|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Hyperoxia may contribute to lung disease in newborns through effects on alveolar neutrophils which predominate in respiratory distress syndrome and other acute lung injuries. Neutrophil chemokines such as interleukin-8 (IL-8) regulate chemoattraction, and are elevated in tracheal aspirates of newborns who develop bronchopulmonary dysplasia (BPD). Blockade of neutrophil chemokines may reduce hyperoxia-induced inflammatory lung injury and BPD. We therefore tested the hypothesis that hyperoxia contributes to elevations of rat neutrophil chemokines, cytokine- induced neutrophil chemoattractant-1 (CINC-1), and macrophage inflammatory protein-2 (MIP-2) in newborn rat lung. Newborn rats were exposed to air or 95% O2 for 8 d. CINC-1 and MIP-2 were measured in whole lung homogenates by ELISA. Newborn 95% O2-exposed animals were given anti-CINC-1 or anti-MIP-2, 1, 5, or 10 µg on Days 3 and 4 of 95% O2 exposure. Bronchoalveolar lavage (BAL) was performed after perfusion on Day 6 to evaluate airway neutrophils, and myeloperoxidase (MPO) was measured in perfused whole lung. Lungs were examined histologically and immunohistochemically for effects of 95% O2 ± antichemokine. CINC-1 and MIP-2 increased nearly tenfold by Day 8 95% O2 treatment versus air control. CINC-1 and MIP-2 immunolabeling was increased in alveolar macrophages and alveolar epithelium in 95% O2. Anti-CINC-1 and anti-MIP-2 treatment at every dose reduced neutrophil number > 90% in BAL. Anti-CINC-1 10 µg reduced tissue MPO by 50%. Antichemokine treatment on Days 3 and 4 prevented alveolar septal thickening and reduced chemokine immunolabeling on Day 6. Hyperoxia-induced neutrophil influx is mediated in part by CINC-1 and MIP-2 in newborn rats and can be partially prevented by treatment with anti-CINC-1 and anti-MIP-2.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Hyperoxia-mediated injury is known to be important in the pathophysiology of bronchopulmonary dysplasia (BPD) in newborns (1). Oxygen exposure may itself initiate cellular injury, humoral mediator release, and neutrophil chemokinesis (2). Further injury may be propagated by neutrophilic alveolitis, through release of neutrophil proteases and neutrophil peroxidases (5).
Inflammation is believed to play a central role in pathophysiology of BPD in premature newborns. Elevations of neutrophil chemokines such as interleukin-8 (IL-8) and other proinflammatory cytokines have been observed in tracheal aspirates of patients who later develop BPD (6). Glucocorticoids have been a mainstay of clinical treatment of established BPD and are believed to work in part by reducing inflammation, but are nonselective and have serious side effects, prompting the search for more selective targeting of anti- inflammatory strategies (9).
IL-8 is the most potent neutrophil chemokine, acting
through the C-X-C chemokine receptor-2 (CXCR2) receptor
(10). In rats, cytokine-induced neutrophil chemoattractant-1
(CINC-1) acts through rat CXCR2 which is > 85% homologous to human CXCR2 (formerly termed IL-8RB) (11, 12).
Macrophage inflammatory protein-2 (MIP-2) is a second potent rat neutrophil chemokine, a CXCR2 ligand homologous to human GRO-
, and less potent than CINC-1 (13). The role
of the C-X-C chemokines in acute inflammatory conditions
such as lipopolysaccharide (LPS) treatment has been extensively studied (14). Early elevations of cytokines IL-1 and tumor necrosis factor-alpha (TNF-
) induce sustained elevations of C-X-C chemokines, such as CINC-1 and MIP-2 in rats,
IL-8 and GRO- in humans, and neutrophil activating protein-2 (NAP-2) in guinea pigs (15). Neutralization of CINC-1
or MIP-2 using affinity-purified antichemokine antibodies
substantially inhibits neutrophil influx when coadministered with LPS, and is more effective at blocking neutrophil influx than immunotherapy targeting IL-1 or TNF- (16).
On the basis of the aforementioned studies, we proposed the hypothesis that blockade of neutrophil C-X-C chemokines during hyperoxia would be useful for prevention and treatment of BPD. We sought to evaluate the effects of acute hyperoxia on the potent neutrophil chemokines CINC-1 and MIP-2 in a newborn rat model of acute hyperoxia exposure, and the effects of neutralizing anti-CINC-1 and anti-MIP-2 on the development of lung inflammation during hyperoxia exposure.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Hyperoxia Exposure
Newborn rats were exposed in 95% O2-5% air or air alone, beginning on the day of birth, within 10 h of delivery, as previously described in detail by Bruce and colleagues (19). Randomly assorted litters were exposed to 95% O2 at 0.1 L/min in sealed Plexiglas cages 60 × 45 × 25 cm, which we have previously described in detail (20). Oxygen was measured in each cage every 15 min with an analyzer (model 572; Servomex, Norwood, MA). CO2 was removed with a soda-lime trap. Nursing rat dams were switched between air and 95% O2 every 24 h. Exposure to ambient oxygen was < 10 min/d for weighing and cage cleaning. Litter size was adjusted to 10 pups using air-exposed control animals throughout the exposure to control for effects of litter size on nutrition and growth. Two litters were exposed in each treatment condition.
