Hemorrhagic shock induces G-CSF expression in bronchial epithelium

Christian Hierholzer1, Edward Kelly1, Katsuhiko Tsukada1, Eric Loeffert2, Simon Watkins2, Timothy R. Billiar1, and David J. Tweardy3,4

Departments of 1 Surgery, 2 Cell Biology, 3 Medicine, and 4 Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hemorrhagic shock (HS) initiates a series of inflammatory processes that includes the activation of polymorphonuclear granulocytic neutrophils (PMN). We tested the hypothesis that HS induces granulocyte colony-stimulating factor (G-CSF), a cytokine that augments PMN effector functions, in the lungs of rats. Sprague-Dawley rats were subjected to compensated or decompensated HS followed by resuscitation and death at 4 or 8 h. Animals subjected to HS demonstrated acute lung injury with PMN infiltration, edema, and hypoxia. Using semiquantitative reverse transcriptase-polymerase chain reaction, we detected a 1.9- to 7.1-fold increase in G-CSF mRNA levels in the lung of animals subjected to HS compared with sham controls. Levels of G-CSF mRNA increased with increased duration of the ischemic phase of resuscitated shock. In situ hybridization revealed that bronchoepithelial cells were the major cellular site of G-CSF mRNA. Thus production of G-CSF mRNA by bronchoepithelial cells is dramatically increased in a rat model of HS that also demonstrated lung injury. Increased local G-CSF levels may contribute to PMN recruitment and activation and resultant lung injury in HS.

inflammation; bronchoepithelial cell; lung; granulocyte colony-stimulating factor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HEMORRHAGIC SHOCK (HS) initiates a cascade of inflammatory events that adversely affects patient survival after successful resuscitation. Polymorphonuclear neutrophilic granulocytes (PMN) are the major cellular element that mediates this inflammatory process in HS (6). Activation of PMN in HS leads to enhanced adhesiveness of PMN to endothelium, to production and release of reactive oxygen intermediates (ROI; see Ref. 10), and to degranulation and protease release (16). The central role of activated PMN in lethal HS has been established in rat and rabbit models of HS. Depletion of PMN before HS prevents death in rats subjected to HS (2). In addition, pretreatment of rabbits with antibody against CD18 (beta 2-integrin subunit) prevents PMN adherence and almost completely abrogates tissue injury in HS (25).

Cytokines known to activate PMN have been shown to be produced in HS. Studies thus far have focused on the proinflammatory cytokines tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, and IL-6 and the immunosuppressive cytokine transforming growth factor-beta (4). Little attention has been directed, to date, on the hematopoietic growth factors specific for neutrophils (PMN), such as granulocyte colony-stimulating factor (G-CSF). G-CSF is essential and unique for the production of neutrophils (8). In addition, G-CSF may promote the inflammatory process through its direct effects on PMN, including degranulation, increased production of reactive oxygen species, and chemotaxis.

The current study was designed to determine if G-CSF production occurs in a model of HS accompanied by lung injury. Using reverse transcriptase (RT)-polymerase chain reaction (PCR), we demonstrated that the lungs from rats subjected to HS express elevated levels of G-CSF mRNA. In situ hybridization of lung revealed that the major cellular site of G-CSF mRNA production was bronchoepithelial cells. Bronchoepithelial cells lining distal airways produced more G-CSF mRNA than cells lining proximal airways. This novel cellular source of G-CSF production in HS was confirmed by in vitro studies of freshly isolated rat respiratory cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HS Protocol

This study was approved by the University of Pittsburgh Institutional Review Board for animal experimentation and conforms to National Institutes of Health guidelines for the care and use of laboratory animals. Fasted male Sprague-Dawley rats (Charles River Breeding Laboratory, Cambridge, MA) were used for all phases of this study. The mean weight of rats was 274 ± 3.7 (SE) g. Animals were randomly subjected to either the shock or sham protocol. For initial anesthesia, penthrane inhalation was used. The animals were orally intubated with a 14-gauge cannula. The right carotid artery and left jugular vein were cannulated with 21-gauge tubing after surgical preparation and isolation. The cannulas, syringes, and tubing were flushed with heparin sodium (1,000 U/ml) before all procedures. Arterial blood pressure was continuously monitored with a Spacelab 514 multimonitor (Spacelabs, Hillsboro, OR). A Harvard small- animal ventilator (Braintree Scientific, Braintree, MA) was used to administer a 2.5-ml tidal volume of room air at 72 strokes/min. After vascular cannulation, the animals received intravenous anesthesia (50 mg/kg pentobarbital sodium).

