Febrile-range hyperthermia modifies endothelial and neutrophilic functions to promote extravasation.

Acute respiratory distress syndrome (ARDS) is a neutrophil (polymorphonuclear leukocyte; PMN)-driven lung injury that is associated with fever and heat-stroke, and involves approximately 40% mortality. In murine models of acute lung injury (ALI), febrile-range hyperthermia (FRH) enhanced PMN accumulation, vascular permeability, and epithelial injury, in part by augmenting pulmonary cysteine-x-cysteine (CXC) chemokine expression. To determine whether FRH increases chemokine responsiveness within the lung, we used in vivo and in vitro models that bypass the endogenous generation of chemokines. We measured PMN transalveolar migration (TAM) in mice after intratracheal instillations of the human CXC chemokine IL-8 in vivo, and of IL-8-directed PMN transendothelial migration (TEM) through human lung microvascular endothelial cell (HMVEC-L) monolayers in vitro. Pre-exposure to FRH increased in vivo IL-8-directed PMN TAM by 23.5-fold and in vitro TEM by 7-fold. Adoptive PMN transfer demonstrated that enhanced PMN TAM required both PMN donors and recipients to be exposed to FRH, suggesting interdependent effects on PMNs and endothelium. FRH exposure caused the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in lung homogenates and circulating PMNs, with an associated increase in HSP27 phosphorylation and stress-fiber formation. The inhibition of these signaling pathways with U0126 and SB203580 blocked the effects of FRH on PMN extravasation in vivo and in vitro. Collectively, these results (1) demonstrate that FRH augments chemokine-directed PMN extravasation through direct effects on endothelium and PMNs, (2) identify ERK and p38 signaling pathways in the effect, and (3) underscore the complex effects of physiologic temperature change on innate immune function and its potential consequences for lung injury.

Surfactant protein-A limits Ureaplasma-mediated lung inflammation in a murine pneumonia model.

Ureaplasma respiratory tract colonization stimulates prolonged, dysregulated inflammation in the lungs of preterm infants, contributing to bronchopulmonary dysplasia (BPD) pathogenesis. Surfactant protein-A (SP-A), a lung collectin critical for bacterial clearance and regulating inflammation, is deficient in the preterm lung. To analyze the role of SP-A in modulating Ureaplasma-mediated lung inflammation, SP-A deficient (SP-A-/-) and WT mice were inoculated intratracheally with a mouse-adapted U. parvum isolate and indices of inflammation were sequentially assessed up to 28 d postinoculation. Compared with infected WT and noninfected controls, Ureaplasma-infected SP-A-/- mice exhibited an exaggerated inflammatory response evidenced by rapid influx of neutrophils and macrophages into the lung, and higher bronchoalveolar lavage TNF-alpha, mouse analogue of human growth-related protein alpha (KC), and monocyte chemotactic factor (MCP-1) concentrations. However, nitrite generation in response to Ureaplasma infection was blunted at 24 h and Ureaplasma clearance was delayed in SP-A-/- mice compared with WT mice. Coadministration of human SP-A with the Ureaplasma inoculum to SP-A-/- mice reduced the inflammatory response, but did not improve the bacterial clearance rate. SP-A deficiency may contribute to the prolonged inflammatory response in the Ureaplasma-infected preterm lung, but other factors may contribute to the impaired Ureaplasma clearance.

Heat shock co-activates interleukin-8 transcription.

The heat shock (HS) response is a phylogenetically ancient cellular response to stress, including heat, that shifts gene expression to a set of conserved HS protein (HSP) genes. In our earlier studies, febrile-range hyperthermia (FRH) not only activated HSP gene expression, but also increased expression of CXC chemokines in mice, leading us to hypothesize that the CXC chemokine family of genes might be HS-responsive. To address this hypothesis we analyzed the effect of HS on the expression of IL-8/CXCL-8, a member of the human CXC family of ELR(+) chemokines. HS markedly enhanced TNF-alpha-induced IL-8 secretion in human A549 respiratory epithelial-like cells and in primary human small airway epithelial cells. IL-8 mRNA was also up-regulated by HS, but the stability of IL-8 mRNA was not affected. TNF-alpha-induced reporter activity of an IL-8 promoter construct IL8(-1471/+44)-luc stably transfected in A549 cells was also enhanced by HS. Electrophoretic mobility and chromatin immunoprecipitation assays showed that the stress-activated transcription factor heat shock factor-1 (HSF-1) binds to at least two putative heat shock response elements (HSE) present in the IL-8 promoter. Deletional reporter constructs lacking either one or both of these sites showed reduced HS responsiveness. Furthermore, depletion of HSF-1 using siRNA also reduced the effects HS on TNF-alpha-induced IL-8 expression, demonstrating that HSF-1 could also act to regulate IL-8 gene transcription. We speculate that during evolution the CXC chemokine genes may have co-opted elements of the HS response to amplify their expression and enhance neutrophil delivery during febrile illnesses.

Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells.

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) = 49 degrees 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 +/- 6.1% and 21.1 +/- 6.5%, respectively. Ex vivo induction of HSP72 in PBMCs exposed to heat shock (43 degrees C) was blunted on day 10 compared with day 1. A correlation was identified (r(2) = 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.

Antenatal Ureaplasma urealyticum respiratory tract infection stimulates proinflammatory, profibrotic responses in the preterm baboon lung.

Chronic inflammation and fibrosis are hallmarks of lung pathology of newborn Ureaplasma infection. We hypothesized that antenatally acquired Ureaplasma stimulates a chronic inflammatory, profibrotic immune response that contributes to lung injury, altered developmental signaling, and fibrosis. Lung specimens from 125-d gestation baboon newborns ventilated for 14 d that were either infected antenatally with Ureaplasma serovar 1 or noninfected, and 125-d and 140-d gestational controls were obtained from the Baboon BPD Resource Center (San Antonio, TX). Trichrome stain to assess fibrosis and immunohistochemistry for alpha-smooth muscle actin (alpha-SMA) and transforming growth factor beta1 (TGFbeta1) were performed. Lung homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA) for cytokines [tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, TGFbeta1, oncostatin M (OSM), IL-10, and interferon gamma (IFNgamma)] and the chemokine MCP-1 and by Western blot for Smad2, Smad3, and Smad7. Compared with noninfected ventilated and gestational controls, Ureaplasma-infected lungs demonstrated more extensive fibrosis, increased alpha-SMA and TGFbeta1 immunostaining, and higher concentrations of active TGFbeta1, IL-1beta, and OSM, but no difference in IL-10 levels. There was a trend toward higher Smad2/Smad7 and Smad3/Smad7 ratios in Ureaplasma lung homogenates, consistent with up-regulation of TGFbeta1 signaling. Collectively, these data suggest that a prolonged proinflammatory response initiated by intrauterine Ureaplasma infection contributes to early fibrosis and altered developmental signaling in the immature lung.

Disordered pulmonary myofibroblast distribution and elastin expression in preterm infants with Ureaplasma urealyticum pneumonitis.

Respiratory colonization of preterm infants with Ureaplasma urealyticum is a significant risk factor for bronchopulmonary dysplasia, a chronic lung disease characterized by arrest of alveolar development, variable interstitial fibrosis, and disordered elastic fibers in the distal airspaces. As indicated in previous studies, moderate to severe fibrosis is a hallmark of pathology in the Ureaplasma-infected preterm lung. To further characterize the preterm lung's response to Ureaplasma, lung specimens from 4 gestational controls (GC), 12 other pneumonia and 5 Ureaplasma-infected infants were analyzed by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and transforming growth factor beta1 (TGFbeta1), Hart's elastin staining, and in situ hybridization for tropoelastin (TE) expression. Cells positive for alphaSMA were observed in thickened, extensive bundles surrounding terminal airspaces in Ureaplasma and other pneumonia cases compared to individual myofibroblasts in GC. The myofibroblast pattern correlated with the severity of fibrosis, but not duration of ventilation. Transforming growth factor beta1 immunostaining was primarily localized to alveolar macrophages and was increased in Ureaplasma more than in other pneumonia cases. Elastic fibers and TE-expressing cells were spatially limited to emerging septal tips in GC. In pneumonia cases, increased deposition of elastic fibers was observed surrounding terminal airspaces, but TE expression was similar to GC. In Ureaplasma specimens, accumulation of elastic fibers correlated with duration of ventilation, and TE expression was extensive throughout the walls of terminal airspaces. These findings suggest that Ureaplasma is associated with alveolar macrophage TGFbeta1 immunostaining and myofibroblast proliferation contributing to abnormal septation, interstitial fibrosis, and a prolonged and strong elastogenic response in the preterm lung.

G-CSF, but not corticosterone, mediates circulating neutrophilia induced by febrile-range hyperthermia.