At 2, 4, 6, and 8 d, pups were anesthetized with sodium pentobarbital 150 mg/kg intraperitoneally and a tracheal cannula was placed. An abdominal incision was made, the diaphragm punctured carefully to collapse the lungs. After midline thoracotomy, the pulmonary artery was cannulated with a 25-gauge needle and the left atrial appendage was clipped. The lungs were gently perfused with 10 ml of 0.9% NaCl 1 mM ethylenediaminetetraacetic acid (EDTA) pH = 7.4 to remove blood. In some animals, both lungs were slowly lavaged via the tracheal cannula five times with 0.5 ml 0.9% NaCl 1 mM EDTA.
Neutrophil Measurements
Lavage volumes were recorded, and cell counts measured with a
hemocytometer. Differential counts of 
200 cells were obtained using Wright-Giemsa stain. Lungs were removed, weighed using an analytical balance, and flash frozen. Neutrophil accumulation in lung parenchyma was evaluated using myeloperoxidase (MPO) activity as
determined by o-anisidine absorbance at 504 nm using a microplate method as previously described (21). The lungs of five animals per
treatment group were analyzed.
CINC-1 and MIP-2 ELISA
CINC-1 measurements were performed using the supernatants obtained from whole lung homogenates from animals which were perfused, but not lavaged. We used a sandwich ELISA previously described with the following modifications (22). Plates were coated with affinity-purified polyclonal goat anti-rat CINC-1 at 10 µg/ml (kind gift
of Dr. John Zagorski), and standard curves prepared in duplicate using
rat CINC-1 (R&D Systems, Minneapolis, MN). Rabbit anti-rat CINC-1
was used as the detection antibody 1 µg/ml (Chemicon, Temecula,
CA), and was detected with anti-rabbit-horseradish peroxidase conjugate (Sigma, St. Louis, MO), and o-phenylenediamine-HCl substrate at
492 nm in a microplate reader. Concentrations were calculated using
software supplied with the plate reader according to the manufacturer's
directions (BioTek, Winooski, VT). Likewise, a commercial ELISA kit
was used to measure MIP-2 in whole lung homogenates according to
the manufacturer's directions (R&D Systems, Minneapolis, MN). The
usable ranges of both assays were 50 pg/ml to 25 ng/ml. Standards were
assayed in triplicate and samples were assayed in duplicate. Results for
the assay of standards or samples were accepted when the coefficient of
variation was 
10%. CINC-1 and MIP-2 measurements were normalized to whole lung. Five animals per treatment group were analyzed.
Histologic Evaluation
Lungs were inflation fixed at 20 cm H2O for 30 min in 10% phosphate-buffered formalin, immersed in fixative overnight, paraffin embedded, and cut at 5 µm thickness. Sagittal sections through the center of the lung were dewaxed in xylene and rehydrated in graded ethanols. Sagittal sections were obtained from the center of the block and encompassed at least three of four lobes in order to be representative, and were stained with hematoxylin-eosin. Two sections were obtained from each of four animals per treatment group and a minimum of 10 fields at ×200 magnification were examined.
CINC-1 and MIP-2 Immunohistochemistry
Sections from anti-CINC-1 and anti-MIP-2 treated 95% O2-exposed pups at 6 d were studied to evaluate the effect of antichemokine treatment on chemokine expression. Sections from 8 d air and 8 d 95% O2- exposed pups were used to immunolocalize CINC-1 and MIP-2 expression, because whole lung cytokine measurements were highest at 8 d. Sections were incubated in goat anti-rat CINC-1, or goat anti-mouse MIP-2 (R&D Systems) 1 µg/ml in phosphate-buffered saline (PBS) 0.01% Tween 20 for 2 h, washed in buffer three times, then detected with rabbit anti-goat peroxidase conjugate 1:500 (Sigma). Immunoaffinity purified anti-mouse MIP-2 cross-reacts completely with rat MIP-2 (23). Substrate was added according to the manufacturer's instructions (VIP; Vector, Burlingame, CA), and sections were counterstained with methyl green. Slides were photographed with a Vanox-S AH-2 microscope (Olympus America, Melville, NY).
