Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Gloria S. Pryhuber1,2, David P. O'Brien1,3, Raymond Baggs2, Richard Phipps2,4, Heidie Huyck1, Inaki Sanz3, and Moon H. Nahm1,3,4,5

Departments of 1 Pediatrics, 2 Environmental Medicine, 3 Medicine, and 5 Pathology and 4 The Cancer Center, Strong Children's Research Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta , IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta , MIP-2, interferon-gamma -induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha . However, IL-1beta , IL-6, macrophage migration inhibitory factor, MIP-1alpha , MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.

oxygen toxicity; C57BL/6 mice; tumor necrosis factor receptor I knockout; tumor necrosis factor receptor II; lung inflammation; manganese superoxide dismutase; intercellular adhesion molecule-1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LUNGS OF ADULT ANIMALS, including humans, develop acute lung injury when exposed to greater than ambient oxygen concentrations. Because many patients require oxygen therapy to survive illness or injury, pulmonary oxygen toxicity and its prevention are significant clinical issues. Exogenous delivery or stimulation of endogenous antioxidants correlate with decreased oxygen toxicity, supporting a role of reactive oxygen species (ROS) in hyperoxia-induced lung injury (10, 28). High-dose oxygen and ROS also induce an inflammatory response observed as an influx and activation of polymorphonuclear cells (PMN) and macrophages and increased expression of cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta .

TNF-alpha is a cytokine produced primarily by activated macrophages in response to numerous stimuli including hyperoxia (11). TNF-alpha induces acute inflammation by enhancing endothelial permeability, adherence, and transmigration of inflammatory cells into lung interstitium, as well as by increasing production of superoxide anion in PMN and of additional cytokines and chemokines by several cell types (for review, see Ref. 4). TNF-alpha also causes endothelial injury, platelet aggregation, and sequestration, resulting in thrombosis and edema (2, 18). In addition, TNF-alpha is recognized as a classic regulator of cell survival or death by apoptosis or necrosis. In this context, it is not surprising that anti-TNF-alpha antibodies reduced hyperoxia-induced lung injury, supporting a role for TNF-alpha in the pathogenesis of pulmonary toxicity. In contrast, however, low-dose administration of TNF-alpha to rats and mice also reduced oxygen-induced interstitial pneumonitis, edema, and lethality induced by subsequent exposure to 100% oxygen for up to 3 days (36). The protective effect of TNF-alpha is thought to be related to induction of manganese superoxide dismutase (Mn SOD), an enzyme that scavenges the extremely reactive superoxide radicals in the mitochondria.

The role of TNF-alpha in hyperoxic lung injury remains particularly unclear due to a seemingly paradoxical effect of TNF-alpha on susceptibility to oxygen toxicity (13). Treatment with either TNF-alpha or antibodies to TNF-alpha resulted in increased survival in mice exposed to high-dose oxygen (13, 32, 36). The current study utilized mouse models in which the receptors through which TNF-alpha functions, TNFR-I (p55) and TNFR-II (p75), were eliminated by targeted gene ablation to test their role in pulmonary oxygen toxicity. TNFR-I is the most abundant TNF receptor in the lung and has been most tightly linked with TNF-alpha -induced cytolysis, apoptosis, and induction of cellular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) (19, 20). The function of TNFR-II is less well defined and may depend on interactions with TNFR-I (8, 31). The goals of the current study were to determine the importance of signaling through the predominant TNFR-I p55 receptor in hyperoxia-induced acute lung injury and to define differences in the inflammatory and epithelial cell response to hyperoxia in the presence and the absence of the p55 receptor.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Control C57BL/6 and TNFR-II(-/-) (9) strains were purchased as breeding pairs from Jackson Laboratories (Bar Harbor, ME). The TNFR-I(-/-) strain was generated by homologous recombination in C57BL/6-derived embryonic stem cells as previously described (22). Age- and sex-matched 8- to 10-wk-old mice utilized in the current protocol were bred and maintained in microisolator cages in specific pathogen-free rooms in the animal care facility at the University of Rochester Medical Center (Rochester, NY). Sentinel animals maintained in the same rooms, on bedding mixed with bedding taken from other cages within the room, routinely tested negative for common murine pathogens including murine hepatitis, pinworm, and Sendai virus. All animal care and experimental protocols were approved by the University of Rochester Committee on Animal Research and follow the guidelines of International Animal Care and Use Committee.

Intratracheal TNF-alpha exposure. Instillation of a 50-µl volume of control, sterile, nonpyrogenic saline or recombinant murine TNF-alpha diluted in the same saline (rmTNF-alpha ; 5 µg/mouse; R&D Systems, Minneapolis, MN) was accomplished by blunt needle endotracheal intubation. The mice were anesthetized briefly with isoflurane before being gently suspended by the incisors. The mouth was opened, and the tongue was displaced by forceps. Intubation was detected by the feel of the cartilaginous rings of the trachea and was documented to be reliable by vital dye instillation in >90% of instillations. Greater than 95% of the mice recovered without incident. Total lung RNA was isolated 18 h after delivery of TNF-alpha .

