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From the Divisions of Pulmonary and Critical Care Medicine* and Infectious Disease, the Department of Medicine, the Division of Neonatology, the Department of Pediatrics, the Department of Pathology, University of Maryland, Baltimore, Maryland; Medical and Research Services of the Baltimore Veterans Administration Medical Center,¶ Baltimore, Maryland; and the Division of Pulmonary and Critical Care Medicine,|| University of Michigan, Ann Arbor, Michigan
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Abstract |
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In the retrospective studies of human infections, the association of fever with survival was lost as the acuity of the patient population increased.4 Furthermore, in patients with Escherichia coli and Pseudomonas aeroginosa sepsis, administration of the antipyretic acetaminophen was associated with increased survival.5,6 Collectively, these studies suggest that fever might be harmful when it occurs in certain clinical settings. In our experimental murine Klebsiella peritonitis model,3 bacterial burden at time of death was much lower in the mice exposed to FRH than the euthermic mice, suggesting that mechanisms other than the infection contributed to death in the warmer mice. A similar effect of FRH on the relationship between survival and pathogen burden occurs in rabbits with experimental S. pneumoniae peritonitis in which FRH (41°C) shortens survival time despite reducing levels of bacteremia compared with infected euthermic (39°C) animals.7 Based on these studies, we reasoned that the ultimate effect of fever is determined by a dynamic balance between accelerated pathogen clearance and augmented collateral host tissue injury. Specifically, fever will be beneficial if it sufficiently shortens the course of an infection, thereby reducing the risk for tissue injury.
The evolutionary persistence of fever indicates that this strategy is usually effective. However, until twentieth-century man, increases in body temperature occurred almost exclusively during active infections or in situations associated with enhanced risk of infection, including traumatic injury, exertion, or flight or fight responses. In modern man, the acute phase response comprising fever and inflammation, is often activated by stimuli other than replicating pathogens, including antibiotic-treated infections and noninfectious causes.8 We hypothesized that in such situations, the increased rate of host tissue injury stimulated by fever would not be counterbalanced by accelerated pathogen clearance, thereby causing harm rather than conferring protection.
We chose pulmonary oxygen toxicity, a highly clinically relevant, noninfectious form of injury that is unique to modern medicine to test this hypothesis. Exposure to artificially high concentrations of oxygen is often essential for patient survival during severe illnesses.9
Unfortunately, such exposure is associated with severe, often lethal lung injury in man,10
other primates,11
and rodents.12-22
The mechanisms of acute hyperoxic lung injury have not been definitively elucidated, but it is widely believed that generation of reactive oxygen species (ROS) plays a key role.23
The contribution of neutrophils (PMN) to pulmonary oxygen toxicity is less clear. PMN recruitment to the hyperoxic lung occurs late in the course of lung injury.24,25
Furthermore, PMN depletion produces inconsistent effects on hyperoxic lung injury with as many studies showing protection19,26,27
as showing no effect24,28-30
or even worsening of pulmonary oxygen toxicity.24
The cytokines, tumor necrosis factor (TNF)-
, interleukin (IL)-1ß, IL-3, and IL-6, as well as the chemokines monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1, MIP-2, and 
-interferon-inducible protein (IP)-1018,20,31
each accumulate, and expression of both adhesion molecules, intercellular adhesion molecule (ICAM)-1 and E-selectin increase32
in the hyperoxic lung. Collectively, these studies suggest that despite its non-biological nature, hyperoxia appears to cause lung injury in part by activating the innate immune response.
We used a conscious, temperature-controlled mouse model to determine how co-exposure to FRH modifies pulmonary oxygen toxicity. We showed that maintaining core temperature between 39°C and 40°C (by increasing ambient temperature from 24°C to 34°C) rather than at normal basal levels (36.5°C to 37°C) in mice exposed to hyperoxia accelerates lethal pulmonary vascular endothelial injury, reduces the threshold of inspired oxygen level for lethality, induces expression of the PMN growth factor granulocyte-colony stimulating factor (G-CSF), and expands the circulating PMN pool. Furthermore, FRH was shown to augment pulmonary expression of the glutamic acid-leucine-arginine motif-positive (ELR+) CXC chemokine genes KC and LPS-induced CXC chemokine (LIX), enhance CXC receptor-2 (CXCR2)-dependent recruitment of PMN, and change the histological pattern of lung injury to a neutrophilic interstitial pneumonitis.
