Febrile-range hyperthermia augments lipopolysaccharide-induced lung injury by a mechanism of enhanced alveolar epithelial apoptosis

Abstract

Fever is common in critically ill patients and is associated with worse clinical outcomes, including increased intensive care unit mortality. In animal models, febrile-range hyperthermia (FRH) worsens acute lung injury, but the mechanisms by which this occurs remain uncertain. We hypothesized that FRH augments the response of the alveolar epithelium to TNF-alpha receptor family signaling. We found that FRH augmented LPS-induced lung injury and increased LPS-induced mortality in mice. At 24 h, animals exposed to hyperthermia and LPS had significant increases in alveolar permeability without changes in inflammatory cells in bronchoalveolar lavage fluid or lung tissue as compared with animals exposed to LPS alone. The increase in alveolar permeability was associated with an increase in alveolar epithelial apoptosis and was attenuated by caspase inhibition with zVAD.fmk. At 48 h, the animals exposed to hyperthermia and LPS had an enhanced lung inflammatory response. In murine lung epithelial cell lines (MLE-15, LA-4) and in primary type II alveolar epithelial cells, FRH enhanced apoptosis in response to TNF-alpha but not Fas ligand. The increase in apoptosis was caspase-8 dependent and associated with suppression of NF-kappaB activity. The FRH-associated NF-kappaB suppression was not associated with persistence of IkappaB-alpha, suggesting that FRH-mediated suppression of NF-kappaB occurs by means other than alteration of IkappaB-alpha kinetics. These data show for the first time that FRH promotes lung injury in part by increasing lung epithelial apoptosis. The enhanced apoptotic response might relate to FRH-mediated suppression of NF-kappaB activity in the alveolar epithelium with a resultant increase in susceptibility to TNF-alpha-mediated cell death.

FIGURE 1

FRH is associated with increased LPS-induced mortality in an animal model. A, Exposure to ambient hyperthermia (35°C) generates febrile-range core body temperatures (39.5–40°C) in mice as compared with animals that were maintained at room temperature (23°C) independent of LPS exposure. B, Exposure to IT LPS and FRH results in increased mortality in mice. Animals treated with LPS alone or FRH alone had no mortality by 48 h, whereas animals treated with FRH plus LPS had 62% mortality. The FRH plus LPS survival curve shown reflects an LPS dose of 50 µg. Data from 5 and 25 µg doses LPS plus FRH are not shown but also demonstrate increased mortality. ^†p< 0.0001.

FIGURE 2

FRH augments alveolar permeability and delayed neutrophil recruitment in an LPS injury model. Lung weight (A), total protein in BAL fluid (B), and IgM concentration in BAL fluid (C) are increased in animals exposed to FRH and LPS as compared with animals exposed to LPS and euthermia. The changes in alveolar permeability precede FRH-associated changes in LPS-induced inflammation. Total cells in BAL fluid (D), total BAL fluid neutrophils (E), and tissue MPO activity in lung homogenates (F) are increased in animals exposed to FRH and LPS as compared with LPS alone after 48 h. For all groups, *p< 0.05; **p< 0.01; ^†p< 0.001.

FIGURE 3

FRH causes histologic evidence of LPS-induced lung injury prior to neutrophil recruitment. H&E stains of paraffin-embedded lungs of animals exposed to LPS 50 µg and euthermia for 24 h, original magnifications ×200 (A) and ×400 (B), or hyperthermia, ×200 (C) and ×400 (D). The animals exposed to LPS and hyperthermia have more airway edema, alveolar septal thickening, and pyknotic nuclei (arrows) in the alveolar walls as compared with LPS alone. After 48 h, the lungs of animals exposed to LPS 50 µg and euthermia, original magnifications ×200 (E) and ×400 (F), or hyperthermia, ×200 (G) and ×400 (H) show that hyperthermia exaggerates delayed neutrophil recruitment.

FIGURE 4

FRH enhances alveolar epithelial apoptosis. The lungs of mice exposed to LPS 50 µg and FRH (B) show an increase in TUNEL-positive cells as compared with LPS alone (A), original magnification ×100. Differential interference contrast microscopic images merged with the fluorescent TUNEL images (original magnification ×400) in euthermic (C) or hyperthermic (D) mice show that the fluorescence localizes to the alveolar walls. E, At 24 and 48 h, the number of TUNEL-positive cells per high-power field in hyperthermic animals exposed to LPS is significantly higher than in euthermic animals exposed to LPS. F, Many of the TUNEL-positive cells localize to the corners of alveoli, original magnification ×1000. ^†p< 0.0001; **p< 0.01.