Oxygen exposure. The mice were exposed to >95% oxygen (hereafter referred to as 100% oxygen) by delivery of 100% oxygen at 4 l/min to standard plastic housing cages (10 in. × 8 in. × 12 in.) with metal wire covers as previously described (1). Free access to food and water was provided. Three cages (3 or 4 mice/cage) were placed inside a Plexiglas chamber. The oxygen gas was humidified and filtered through a 0.22-µm filter before passage into the chamber. The percentage of inspired oxygen within the animal cage was >95% oxygen at all times as verified by an oxygen analyzer calibrated to room air and to a pure source of 100% oxygen. Pressure within the cages remains atmospheric. The chambers themselves were further isolated from the external environment by placement within existing "Rochester chambers" located in the Department of Environmental Medicine at the University of Rochester. The gas exposure system is a single-pass system with exhaust air vented to the outside through the chamber vents. Control mice exposed to room air were maintained in plastic cages in a Rochester chamber adjacent to the oxygen exposure chamber. After the indicated exposures, the mice were euthanized by intraperitoneal injection of pentobarbital sodium (150 mg/kg; Abbott Laboratories, Chicago, IL) and transection of the abdominal aorta. The lungs were allowed to deflate by opening the diaphragm, and the trachea was cannulated by 20-gauge catheter. The sternum was removed, and the left bronchus was isolated by a hemostat. The left lung was removed, immediately frozen in liquid nitrogen, and stored at -80°C before isolation of total RNA. After removal of the left lung, the right lung was inflation fixed via the trachea with 2% glutaraldehyde in 0.1 M cacodylic acid (pH 7.4; Sigma, St. Louis, MO) at 10 cmH2O pressure for 10 min. After ligation of the trachea, the lung was removed en bloc, immersion fixed for 16-20 h, washed, then stored in 0.1 M cacodylic acid at 4°C until it was dehydrated and embedded in paraffin. Sections (4 µm) were analyzed by routine hematoxylin and eosin stain and light microscopy for evidence of inflammation and oxygen toxicity. The tissue sections, identified by sequential code number, were examined without knowledge of the experimental exposure. An oxygen toxicity score of 0 (no histopathology) to 4+ (maximum severity seen in previous hyperoxia exposed murine lung) was assigned to each tissue based on the degree of alveolitis, bronchiolitis, bronchitis, fibrosis, and extent of diffuse involvement. All histological comparisons of WT to TNFR-I(-/-) lungs presented were performed on tissues harvested, processed, and stained concurrently.

Analysis of receptor, cytokine, chemokine, ICAM-1, and Mn SOD mRNA. Total lung RNA was isolated from left lung lobes that were homogenized (Tekmar tissuemizer) in 1 ml of 4 M guanidine isothiocyanate (Kodak Chemical, Rochester, NY), 0.5% N-lauroyl sarcosine (Sigma), and 25 mM sodium citrate and stored at -80°C. Total RNA was recovered by an acid phenol extraction method utilizing Phase Lock Gel II (5 Prime right-arrow 3 Prime, Boulder, CO). The RNA was quantified by absorbance at 260 nm in 10 mM Tris and 0.1 mM EDTA, pH 8.0. Cytokine and chemokine mRNAs were detected in total lung RNA by RNase protection assay (RPA) utilizing commercially available DNA templates and protocol (Riboquant, mCK5, mCK3b, mCK2, mCR4; PharMingen, San Diego, CA). Radiolabeled, single-strand cRNA for the indicated genes was synthesized at room temperature utilizing [alpha -32P]UTP (3,000 Ci/mmol; EasyTides, New England Nuclear) and T7 polymerase. RNA samples (5-10 µg), including murine control RNA and yeast tRNA (2 µg) as positive and negative controls, were dried, then resuspended in 8 µl of hybridization buffer and 2 µl of radiolabeled probe (~3 × 105 cpm/µl). The hybridization mixtures were overlaid with mineral oil, denatured at 90°C, and incubated for 16-18 h in a prewarmed 56°C oven. After incubation, single-strand RNA was digested in an RNase A/T1 cocktail, followed by proteinase K digestion. The remaining radiolabeled RNA fragments, protected from degradation by hybridization to homologous cellular mRNA, were electrophoresed on a 6% acrylamide-urea gel (GIBCO BRL) with a radiolabeled probe set (1-2 × 103 cpm) as size markers. The gels were dried and analyzed by phosphorimaging. The data were normalized to ribosome-associated protein L32 (rpL32) mRNA content.