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Materials and Methods |
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Eight- to ten-week-old male outbred CD-1 mice, weighing 25 to 30 g were purchased from Harlan-Sprague Co. (Indianapolis, IN), housed in the Baltimore Veterans Administration Medical Center animal facility under the supervision of a full-time veterinarian, and used within 4 weeks of delivery. Mice were adapted to standard plastic cages for at least 4 days before study. To avoid the influence of diurnal cycling, all experiments were started at approximately the same time each day (between 8:00 a.m. and 10:00 a.m.). One week before each experiment, sentinel mice were implanted with telemetric intraperitoneal temperature sensors (Mini Mitter 1.05g in vivo temperature sensor model 100-0035; Bend, OR).
Experimental mice were housed in modified plastic cages to allow continuous inflow of air/oxygen mixtures and outflow through a 1 cm2 outflow port. The oxygen level in each cage was measured twice daily using an oxygen analyzer (Vascular Technology; Chelmsford, MA). Groups of three to four mice, including one thermister-implanted sentinel mouse per group, were placed in each cage. The cages were placed in modified infant isolettes with temperature set to 24°C to maintain euthermia or 34°C to 34.5°C to maintain FRH and core temperatures of the sentinel mice were continuously monitored using the Mini Mitter Automated Data Acquisition System. Except for the ambient temperature, handling of the euthermic and hyperthermic mice was identical. Exposures to hyperthermia and hyperoxia were initiated simultaneously. All procedures were approved by the University of Maryland Baltimore Animal Care and Use Committee.
Lung Lavage and Blood Processing
At selected times, groups of mice were anesthetized by 10 to 30 second exposure to isoflurane, euthanized by cervical dislocation, and lung lavage was performed in situ through an 18g blunt-end needle secured in the trachea, using 1 ml phosphate-buffered saline (PBS) instilled and withdrawn twice, followed by instillation and recovery of a second 1 ml of PBS. The two aliquots of lung lavage were pooled, cells were collected by centrifugation at 1000 x g for 3 minutes, and cell-free supernatants were stored at -80°C for analysis of total protein and cytokine concentrations. Total cell counts were performed manually using a hemacytometer and differential cell counts of Diff-Quick-stained cytopreparations were performed by two blinded observers (PR and RMV) using morphological criteria. Heparinized blood was collected via cardiac puncture. Total blood PMN count was determined by manually counting total leukocytes after lysis of erythrocytes with Tris-buffered ammonium chloride and manually analyzing the leukocyte composition in Diff Quik-stained blood smears. Plasma was stored at -80°C for analysis of cytokine concentrations.
CXCR2 Blockade and G-CSF Neutralization
In experiments to analyze the requirement for CXCR2 for PMN recruitment, mice received a single i.p. injection of 0.5 ml goat anti-mouse CXCR2 antiserum, as previously described,33 2 hours before exposure to hyperoxia and FRH and were either sacrificed 40 hours later for analysis of bronchoalveolar lavage fluid (BALF) and lung tissue or were monitored for survival. Control animals received an equal volume of pre-immune goat serum. To determine whether G-CSF was required for FRH-induced neutrophilia, mice were treated with 0.5 ml rabbit anti-G-CSF (T. Hartung; University of Konstanz; Konstanz, Germany) diluted 1:5 in PBS and administered i.p. 2 hours prior to 24-hour exposure to FRH or euthermia. Control mice received an equal volume of diluted rabbit serum. Mice were euthanized, blood was collected by cardiac puncture, and the PMN were manually counted.
Measurement of Cytokine Concentration
Cytokines were measured in the University of Maryland Baltimore Cytokine Core Laboratory using standard two-antibody ELISAs with commercial antibody pairs and recombinant standards from R&D; Minneapolis, MN as previously described.3 The IL-6, KC, MIP-2, LIX, G-CSF, and GM-CSF assays had lower detection limits of 3, 15, 62, 31, 15, and 12.5 pg/ml, respectively.