Anti-CINC-1 and MIP-2 Treatment
On Days 3 and 4, hyperoxia-exposed pups were injected with immunoaffinity-purified goat anti-rat CINC-1, at 1, 5, or 10 µg (approximately 0.2, 1, and 2 mg/kg body weight, respectively). Five pups were injected at each dose, and an additional five were injected with nonimmune goat IgG, 5 µg (approximately 1 mg/kg). Animals were killed on Day 6, and lungs were perfused, lavaged, and removed as noted previously for protein analysis and analyzed for leukocyte accumulation as previously described. The procedure was repeated with the same dose of goat anti-mouse MIP-2. Lungs from five animals per treatment group were analyzed.
Statistical Analysis
Grouped data were expressed as mean ± SEM. Between-group analysis was tested using single-factor analysis of variance (ANOVA). Pairwise comparisons were made using Tukey-Kramer analysis. Survival was analyzed using the Kaplan-Meier method. Results were
computed using statistical software (JMP, Cary, NC). Significance was
accepted at p < 0.05, assuming an error of 0.05 and a error of 0.10.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Effects of Hyperoxia on Chemokine and Neutrophil Accumulation
CXC chemokine assays. By ELISA, whole lung CINC-1 increased more than 5- and 10-fold at 6 and 8 d of 95% O2 exposure compared with air-exposed controls. (Figure 1). There were parallel increases in MIP-2 in 6- and 8-d 95% O2- exposed rats. The overall amount of MIP-2 accumulation was nearly 10 times that of CINC-1, the more potent of the two neutrophil chemokines.
|
Neutrophil accumulation. Neutrophil accumulation in 95% O2-exposed newborn rats was parallel to the increases in CINC-1 and MIP-2. Neutrophil content in bronchoalveolar lavage (BAL) increased markedly with duration of oxygen exposure, but was nearly undetectable in BAL of 6- and 8-d air-exposed rats (Figure 2). Whole lung MPO also increased with oxygen exposure duration, but to a much lesser extent. MPO concentrations in 6- and 8-d 95% O2-exposed rats were approximately double the concentrations in air-exposed controls (Figure 2).
|
95% O2 Effects on Chemokine Immunolabeling at 8 d
There was prominent CINC-1 labeling in alveolar macrophages in 8-d 95% O2 (Figure 3). Labeled alveolar macrophages were rare in the air-exposed group. Alveolar epithelial labeling was also observed, but was much less intense and was not uniform. Unlike macrophages, alveolar epithelium did not show noticeably increased CINC-1 labeling at 8-d 95% O2 exposure compared with 8-d air exposure although this was not quantified.
|
As with CINC-1, macrophages were abundant and intensely labeled with anti-MIP-2 in the 8-d 95% O2-treated group, particularly in areas of lung collapse and injury (Figure 3). Few alveolar macrophages were seen in the air control group, and none showed positive labeling. Alveolar epithelial labeling was also seen to a much lesser extent. There were no apparent effects at 8 d of 95% O2 on alveolar epithelial signal intensity compared with air controls.
Effects of Antichemokine Treatment on Survival and Growth
The survival in the air-exposed animals at 6 d was 100%. The survival among the 95% O2-exposed anti-CINC-1-treated group as a whole was 83 ± 8%, and did not differ among dosage groups. Survival in the anti-MIP-2-treated group was 75 ± 7% and did not differ among dosage groups. There was no statistically significant effect on survival when any treatment group was compared with nonimmune 95% O2-exposed IgG control animals, 75 ± 8%.
95% O2-exposed animals (n = 20) were smaller than air-exposed animals (n = 24) at 6 d, 12.4 ± 1 g versus 15.3 ± 0.8 g, p = 0.04. The effect of oxygen on growth was unaffected by anti-CINC-1 or anti-MIP-2 treatment when measured at 6 d, although there was a trend toward increased weight in the 10 µg anti-CINC group, (n = 10) 14 ± 0.03 g, p = 0.10.
Effects of Antichemokine Treatment on Lung Histology
Air-exposed animals showed normal architecture and no cellular influx. All 95% O2 ± IgG control lungs demonstrated atelectasis, septal thickening, and inflammatory cellular influx. Atelectasis and cellular influx were typically nonhomogeneously distributed. 95% + anti-CINC-1 and 95% + anti-MIP-2 treated groups showed reduced cellular influx in alveolar spaces, and showed decreased septal thickening as well, in all dosage groups, particularly in the 10-µg treatment groups (Figure 4). There were no differences apparent in elastin staining using Masson's trichrome between 95% O2 + IgG control and 95% O2 + antichemokine treatment groups at 6 d (data not shown).
|
Effects of Antichemokine Treatment on Neutrophil Accumulation: BAL and MPO
Treatment with anti-CINC-1 on Days 3 and 4 led to tenfold decline in leukocytes, and in particular neutrophils on Day 6 in all treatment groups (p < 0.001), when compared with nonimmune IgG-treated O2-exposed animals (Figure 5). Leukocytes in BAL were similar in number to air controls. Similarly, MPO accumulation in whole lung homogenates showed parallel, dose-related decrements, although the changes in MPO were not as pronounced as the effects on BAL neutrophils (Figure 5). MPO concentrations in the 5- and 10-µg treatment groups were intermediate between MPO in 95% O2 and air groups.