Mn SOD and ICAM-1 mRNA levels were quantified after oxygen exposure by Northern analysis and normalized to rpL32 mRNA content. For Northern analysis, equal amounts of purified RNA samples were fractionated on a 1.2% agarose-7% formaldehyde gel and transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH). After ultraviolet cross-linking, the Nytran filters were stained with 1% methylene blue in 0.3 M sodium acetate to assess the integrity of the RNA and to verify the uniformity of loading. The Nytran was then prehybridized for 3-4 h in a 65°C hybridization oven (Robbins Scientific, Sunnyvale, CA) in 500 mM NaH2PO4-Na2HPO4 (0.73:1; pH 7.0), 1 mM EDTA, 1% BSA, and 7% SDS. Hybridization was performed at 65°C overnight in the same solution with addition of random-primed [alpha -32P]dCTP-radiolabeled human Mn SOD cDNA (kind gift from Dr. J. Wispe, Childrens Hospital, Cincinnati, OH) or 1.39-kb Hind III-EcoR I DNA fragment representing the 3'-half of murine ICAM-1 cDNA. After hybridization, blots were washed once at room temperature and again at 65°C, both for 20 min, in 40 mM NaH2PO4-Na2HPO4, 1 mM EDTA, and 1% SDS. All filters were exposed to a phosphorimaging storage screen for 1-24 h. The hybridization signal was quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). After quantification by phosphorimaging, the filters were stripped and rehybridized with alpha -32P-labeled rpL32 cDNA, utilized as internal control for RNA integrity and loading.

Quantitative Northern and RPA data were analyzed by ANOVA and Fisher's protected least significant difference statistic with Statview 4.0 statistical analysis software (SAS Institute, Cary, NC) on a Macintosh computer system. The experimental data are expressed as a percent of the mRNA detected in the control lungs normalized to rpL32 mRNA content.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF receptor mRNA expression in murine lung. Expected absence of TNF receptor mRNA was confirmed in TNFR-I(-/-) and TNFR-II(-/-) mouse total lung RNA analyzed by RPA (Fig. 1). Compensatory increase in TNFR-II or I mRNA in the TNFR-I or II(-/-) mice was not detected. WT mouse lung expressed predominantly TNFR-I mRNA and detectable, but much less abundant, TNFR-II. There was no significant change in either TNFR-I or TNFR-II mRNA levels in any of the three strains exposed to 100% oxygen for up to 84 h.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Tumor necrosis factor (TNF) receptor mRNA levels in wild-type (WT; C57BL/6) and TNF receptor-ablated mice. A: representative RNase protection assay (RPA) of TNF receptor type I (TNFR-I; bottom band) and TNF receptor type II (TNFR-II; top band) mRNAs in WT, TNFR-I(-/-) [RI(-/-)], and TNFR-II(-/-) [RII(-/-)] murine lungs after exposure to room air or 100% oxygen for 84 h.

Lack of cytokine and chemokine response to intratracheal TNF-alpha in TNFR-I-ablated mice. TNF-alpha is an early response cytokine, initiating an inflammatory cascade by induction of other cytokines and chemokines such as IL-1beta , macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein (MIP)-1beta , monocyte chemoattractant protein (MCP)-1, and interferon (IFN)-gamma -induced protein-10 (IP-10), factors also known to be induced in hyperoxic models of lung injury (4, 7). To determine if the TNFR-I is necessary for TNF-alpha -mediated induction of these genes, total RNA was isolated from lungs of WT and TNFR-I(-/-) mice 18 h after instillation of rmTNF-alpha and subjected to RPA utilizing template sets represented in Fig. 2, A and B. The regulated on activation normal T cell expressed and secreted (RANTES) chemokine and macrophage migration inhibitory factor (MIF) cytokine were constitutively expressed in both strains of mice. IL-1beta , IL-1 receptor antagonist (RA), MIP-1beta , MIP-2, IP-10, and MCP-1 mRNAs were induced by intratracheal TNF-alpha in lungs of WT mice but not in TNFR-I(-/-) mice (Fig. 2, C and D).


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 2.   Increased cytokine (A) and chemokine (B) mRNA levels in WT or TNF receptor ablated mice after intratracheal (IT) TNF-alpha . A and B: representative RPA analysis of total lung RNAs (5 µg) of WT and TNFR-I(-/-) mice treated with IT recombinant murine TNF-alpha (5 µg/mouse; +) or control saline (-) 18 h before harvest of lung. Lane P, radiolabeled RPA template set undigested with RNase and utilized as size markers for RPA. Templates are 29 nucleotides larger than fragments (arrowheads) protected by mRNA of interest. IL, interleukin; IL-1RA, IL-1 receptor antagonist; MIP-2, macrophage inflammatory protein-2; IFN-gamma , interferon-gamma ; IGIF, IFN-gamma -inducing factor; MIF, macrophage migration inhibitory factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RANTES, regulated on activation normal T cells expressed and secreted; IP-10, IFN-gamma -induced protein-10; MCP-1, monocyte chemoattractant protein-1; TCA-3, T-cell activation gene 3; rmTNF-alpha , recombinant murine TNF-alpha . C and D: cumulative data of mRNAs in WT and RI(-/-) mice 18 h after IT saline (solid bars) or TNF-alpha (open bars) treatment. Values are means ± SD; n = 3 mice for each strain and treatment. Analysis was by ANOVA with Fisher's protected least significant difference (PLSD) post hoc test. * P < 0.05 vs. saline control of same strain.