The protocol was designed to study compensated and decompensated HS. The HS procedure started with an initial bleed of 2.25 ml/100 g body wt over 10 min. The target mean arterial blood pressure (MAP) was 40 mmHg. During the compensation phase, a mean shed blood volume of 7.8 ml was withdrawn to maintain the target MAP. The end of compensation was reached when the MAP dropped below 40 mmHg and replacement of heparinized shed blood was required to maintain the blood pressure at 40 mmHg. Two groups (5 animals each) were resuscitated at compensation end point [66.1 ± 1.1 min; 0% shed blood return (SBR)] with heparinized shed blood and crystalloid solution to achieve an MAP of 80 mmHg. One group was killed 4 h after resuscitation, and the other group was killed 8 h after resuscitation. Two additional groups (5 animals each) were subjected to prolonged shock (157.3 ± 2.3 min), during which the blood pressure was maintained at 40 mmHg after compensation end point by returning heparinized shed blood (2.7 ml; 35% SBR). Both groups were resuscitated to an MAP of 80 mmHg by returning the remaining heparinized shed blood plus crystalloid solution in the amount equal to two times the shed blood volume. One group was killed at 4 h after resuscitation, and the other group was killed at 8 h after resuscitation. We previously determined that the 24-h survival rate of animals subjected to the HS protocol and resuscitated at compensation end point was 100%, whereas the survival rate of animals subjected to the decompensated HS protocol was 20% (12).

Sampling of Arterial Blood, Organs, and Cells

For determination of arterial blood gases, 1 ml of arterial blood was aspirated from the abdominal aorta after cannulation and before the organs were flushed and harvested. Analysis of arterial blood gases included measurement of PO2, PCO2, and pH using a blood gas analyzer (Radiometer ABL 505; Radiometer, Copenhagen, Denmark). After the carcasses were flushed with cold (4°C) isotonic saline solution, the lungs were removed and were used for wet-to-dry determination. Samples of lung were immediately frozen in liquid N2 and were stored at -80°C. Total cellular RNA was extracted from the samples using the method of Chomczynski and Sacchi (5). In addition, samples of lung were sectioned and stained for myeloperoxidase (MPO) as previously described (11, 13). The stained slides were examined at ×400 magnification, and 10 random fields of each lung specimen were blindly scored for number of intensely MPO-positive PMN as previously described (13). Bronchoepithelial cells were obtained from freshly isolated rat trachea cells. Cells were scraped off freshly harvested tracheas (5 animals) treated with protease (1 h at room temperature) and were grown in Eagle's minimum essential medium (Fisher) for 16 h before incubation with or without recombinant human IL-1beta (Biological Response Modifiers Program; National Cancer Institute, Frederick, MD).

RT-PCR Amplification

Total RNA (2.5 µg) was subjected to first-strand cDNA synthesis using oligo(dT) primer and Moloney murine leukemia virus RT (20). The choice of primers for amplification of rat G-CSF (rG-CSF) cDNA was based on two regions within the human and murine cDNA sequence that were each completely identical over an area of at least 30 nucleotides. These regions were located on different exons and were separated by ~500-600 base pairs (bp). Primers were designed within these two regions with the assistance of a PCR primer design program, PCR Plan (Intelligenetics, Mountain View, CA). The 5'-primer sequence, based on nucleotides 513-530 of murine sequence (23), was TTGCCACCACCATCTGGC. The 3'-primer sequence, based on nucleotides 1048-1072 of the murine sequence (23), was ACTGCTGTTTAAATATTAAACAGGG. The primers amplified a product of ~560 bp in length. PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 57°C for 2 min, and polymerization at 72°C for 3 min in a Perkin-Elmer 480 thermocycler. PCR reactions were performed using different numbers of cycles to detect a linear range of input RNA. The optimized cycle number was identified at 30 cycles. Rat peritoneal macrophages elicited with thioglycolate and RAW 264.7 cells stimulated in vitro with lipopolysaccharide served as positive control for rG-CSF mRNA. The negative control for each set of PCR reactions contained water instead of DNA template. PCR product (20% of the reaction volume) was electrophoretically separated on a 2% agarose gel and was stained with ethidium bromide.