|
Anti-MIP-2 treatment on Days 3 and 4 had a similar effect at preventing BAL leukocyte and neutrophil accumulation at 6 d 95% O2, as shown in Figure 6. Macrophages were slightly increased in the 10-µg group compared with the 1- and 5-µg treatment groups, but were significantly decreased in all antibody treatment groups compared with the 95% O2 group. In contrast to anti-CINC-1 treatment, however, no significant effects of anti-MIP-2 treatment were detected on whole lung MPO accumulation at 6 d 95% O2.
|
Effects of Antichemokine Treatment on Chemokine Immunolabeling at 6 d
The most intense CINC-1 labeling was apparent in macrophages, which were fewer in number in the anti-CINC 5- and 10-µg treatment groups, consistent with the BAL results. Results for the anti-CINC 10-µg treatment group versus control are shown in Figure 7. Alveolar epithelial labeling was also diminished in the anti-CINC-1-treated groups. Bronchiolar epithelial labeling was evident at 6 d, and was diminished by anti-CINC-1 treatment, particularly in the 10-µg treatment group shown in Figure 7. The predominant effect of anti-MIP-2 was on MIP-2 labeling in macrophages. In contrast to CINC-1, MIP-2 labeling in alveolar epithelium was much less apparent. At 6 d, there was faint bronchiolar epithelial labeling in both control and anti-MIP-2-treated lung. Faint CINC-1 and MIP-2 labeling was seen in bronchiolar epithelium at 8 d, in both 95% O2 and air-treated groups.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Our results show that pulmonary neutrophil influx during hyperoxia in newborn rats is regulated in part by CXC neutrophil chemokines CINC-1 and MIP-2. Pulmonary neutrophil accumulation can be prevented by treatment with antichemokine neutralizing antibodies before high concentrations of chemokines accumulate during hyperoxia. Alveolar septal thickness was preserved by antichemokine treatment, consistent with reduced alveolar-capillary leak and edema, although this was not quantified. The newborn rat model of high oxygen exposure shares a similar early inflammatory response pattern with primates with severe respiratory distress syndrome, namely acute neutrophil influx followed by macrophage influx (24).
Studies of inflammatory cytokines in premature newborns
have shown that tracheal aspirate elevations of IL-1, TNF-,
IL-6, and IL-8 among others predict development of BPD,
and may be evident on the day of birth (6). This finding suggests that perinatal factors preceding direct lung injury by hyperoxia or mechanical ventilation may contribute to the overall inflammatory state. The use of high partial pressures of
oxygen in neonatal lung has long been suspected to induce inflammatory mediators, which contribute to the propagation of
injury (9). In a recent study of chronic lung disease in premature baboons, Coalson and colleagues found that increased
tracheal aspirate IL-8 and neutrophils preceded morphometric changes consistent with human chronic lung disease of prematurity (25).
Our investigations focused on CINC-1 and MIP-2, ligands
of the CXCR2 receptor, because previous experiments evaluating acute inflammation in LPS-induced lung injury in adult
rats demonstrated marked reductions in inflammatory responses after coadministration of anti-CINC-1 and anti-MIP-2,
despite elevations in other inflammatory cytokines, such as
IL-1 and TNF- (16, 18, 26). This finding indicates the importance of the neutrophil chemokine pathway in the propagation of acute lung injury. Proinflammatory cytokines IL-1
and TNF- induce the expression of neutrophil chemokines IL-8, GRO-, GRO-, NAP-2, CINC-1, and MIP-2 (14). The
CXCR2 murine knockout model demonstrates a marked inability to mobilize neutrophils during inflammatory stimuli despite adequate bone marrow precursors, further corroborating
the dominance of this pathway in neutrophil chemotaxis (27).
CINC-1 and MIP-2 were induced by 95% O2. Neutrophil chemokines IL-8 and MIP-2 have also been shown to be induced in newborn mice and rabbits (28, 29). Tissue elevations in CINC-1 and MIP-2 occurred in parallel with BAL leukocyte accumulation, and MPO accumulation in whole lung. Chemokine levels in 95% O2-exposed newborn rats were significantly increased at 4 d, and continued to increase sharply at 6 and 8 d. The BAL neutrophil accumulation observed after 95% O2 for 8 d was comparable to BAL neutrophil counts after LPS administration (16), suggesting similar degrees of neutrophil alveolitis. Alveolar macrophages were the predominant site of increased cellular CINC-1 and MIP-2 expression as measured by our immunohistochemical studies. There were also hyperoxia-induced increases in CINC-1 immunolabeling of alveolar epithelium apparent at 6 and 8 d. Hyperoxia can induce type II cell hyperplasia and hypertrophy, which may account for the increased chemokine expression in alveolar epithelium. Anti-CINC-1 staining was more evident in 95% O2-exposed 6 d alveolar epithelium than at 8 d, possibly because chemokine expression was downregulated by 8 d in alveolar epithelium, although this was not tested. Although the most intense immunolabeling for CINC-1 and MIP-2 was noted in alveolar macrophages, the relative contributions of alveolar macrophage and alveolar epithelium populations to lung chemokine concentrations are unknown. Local variations in chemokine expression may influence local inflammation and responses to antichemokine treatment.
