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

Abstract

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.

Figures

Figure 1.

Effects of exposure to febrile-range hyperthermia (FRH) on IL-8–directed transalveolar polymorphonuclear leukocyte (PMN) migration. (A) Time course of core temperature in normothermic and FRH-exposed mice. Mean ± SE, n = 4 per group. The curves are different (P < 0.05 according to MANOVA). (B) Mice were exposed to FRH for the indicated times and returned to normothermia, intratracheal IL-8 was instilled, and 4 hours later, lung lavage PMN and macrophage (MAC) content was measured. BALF, bronchoalveolar lavage fluid. (C) Mice were exposed to FRH for 24 hours, and allowed to recover at normothermia for the indicated times before IL-8–directed transalveolar migration (TAM) assay. Control mice remained normothermic. Mean ± SEM; eight mice per group. *P < 0.05 versus time 0 (B) or control (C). (D and E) Inflation-fixed lungs from normothermic (NT) and 24-hour FRH-exposed mice without intratracheal IL-8 instillation or 4 hours after intratracheal IL-8 instillation were stained for granulocyte receptor-1 (GR-1) and vascular endothelial (VE)-cadherin, and analyzed by confocal microscopy for numbers of total (D) and extravasating (E) PMNs per ×60 high-power field (hpf) and (F) PMN elongation. Mean ± SEM; five fields per mouse, four mice per group. *P < 0.05, versus NT mice without IL-8. †P < 0.05, versus FRH mice without IL-8. ¶P < 0.05, versus NT mice with IL-8, respectively.

Figure 2.

Effects of FRH exposure on PMNs and endothelia. (A and B) Recipient or donor mice were exposed to FRH for 24 hours, and donor PMNs were isolated, fluorescently labeled, and injected via the tail vein, and IL-8–directed donor PMN TAM was determined by flow cytometry. The total donor PMN content in lung lavage (A) and the proportion of lung-lavage PMNs of donor origin (B) are shown for the indicated transfers between FRH-exposed and normothermic (NT) mice. Mean ± SE; six mice per group. *P < 0.05, versus all other groups. (C) Blood was collected from normothermic or 24-hour FRH-exposed mice, and the percentages of PMNs expressing CD11a, CD11b, or CD18 were analyzed according to flow cytometry by gating on GR-1–stained cells. Mean ± SE; four mice per group. *P < 0.05 versus normothermic mice. (D) After erythrocyte lysis, the binding of intercellular adhesion molecule-1 (ICAM-1)–coated fluorescent microbeads during 30-minute incubation at 37°C was analyzed by flow cytometry and expressed as mean number of beads per GR-1–stained PMN. Mean ± SE; four replicates. (E) Lung homogenates from six normothermic and six 24-hour FRH-exposed mice were analyzed for ICAM-1 and ICAM-2 expression by immunoblotting, normalized to β-tubulin levels, as expressed relative to normothermic levels; mean ± SE.

Figure 3.

Effects of FRH on activation of extracellular regulated kinase (ERK) and p38 in vivo. (A) Lung homogenates from normothermic mice (lanes 1–3) and mice exposed to FRH for 9 hours (ERK) or 6 hours (p38) (lanes 4–7) were analyzed for phosphorylated (p) and total (t) ERK and p38 by immunoblotting. (B) Lung homogenates from normothermic mice and mice exposed to FRH for the indicated times were analyzed for phosphorylated and total HSP27 by immunoblotting. (C and D) Blood was collected from four normothermic mice and four mice exposed to FRH for the indicated times, their erythrocytes were lysed, cells were stained for GR-1 and total or phosphorylated ERK (C) or p38 (D), and percentages of labeled PMNs were analyzed by flow cytometry. Mean ± SE. *P < 0.05 versus time 0. (E) Groups of eight mice were untreated or treated with 2% DMSO (sham), 200 μg U0126, or 1 mg SB203580 (SB), 30 minutes before 16-hour FRH or normothermic exposure, and their IL-8–directed PMN TAM was measured. Mean ± SE. *P < 0.05, versus normothermic mice. †P < 0.05, versus untreated mice.

Figure 4.

Effects of FRH on PMN transendothelial migration (TEM) through human lung microvascular endothelial cell (HMVEC-L) monolayers. (A) HMVEC-Ls were incubated at 39.5°C for the indicated times or treated with 1 ng/ml TNF-α for 6 hours at 37°C, and the IL-8–directed TEM of acetomethoxy calcein (calcein AM)–stained human PMNs was measured for 2 hours at 37°C and standardized to untreated HMVEC-Ls. Mean ± SE; four experiments. †P < 0.05 versus time 0. †P < 0.05 versus TNF-α. (B) HMVEC-L monolayers on Matrigel-coated plastic were incubated at 37°C or 39.5°C for 6 hours, lysed and immunoblotted for ICAM-1 and ICAM-2, normalized to β-tubulin, and expressed relative to levels in 37°C cells; mean ± SE. (C) HMVEC-Ls were untreated (Control) or pretreated for 30 minutes with DMSO, U0126, or SB203580, and incubated for 24 hours at 37°C or 39.5°C, and the PMN TEM was measured. Mean ± SE; four experiments. *P < 0.05, versus 37°C. †P < 0.05, versus untreated at 39.5°C. (D) Postconfluent HMVEC-L monolayers were serum-starved for 12 hours, incubated at 39.5°C for 1, 2, 3, 6, or 24 hours under serum-free conditions, and immunoblotted for phosphorylated (p) and total (t) p38 and ERK. Controls include 24 hours at 37°C, and 30 minutes with 1 ng/ml TNF-α at 37°C. Representatives of four similar blots are shown. (E) Postconfluent HMVEC-L monolayers were incubated at 37°C or 39.5°C for 6 hours, stained with AlexaFluor488-coupled phalloidin, and imaged by confocal microscopy. Representatives of four similar sets of coverslips are shown. (F) HMVEC-Ls were serum-starved for 12 hours, incubated at 37°C or 39.5°C for 24 hours, and subjected to ICAM-1 crosslinking at 37°C by incubating with mouse anti–ICAM-1 for 30 minutes (time 0) and anti-mouse IgG for the indicated times. Lysed cells were immunoblotted for p-p38 and t-p38 and p-ERK and t-ERK. Representatives of four similar immunoblots are shown.