For semiquantitative RT-PCR (14) gamma -32P-end-labeled 5'-primer was used. Fifteen microliters of the PCR reaction were separated on a 10% polyacrylamide gel. After gel drying and exposure to a PhosphorImager screen (Molecular Dynamics PhosphorImager, Sunnyvale, CA), the relative radioactivity of the bands was determined by volume integration using laser scanning densitometry. Each gel contained the same positive control, which permitted normalization of samples and comparison between gels.

DNA Subcloning and Sequencing

The fragment of 560 bp, amplified using the G-CSF primers, was subcloned into the EcoR V site of a pBluescript (Stratagene, La Jolla, CA). The insert was sequenced using a ABI PRISM 377 DNA sequencing system (Perkin-Elmer Cetus).

In Situ Hybridization

Tissue and cell preparation for light microscopy. For standard light microscopy of cryostat sections, tissues were lightly fixed in 2% paraformaldehyde, infused with 30% sucrose overnight, and frozen in liquid N2-cooled isopentane. Five-micrometer sections were cut on a cryostat microtome and were mounted on positively charged "Superfrost" slides (Fisher).

Riboprobe synthesis and light in situ hybridization. Both antisense and sense riboprobes were made using radioactively labeled nucleotides. Purified, linearized rG-CSF DNA in pBluescript was incubated in the presence of 35S-labeled UTP, unlabeled CTP, ATP, and GTP along with the relevant polymerase for 2 h at 37°C. The labeled RNA was hydrolyzed via alkaline/heat hydrolysis. The RNA was then column purified and was precipitated with potassium acetate and cold ethanol. The resulting RNA pellet was dried and resuspended in a hybridization buffer containing 50% formamide, 0.3 M NaCl, 20 mM EDTA, 5 mM NaH2PO4, 10% dextran sulfate, 1× Denhardt's solution, and 0.5 mg/ml yeast tRNA. Hybridization of the probe with the tissue was performed essentially as follows. Frozen sections were cut, dried onto slides at room temperature overnight, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; 20 min), permeabilized in 2% paraformaldehyde in PBS containing 0.1% Triton X-100 (10 min), washed twice in PBS, digested with 10 mg/ml proteinase K (10 min), washed in PBS containing 1% glycine, and acetylated. The blocking solution contained dithiothreitol, iodoacetamide, and N-ethylmaleimide. After dehydration through graded alcohols, the sections were hybridized overnight at 50°C in labeled specific antisense riboprobe. Controls included the sense strand of the probe and a no probe. Sections were then washed two times in 50% formamide-2× sodium chloride-sodium citrate (SSC) for 15 min at 50°C and nonspecific probe binding digested in ribonuclease for 30 min at 37°C. After further washes in RNA wash solution 1 and in 2× SSC, sections were washed in Genius buffer I (Boehringer Mannheim), Genius buffer II, and Genius buffer III and then were developed in nitro blue tetrazolium overnight. The specimen were washed in PBS, dehydrated, mounted in permount, and visualized with a Nikon FXA photomicroscope.

Statistics

Unless otherwise indicated, data are presented as means ± SE. Comparisons of means were performed using the Mann-Whitney U-test contained within the StatView 4.1 program (Abacus Concepts, Berkeley, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Lungs of Rats Subjected to HS Show Signs of Acute Lung Injury With Edema and PMN Infiltration

To determine if the lungs of rats subjected to our HS protocol demonstrated evidence of acute lung damage, we examined MPO-stained lung sections to determine if there was PMN infiltration into interstitium and alveolar space (Table 1). We also measured the wet-to-dry ratio as an indicator of pulmonary edema and examined arterial blood gases. The lungs of animals from all four shock groups demonstrated increased PMN infiltration and wet-to-dry ratios compared with their corresponding sham group. There was a greater increase in PMN infiltration and wet-to-dry ratios for animals subjected to decompensated HS and killed 8 h after resuscitation compared with the corresponding compensated group (P < 0.01 for both parameters). The sham procedure caused a small, but significant, increase in PMN infiltration, but the wet-to-dry ratio was not increased compared with normal control animals.