The efficacy of systemic antibody treatment may depend on the relationship between the antibody dose and the concentration and location of the target chemokines, as recently suggested by Blackwell and colleagues (30). We did not measure serum or BAL chemokine levels and are not able to address whether the hyperoxia exposure we used caused a systemic rather than mainly local inflammatory chemokine response. Because a prominent source of CINC-1 and MIP-2 in acute lung inflammation is the macrophage, we presume that BAL concentrations of chemokines would be reduced in parallel with the antichemokine effects on macrophage numbers in BAL, although this was not tested. Endothelial leak associated with hyperoxia may have permitted sufficient neutralizing antichemokine antibody to escape the vessels and neutralize the chemokine gradient between the intravascular and intrapulmonary compartments. Expression of leukocyte adhesion molecules such as E-selectin is dependent on CXCR2 ligands such as CINC-1 and IL-8 (31). Antibody neutralization may eliminate chemokine-induced expression of adhesion molecules, reducing neutrophil vascular adhesion and extravascular migration. Oxidant release in activated neutrophils is also induced by chemokines and may be reduced by antichemokine treatment (14).
The different effects of anti-CINC-1 and anti-MIP-2 on tissue MPO may be related to the differences in chemokine potency. CINC-1-mediated effects on leukocyte adhesion may persist despite anti-MIP-2 treatment, accounting for retention of neutrophils in pulmonary vasculature and higher MPO tissue levels compared with anti-CINC-1-treated animals. Blood retained in the perfused lung may interfere with MPO activity measurements of whole lung homogenates, which is likely to be more pronounced in small, newborn rat lungs.
Antichemokine antibody treatment on Days 3 and 4 also decreased chemokine expression as measured by immunohistology at Day 6. Macrophage labeling for CINC-1 and MIP-2 on Day 6 was decreased after antichemokine treatment on Days 3 and 4. Alveolar macrophages were also fewer in number. The sustained effect of antichemokine antibodies on neutrophil accumulation may be explained by the decrease in chemokine immunohistochemical expression that we observed. We speculate that prevention of neutrophil accumulation protected the lungs from injury sufficient to avoid release of proinflammatory mediators that induce chemokine expression.
To evaluate effects of antichemokine treatment on lung injury, we performed histologic examinations of antibody and control treated animals. Hyperoxia effects on inflammation and injury vary with fraction of inspired oxygen (FIO2) and duration. Because the exposure was relatively brief, we did not grossly observe severe edema or hemorrhage in any of the 95% O2-treated groups. Our studies were designed to evaluate the mechanisms of acute neutrophil influx after hyperoxia, and therefore our histologic examination was focused on airway and parenchymal inflammation. Alveolar macrophages were prominent in all the control 95% O2-exposed animals, and were in general less abundant in the antichemokine-treated animals, consistent with the BAL results. Parenchymal neutrophils were evident in small numbers in the hyperoxia-exposed animals, and were less apparent in the 5- and 10-µg antichemokine treatment groups. Alveolar size and septal thickness were relatively preserved in the 5- and 10-µg antichemokine treatment groups. Quantitative morphometric analysis should clarify whether normal alveolar development is in fact preserved by antichemokine treatment.
The doses of anti-CINC-1 and anti-MIP-2 chosen were similar to those previously used in LPS administration experiments. We had initially postulated that oxygen-induced inflammation would be more indolent than the acute LPS effects on CINC-1 and MIP-2 expression. Higher or more frequent doses of antineutrophil chemokines may prove more effective. Because the predominant neutrophil chemotactic effects of C-X-C chemokines are exerted through the CXCR2 receptor, it would be interesting to test the effects of blocking all potential agonists by using a recently identified CXCR2 receptor antagonist (32). Disruption of the CXCR2 gene, for example, completely eliminates neutrophil chemokinesis in knockout mice (27).
In the future, it will be important to further evaluate the extent of lung injury to determine whether the short-term antichemokine effects on inflammation conferred any sustained benefits to lung function or development. If decreased neutrophil deposition in the lung confers similar protection to that of antichemokine treatment in LPS lung injury models, there may be attenuation of capillary leak, improved lung compliance, and improved lung development as a consequence of reduced injury. Repeated dosing may be necessary to achieve sustained protection against neutrophil-mediated injury.