Survival of TNFR-I- and TNFR-II-ablated mice in hyperoxia. To determine the importance of the two TNF receptors in oxygen-induced mortality, the length of survival of TNFR-I(-/-), TNFR-II(-/-), and control, WT C57BL/6J mice exposed simultaneously to 100% oxygen was compared. As demonstrated in Fig. 3A, there was no strain difference in overall survival (P = 0.4 by Mantel-Cox log rank test). The average time of death for WT mice was 103 ± 14.6 (SD) h (range 76-132 h) compared with 110 ± 9.3 h (range 92-124 h) for the TNFR-I(-/-) mice and 108 ± 16.5 h (range 76-132 h) for TNFR-II(-/-) mice. Analysis of the natural log cumulative hazard plot (Fig. 3B), which considers the natural log of the cumulative hazard as a function of the natural log of the time of death and allows an estimation of relative hazard of each strain over time, suggests that the early survival of the TNF receptor-deficient mice was improved in comparison with WT and TNFR-II(-/-) mice (5).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Murine survival in 100% oxygen. C57BL/6J (n = 20; ), TNFR-I(-/-) (n = 20; ) and TNFR-II(-/-) (n = 10; star ) mice were exposed to 100% oxygen as described in MATERIALS AND METHODS. Surviving mice were counted every 3 h. A: Kaplan-Meier cumulative survival plot (P = 0.4 by Mantel-Cox log rank test). B: natural log (ln) cumulative (cum) hazard plot.

Histological evidence of oxygen toxicity in TNFR-I-ablated mice. After the survival analysis, an additional 74 mice were exposed to 100% oxygen for defined periods of time [37 TNFR-I(-/-) and 37 C57BL/6J mice]. Of these animals, four TNFR-I(-/-) mice died before planned time point [64 h (n = 1), 69 h (n = 1), and 90 h (n = 2)] while five WT died prematurely [64 h (n = 1), 83 h (n = 1), and 92 h (n = 3)]. Tissues from these mice were excluded from further analysis. TNFR-II(-/-) mice were not further studied at this time because there was no difference in survival characteristics compared with WT mice.

Histopathology was assessed by light microscopy after routine hematoxylin and eosin staining. Representative, comparative photomicrographs of distal airway and alveolar regions of WT and TNFR-I(-/-) mice exposed to 100% oxygen for 48 and 84 h are shown in Fig. 4. No histological differences were detected by light microscopy between lungs of WT and TNFR-I(-/-) mice exposed only to room air. At 48 h, airway debris, PMN, and macrophages, as well as peribronchiolar edema and inflammation, are evident in C57BL/6J mice. Mild alveolar septal thickening was seen in both strains at 48 h, appearing more diffusely in C57BL/6J mice. By 84 h, distal bronchiolar epithelium was denuded in both strains, but the lungs of TNFR-I(-/-) mice appeared more severely injured than WT lungs, with more diffuse, amorphous, intra-alveolar debris and periarteriolar edema and inflammatory cell infiltrate in the TNFR-I(-/-) lungs. A toxicity score, based on appearance and extent of alveolitis, bronchiolitis, bronchitis, and fibrosis, was assigned to each experimental tissue sample (1). Cumulative toxicity scores, depicted in Fig. 5, support the observation that lungs of TNFR-I(-/-) mice were less injured at 48 h, yet more severely injured at 93 h, than simultaneously exposed, sex- and age-matched C57BL/6J control mice.


View larger version (100K):
[in this window]
[in a new window]
 
Fig. 4.   Histopathology in C57BL/6J and TNFR-I(-/-) mouse lungs after 48 and 84 h of hyperoxia. Representative lung sections were taken from C57BL/6J and TNFR-I(-/-) mice at 0, 48, and 84 h of oxygen exposure. Staining of sections was by standard hematoxylin and eosin procedure. Nos. on left, original magnification.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Temporal pattern of pulmonary oxygen toxicity depends on presence of TNFR-I. Groups (3-6 per genotype per time point) of C57BL/6J (open bars) and TNFR-I(-/-) mice (solid bars) were exposed for up to 93 h to room air or 100% oxygen. Lung sections were assigned a toxicity score based on light-microscopic assessment of degree of injury to alveoli, bronchioles, and bronchi as described in MATERIALS AND METHODS. Values are means ± SD. Analysis was by ANOVA with PLSD post hoc test. # P < 0.01 for WT vs. TNFR-I(-/-) mice at given time point.

Induction of cytokines TNF-alpha , IL-1beta , MIF, and IL-6 mRNAs by hyperoxia. TNF-alpha mRNA was mildly induced by 72-84 h of exposure to hyperoxia in both WT and TNFR-I(-/-) mice (Fig. 6A). To determine if differences in oxygen susceptibility were due to differential induction of cytokines previously shown to be induced under hyperoxic conditions (14), oxygen-induced changes in mRNAs for MIF, IL-1beta , and IL-6 were measured by RPA (Fig. 6B). MIF mRNA (inhibitor of macrophage migration) was consistently elevated by 48 h of oxygen exposure in both WT and TNFR-I(-/-) mice. IL-1beta and IL-6 mRNAs were also induced in some animals of both strains at 84 h. However, the increase in IL-1beta and IL-6 was animal dependent, and the cytokine induction pattern was not significantly different between WT and TNFR-I(-/-) mice. Levels of IL-1alpha , IL-1RA, the IL-6 receptor IFN-gamma -inducing factor, TNF-beta , lymphotoxin-beta , IFN-beta , transforming growth factor (TGF)-beta 1, and TGF-beta 3 mRNAs were not significantly altered by hyperoxia in either strain of mice. IFN-gamma mRNA was significantly decreased and TGF-beta 2 mRNA was increased by 84 h of exposure to hyperoxia (P < 0.01), with no significant difference between strains of mice. All analyses were performed by ANOVA on data normalized by rpL32 mRNA content.