                              
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Table 1.   Acute lung injury of animals subjected to HS

Hypoxia occurred in each group (PO2 = 88 ± 3.2 mmHg for 4-h compensated, 91.5 ± 2.3 mmHg for 8-h compensated, 73.5 ± 2.5 mmHg for 4-h decompensated, and 59.6 ± 3.6 mmHg for 8-h decompensated) compared with normal controls (P <=  0.01 for all groups). Arterial blood gases in animals within the four sham groups (PO2 = 98.5 ± 3.5 mmHg, PCO2 = 39.8 ± 2.4 mmHg, and pH 7.38 ± 0.02) were not different from levels measured in normal control animals (PO2 = 102 ± 1.5 mmHg, PCO2 = 38.9 ± 1.7 mmHg, and pH 7.39 ± 0.01). Hypercapnia (PCO2 = 53.2 ± 5.9 mmHg) and acidosis (pH 7.27 ± 0.05) were also observed but only in the 8-h decompensated group, which demonstrated the greatest PMN infiltration and pulmonary edema.

G-CSF mRNA Levels Were Increased in Lungs of Rats Subjected to HS

To determine if G-CSF is expressed locally in the lung after HS where it may contribute to the PMN recruitment, lung edema, and hypoxia, we performed RT-PCR on RNA obtained from whole lung. Because the rG-CSF cDNA has not been cloned, oligodeoxynucleotide primers based on two regions of complete homology between human and mouse G-CSF cDNA sequence were designed to amplify a fragment of rG-CSF cDNA of ~560 bp. Ethidium bromide-stained agarose gels of RT-PCR reactions of whole lung RNA using G-CSF primers revealed a prominent G-CSF amplification product of 560 bp in each animal in all shock groups (Fig. 1). This band was either absent or reduced in intensity in the sham control animals compared with shock animals. Total RNA from the lung of normal rats did not demonstrate an amplified G-CSF band (data not shown).


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Fig. 1.   Reverse transcriptase (RT)-polymerase chain reaction (PCR) of lung RNA isolated from rats subjected to hemorrhagic shock (HS). Total RNA was isolated at 4 or 8 h from lungs of rats subjected to the compensated [0% shed blood return (SBR)] or decompensated (35% SBR) HS protocol (H) or to the corresponding sham control protocol (C). Reaction products were separated on agarose gels and were stained with ethidium bromide after RT-PCR was performed using either the granulocyte colony-stimulating factor (G-CSF) amplification primers (top) or actin primers (bottom).

Southern blot analysis of the agarose gels demonstrated that the 560-bp band cross-hybridized with the human G-CSF cDNA (data not shown). To confirm the identity of the 560-bp band, the fragment was subcloned into pBluescript and was sequenced. DNA sequencing revealed 91 and 71% homology to the murine and human G-CSF cDNA, respectively (Fig. 2). In the appropriate open reading frame, the rG-CSF cDNA encodes 59 amino acid residues identical in length to the corresponding sequence in the murine cDNA and 2 amino acid residues shorter than the corresponding human cDNA. The protein sequence over this region is 86 and 71% homologous to the murine and human sequences, respectively.


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Fig. 2.   Nucleotide sequence and corresponding amino acid sequence of the rat (r) G-CSF fragment. The sequence is aligned with the corresponding region of murine (m) G-CSF cDNA (23), nucleotides 513-1072, and human (h) G-CSF cDNA (18), nucleotides 468-1026. Differences in the nucleotide sequence between rat and murine and rat and human are indicated by a lower case letter. Differences in amino acid residues are indicated under each sequence. Nucleotide sequence on which each amplification primer was based is underlined.

To determine differences between shock groups and sham control groups more quantitatively, we employed RT-PCR using end-labeled 5'-primer. This approach was pursued when Northern blot analysis of up to 20 µg of total lung RNA from HS animals, using the 560-bp rG-CSF cDNA as probe, did not reveal a hybridization signal. The relative intensity of the band amplified by RT-PCR was quantitated by volume integration using a PhosphorImager (Fig. 3). Normal animals demonstrated low levels of amplified G-CSF mRNA. In sham control animals, levels of amplified G-CSF mRNA were elevated 1.6- to 4-fold compared with normal control animals (P <=  0.01). However, in the compensated shock animals, the amount of amplified G-CSF mRNA was elevated 7.5-fold at 4 h (P < 0.01) and 9.5-fold at 8 h (P = 0.01) after resuscitation compared with normal control animals and 6-fold at 4 h (P < 0.01) and 1.9-fold at 8 h (P = 0.03) after resuscitation compared with sham control animals. In decompensated shock animals, the amount of amplified G-CSF fragment was further elevated 12.8-fold at 4 h (P < 0.01) and 17.5-fold at 8 h (P = 0.01) after resuscitation compared with normal control animals and 7.1-fold at 4 h (P < 0.01) and 4.6-fold at 8 h (P = 0.02) after resuscitation compared with sham animals. Comparison of G-CSF mRNA production between compensated and decompensated shock animals revealed that, in animals subjected to decompensated HS, levels of G-CSF mRNA were increased 1.9-fold compared with animals subjected to compensated HS at 4 h (P = 0.01) and 1.7-fold at 8 h (P = 0.03) after resuscitation.