The potential therapeutic application of cytokine blockade has been stimulated by recent successful uses in the treatment of arthritis and inflammatory bowel disease (33). Kotecha and others have suggested cytokine blockade for the prevention and treatment of chronic lung disease in newborns (8). Present treatments directed against inflammation have almost exclusively used glucocorticoids. The large number of undesirable side effects, including adverse effects on lung (36) and somatic growth (37), decreased resistance to infection, and others have intensified the search for better targeted therapies.
In summary, 95% O2 induces CINC-1 and MIP-2 accumulation in neonatal rat lung, in parallel with alveolar neutrophil accumulation. Cellular expression of CINC-1 and MIP-1 in hyperoxia is overwhelmingly in alveolar macrophages, but is found in alveolar and bronchiolar epithelium. Neutralizing anti-CINC-1 and anti-MIP-2 treatment given before the 95% O2-induced rise in CINC-1 and MIP-2 partially prevented neutrophil accumulation in BAL from 95% O2-treated rats. Anti-CINC-1 also partially prevented neutrophil sequestration in lung tissue. Alveolar septal thickening and inflammatory cell influx evaluated histologically were prevented in antichemokine-treated animals. Anti-CINC-1 and anti-MIP-2 treatment on Days 3 and 4 reduced CINC-1 and MIP-2 labeling in alveolar and bronchiolar epithelium, as well as reducing intensely labeled macrophages. We conclude that hyperoxia-induced neutrophilic inflammation in newborn rats is mediated in part by CINC-1 and MIP-2. Blockade of the C-X-C pathway may prevent inflammation-associated lung injury in hyperoxia-exposed newborns.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Richard L. Auten, Jr., M.D., DUMC Box 3179, Durham, NC 27710. E-mail: auten{at}duke.edu
(Received in original form November 3, 1999 and in revised form April 27, 2000).
Acknowledgments: The authors gratefully acknowledge Drs. Ronald Goldberg and Claude Piantadosi for advice in conducting the present studies and for the careful reading of this manuscript, and Dr. John Zagorski for his gift of the anti-CINC-1 antibodies.
Supported by the North Carolina American Lung Association, the Children's Miracle Network, and the Walker P. Inman Grant.
| |
References |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1. Saugstad OD. Chronic lung disease: the role of oxidative stress. Biol Neonate 1998;74(Suppl 1):21-28.
2. Merritt TA. Oxygen exposure in the newborn guinea pig lung lavage cell chemotactic and elastase response: a possible relationship to bronchopulmonary dysplasia. Pediatr Res 1982; 16: 798-805 [Medline].
3. D'Angio CT, Sinkin RA, LoMonaco MB, Finkelstein JN. Interleukin-8 and monocyte chemoattractant protein-1 mRNAs in oxygen-injured rabbit lung. Am J Physiol 1995;268(5, Pt 1):L826-L831.
4. Johnston CJ, Wright TW, Reed CK, Finkelstein JN. Comparison of adult and newborn pulmonary cytokine mRNA expression after hyperoxia. Exp Lung Res 1997; 23: 537-552 [Medline].
5. Strieter RM, Kunkel SL. Acute lung injury: the role of cytokines in the elicitation of neutrophils. J Investig Med 1994; 42: 640-651 [Medline].
6.
Kotecha S,
Chan B,
Azam N,
Silverman M,
Shaw RJ.
Increase in interleukin-8 and soluble intercellular adhesion molecule-1 in bronchoalveolar lavage fluid from premature infants who develop chronic lung
disease.
Arch Dis Child Fetal Neonatal Ed
1995;
72:
F90-F96
7. Kotecha S, Wilson L, Wangoo A, Silverman M, Shaw RJ. Increase in interleukin (IL)-1 beta and IL-6 in bronchoalveolar lavage fluid obtained from infants with chronic lung disease of prematurity. Pediatr Res 1996; 40: 250-256 [Medline].
8. Kotecha S. Cytokines in chronic lung disease of prematurity. Eur J Pediatr 1996;155(Suppl 2):S14-S17.
9. Jobe AH, Ikegami M. Mechanisms initiating lung injury in the preterm. Early Human Develop 1998; 53: 81-94 [Medline].
10. Murphy PM. Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol 1997; 34: 311-318 [Medline].
11. Zagorski J, DeLarco JE. Rat CINC (cytokine-induced neutrophil chemoattractant) is the homolog of the human GRO proteins but is encoded by a single gene. Biochem Biophys Res Commun 1993; 190: 104-110 [Medline].
12.
Dunstan CAN,
Salafranca MN,
Adhikari S,
Xia Y,
Feng L,
Harrison JK.
Identification of two rat genes orthologous to the human interleukin-8
receptors.
J Biol Chem
1996;
271:
32770-32776
13. Driscoll KE, Hassenbein DG, Howard BW, Isfort RJ, Cody D, Tindal MH, Suchanek M, Carter JM. Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol 1995; 58: 359-364 [Abstract].