Measurement of Wet:Dry Weight
Lungs were excised, washed of surface blood, blotted dry, and the mediastinum and connective tissue removed. After weighing both lungs, the tissue was dried at 100°C until the final stable dry weight was measured.
Histological Analysis
After euthanasia, the anterior chest wall was removed, the trachea cannulated with an 18g blunt needle, the lungs inflated in situ with 10% buffered formalin at 20 cm H2O pressure, and the lungs and mediastinum removed en bloc and fixed in 10% buffered formaldehyde. Gross sections of the trachea, esophagus, and lung parenchyma at the level of mid-bronchial bifurcation were paraffin-embedded and 5-µm sections were processed routinely for hematoxylin and eosin staining. A veterinary pathologist (S.R.) evaluated the sections in a blinded fashion for morphological signs of injury and inflammation. For immunohistologic analysis, lungs were harvested for frozen sectioning exactly as described except lung inflation was accomplished by inflating with a fixed 0.7 ml volume of 50% OCT (v/v in PBS). The inflated lungs were embedded in neat OCT and 8-µm cryosections were acetone-fixed for 3 minutes at -20°C and stored at -80°C for immunostaining. Slides were washed in PBS at 25°C for 20 minutes. Endogenous peroxidase was inactivated by incubating for 10 minutes with 30% hydrogen peroxide in methanol at 25°C. Non-specific signal was reduced by sequentially blocking with 5% serum (v/v) in PBS for 30 minutes, and then with a commercial avidin/biotin blocking kit (Vector; Burlingame, CA) according to the manufacturer’s instructions. The blocked slides were sequentially incubated with primary antibody for 1 hour, a secondary antibody (where indicated) for 30 minutes, a commercial avidin/biotin peroxidase detection system (Vector), and 1 mg/ml DAB (Sigma) in 0.02% hydrogen peroxide in PBS. All blocking and subsequent incubations were at 25°C. Blocking serum, primary, and secondary antibodies used for each cell type were as follows. For macrophages, mouse serum, 1 µg/ml rat anti-mouse Mac-3, and 2.5 µg/ml biotinylated goat anti-rat IgG1/2a (both from BD-Pharmingen; San Diego, CA) were used. For PMN, bovine serum and the 2.5 µg/ml biotinylated rat anti-mouse Gr-1 (BD-Pharmingen) were used. For T lymphocytes, goat serum, 2.5 µg/ml rat anti-mouse CD3, and 2.5 µg/ml biotinylated goat anti-rat IgG2b (both from BD-Pharmingen) were used. For NK cells, goat serum, 1:100 (v/v) dilution of rabbit anti-rat/mouse Aialo-GM, and 1:800 (v/v) dilution of peroxidase-conjugated goat anti-rabbit IgG (both from Accurate Chemical; Westbury, NY) were used. Immunostained slides were counterstained with hematoxylin (BD Sciences) and were scored by two blinded observers (P.R. and J.D.H.). The number of positively stained cells per high power field was manually counted in comparable regions of each section located equidistant from the pleural surface and lung hilum so as to avoid bronchovascular structures. For each mouse, the mean of four fields per section and three sections was calculated.
In Vitro Cell Culture Systems
RAW 264.7 murine macrophages and NIH 3T3 murine lung fibroblasts were obtained from the American Type Culture Collection (Manassas, VA). SVEC-4–10, an SV40-transformed murine endothelial cell line was provided by Dr. A. Passaniti (University of Maryland School of Medicine, Baltimore, MD). RAW 264.7 cells were cultured in RPMI 1640 supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 10 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.3 and containing 10% fetal bovine serum (FBS; Life Technologies; Gaithersburg, MD) at 37°C in 5% CO2-enriched air. NIH 3T3 cells and SVEC4–10 cells were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 50 units/ml penicillin, 50 µg/ml streptomycin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES buffer, pH 7.3 and containing 10% FBS at 37°C in 5% CO2-enriched air. MLE12 cells, a cell line derived from murine distal respiratory epithelial cells were obtained from Jeffrey Whitsett (University of Cincinatti School of Medicine,Cincinnati, OH) and were cultured in 5 µg/ml insulin, 5 µg/ml transferrin, 10 pM hydrocortisone, 10 pM ß-estradiol 10 mmol/L HEPES, pH 7.3, 2 mmol/L L-glutamine, 100 units/ml penicilln, 100 µg/ml streptomycin, and 2% FBS. To determine the capacity of different cell types to increase cytokine generation in response to hyperoxia and hyperthermia, the cells were seeded 2.5 x 105 per ml in 24-well culture plates and cultured in either 95% air/5% CO2 or 95% oxygen/5% CO2 and at either 37°C or 39.5°C for 24 hours and culture supernatants were analyzed for concentration of KC, LIX, and G-CSF by ELISA.