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Fig. 3.   Semiquantitative RT-PCR of RNA from lungs of rats subjected to compensated (0% SBR) or decompensated (35% SBR) HS and killed 4 or 8 h after resuscitation. RT-PCR reactions from shock animals (n = 5, solid bars), sham control animals (n = 5, open bars), and normal animals (NL, n = 5, open bar) were separated on polyacrylamide gels, and the gels were dried. Radioactive signal in the region corresponding to the 560-bp amplified fragment of rG-CSF mRNA was quantitated by PhophorImager analysis. Differences between each shock and sham group were significant (P <=  0.03 for each group). Levels of G-CSF mRNA were increased 1.9-fold in animals subjected to decompensated HS (dcp) compared with animals subjected to compensated HS (cp) at 4 h (* P = 0.01) and 1.7-fold at 8 h (¥ P = 0.03) after resuscitation. CPM, counts per minute.

Bronchoepithelial Cells Are the Major Cellular Site of G-CSF mRNA Production in Lung

G-CSF is known to be produced by macrophages, endothelial cells, fibroblasts, and astroglial cells (24). To determine the cellular site of G-CSF production in the lung in HS, we performed in situ hybridization of lung specimens incubated with antisense or sense rG-CSF probe. After incubation with antisense probe, examination of the specimens was most notable for concentration of labeled probe within cells lining the luminal side of bronchioles, indicating that bronchial epithelial cells produce G-CSF mRNA (Fig. 4). A gradient of antisense G-CSF probe concentration was observed. Probe was more highly concentrated in cells lining the distal airways (Fig. 4, A and B) than in cells lining the proximal airways (Fig. 4, C and D). These findings indicate that the major cellular site of G-CSF expression in the lung in HS are distal bronchoepithelial cells. Hybridization of sham animals with the antisense probe and hybridization of shock animals with the sense probe were negative for concentration of probe within any cells of the lung, including bronchoepithelial cells (data not shown).


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Fig. 4.   G-CSF in situ hybridization results of lungs from rats subjected to HS (35% SBR for 4 h) hybridized with rat G-CSF antisense probe. Photographs of darkfield views (×400; A, C, and E) and brightfield views (×400; B, D, and F) are shown. A and B: distal bronchiole; arrows, cells lining the bronchiole. C and D: proximal bronchiole; white and black arrows, cells lining the airway; open arrows, cartilage. E and F: arterial blood vessel and terminal alveoli; solid arrows, vascular endothelial cells; open arrows, cells lining the alveoli. Bar, 100 µm.

To confirm respiratory epithelial cells as the cellular site of G-CSF mRNA expression, we performed RT-PCR of freshly isolated rat tracheal epithelial cells cultured for 6 h without or with IL-1beta , a cytokine known to induce G-CSF by a variety of cells (Fig. 5; see Refs. 2, 21, and 24). An amplified cDNA fragment of G-CSF was readily detected in cells incubated either without or with IL-1beta .


