bone marrow cells induced to differentiate in vitro, TNF-alpha decreased the IFN-gamma-induced MHC class II expression in a maturation-dependent fashion. These results provide a rational explanation for the conflicting reports regarding the effect of TNF-alpha on IFN-gamma-induced class II expression. But more importantly they may be relevant to the biologic function of TNF-alpha. Thus, we show that TNF-alpha-treated mice have reduced level of Ia expression on peritoneal macrophages and in vivo treatment with TNF-alpha antagonizes the ability of IFN-gamma to induce class II expression on these macrophages.     

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.