14. Strieter RM, Kunkel SL, Keane MP, Standiford TJ. Chemokines in lung injury: Thomas A. Neff Lecture. Chest 1999;116(Suppl 1):103S-110S.
15.
Luster AD.
Chemokines
chemotactic cytokines that mediate inflammation.
N Eng J Med
1998;
338:
436-445
16. Ulich TR, Howard SC, Remick DG, Wittwer A, Yi ES, Yin S, Guo K, Welply JK, Williams JH. Intratracheal administration of endotoxin and cytokines: VI. Antiserum to CINC inhibits acute inflammation. Am J Physiol 1995;268(2, Pt 1):L245-L250.
17. Tang WW, Yi ES, Remick DG, Wittwer A, Yin S, Qi M, Ulich TR. Intratracheal injection of endotoxin and cytokines: IX. Contribution of CD11a/ICAM-1 to neutrophil emigration. Am J Physiol 1995;269(5, Pt 1):L653-L659.
18. Schmal H, Shanley TP, Jones ML, Friedl HP, Ward PA. Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats. J Immunol 1996; 156: 1963-1972 [Abstract].
19. Bruce MC, Bruce EN, Janiga K, Chetty A. Hyperoxic exposure of developing rat lung decreases tropoelastin mRNA levels that rebound postexposure. Am J Physiol 1993;265(3, Pt 1):L293-L300.
20.
Aderibigbe A,
Thomas R,
Auten R.
Expression of surfactant protein D
(SP-D) in hyperoxia exposed rat lung.
Am J Respir Cell Mol Biol
1999;
20:
219-227
21. Lundberg C, Arfors KE. Polymorphonuclear leukocyte accumulation in inflammatory dermal sites as measured by 51Cr-labeled cells and myeloperoxidase. Inflammation 1983; 7: 247-255 [Medline].
22. Frevert CW, Huang S, Danaee H, Paulauskis JD, Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 1995; 154: 335-344 [Abstract].
23. Standiford TJ, Kunkel SL, Greenberger MJ, Laichalk LL, Strieter RM. Expression and regulation of chemokines in bacterial pneumonia. J Leukoc Biol 1996; 59: 24-28 [Abstract].
24. Jackson JC, Chi EY, Wilson CB, Truog WE, Teh EC, Hodson WA. Sequence of inflammatory cell migration into lung during recovery from hyaline membrane disease in premature newborn monkeys. Am Rev Respir Dis 1987; 135: 937-940 [Medline].
25.
Coalson JJ,
Winter VT,
Siler-Khodr T,
Yoder BA.
Neonatal chronic
lung disease in extremely premature baboons.
Am J Respir Crit Care
Med
1999;
160:
1333-1346
26. Bless, NM, Warner RL, Padgaonkar VA, Lentsch AB, Czermak BJ, Schmal H, Friedl HP, Ward PA. Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion. Am J Physiol 1999;276(1, Pt 1):L57-L63.
27.
Cacalano G,
Lee J,
Kikly K,
Ryan AM,
Pitts-Meek S,
Hultgren B,
Wood WI,
Moore MW.
Neutrophil and B cell expansion in mice that lack the
murine IL-8 receptor homolog.
Science
1994;
265:
682-684
28. D'Angio CT, LoMonaco MB, Chaudhry SA, Paxhia A, Ryan RM. Discordant pulmonary proinflammatory cytokine expression during acute hyperoxia in the newborn rabbit. Exp Lung Res 1999; 25: 443-465 [Medline].
29. D'Angio CT, Johnston CJ, Wright TW, Reed CK, Finkelstein JN. Chemokine mRNA alterations in newborn and adult mouse lung during acute hyperoxia. Exp Lung Res 1998; 24: 685-702 [Medline].
30.
Blackwell TS,
Lancaster LH,
Blackwell TR,
Venkatakrishnan A,
Christman JW.
Chemotactic gradients predict neutrophilic alveolitis in endotoxin-treated rats.
Am J Respir Crit Care Med
1999;
159:
1644-1652
31. Harris JG, Flower RJ, Watanabe K, Tsurufuji S, Wolitzky BA, Perretti M. Relative contribution of the selectins in the neutrophil recruitment caused by the chemokine cytokine-induced neutrophil chemoattractant (CINC). Biochem Biophys Res Commun 1996; 221: 692-696 [Medline].
32.
White JR,
Lee JM,
Young PR,
Hertzberg RP,
Jurewicz AJ,
Chaikin MA,
Widdowson K,
Foley JJ,
Martin LD,
Griswold DE,
Sarau HM.
Identification of a potent, selective non-peptide CXCR2 antagonist
that inhibits interleukin-8-induced neutrophil migration.
