IntroductionClinical conditions that induce impaired cell-mediated immunity like AIDS, malignancy, and immunosuppressive therapy result in impaired MHC class II-mediated delayed-type hypersensitivity (DTH) reaction (1, 2). Moreover, long-standing clinical observations have established that certain diseases that do not induce a generalized immunosuppressive state, also induce impaired DTH reaction to specific antigens, a state clinically defined as "anergy." Classical paradigms of these diseases include tuberculosis (TB), sarcoidosis, and Hodgkin's disease.TB is the leading cause of death from infectious diseases worldwide (3), accounting for eight million new cases and three million deaths annually (6). The lethality of TB is due to both the absence of an effective vaccine and to the poor understanding of how the mycobacteria escape immune surveillance. Anergy in the setting of TB refers to the paradoxical absence of dermal reactivity to intradermal injection with tuberculin purified protein derivative (PPD) in infected persons. It occurs in about 15% of patients with active pulmonary disease and is associated with absence of granuloma formation and all other manifestations of cellular hypersensitivity (3-5). We thus chose to examine the biochemical events that regulate the induction of TB anergy since this understanding may also provide insights into the pathophysiology of this disease.Anergy in vitro and its in vivo counterpart, tolerance, are immunologically defined as the inability of antigenspecific T cells to produce IL-2 and clonally expand on rechallenge with fully competent antigen-presenting cells (APC) (7,8). Induction of anergy is an active signaling process induced when T-cell receptor (TCR) is ligated by antigen without costimulation. Anergy can also be induced in the presence of costimulation if the TCR is ligated by superantigen or by altered peptide ligands that bear a single amino acid substitution in the sequence of the agonistic peptide (9). Although quite distinct, these three approaches to induce anergy appear to share common biochemical events characterized by hypophosphorylation of TCRζ and defective activation of ZAP-70 and Ras (10-15), indicating the generalized significance of these findings in the anergic state. Recently, IL-10 in The lethality of Mycobacterium tuberculosis remains the highest among infectious organisms and is linked to inadequate immune response of the host. Containment and cure of tuberculosis requires an effective cell-mediated immune response, and the absence, during active tuberculosis infection, of delayedtype hypersensitivity (DTH) responses to mycobacterial antigens, defined as anergy, is associated with poor clinical outcome. To investigate the biochemical events associated with this anergy, we screened 206 patients with pulmonary tuberculosis and identified anergic patients by their lack of dermal reactivity to tuberculin purified protein derivative (PPD). In vitro stimulation of T cells with PPD induced production of IL-10, IFN-γ, and proliferation in PPD +...

The tumor necrosis factor alpha (TNF-␣) gene is rapidly activated by lipopolysaccharide (LPS). Here, we show that extracellular signal-regulated kinase (ERK) kinase activity but not calcineurin phosphatase activity is required for LPS-stimulated TNF-␣ gene expression. In LPS-stimulated macrophages, the ERK substrates Ets and Elk-1 bind to the TNF-␣ promoter in vivo. Strikingly, Ets and Elk-1 bind to two TNF-␣ nuclear factor of activated T cells (NFAT)-binding sites, which are required for calcineurin and NFAT-dependent TNF-␣ gene expression in lymphocytes. The transcription factors ATF-2, c-jun, Egr-1, and Sp1 are also inducibly recruited to the TNF-␣ promoter in vivo, and the binding sites for each of these activators are required for LPSstimulated TNF-␣ gene expression. Furthermore, assembly of the LPS-stimulated TNF-␣ enhancer complex is dependent upon the coactivator proteins CREB binding protein and p300. The finding that a distinct set of transcription factors associates with a fixed set of binding sites on the TNF-␣ promoter in response to LPS stimulation lends new insights into the mechanisms by which complex patterns of gene regulation are achieved.Tumor necrosis factor alpha (TNF-␣) is a proinflammatory cytokine that activates multiple-signal transduction pathways and influences a broad range of immunological processes. Multiple extracellular stimuli induce the synthesis of TNF-␣ in a wide variety of cell types, including T and B cells, monocytes and macrophages, mast cells, and fibroblasts (reviewed in reference 1). We have shown that induction of TNF-␣ gene transcription by T or B cell receptor engagement, virus infection, and treatment with a calcium ionophore depends upon the activity of the phosphatase calcineurin (15,18,20). Calcineurin targets the nuclear factor of activated T cells (NFAT) family of proteins (reviewed in references 11 and 38), which are critical for TNF-␣ gene expression by calcineurin-dependent signal transduction pathways (15,48,49).Production of TNF-␣ in response to lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is of particular clinical importance because TNF-␣ is a mediator of septic shock (reviewed in reference 1). Exposure of monocytes and macrophages to LPS results in activation of the mitogen-activated protein kinase (MAPK) pathway, including the extracellular signal-related kinase (ERK), c-jun NH 2 -terminal kinase (JNK), and p38 cascades (reviewed in reference 12).Here, we show that ERK, but not calcineurin or p38, is required for full transcriptional induction of TNF-␣ gene expression by LPS. We identify TNF-␣ promoter elements critical for LPS induction of the gene and demonstrate that two Sp1 binding sites and three Ets binding sites, in addition to a cyclic AMP response element (CRE)-like site and an Egr site, are critical for LPS induction of the TNF-␣ gene. Consistent with this functional analysis of the TNF-␣ promoter, using chromatin immunoprecipitation and formaldehyde crosslinking (ChIP) assays, we directly detect LPS-induc...

The human tumor necrosis factor alpha (TNF-␣) gene is one of the earliest genes expressed upon the activation of a T or B cell through its antigen receptor. Previous experiments have demonstrated that in stimulated T cells, a TNF-␣ promoter element, 3, which binds NFATp, is required for the cyclosporin A-sensitive transcriptional activation of the gene. Here, we demonstrate that a cyclic AMP response element (CRE), which lies immediately upstream of the 3 site, is also required for induction of TNF-␣ gene transcription in T cells stimulated by calcium ionophore or T-cell receptor ligands. The CRE binds ATF-2 and Jun proteins in association with NFATp bound to 3. These proteins bind noncooperatively in vitro; however, the transcriptional activity of the CRE/3 composite site is dramatically higher than the activity of the 3 site alone, indicating that the two sites cooperate in vivo. This study is the first demonstration of a role for ATF-2 in TNF-␣ gene transcription and of a functional interaction between ATF-2/Jun and NFATp. This novel pairing of NFATp with ATF-2/Jun may account for the specific and immediate pattern of TNF-␣ gene transcription in stimulated T cells.

The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes transcribed after the stimulation of a B cell through its antigen receptor or via the CD-40 pathway. In both cases, induction of TNF-alpha gene transcription can be blocked by the immunosuppressants cyclosporin A and FK506, which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells. Furthermore, in T cells, two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immediately adjacent cyclic AMP response element (CRE) site. Here, using the murine B-cell lymphoma cell line A20, we show that the TNF-alpha gene is regulated in a cell-type-specific manner. In A20 B cells, the TNF-alpha gene is not regulated by NFATp bound to the kappa 3 element. Instead, ATF-2 and Jun proteins bind to the composite kappa 3/CRE site and NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site. This new site plays a critical role in the calcium-mediated, cyclosporin A-sensitive induction of TNF-alpha in both A20 B cells and Ar-5 cells. Consistent with these results, quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demonstrated that the -76 site binds to NFATp with a higher affinity than the kappa 3 site. Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analysis. Thus, through the differential use of the same promoter element, the composite kappa 3/CRE site, the TNF-alpha gene is regulated in a cell-type-specific manner in response to the same extracellular signal.