Statistical Analysis
All data are presented as mean ± SE. Differences between two groups were tested using an unpaired Student’s t-test. Differences among more than two groups were tested by a Fisher protected least-squares difference (PLSD) test applied to a one-way analysis of variance. Survival was analyzed using a log-rank test.
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Results |
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Mice housed at usual laboratory temperatures of 24°C and exposed to FIO2 > 0.95 maintained a core temperature within the usual basal range (36.5°C to 37°C). As we previously reported,3
because mice have a limited capacity to eliminate heat, increasing the murine environment from 24°C to only 34°C is sufficient to cause a rapid increase in core temperature that was maintained within the febrile range (39°C to 40°C) (Figure 1A)
. For the remainder of this report, we will refer to mice housed at 24°C as euthermic and those housed at 34°C as hyperthermic. Air-breathing control mice tolerated FRH for 2 weeks without detectable distress. While exposure to FIO2 > 0.95 was uniformly lethal in both groups (Figure 1B)
, survival time in hyperthermic mice was reduced by 50% relative to euthermic, hyperoxia-exposed mice (median survival 2.2 versus 4.5 days; P < 0.001). To determine whether FRH lowered the threshold for oxygen-induced lethality, euthermic and hyperthermic mice were exposed to FIO2 between 0.50 and 0.95 for up to 2 weeks. The LD50 for oxygen was reduced from 0.8 to 0.9 in euthermic mice to 0.6 to 0.7 in hyperthermic mice (Figure 2)
. At necropsy, the lungs in both groups of mice exposed to lethal levels of hyperoxia were grossly edematous and hyperemic (data not shown).
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Reduced survival time in the hyperthermic mice exposed to FIO2 > 0.95 was associated with an earlier increase in both BALF protein concentration (Figure 3A)
and lung wet:dry weight ratios (Figure 3B)
compared with euthermic mice, indicating that pulmonary vascular endothelial injury was accelerated in the warmer animals. The FRH mice also exhibited a more rapid rise in BALF concentration of IL-6 (Figure 3C)
, a ubiquitously expressed marker of inflammation and tissue injury. In air-breathing mice exposed to FRH for 36 or 60 hours, neither BALF IL-6 nor total protein concentrations were increased compared with baseline levels. Mice exposed to hyperoxia and FRH did not survive beyond 60 hours.
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. After 108 hours of exposure to hyperoxia, the lungs from euthermic mice showed multifocal hyaline membrane accumulation, alveolar hemorrhage, and perivascular edema (Figure 4B)
. The pulmonary vasculature was diffusely expanded by accumulation of PMN and lesser numbers of mononuclear cells. The bronchial mucosa demonstrated multiple foci of denuded epithelium and accumulation of PMN, mononuclear cells, and cell debris within the bronchial lumen. In striking contrast to the findings in euthermic mice, hyperthermic mice exposed to hyperoxia for only 36 hours (Figure 4C)
demonstrated marked PMN-rich interstitial pneumonitis. The interstitium was two to three times normal thickness and contained primarily PMN and large mononuclear cells. There were multiple foci of bronchial epithelial hyperplasia with intraluminal accumulation of PAS-positive material, areas of type II pneumocyte hyperplasia, and other areas of bullous emphysema.
