Cell density control of staphylococcal virulence mediated by an octapeptide pheromone.

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

Interleukin 12 suppresses autoantibody production by reversing helper T-cell phenotype in hepatitis B e antigen transgenic mice.

Helper T (Th) cells are classified as Th1 or Th2 cells by virtue of cytokine secretion and function as mediators of cellular or humoral immunity, respectively. Cytokines also regulate the differentiation of Th cells. For example, interleukin (IL)-12 promotes Th1 and suppresses Th2 cell development, suggesting that IL-12 may be useful therapeutically in Th2-mediated autoimmune and allergic disorders. Therefore, the effect of systemic IL-12 treatment on in vivo autoantibody synthesis in hepatitis B e antigen (HBeAg)-expressing transgenic mice, which is dependent on self-reactive Th2 cells, was examined. Low-dose IL-12 significantly inhibited autoantibody production by shifting the Th2-mediated response toward Th1 predominance. Additionally, previous studies suggest that a predominance of HBeAg-specific Th2-type cells may contribute to chronicity in hepatitis B virus infection. Therefore, IL-12 may also prove beneficial in modulating the HBeAg-specific Th response to favor viral clearance in chronic hepatitis B virus infection.

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.