View larger version (73K):
[in this window]
[in a new window]
 
Fig. 6.   Cytokine mRNA abundance after hyperoxia. Representative RPA of 2 templates, mCK-3b (A) and mCK-2b (B), utilized to measure changes in mRNA encoding the cytokines and transforming growth factor (TGF)-beta 1, -beta 2 and -beta 3 as indicated in WT and TNFR-I(-/-) mice exposed to >95% oxygen for 0, 48, or 84 h. L32 and GAPDH mRNA are included as internal controls. Figure is representative of 3 mice of each genotype in each treatment group. LTbeta , lymphotoxin-beta .

Induction of chemokine mRNA by hyperoxia. To determine if differences in oxygen susceptibility between strains were due to differential induction of chemokine mRNA previously shown to be elevated by hyperoxia, RPA was used to determine changes in expression levels of RANTES, eotaxin, MIP-1beta , MIP-1alpha , MIP-2, IP-10, and MCP-1 (Fig. 7). Notably, RANTES and eotaxin mRNA levels were constitutively expressed independent of oxygen exposure and of genotype. MIP-1alpha , MIP-2, MCP-1, and, to a lesser extent, IP-10 mRNAs were nondetectable at 0 h but induced in all animals by 72 h and persistently elevated at 93 h of exposure to hyperoxia (data not shown). There was a trend toward earlier increase in MIP-1alpha and MCP-1 in TNFR-I(-/-) mice and greater increase in MIP-2 in WT mice at 84 h; however, these were not significant differences (Fig. 7B). All analyses were performed on data normalized by rpL32 mRNA content.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 7.   TNFR-I-independent induction of chemokine mRNA by hyperoxia. The indicated chemokine mRNA levels were measured by RPA in total RNA isolated from left lung lobes as described in MATERIALS AND METHODS. A: phosphorimages of representative RPA (mCK-5). B: cumulative data: MIP-2, MCP-1, and MIP-1alpha mRNA levels in oxygen-exposed C57BL/6J (solid bars) and TNFR-I(-/-) mice (open bars) by 48 h. Values are means ± SE; n = 3-5 mice. Analysis was by ANOVA with PLSD post hoc statistic. * P < 0.05 vs. 0-h control of same strain. There were no significant differences between WT and TNFR-I(-/-) chemokine mRNA levels at the given time points.

Induction of Mn SOD and ICAM-1 mRNA. Because previous studies (10, 16, 34) suggested that TNF-alpha -mediated protection from hyperoxia was due to induction of Mn SOD, mRNA levels of the antioxidant enzyme were quantified by Northern analysis, normalized to rpL32 mRNA content (Fig. 8A). Likewise, TNF-alpha is implicated in hyperoxia-induced inflammation by induction of ICAM-1 mRNA levels (Fig. 8B). Induction of Mn SOD mRNA following 48 and 84 h exposure to 100% oxygen was dependent on genotype, suggesting that induction of the enzyme was dependent on signal transduction via TNFR-I. Worsening toxicity in TNFR-I(-/-) mice at later time points may be due to failure to induce Mn SOD. Likewise, ICAM-1 mRNA was increased in the majority of WT mice by 48-84 h of oxygen exposure. No increase in ICAM-1 mRNA was detected in TNFR-I(-/-) mice, suggesting that induction of the adhesion molecule is also TNFR-I dependent.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 8.   Failure of manganese superoxide dismutase (Mn SOD) and intercellular adhesion molecule-1 (ICAM-1) mRNA induction in TNFR-I(-/-) mice after hyperoxia. Mn SOD (A) and ICAM-1 (B) mRNA were measured in C57BL/6J (solid symbols) and TNFR-I(-/-) mice (open symbols) exposed for 0 (triangles), 48 (circles), or 84 (squares) h to >95% oxygen by Northern analysis and quantified by phosphorimaging. Data are normalized to rpL32 mRNA content. Each point represents data from 1 animal. Group mean refers to the average induction compared with room air control at each time point. Analysis was by ANOVA with PLSD post hoc statistic. * P < 0.01 vs. 0-h control for each strain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperoxia-induced lung injury is the product of direct toxicity of ROS and indirect effects of inflammatory cell activation and resulting synthesis of cytokines. The current study and others confirm the increase in TNF-alpha mRNA within 48-72 h of exposure to 100% oxygen (11, 25, 32). The role that TNF-alpha plays in oxygen-induced lung injury remains unclear because the cytokine is multifunctional, inducing, for example, both inflammatory and protective proteins such as ICAM-1 and Mn SOD. TNFR-I mRNA is the predominant TNF receptor mRNA detected in murine lung tissue and, based on current knowledge, TNFR-I is the dominant effector in TNF-alpha biology capable of inducing apoptosis, tissue necrosis, and nonspecific immunity (9, 12, 23). TNFR-II is implicated in fewer activities such as thymocyte growth, T-cell cytotoxicity, and granulocyte-macrophage colony-stimulating factor expression (29, 30, 33). In the current study, mice lacking either TNF receptor, TNFR-I or TNFR-II, remained susceptible to hyperoxia, although the early survival of TNFR-I(-/-) tended to be prolonged.