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. The number of GR-1-positive PMN in lung tissue increased 4- to 5-fold over the first 24 to 36 hours of hyperoxic exposure in the hyperthermic mice compared with no increase in euthermic mice exposed to hyperoxia for 108 hours (Figure 5, A, C, and D)
. The number of Mac-3-positive macrophages remained unchanged during hyperoxic exposure in both euthermic and hyperthermic mice (Figure 5B)
. The large mononuclear cells within the alveolar septal walls in the hyperthermic mice did not immunostain for Mac-3 and based on their location, were most likely type II pneumocytes. Few normal type I pneumocytes were seen in the hyperthermic lung. NK cells and T cells were undetectable in the lungs of all of the animals studied, but were detectable in associated mediastinal lymph nodes (data not shown).
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. BALF macrophage content increased 2.5-fold after 36 to 60 hours of hyperoxic exposure in the hyperthermic mice (Figure 6B)
, but never exceeded baseline levels in the euthermic mice.
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To determine whether FRH augmented PMN recruitment to the lung, in part through expansion of the circulating PMN pool, the effects of FRH on PMN levels in circulating blood were analyzed. Twenty-four hour exposure to FRH in air-breathing mice increased the mean circulating leukocyte count 1.8-fold (Figure 7A)
and the mean PMN count 1.9-fold (Figure 7B)
. In mice exposed to hyperoxia for 24 hours, co-exposure to FRH increased mean circulating PMN count 3.9-fold compared with euthermic hyperoxic mice. In the same mice, 24 hours of exposure to hyperoxia in the absence or presence of FRH or to FRH alone stimulated 7.1-, 8.1-, and 7.1-fold increases, respectively, in mean plasma G-CSF concentration compared with euthermic, air-breathing mice (Figure 7C)
. Plasma GM-CSF was not detectable in any of the four groups of mice. To determine whether G-CSF was required for the FRH-induced neutrophilia, the effect of neutralizing G-CSF was measured in normoxic mice exposed to FRH for 24 hours (Figure 7D)
. Mice were treated with rabbit anti-mouse G-CSF antibody or sham-treated with pre-immune rabbit antiserum 2 hours before initiation of exposure to FRH. Compared with sham-treated controls, treatment with anti-G-CSF antibody reduced the circulating PMN count by 70% in FRH-exposed mice to levels below those in sham-treated euthermic mice. Treatment with anti-G-CSF also tended to reduce the number of circulating PMNs in euthermic mice, likely reflecting the short half-life of circulating PMN.
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To begin to determine how FRH enhances accumulation of PMN in the hyperoxic lung, the concentration of the three known ELR+ CXC chemokines, KC, LIX, and MIP-2, as well as two PMN survival factors, G-CSF and GM-CSF in BALF were measured (Figure 8)
. The concentration of KC peaked after a 24-hour co-exposure to FRH and hyperoxia (FIO2 > 0.95) at levels 10-fold higher than in air-breathing euthermic mice (Figure 8A)
and coincided with the appearance of PMN in lung (see Figure 6
). In comparison, BALF KC levels did not increase in the euthermic mice until 60 hours of exposure to hyperoxia, and increased only 2.2-fold relative to levels in normoxic, euthermic mice. BALF in mice exposed for 24 hours to hyperoxia in the presence or absence of FRH or to FRH alone was further analyzed for additional cytokine composition (Figure 8, B and C)
. Like KC, the BALF concentration of LIX increased 4.2-fold in mice co-exposed to hyperoxia and FRH compared with air-breathing euthermic mice; whereas exposure to either stimulus alone failed to stimulate an increase in LIX concentration in BALF (Figure 8B)
. The mean BALF levels of MIP-2 were modestly increased by approximately 1.7- to 2-fold in mice exposed for 24 hours to either stimulus alone, but, in contrast with the BALF levels of KC and LIX, exposure to FRH and hyperoxia failed to exert additive or synergistic effects on BALF levels of MIP-2 (Figure 8C)
. Neither G-CSF nor GM-CSF was detectable in the BALF from any of the four groups of mice.
