Tumor necrosis factor-␣ (TNF␣) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40°C) markedly attenuates TNF␣ expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNF␣ gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5-UTR of the TNF␣ gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNF␣ gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNF␣ promoter (؊85/؉138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (؊1080/؉138) TNF␣ promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions ؊1080/ ؊845, ؊533/؊196, and ؊326/؊39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal ؊85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNF␣ promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5°C. This suggests that HSF-1 might repress TNF␣ transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1. Tumor necrosis factor-␣ (TNF␣)1 is an early pivotal mediator expressed in response to infection and injury (1). Although TNF␣ is essential for optimal host defense, persistent or inappropriately high TNF␣ expression has grave consequences, including multiorgan failure and death (2-5). The pleiotropic nature of TNF␣ has lead to the evolution of stringent and redundant regulatory mechanisms imposed at transcriptional, translational, and posttranslational levels (6 -11). We reported that exposure to febrile range hyperthermia suppresses TNF␣ expression in murine peritoneal macrophages, Kupffer cells, precision-cut liver slices, the murine Raw 264.7 macrophage cell line, human monocyte-derived macrophages, and the THP1 monocyte cell line (10,(12)(13)(14)(15)(16). We showed that the predominant mechanism of suppression of TNF␣ expression is by an abrupt and early cessation of TNF␣ transcription, and that the TNF␣ gene sequence between Ϫ85 and ϩ138 is sufficient to confer temperature responsiveness in murine macrophages (15). We also showed that the heat stressactivated transcription factor, heat shock transcription factor 1 (HSF-1) is activated at febr...

Fever, a nonspecific acute-phase response, has been associated with improved survival and shortened disease duration in infections, but the mechanisms of these beneficial responses are poorly understood. We previously reported that increasing core temperature of bacterial endotoxin (LPS)-challenged mice to the normal febrile range modified expression of tumor necrosis factor alpha (TNF-␣), interleukin 1␤ (IL-1␤), and IL-6, three cytokines critical to mounting an initial defense against microbial pathogens, but survival was not improved in the warmer animals. We speculated that our inability to show a survival benefit of optimized cytokine expression in the warmer animals reflected our use of LPS, a nonreplicating agonist, rather than an infection with viable pathogens. The objective of this study was to determine if increasing murine core temperature altered cytokine expression and improved survival in an experimental bacterial peritonitis model. We showed that housing mice at 35.5°C rather than 23°C increased core temperature from 36.5 to 37.5°C to 39.2 to 39.7°C, suppressed plasma TNF-␣ expression for the initial 48 h, delayed gamma interferon expression, improved survival, and reduced the bacterial load in mice infected with Klebsiella pneumoniae peritonitis. We showed that the reduced bacterial load was not caused by a direct effect on bacterial proliferation and probably reflected enhanced host defense. These data suggest that the increase in core temperature that occurs during bacterial infections is essential for optimal antimicrobial host defense.

McClung JP, Hasday JD, He J, Montain SJ, Cheuvront SN, Sawka MN, Singh IS. Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells. Am J Physiol Regul Integr Comp Physiol 294: R185-R191, 2008. First published October 31, 2007 doi:10.1152/ajpregu.00532.2007.-The induction of cellular acquired thermal tolerance (ATT) during heat acclimation (HA) in humans is not well described. This study determined whether exercise-HA modifies the human heat shock protein (HSP)72 and HSP90 responses and whether changes are correlated with physiological adaptations to HA. Using a 10-day HA protocol comprising daily exercise (treadmill walking) in a hot environment (T a ϭ 49°C, 20% RH), we analyzed baseline and ex vivo heat-induced expression of HSP72 and HSP90 in peripheral blood mononuclear cells (PBMCs) isolated prior to exercise from eight subjects on day 1 and 10 of the HA protocol. Classical physiological responses to HA were observed, including significantly reduced heart rate and core body temperature, and significantly increased sweating rate. Baseline levels of HSP72 and HSP90 were significantly increased following acclimation by 17.7 Ϯ 6.1% and 21.1 Ϯ 6.5%, respectively. Ex vivo induction of HSP72 in PBMCs exposed to heat shock (43°C) was blunted on day 10 compared with day 1. A correlation was identified (r 2 ϭ 0.89) between changes in core temperature elevation and ex vivo HSP90 responses to heat shock between days 1 and 10, indicating that volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. In summary, 1) exercise-HA resulted in increased baseline levels of HSP72 and HSP90, 2) ex vivo heat inducibility of HSP72 was blunted after HA, and 3) volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. These data demonstrate that physiological adaptations in humans undergoing HA are accompanied by both increases in baseline levels and changes in regulation of cytoprotective HSPs. stress; hyperthermia; adaptation; exertional tolerance HEAT ACCLIMATION (HA) REFERS to biological adaptations that reduce physiological strain (e.g., heart rate and body temperature), improve comfort, and improve physical exercise capabilities after repeated days of heat exposure (28, 30). Acquired thermal tolerance (ATT) refers to the cellular changes induced by repeated exposure to heat that confer cytoprotection against subsequent, more extreme, and potentially lethal heat exposure (11,17,33). For example, rodents with fully developed ATT can survive a 60% greater heat load compared with heat-naïve animals (21). Heat acclimation and ATT are complementary with the former reducing physiological strain and the latter providing cellular protection against serious heat injury for any degree of physiological strain. Few studies have examined the coincident induction of HA and ATT ...

. TNF-␣ increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia.

Levels of pulmonary and activation-regulated chemokine (PARC) mRNA and protein are increased in the lungs of patients with pulmonary fibrosis. The purpose of this study was to establish whether PARC could be directly involved in development of pulmonary fibrosis by stimulating collagen production in lung fibroblasts. Exposure to PARC increased production of collagen mRNA and protein by 3- to 4-fold in normal adult lung and dermal fibroblast cells. Collagen mRNA transiently increased after 3-6 h of activation with PARC, with an increase in collagen protein detected after 24 h of activation. At the same time, PARC had less pronounced effect on fibroblast proliferation, not exceeding 50% increase over control nonstimulated cells. PARC intracellular signaling led to activation of ERK1/2, but not p38, in fibroblasts; pharmacologic inhibition of ERK, but not p38, also blocked PARC's effect on collagen production. Inhibition experiments with pertussis toxin suggested that PARC receptor is G protein-coupled. Thus, PARC is a member of the CC chemokine family that acts directly as a profibrotic factor.