Several cell types can express the cytokine, but the major source of TNF-alpha in the lung is the alveolar macrophage (11). Consistent with the results of the current study, macrophage depletion, expected to decrease pulmonary TNF-alpha release, modestly increased survival of rats in 100% oxygen (3). Likewise, pentoxifylline, a methylxanthine that inhibits both TNF-alpha synthesis and function, attenuated oxygen toxicity in rats exposed to continuous hyperoxia (17). Anti-TNF-alpha antibodies also increased survival and lessened injury in rats exposed to hyperoxia (13, 32). However, pretreatment or even concurrent treatment with TNF-alpha also decreased oxygen toxicity (36). In the current study, we sought to determine if the protective and toxic effects of TNF-alpha in the hyperoxia model segregates by function of the two TNF receptors. Our results indicate that under hyperoxic conditions, the TNFR-I receptor mediates both favorable and nonfavorable effects.

mRNA levels of several genes, induced by high doses of oxygen and regulated by TNF-alpha , were measured to determine if hyperoxia-induced changes in these mRNAs are TNFR-I dependent. IL-1beta , IL-6, IL-8, and TNF-alpha have been implicated in mediating pulmonary oxygen toxicity due to their early induction during oxygen exposure and their pleiotropic effects in the lung. IL-8 is not found in mice but is functionally replaced by MIP-2. Cytokines IL-1alpha , IL-1beta , and IL-6 were previously demonstrated to be increased in mice exposed to hyperoxia for more than 48 h (14). In the current study, the increase in these cytokine mRNA levels was highly variable, appearing to be animal specific and independent of the function of TNFR- I(-/-). No correlation was found between severity of oxygen toxicity and level of cytokine mRNA.

Chemokines MCP-1, MIP-2, and MIP-1alpha were induced in WT and TNFR-I(-/-) mice beginning at 48 h and consistently by 72 h of exposure. This finding is in agreement with previous reports of MCP-1, MIP-1alpha , and MIP-2 induction in adult mice within 72 h of beginning exposure to >95% oxygen (7). The chemokine induction patterns tended to differ between WT and TNFR-I(-/-) mice in the current study. The increase in MIP-1alpha and MCP-1 occurred slightly earlier in TNFR-I(-/-) mice, and the level of MIP-2 mRNA was greater in WT mice by 84 h; however, these strain differences were not statistically significant given the numbers of animals tested. Overall, the extent of induction of these chemokine mRNAs was TNFR-I independent, consistent with the increase in PMN and macrophages noted histologically in both murine strains.

Several lines of evidence support a protective role for Mn SOD in pulmonary oxygen toxicity (6). In vitro studies demonstrated that TNF-alpha -dependent induction of Mn SOD was protective against oxidant-induced death (34). Overexpression of Mn SOD by transfection also protected endothelial cells from hyperoxic damage (16). In an adult baboon model of hyperoxia, aerosolized recombinant Mn SOD preserved gas exchange and attenuated toxicity (26, 35). The amelioration of injury and prolonged survival in hyperoxia induced by IL-1 and TNF-alpha treatment was associated with increases in antioxidant enzymes (36). In the current study, we demonstrate that induction of Mn SOD mRNA in hyperoxia is TNFR-I dependent. We speculate that the increase in alveolar debris and inflammation reported in the TNFR-I(-/-) mice at the late time points of oxygen exposure reflects increased injury due to failure of Mn SOD induction. Length of survival in hyperoxia, however, was independent of induction of Mn SOD mRNA.

ICAM-1 is a protein involved in the migration, binding, and activation of leukocytes, in particular neutrophils. ICAM-1 was increased in pulmonary artery and umbilical cord endothelial cells exposed to hyperoxia in vitro in association with enhanced neutrophil adhesion (27). In the current study, the modest but significant increase in ICAM-1 mRNA in WT mice is consistent with previous reports (15, 24). In a few of our WT animals, no increase in ICAM-1 mRNA was seen, which may reflect mouse to mouse variability or the lack of sensitivity inherent in analyzing cell-specific changes in mRNA levels in total lung RNA preparations. No increase in ICAM-1 mRNA was detected in any of the TNFR-I-ablated mice, demonstrating the importance of this receptor in regulation of the adhesion molecule. The lack of ICAM-1 induction and resulting decreased translocation of leukocytes may contribute to the reduction in early lung injury (48 h) in the TNFR-I(-/-) mice. It is also possible that the lack of ICAM-1 expression in the presence of increased neutrophil chemoattractants, such as MIP-2, accounts for later increased alveolar and airway PMN in the TNFR-I(-/-) mice. PMN that are attracted to the lung may be more apparent in TNFR-I(-/-) mice because the adhesion molecule that keeps them closely associated with the interstitium and epithelium is not active. The current study does suggest that induction of ICAM-1 mRNA is not critical for the development of oxygen-induced lung injury or mortality.