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and in lung tissue (Figure 9, B and C)
. Goat anti-mouse CXCR2 antiserum33
or preimmune goat serum was administered 2 hours before a 40-hour co-exposure to FRH and FIO2 > 0.95. Based on the kinetics of pulmonary vascular endothelial injury (Figure 3)
and accumulation of PMN in BALF (Figure 6)
, we chose a 40-hour co-exposure to ensure detectable lung injury in the sham-treated mice. Seven of 14 control animals died during the 40-hour exposure to FRH and hyperoxia compared with only one death in 14 mice pre-infused with anti-CXCR2 antiserum (P = 0.02; Fisher exact test). Necropsy revealed the lungs to be edematous and hyperemic in all mice exposed to lethal hyperoxia. In those mice surviving a 40-hour exposure to hyperoxia and hyperthermia, mean PMN content in BALF (Figure 9A)
and in immunostained lung tissue (Figure 9, B and C)
was reduced by 98% and 83%, respectively, in the anti-CXCR2 antibody-treated mice compared with the sham-treated mice and were similar to levels in euthermic air-breathing controls. The BALF protein concentration in the CXCR2-treated mice was reduced by 72% (196 ± 65 versus 691 ± 165 µg/ml; P < 0.05) compared with the sham-treated controls, but the number of macrophages in BALF or lung interstitium was not different between the anti-CXCR2-treated and control mice (data not shown). Within groups of nine mice exposed to hyperoxia and FRH, median survival time increased to 60 hours in the CXCR2 antibody-treated mice compared to 36 hours in the sham-treated mice (Figure 9D)
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. Hyperoxia alone and in combination with hyperthermia approximately doubled generation of G-CSF compared with 37°C cell culture, but hyperthermia failed to increase G-CSF expression in normoxic SVEC-4–10 cell culture. In NIH 3T3 fibroblast cultures, hyperthermia alone increased KC generation 10% and co-exposure to hyperoxia and hyperthermia increased KC generation by 27%, but hyperoxia alone did not alter KC generation compared with normoxic, euthermic cells. In the same cells, hyperoxia increased G-CSF generation by 35% and co-exposure to hyperoxia and hyperthermia augmented G-CSF generation by 65%, but exposure to hyperthermia alone did not alter G-CSF generation compared with normoxic, euthermic cells. LIX generation by these cells was not modified by exposure to hyperoxia or hyperthermia, alone or in combination.
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Discussion |
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Using this model, we showed that FRH accelerated hyperoxic lung injury, shortened survival time, and reduced the threshold fractional index of oxygen required for lethality. We used three measures of lung injury. In mice exposed to lethal levels of hyperoxia, both the lung wet:dry weight ratio and BALF protein concentration increased earlier in the hyperthermic mice than in the euthermic mice, indicating an accelerated loss of pulmonary vascular endothelial barrier integrity and pulmonary edema formation in the warmer animals. This was accompanied by an earlier increase in BALF levels of IL-6, a pleiotropic cytokine that is expressed at sites of inflammation and tissue injury and is a predictor of mortality in sepsis.40
Histological evidence of lung injury during exposure to hyperoxia was detectable 2 to 3 days earlier in hyperthermic than in the euthermic mice and was temporally associated with the increase in BALF IL-6 concentration and death in both groups of mice. Once injury commenced, the histological patterns of injury were profoundly different in the euthermic and hyperthermic mice, suggesting that co-exposure to FRH might augment pulmonary oxygen toxicity by activating distinct cytotoxic pathways that are not activated by hyperoxia alone. Specifically, co-exposure to FRH altered the kinetics of PMN recruitment, the distribution of PMN within the hyperoxic lung, and the apparent contribution of PMN to lung injury. The histological changes in the euthermic mice after 108 hours of exposure to hyperoxia were similar to those reported by others19,41 and characterized by diffuse hyaline membranes, perivascular edema, intra-alveolar hemorrhage, and attenuation of bronchial epithelium. In striking contrast, after only 36 hours of exposure to hyperoxia, the hyperthermic mice developed a marked PMN-rich interstitial pneumonitis. Total lung PMN content as determined by counting GR-1-immunostaining cells peaked more rapidly (36 hours versus 108 hours) and at 2.5-fold higher levels in FRH than euthermic mice. In studies of fever in a lizard model of gram-negative bacterial infection, the accelerated pathogen clearance and improved host survival found in the febrile animals was associated with augmented accumulation of granulocytes at the subcutaneous inoculation site.42 Collectively, these results suggest that enhanced PMN recruitment might be a common and evolutionarily conserved pathway through which fever augments host defenses. In contrast, FRH and hyperoxia exerted a more modest effect on pulmonary macrophages. Although co-exposure to FRH and hyperoxia was associated with a moderate increase in macrophages content in BALF, the total number of macrophages detected by analyzing anti-Mac-3-stained lung tissue was not altered in these mice. While the majority of intrapulmonary macrophages are located within the bronchoalveolar compartment, the efficiency of macrophage recovery in BALF is variable.43 It is possible that co-exposure to FRH and hyperoxia might stimulate a redistribution of interstitial macrophages to the bronchoalveolar compartment or reduce macrophage adherence to the bronchoalveolar epithelial surface, thereby increasing the efficiency of cell recovery in BALF.