J Biol Chem
1998;
273:
10095-10098
33. Rutgeerts P. Medical therapy of inflammatory bowel disease. Digestion 1998;59:453-469.
34. Moreland LW. Soluble tumor necrosis factor receptor (p75) fusion protein (ENBREL) as a therapy for rheumatoid arthritis. Rheum Dis Clin North Am 1998; 24: 579-591 [Medline].
35.
Lovell D,
Gianni EH,
Reiff A,
Cawkwell GD,
Silverman ED,
Nocton JJ,
Stein LD,
Gedalia A,
Ilowite NT,
Wallace CA,
Whitmore J,
Finck BK.
Etanercept in children with polyarticular juvenile rheumatoid arthritis.
N Engl J Med
2000;
342:
763-769
36. Tschanz SA, Burri PH. Postnatal lung development and its impairment by glucocorticoids. Pediatr Pulmonol Suppl 1997; 16: 247-249 [Medline].
37. Van Goudoever JB, Wattimena JD, Carnielli VP, Sulkers EJ, Degenhart HJ, Sauer PJ. Effect of dexamethasone on protein metabolism in infants with bronchopulmonary dysplasia. J Pediatr 1994; 124: 112-118 [Medline].
This article has been cited by other articles:
 |
|
 |
|
  N. H. Hillman, T. J. M. Moss, S. G. Kallapur, C. Bachurski, J. J. Pillow, G. R. Polglase, I. Nitsos, B. W. Kramer, and A. H. Jobe Brief, Large Tidal Volume Ventilation Initiates Lung Injury and a Systemic Response in Fetal Sheep Am. J. Respir. Crit. Care Med., September 15, 2007; 176(6): 575 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bry, J. A. Whitsett, and U. Lappalainen IL-1beta Disrupts Postnatal Lung Morphogenesis in the Mouse Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 32 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Tarlowe, A. Duffy, K. B. Kannan, K. Itagaki, R. F. Lavery, D. H. Livingston, P. Bankey, and C. J. Hauser Prospective Study of Neutrophil Chemokine Responses in Trauma Patients at Risk for Pneumonia Am. J. Respir. Crit. Care Med., April 1, 2005; 171(7): 753 - 759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yi, R. P. Jankov, R. Belcastro, D. Humes, I. Copland, S. Shek, N. B. Sweezey, M. Post, K. H. Albertine, R. L. Auten, et al. Opposing Effects of 60% Oxygen and Neutrophil Influx on Alveologenesis in the Neonatal Rat Am. J. Respir. Crit. Care Med., December 1, 2004; 170(11): 1188 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Sue, J. A. Belperio, M. D. Burdick, L. A. Murray, Y. Y. Xue, M. C. Dy, J. J. Kwon, M. P. Keane, and R. M. Strieter CXCR2 Is Critical to Hyperoxia-Induced Lung Injury J. Immunol., March 15, 2004; 172(6): 3860 - 3868. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Vozzelli, S. N. Mason, M. H. Whorton, and R. L. Auten Jr. Antimacrophage chemokine treatment prevents neutrophil and macrophage influx in hyperoxia-exposed newborn rat lung Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L488 - L493. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mukaida Pathophysiological roles of interleukin-8/CXCL8 in pulmonary diseases Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L566 - L577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Ahmed, H. B. Suliman, R. J. Folz, E. Nozik-Grayck, M. L. Golson, S. N. Mason, and R. L. Auten Extracellular Superoxide Dismutase Protects Lung Development in Hyperoxia-exposed Newborn Mice Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 400 - 405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Frossard, A. K. Saluja, N. Mach, H. S. Lee, L. Bhagat, A. Hadenque, L. Rubbia-Brandt, G. Dranoff, and M. L. Steer In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L541 - L548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Auten, M. H. Whorton, and S. Nicholas Mason Blocking Neutrophil Influx Reduces DNA Damage in Hyperoxia-Exposed Newborn Rat Lung Am. J. Respir. Cell Mol. Biol., April 1, 2002; 26(4): 391 - 397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. A. Quintero and J. R. Wright Clearance of surfactant lipids by neutrophils and macrophages isolated from the acutely inflamed lung Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L330 - L339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. TOBIN Pediatrics, Surfactant, and Cystic Fibrosis in AJRCCM 2000 Am. J. Respir. Crit. Care Med., November 1, 2001; 164(9): 1581 - 1594. [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Auten, R. M. Richardson, J. R. White, S. N. Mason, M. A. Vozzelli, and M. H. Whorton Nonpeptide CXCR2 Antagonist Prevents Neutrophil Accumulation in Hyperoxia-Exposed Newborn Rats J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 90 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Auten Jr., S. N. Mason, D. T. Tanaka, K. Welty-Wolf, and M. H. Whorton Anti-neutrophil chemokine preserves alveolar development in hyperoxia-exposed newborn rats Am J Physiol Lung Cell Mol Physiol, August 1, 2001; 281(2): L336 - L344. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Cell Mol. Biol. |