Many critically ill patients are supported with high doses of oxygen at the expense of oxygen-induced lung injury. To minimize this iatrogenic disease, it is necessary to understand the toxic and protective functions of endogenous factors stimulated by oxygen exposure. Agents that provide selective blockade of function of specific cytokines or receptors are or will soon be available for therapeutic use (21). The current study supports the prediction that global blockade of TNF-alpha signaling or more specifically of TNFR-I signal transduction will not protect against hyperoxia-induced pulmonary inflammation and toxicity.


    ACKNOWLEDGEMENTS

We thank J. Peschon, Immunex Research & Development (Seattle, WA), for providing p55 knockout mice and reviewing the manuscript. We thank Nancy Corson, Department of Environmental Medicine, University of Rochester (Rochester, NY), for help in intratracheal administration of tumor necrosis factor-alpha .


    FOOTNOTES

Funding of this work by National Heart, Lung, and Blood Institute Grants K08-HL-03203 (to G. S. Pryhuber) and HL-56002 (to R. Phipps); National Institute of Environmental Health Sciences Grant P30-ES-01247 (to R. Phipps and R. Baggs); National Cancer Institute Grant CA-11198 (to R. Phipps); and National Institute of Allergy and Infectious Diseases Grant AI-31473 (to M. H. Nahm) is gratefully acknowledged.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. S. Pryhuber, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 651, Rochester, NY 14642 (E-mail: pryh{at}uhura.cc.rochester.edu).

Received 15 June 1999; accepted in final form 15 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adawi, A, Zhang Y, Baggs R, Finkelstein J, and Phipps RP. Disruption of the CD40-CD40 ligand system prevents an oxygen-induced respiratory distress syndrome. Am J Pathol 152: 651-657, 1998[Abstract].

2.   Barazzone, C, Tacchini-Cottier F, Vesin C, Rochat AF, and Piguet PF. Hyperoxia induces platelet activation and lung sequestration: an event dependent on tumor necrosis factor-alpha and CD11a. Am J Respir Cell Mol Biol 15: 107-114, 1996[Abstract].

3.   Berg, JT, White JE, and Tsan MF. Response of alveolar macrophage-depleted rats to hyperoxia. Exp Lung Res 21: 175-185, 1995[ISI][Medline].

4.   Beutler, B, and Grau GE. Tumor necrosis factor in the pathogenesis of infectious diseases. Crit Care Med 21: S423-S435, 1993[ISI][Medline].

5.   Caldarola, J, Dilmaghani A, Gagnon J, Haycock K, Roth J, Soper C, and Wasserman E. StatView Reference (5th ed.). Cary, NC: SAS Institute, 1998.

6.   Clerch, LB, Wright AE, and Coalson JJ. Lung manganese superoxide dismutase protein expression increases in the baboon model of bronchopulmonary dysplasia and is regulated at a posttranscriptional level. Pediatr Res 39: 253-258, 1996[Abstract].

7.   D'Angio, CT, Johnston CJ, Wright TW, Reed CK, and Finkelstein JN. Chemokine mRNA alterations in newborn and adult mouse lung during acute hyperoxia. Exp Lung Res 24: 685-702, 1998[ISI][Medline].

8.   Declercq, W, Denecker G, Fiers W, and Vandenabeele P. Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75. J Immunol 161: 390-399, 1998[Abstract/Free Full Text].

9.   Erickson, SL, de Sauvage FJ, Kikly K, Carver-Moore K, Pitts-Meek S, Gillett N, Sheehan KC, Schreiber RD, Goeddel DV, and Moore MW. Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature 372: 560-563, 1994[ISI][Medline].

10.   Frank, L, Summerville J, and Massaro D. Protection from oxygen toxicity with endotoxin. Role of the endogenous antioxidant enzymes of the lung. J Clin Invest 65: 1104-1110, 1980[ISI][Medline].

11.   Horinouchi, H, Wang CC, Shepherd KE, and Jones R. TNF alpha gene and protein expression in alveolar macrophages in acute and chronic hyperoxia-induced lung injury. Am J Respir Cell Mol Biol 14: 548-555, 1996[Abstract].

12.   Hsu, H, Shu HB, Pan MG, and Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84: 299-308, 1996[ISI][Medline].

13.   Jensen, JC, Pogrebniak HW, Pass HI, Buresh C, Merino MJ, Kauffman D, Venzon D, Langstein HN, and Norton JA. Role of tumor necrosis factor in oxygen toxicity. J Appl Physiol 72: 1902-1907, 1992[Abstract/Free Full Text].

14.   Johnston, CJ, Mango GW, Finkelstein JN, and Stripp BR. Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice. Am J Respir Cell Mol Biol 17: 147-155, 1997[Abstract/Free Full Text].

15.   Kang, BH, Crapo JD, Wegner CD, Letts LG, and Chang LY. Intercellular adhesion molecule-1 expression on the alveolar epithelium and its modification by hyperoxia. Am J Respir Cell Mol Biol 9: 350-355, 1993[ISI][Medline].

16.   Lindau-Shepard, B, Shaffer JB, and Del Vecchio PJ. Overexpression of manganous superoxide dismutase (MnSOD) in pulmonary endothelial cells confers resistance to hyperoxia. J Cell Physiol 161: 237-242, 1994[ISI][Medline].