We previously showed that sustained exposure to febrile-range hyperthermia (FRH) for 24 h caused an increase in circulating granulocyte colony-stimulating factor (G-CSF) levels and a peripheral neutrophilia in mice (Hasday J, Garrison A, Singh I, Standiford T, Ellis G, Rao S, He JR, Rice P, Frank M, Goldblum S, and Viscardi R. Am J Pathol 162: 2005-2017, 2003). In this study, we utilized a conscious temperature-clamped mouse model to analyze the kinetics of G-CSF expression and peripheral neutrophil expansion and the contributions of FRH-induced G-CSF expression, glucocorticoid generation, and catecholamine-induced neutrophil demargination. In conscious mice housed at an ambient temperature of 34.5 degrees C, core temperature rapidly equilibrated at 39.5-40 degrees C. Peripheral neutrophil counts increased 2-fold after 24-h exposure to hyperthermia, peaked at 3.6-fold baseline levels after 36-h exposure to FRH, and returned to baseline levels after 42 h of sustained hyperthermia. Plasma G-CSF levels were increased by 6.8-fold after 24 h and peaked at 40-fold baseline levels after 36 h in the hyperthermic mice. Plasma corticosterone levels peaked at 3.3-fold baseline levels after 30-h sustained hyperthermia and returned to baseline by 42 h. Immunoneutralization of G-CSF blocked FRH-induced peripheral neutrophilia, but blockade of the glucocorticoid receptor with mifepristone failed to modify FRH-induced neutrophilia. Epinephrine induced similar increases in peripheral blood absolute neutrophil counts in euthermic mice (2.2-fold increase) and mice exposed to FRH for 36 h (1.8-fold increase). Collectively, these data suggest that FRH-induced expression of G-CSF drives the sustained peripheral neutrophilia that occurs during sustained (36 h) hyperthermia, whereas glucocorticoid generation and catecholamine-induced demargination play little role in this response.

Febrile-range hyperthermia augments neutrophil accumulation and enhances lung injury in experimental gram-negative bacterial pneumonia.

We previously demonstrated that exposure to febrile-range hyperthermia (FRH) accelerates pathogen clearance and increases survival in murine experimental Klebsiella pneumoniae peritonitis. However, FRH accelerates lethal lung injury in a mouse model of pulmonary oxygen toxicity, suggesting that the lung may be particularly susceptible to injurious effects of FRH. In the present study, we tested the hypothesis that, in contrast with the salutary effect of FRH in Gram-negative peritonitis, FRH would be detrimental in multilobar Gram-negative pneumonia. Using a conscious, temperature-clamped mouse model and intratracheal inoculation with K. pneumoniae Caroli strain, we showed that FRH tended to reduce survival despite reducing the 3 day-postinoculation pulmonary pathogen burden by 400-fold. We showed that antibiotic treatment rescued the euthermic mice, but did not reduce lethality in the FRH mice. Using an intratracheal bacterial endotoxin LPS challenge model, we found that the reduced survival in FRH-treated mice was accompanied by increased pulmonary vascular endothelial injury, enhanced pulmonary accumulation of neutrophils, increased levels of IL-1beta, MIP-2/CXCL213, GM-CSF, and KC/CXCL1 in the bronchoalveolar lavage fluid, and bronchiolar epithelial necrosis. These results suggest that FRH enhances innate host defense against infection, in part, by augmenting polymorphonuclear cell delivery to the site of infection. The ultimate effect of FRH is determined by the balance between accelerated pathogen clearance and collateral tissue injury, which is determined, in part, by the site of infection.

Bacterial endotoxin modifies heat shock factor-1 activity in RAW 264.7 cells: implications for TNF-alpha regulation during exposure to febrile range temperatures.

Recent studies have identified heat shock factor (HSF)-1, the predominant heat/stress-stimulated transcriptional activator of heat shock protein genes as a repressor of certain cytokine genes, including TNF-alpha and IL-1beta. We previously showed that exposing macrophages to febrile-range temperature (FRT; 39.5 degrees C) activates HSF-1 to a DNA binding form that does not activate heat shock protein gene transcription, but apparently represses TNF-alpha and IL-1beta transcription. Prewarming macrophages to 39.5 degrees C for 30 min prior to stimulation with bacterial lipopolysaccharide (LPS) does not change the induction of TNF-alpha transcription, but markedly reduces its duration. This raised the question of how TNF-alpha transcription could occur at all in the presence of activated HSF-1. We used RAW 264.7 cells to test the hypothesis that macrophage activation triggers a transient reversal of HSF-1-mediated repression, thereby allowing induction of TNF-alpha transcription. Electrophoretic mobility shift assays revealed that LPS triggers a transient inactivation of HSF-1 that temporally correlates with TNF-alpha transcription and was associated with a transient increase in HSF-1 molecular weight, a decrease in its pI, and appearance of HSF-1 phosphorylating activity. The serine/threonine phosphatase inhibitor, calyculin A, blocked the inhibitory affect of FRT on LPS-induced TNF-alpha generation and prevented the re-activation of HSF-1. We propose that LPS stimulation of FRT-exposed macrophages stimulates a sequential phosphorylation and dephosphorylation of HSF-1, causing a cycle of inactivation and reactivation of HSF-1 repressor activity that allows a temporally-limited period of gene transcription.