FIGURE 5

Incubation temperature modifies TNF-α, but not Fas-mediated, death in alveolar epithelial cell lines. Both MLE-15 (A) and LA-4 cells (C) exposed to TNF-α show increased sensitivity to TNF-α mediated death as incubation temperatures increase. B, MLE-15 cells are insensitive to Fas ligand-mediated cell death, and incubation temperatures do not affect the sensitivity. D, LA-4 cells are sensitive to Fas-mediated cell death, and this response is not affected by incubation temperatures. *p< 0.05; **p< 0.01; ^†p< 0.001.

FIGURE 6

Febrile-range temperature enhances apoptosis in MLE-15 cells exposed to TNF-α. A, Temperature alone does not affect Annexin V-FITC binding in MLE-15 cells. B, MLE-15 cells exposed to TNF-α 100 ng/ml and 34, 37, or 39.5°C for 6 h demonstrate Annexin V-FITC binding increases as a function of temperature. C, Caspase-3/7 activity in MLE-15 cells exposed to TNF-α and 34, 37, or 39.5°C for 2 h shows that caspase-3/7 activity generated by cells exposed to TNF-α increases as a function of incubation temperature. ^†p< 0.05.

FIGURE 7

FRH enhances TNF-α–mediated cell death in a caspase-dependent manner. A, zVAD.fmk suppresses caspase-3/7 activity in MLE-15 cells exposed to TNF-α 5 ng/ml, 34, 37, or 39.5°C at low doses. B, zVAD.fmk improves MLE-15 survival posttreatment with TNF-α 5 ng/ml and 39.5°C. C, A strong dose response exists between zVAD.fmk and MLE-15 cell survival at 39.5°C. FA.fmk does not improve MLE-15 cell survival after TNF-α exposure at 39.5°C. **p< 0.01.

FIGURE 8

FRH augments TNF-α–mediated cell death via the death-receptor pathway. Caspase-8 (A) and caspase-9 (B) activity increase as a function of TNF-α dose and incubation temperature. C, The chemical caspase-8 inhibitor, zIETD, rescued MLE-15 cells from FRH-augmented death at TNF-α 5 ng/ml. Neither the chemical caspase-9 inhibitor, zLEHD (D), nor the inactive chemical analog of the inhibitors, FA.fmk (Fig. 7C), improved MLE-15 survival postexposure to TNF-α 5 ng/ml and FRH. *p< 0.05; **p< 0.01; ^†p< 0.001.

FIGURE 9

FRH-augmented cell death is not mediated by a soluble factor. A, The media of MLE-15 cells exposed to TNF-α and various incubation temperatures for 18 h was collected and transferred to MLE-15 cells and then incubated at 37°C for 18 h. No difference in the cell survival at each conditioning dose of TNF-α exists, although a trend for increased mortality from the cell-conditioned media of cells at 34°C exists. This reflects increased production (B) of and increased stability (C) of the TNF-α at 34°C compared with 39.5°C.

FIGURE 10

Temperature does not modify expression of TNFRs on MLE-15 cells. MLE-15 cells were exposed to 34, 37, or 39.5°C, 5% CO₂, and then evaluated for surface expression of TNFR1 and 2. No difference in surface expression of TNFR1 (A) or 2 (B) exists.

FIGURE 11

JNK, but not other MAPKs, contributes to FRH-augmented cell death by TNF-α. Chemical inhibitors of the MAPKs, MEK1/2 (U0126), JNK (SP600125), and p38 (SB202190) at 1 µM concentration were added to MLE-15 cells exposed to TNF-α 5 ng/ml and 34, 37, or 39.5°C. The MAPK inhibitors for MEK1/2 and p38 did not affect cell survival. The JNK inhibitor protected MLE-15 cells from exposure to TNF-α and 39.5°C. **p< 0.01; ^†p< 0.001.

FIGURE 12

FRH suppresses NF-κB activity, which increases MLE-15 susceptibility to TNF-α–mediated death. A, MLE-15 cells were transiently dually transfected with an NF-κB–inducible firefly luciferase and a constitutively active renilla luciferase and then exposed to TNF-α at 34, 37, or 39.5°C. NF-κB activity in MLE-15 cells generated by TNF-α exposure is strongly suppressed as a function of increasing incubation temperature. B, KC production from MLE-15 cells after TNF-α exposure is suppressed by increasing incubation temperatures. C, The IκB-α kinase inhibitor BMS 345541 renders the MLE-15 cells at all incubation temperatures more sensitive to TNF-α–mediated cell death. In the absence of TNF-α, the IκB-α kinase inhibitor does not affect cell viability (data not shown). D, Total IκB-α levels in MLE-15 cells exposed to TNF-α 50 ng/ml fluctuate similarly in all temperature exposures. A representative Western blot is shown with densitometry from three Western blots for total IκB-α normalized for protein loading. **p< 0.01; ^†p< 0.001.