17.   Lindsey, HJ, Kisala JM, Ayala A, Lehman D, Herdon CD, and Chaudry IH. Pentoxifylline attenuates oxygen-induced lung injury. J Surg Res 56: 543-548, 1994[ISI][Medline].

18.   Lo, SK, Everitt J, Gu J, and Malik AB. Tumor necrosis factor mediates experimental pulmonary edema by ICAM-1 and CD18-dependent mechanisms. J Clin Invest 89: 981-988, 1992[ISI][Medline].

19.   Mackay, F, Loetscher H, Stueber D, Gehr G, and Lesslauer W. Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55. J Exp Med 177: 1277-1286, 1993[Abstract].

20.   Neumann, B, Machleidt T, Lifka A, Pfeffer K, Vestweber D, Mak TW, Holzmann B, and Kronke M. Crucial role of 55-kilodalton TNF receptor in TNF-induced adhesion molecule expression and leukocyte organ infiltration. J Immunol 156: 1587-1593, 1996[Abstract].

21.   O'Dell, J. Anticytokine therapy---a new era in the treatment of rheumatoid arthritis? N Engl J Med 340: 310-312, 1999[Free Full Text].

22.   Peschon, JJ, Torrance DS, Stocking KL, Glaccum MB, Otten C, Willis CR, Charrier K, Morrissey PJ, Ware CB, and Mohler KM. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol 160: 943-952, 1998[Abstract/Free Full Text].

23.   Pfeffer, K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, and Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457-467, 1993[ISI][Medline].

24.   Piedboeuf, B, Frenette J, Petrov P, Welty SE, Kazzaz JA, and Horowitz S. In vivo expression of intercellular adhesion molecule 1 in type II pneumocytes during hyperoxia. Am J Respir Cell Mol Biol 15: 71-77, 1996[Abstract].

25.   Shea, LM, Beehler C, Schwartz M, Shenkar R, Tuder R, and Abraham E. Hyperoxia activates NF-kappaB and increases TNF-alpha and IFN-gamma gene expression in mouse pulmonary lymphocytes. J Immunol 157: 3902-3908, 1996[Abstract].

26.   Simonson, SG, Welty-Wolf KE, Huang YCT, Taylor DE, Kantrow SP, Carraway MS, Crapo JD, and Piantadosi CA. Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry. J Appl Physiol 83: 550-558, 1997[Abstract/Free Full Text].

27.   Suzuki, Y, Aoki T, Takeuchi O, Nishio K, Suzuki K, Miyata A, Oyamada Y, Takasugi T, Mori M, Fujita H, and Yamaguchi K. Effect of hyperoxia on adhesion molecule expression in human endothelial cells and neutrophils. Am J Physiol Lung Cell Mol Physiol 272: L418-L425, 1997[Abstract/Free Full Text].

28.   Tang, G, White JE, Gordon RJ, Lumb PD, and Tsan MF. Polyethylene glycol-conjugated superoxide dismutase protects rats against oxygen toxicity. J Appl Physiol 74: 1425-1431, 1993[Abstract].

29.   Tartaglia, LA, and Goeddel DV. Two TNF receptors. Immunol Today 13: 151-153, 1992[ISI][Medline].

30.   Tartaglia, LA, Goeddel DV, Reynolds C, Figari IS, Weber RF, Fendly BM, and Palladino MA, Jr. Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor. J Immunol 151: 4637-4641, 1993[Abstract/Free Full Text].

31.   Tartaglia, LA, Pennica D, and Goeddel DV. Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 268: 18542-18548, 1993[Abstract/Free Full Text].

32.   Tsan, MF, White JE, Michelsen PB, and Wong GH. Pulmonary O2 toxicity: role of endogenous tumor necrosis factor. Exp Lung Res 21: 589-597, 1995[ISI][Medline].

33.   Vandenabeele, P, Declercq W, Vercammen D, Van de Craen M, Grooten J, Loetscher H, Brockhaus M, Lesslauer W, and Fiers W. Functional characterization of the human tumor necrosis factor receptor p75 in a transfected rat/mouse T cell hybridoma. J Exp Med 176: 1015-1024, 1992[Abstract].

34.   Warner, BB, Burhans MS, Clark JC, and Wispe JR. Tumor necrosis factor-alpha increases Mn-SOD expression: protection against oxidant injury. Am J Physiol Lung Cell Mol Physiol 260: L296-L301, 1991[Abstract/Free Full Text].

35.   Welty-Wolf, KE, Simonson SG, Huang YCT, Kantrow SP, Carraway MS, Chang LY, Crapo JD, and Piantadosi CA. Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. II. Morphometric analysis. J Appl Physiol 83: 559-568, 1997[Abstract/Free Full Text].

36.   White, CW, and Ghezzi P. Protection against pulmonary oxygen toxicity by interleukin-1 and tumor necrosis factor: role of antioxidant enzymes and effect of cyclooxygenase inhibitors. Biotherapy 1: 361-367, 1989[Medline].


Am J Physiol Lung Cell Mol Physiol 278(5):L1082-L1090
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society