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Fig. 5.   G-CSF mRNA production by rat tracheal epithelial cells. Cells were prepared and grown as described in MATERIALS AND METHODS and were incubated in the absence (TEC/CONT) or presence (TEC/IL-1) of 1,000 U/ml interleukin (IL)-1beta for 6 h. Total RNA was isolated from cells, and 2 µg were subjected to RT-PCR using either the rG-CSF or rodent actin primer (27). One-half of the PCR reaction was separated on agarose and stained with ethidium bromide (top). The size of each band in the DNA marker lane (100-bp ladder; BP) is indicated on left. PCR reactions containing no DNA amplified with rG-CSF primer (G) or actin primer (A) were included as negative controls. Position of the rG-CSF cross-hybridizing band is demonstrated on right. Gel was blotted and hybridized with rG-CSF cDNA and exposed to Kodak XAR film (bottom).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study provides evidence that G-CSF mRNA is produced in the lung in a rat model of HS accompanied by PMN infiltration, pulmonary edema, and hypoxia. Semiquantitative RT-PCR demonstrated an amplified G-CSF cDNA fragment in shock animals that was elevated two- to sevenfold over the amount amplified in sham animals. Levels of G-CSF mRNA increased with increased duration of the ischemic phase of resuscitated shock as did PMN infiltration, pulmonary edema, and hypoxia. In situ hybridization studies established that bronchoepithelial cells were the major cellular site of G-CSF mRNA, with cells lining the distal airways expressing the greatest amount of G-CSF. RT-PCR of freshly isolated airway epithelial cells confirmed the ability of these cells to produce G-CSF mRNA.

G-CSF has previously been shown to be produced by macrophages, endothelial cells, fibroblasts, and astroglia cells (24). The major cellular site for G-CSF mRNA expression by the lung in HS was determined to be bronchoepithelial cells. G-CSF previously has been detected by enzyme-linked immunosorbent assay in the conditioned medium of cultures of primary human bronchoepithelial cells (7). Because >95% of the cultured cells in this study stained positive for keratin, these results strongly suggest that human bronchial epithelial cells can produce G-CSF when cultured in vitro. G-CSF mRNA and protein production were induced by TNF-alpha in a SV40-transformed human bronchial epithelial cell line, BEAS-2B (17). Our in situ RNA hybridization findings represent the first direct in vivo demonstration of G-CSF mRNA expression by bronchial epithelial cells.

We found that G-CSF mRNA expression was more prominent in distal compared with proximal bronchioles. For G-CSF production by distal bronchial epithelial cells to cause alveolar recruitment of PMN, the cytokine must reach the terminal alveoli. This could occur through movement of G-CSF down the lumen of distal bronchioles to the alveoli, but this is unlikely given the normal movement of the mucous coating in the opposite direction, mediated by the coordinated beating of cilia. Alternatively, and much more plausible, G-CSF may be secreted from the basolateral surface of bronchial epithelial cells and move into alveoli adjacent to the distal bronchioles by diffusion through the interstitial fluid of the lung.

In our model of resuscitated HS, the duration of shock appears to contribute to the levels of G-CSF mRNA expression. Analysis of the results of semiquantitative RT-PCR for the effect of the duration of shock on G-CSF mRNA production indicates that at both time points of death (4 or 8 h after resuscitation) levels of G-CSF mRNA were increased in animals subjected to decompensated HS compared with animals subjected to compensated HS. Elevated G-CSF mRNA levels in the lungs of rats subjected to decompensated HS were accompanied by more severe signs of acute lung injury characterized by PMN infiltration, pulmonary capillary leakage, and hypoxia, suggesting that increased G-CSF mRNA levels may contribute to increased lung damage and hypoxia observed in resuscitated HS.

G-CSF may enhance PMN-mediated lung damage through its effects on PMN accumulation in tissues and its ability to activate PMN at this site. Local production of G-CSF may affect tissue accumulation through its demonstrated ability to enhance PMN adhesion to endothelium via a mechanism involving integrin affinity conversion (15), as well as through its ability to enhance PMN chemotaxis (26) and chemokinesis (22). In addition, G-CSF has been demonstrated to promote PMN survival by suppression of apoptosis (1). Once in the tissue, G-CSF can promote PMN degranulation (9) as well as prime and activate PMN for the production and release of ROI (19). G-CSF has been demonstrated to provide beneficial effects in inflammatory states initiated by bacterial infection (8) through its ability to enhance PMN phagocytosis and bactericidal activity. Our studies suggest that G-CSF may have deleterious effects as part of the systemic inflammatory response to an insult such as HS, which does not involve an infectious agent.

    ACKNOWLEDGEMENTS

This work was supported, in part, by National Institute of General Medical Sciences Grant GM-53789 and by the Deutsche Forschungsgemeinschaft Grant HI 614/1-1.

    FOOTNOTES

Address for reprint requests: D. J. Tweardy, W1052 Biomedical Science Tower, Univ. of Pittsburgh Cancer Institute, 200 Lothrop St., Pittsburgh, PA 15213.

Received 24 February 1997; accepted in final form 11 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(5):L1058-L1064
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