The ELR+ CXC chemokines comprise one of five distinct classes of PMN chemoattractants that activate PMN via distinct G-protein-coupled, 7-transmembrane domain receptors. Mice lack an analogue of the major human ELR+ CXC chemokine, IL-8, but they express three other members of this chemokine family, notably KC/Gro, MIP-2/Groß, and LIX. Mean BALF concentrations of two of the three murine ELR+ CXC chemokines, KC and LIX, increased 10- and 4.2-fold, respectively, during co-exposure to hyperoxia and FRH and BALF levels of MIP-2 were modestly increased by FRH alone, suggesting that FRH might enhance PMN recruitment to the hyperoxic lung, in part, through augmented expression of all three ELR+ CXC chemokines. In mice, the three ELR+ CXC chemokines act through a single receptor, CXCR2.44
Immunoblockade of this receptor completely blocked the increase in PMN accumulation in lung interstitium and in BALF, in mice exposed to hyperoxia and FRH. Collectively, these results indicate that the ELR+ CXC chemokines are required for the early recruitment of PMN to the hyperoxic, hyperthermic lung, and suggest that the increased expression of KC and LIX might directly contribute to the augmented PMN recruitment.
While co-exposure to FRH and hyperoxia stimulated KC and LIX expression within the bronchoalveolar compartment, exposure to FRH alone failed to increase the mean BALF level of either chemokine. KC secretion in 3T3 fibroblast cultures during in vitro exposure to hyperoxia and hyperthermia showed a similar pattern in which co-exposure to 95% oxygen and 39.5°C incubation temperature stimulated a supra-additive increase in KC expression. In contrast, constitutive LIX secretion in 3T3 cell cultures was not augmented by hyperoxia or hyperthermia alone or in combination, demonstrating that the cytokine modifying effects of hyperthermia are gene-specific, even in the same cell. Similarly, constitutive LIX secretion in SVEC-10-4 EC cultures was also unaltered by exposure to hyperoxia or hyperthermia, but LIX generation was blocked by co-exposure to hyperoxia and hyperthermia and these cells failed to secrete detectable KC during in vitro exposure to hyperoxia. We previously reported that incubating human pulmonary artery endothelial cells at 39.5°C augmented TNF--induced expression of IL-8,45
another member of the ELR+ CXC chemokine family; however, exposing the same cells to FRH in the absence of another stimulus (exogenous TNF-) failed to stimulate IL-8 expression. These results identify limitations in extrapolating behavior of in vitro cell cultures to the in vivo environment. For example, hyperoxia and hyperthermia may indirectly activate chemokine gene expression in vivo by activating proximal autocrine/paracrine mediators. Furthermore, cell lines may not faithfully recapitulate in vivo gene regulation. Identification of the cellular source of the augmented cytokine generation in the hyperoxic, hyperthermic mouse and the intermediate mediators required for cytokine expression is presently under investigation in our laboratory. However, the differences in responsiveness of KC, MIP-2, and LIX to hyperoxia and FRH in the present study, as well as the previously reported differences in induction kinetics, tissue distribution, and responsiveness to corticosteroids among these three chemokines,46
suggest that KC, MIP-2, and LIX serve distinct functions and might differentially contribute to PMN recruitment in the presence and absence of fever.
