Tumor necrosis factor alpha (TNF-␣) is a potent cytokine which regulates inflammation via the induction of adhesion molecules and chemokine expression. Its expression is known to be regulated in a complex manner with transcription, message turnover, message splicing, translation, and protein cleavage from the cell surface all being independently regulated. This study examined both cell lines and primary cells to understand the developmental regulation of epigenetic changes at the TNF-␣ locus. We demonstrate that epigenetic modifications of the TNF-␣ locus occur both developmentally and in response to acute stimulation and, importantly, that they actively regulate expression. DNA demethylates early in development, beginning with the hematopoietic stem cell. The TNF-␣ locus migrates from heterochromatin to euchromatin in a progressive fashion, reaching euchromatin slightly later in differentiation. Finally, histone modifications characteristic of a transcriptionally competent gene occur with myeloid differentiation and progress with differentiation. Additional histone modifications characteristic of active gene expression are acquired with stimulation. In each case, manipulation of these epigenetic variables altered the ability of the cell to express TNF-␣. These studies demonstrate the importance of epigenetic regulation in the control of TNF-␣ expression. These findings may have relevance for inflammatory disorders in which TNF-␣ is overproduced.
Tumor necrosis factor alpha (TNF-␣) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-␣ ؊308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-␣ in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the ؊308 site in cells that do not make TNF-␣. Cells competent to produce TNF-␣ have Ets-1 bound to the ؊308 promoter site. Active transcription is accompanied by NF-B and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-␣ promoter, particularly at the ؊308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-␣ repressor identified.Tumor necrosis factor (TNF-␣) is a proinflammatory cytokine which plays a role in humoral immunity, chemokine expression, the regulation of adhesion molecule expression, and apoptosis. The expression of TNF-␣ is regulated at many levels, including transcription, message turnover, protein production, and protein release (15,21,25,36). The transcription of TNF-␣ is complex, with tissue-specific expression and stimulusinducible expression, which in turn can be tissue specific. Some of the TNF-␣ promoter motifs which have been implicated in transcription include binding sites for NF-B, NFAT, ATF-2, Ets-1, C/EBP, cis-acting replication element, AP-1, and AP-2 (13, 14, 24, 26-28, 35, 37, 39, 40, 48, 49, 58, 59). Several promoter polymorphisms have been identified, and a polymorphism at Ϫ308 has been implicated in the regulation of TNF-␣ transcription (24,29,52,56). (This work uses the traditional numbering system for the promoter polymorphisms [55].)The TNF-␣ promoter polymorphism at Ϫ308 involves a biallelic single-base pair transition from G to A. The polymorphic sequence, designated as TNF-␣ Ϫ308A, has gene frequencies of approximately 0.12 to 0.17 in Caucasians and 0.08 in African Americans (9,10,42,45,4). Studies have found associations of TNF-␣ Ϫ308A with systemic lupus erythematosus, sarcoidosis, alopecia areata, rheumatoid arthritis, and dermatitis herpetiformis (9,17,18,34,42,43,45,54). Other studies have found no association of TNF-␣ Ϫ308A with rheumatoid arthritis, ankylosing spondylitis, and Felty's syndrome (5, 51, 53). Certain infectious diseases, such as cerebral malaria and mucocutaneous leishmania, als...
Objectives. Patients with chronic granulomatous disease and carrier mothers of patients with chronic granulomatous disease are predisposed to developing various forms of lupus. This disorder is a neutrophil defect in intracellular killing. Abnormal apoptosis has been described. We hypothesized that abnormal apoptosis occurring in neutrophils of patients made them more immunogenic. Methods. Human patients with chronic granulomatous disease were examined for abnormalities of neutrophil apoptosis by flow cytometry. To model the effect of abnormal apoptosis, a murine model was used. Apoptotic cells from either wild type or mice with chronic granulomatous disease were injected into either wild type or chronic granulomatous disease mice and autoantibodies were determined by ELISA. Results. Our studies found that human and murine neutrophils carrying the gp91 form of chronic granulomatous disease had impaired exposure of phosphatidyl serine on the surface. Other markers of apoptosis were largely normal. Injection of apoptotic neutrophils from gp91 knockout mice into gp91 knockout mice led to the development of characteristic autoantibodies of lupus. Conclusions. Humans with chronic granulomatous disease may be at an increased risk of developing lupus due to abnormal apoptosis and abnormal clearance of apoptotic cells.
In systemic lupus erythematosus, TNFα is elevated in the serum and correlates with disease activity and triglyceride levels. The stimuli that drive TNFα in this setting are incompletely understood. This study was designed to evaluate monocyte chromatin at the TNFα locus to identify semi-permanent changes that might play a role in altered expression of TNFα. SLE patients with relatively quiescent disease (mean Physician Global Assessment=0.6) and healthy controls were recruited for this study. TNFα expression was measured by intracellular cytokine staining of different monocyte subsets in patients (n=24) and controls (n=12). Histone acetylation at the TNFα locus was measured by chromatin immunoprecipitation using a normalized quantitative PCR in patients (n=46) and controls (n=24). There were no differences in the overall fractions of cells expressing CD14 in SLE patients compared to controls, however, the fraction of DR+/CD16+ cells expressing CD14 was slightly higher as was true in the monocyte subset defined by DR+/CD11b+. Within the monocyte population defined by physical characteristics and DR+/CD14+, TNFα expressing cells were more frequent in SLE patients compared to controls. Both the fraction of positive cells and the mean fluorescence intensity were higher in patients than controls. Consistent with this was the finding that monocytes from patients had increased TNFα transcripts and more highly acetylated histones at the TNFα locus compared to controls. Furthermore, patients with the highest levels of TNFα histone acetylation were more likely to have had consistently elevated erythrocyte sedimentation rates, and to have required cytotoxic use. Histone acetylation, associated with increased transcriptional competence of TNFα, may play a role in certain inflammatory aspects of the disease.
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