Febrile-range hyperthermia augments pulmonary neutrophil recruitment and amplifies pulmonary oxygen toxicity.

Febrile-range hyperthermia (FRH) improves survival in experimental infections by accelerating pathogen clearance, but may also increase collateral tissue injury. We hypothesized that FRH would worsen the outcome of inflammation stimulated by a non-replicating agonist and tested this hypothesis in a murine model of pulmonary oxygen toxicity. Using a conscious, temperature-controlled mouse model, we showed that maintaining a core temperature at FRH (39 degrees C to 40 degrees C) rather than at euthermic levels (36.5 degrees C to 37 degrees C) during hyperoxia exposure accelerated lethal pulmonary vascular endothelial injury, reduced the inspired oxygen threshold for lethality, induced expression of granulocyte-colony stimulating factor, and expanded the circulating neutrophil pool. In these same mice, FRH augmented pulmonary expression of the ELR(+) CXC chemokines, KC and LPS-induced CXC chemokine, enhanced recruitment of neutrophils, and changed the histological pattern of lung injury to a neutrophilic interstitial pneumonitis. Immunoblockade of CXC receptor-2 abrogated neutrophil recruitment, reduced pulmonary vascular injury, and delayed death. These combined data demonstrate that FRH may enlist distinct mediators and effector cells to profoundly shift the host response to a defined injurious stimulus, in part by augmenting delivery of neutrophils to sites of inflammation, such as may occur in infections. In certain conditions, such as in the hyperoxic lung, this process may be deleterious.

Characterization of a murine model of Ureaplasma urealyticum pneumonia.

Ureaplasma urealyticum respiratory tract colonization in preterm infants has been associated with a high incidence of pneumonia and the development of bronchopulmonary dysplasia. However, study of this human pathogen has been hampered by the absence of animal models. We have developed the first juvenile mouse model of Ureaplasma pneumonia and characterized the histopathology during the month following inoculation. C3H/HeN mice were inoculated intratracheally with a mouse-adapted clinical Ureaplasma isolate (biovar 2) or sham inoculated with 10B broth. Culture of lung homogenates and PCR of DNA from bronchoalveolar lavage fluid (BAL) confirmed the presence of Ureaplasma in 100% of inoculated animals at 1 day, 60% at 2 days, 50% at 3 days, and 25% at 7 and 14 days. Ureaplasma was undetectable 28 days postinoculation. There were marked changes in BAL and interstitial-cell composition with increased number of polymorphonuclear leukocytes 1 to 2 days and 14 days postinoculation and macrophages at 2 and 14 days postinoculation. The Ureaplasma infection caused a persistent focal loss of airway ciliated epithelium and a mild increase in interstitial cellularity. There were no differences in BAL protein concentration during the first 28 days, suggesting that pulmonary vascular endothelial barrier integrity remained intact. Comparison of BAL cytokine and chemokine concentrations revealed low levels of tumor necrosis factor alpha (TNF-alpha) at 3 days and monocyte chemoattractant protein 1 at 7 days in Ureaplasma-infected mice but a trend toward increased TNF-alpha at 14 days and increased granulocyte-macrophage colony-stimulating factor and interleukin-10 at 28 days. These data suggest that Ureaplasma alone may cause limited inflammation and minimal tissue injury in the early phase of infection but may promote a mild chronic inflammatory response in the later phase of infection (days 14 to 28), similar to the process that occurs in human newborns.

A high affinity HSF-1 binding site in the 5'-untranslated region of the murine tumor necrosis factor-alpha gene is a transcriptional repressor.

Tumor necrosis factor-alpha (TNFalpha) 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 degrees C) markedly attenuates TNFalpha 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 TNFalpha 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 TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha 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) TNFalpha 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 TNFalpha 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 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.