We showed that FRH expands the circulating PMN pool in air-breathing and hyperoxic mice. Such an increase might result from increased release of PMN from the bone marrow and/or through PMN demargination into the circulating pool. Since the lung is a major site of PMN margination,47
the coincidence of increased circulating and intrapulmonary PMN in the hyperoxic hyperthermic mice suggests that the marrow rather than pulmonary demargination is the major source of excess circulating PMN in these mice. We found that exposing mice to FRH for 24 hours increases circulating G-CSF levels 7.1-fold. Uchida et al48
reported that administration of exogenous G-CSF to mice increases proliferation of PMN precursors and reduces marrow transit time of PMN, leading to elevated circulating PMN levels detectable within 1 to 2 days. In fact, we showed that neutralization of G-CSF with anti-G-CSF antibodies abrogated FRH-induced neutrophilia. These combined data suggest that G-CSF-induced expansion of granulopoiesis is a major source of the additional circulating PMN present after 24 hours of exposure to FRH. However, we have not excluded contributions to the expanded circulating pool from other sources. Brenner et al49
report that exercise-induced hyperthermia stimulates catecholamine and corticosteroid release and PMN demargination, suggesting that FRH might induce both a rapid G-CSF-independent and a prolonged G-CSF-dependent expansion of the circulating PMN pool. G-CSF also has been reported to increase PMN degranulation, secretory vesicle mobilization, and surface CD11b expression50
and augment superoxide generation51
and chemotaxis.52
The capacity of FRH to simultaneously expand the circulating PMN pool and to augment generation of chemotactic gradients at sites of inflammation might serve to rapidly mobilize PMN to sites of infection or injury. Interestingly, Rosenspire et al53
recently reported that release of reactive oxygen intermediates and nitric oxide are greatly augmented in human PMN on exposure to FRH in vitro. Thus, FRH might provide both delivery and activation of PMN to sites of inflammation. While this process might be essential to contain infection, it may lead to tissue injury and dysfunction, multiorgan failure, and even death, such as occurs in hyperoxic lung injury. In fact, inhibition of PMN recruitment through prior infusion of hyperthermic, hyperoxic mice with anti-CXCR2 antibody improved survival time. However, median survival time in these animals remained shorter compared with euthermic, hyperoxic mice, suggesting that hyperthermia itself might exert additional injurious effects within the hyperoxic lung. We have previously demonstrated that in vitro exposure to hyperthermia (39.5°C) synergizes with TNF- treatment to disrupt barrier function in cultured human pulmonary EC in the absence of PMN,45
suggesting that hyperoxia and hyperthermia might also cause lung injury by directly disrupting endothelial barrier integrity.
Although the temperature used in this study is below the generally accepted threshold for the heat shock response,39
more recent studies have demonstrated that temperatures in the febrile range can stimulate expression of heat shock proteins in vivo.54,55
Furthermore, inflammatory mediators, including arachidonic acid56
and interferon-,57
lower the thermal threshold for induction of the heat shock response. Thus, it is possible that heat shock proteins were generated in the hyperthermic, hyperoxic mouse lung. However, heat shock proteins usually exert tissue protective58
and anti-inflammatory effects,59
suggesting that if the heat shock response did contribute to the augmented pulmonary oxygen toxicity in the hyperthermic mice, it did so through an atypical mechanism.
In summary, we have shown that exposure to FRH accelerates and enhances pulmonary oxygen toxicity and have provided evidence that this is caused by enhanced recruitment and retention of PMN within the hyperoxic lung. We have also shown that FRH might enhance PMN accumulation by inducing expression of G-CSF, expanding the circulating PMN pool, and augmenting generation of ELR+ CXC chemokines within the hyperoxic lung. The capacity of FRH to augment hyperoxic lung injury in light of its protective effects during life-threatening infections underscores the importance of understanding the context in which biological processes function. The core temperature increase that occurs during febrile illnesses is a powerful immune modulator, the net effects of which depend on the clinical setting. The results of the present study underscore the importance of directly analyzing the effects of core temperature manipulation in critically ill patients.
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Acknowledgements |
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Footnotes |
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Supported by Public Health Service grant no. AI42117 (to J.D.H.), a Veterans Administration Merit Review Award (to J.D.H.), and a Passano Foundation Physician-Scientist Award (to J.D.H.).
Accepted for publication March 13, 2003.
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. J Appl Physiol 2001, 90:90-98This article has been cited by other articles:
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