We hypothesized that CD18 deficiency would impair the ability of neutrophils to emigrate from pulmonary blood vessels during certain pneumonias. To directly compare the abilities of wild-type (WT) and CD18-deficient neutrophils to emigrate, mice with both types of leukocytes in their blood were generated by reconstituting the hemopoietic systems of lethally irradiated C57BL/6 mice with mixtures of fetal liver cells from WT and CD18-deficient mice. Percentages of CD18-deficient neutrophils in the circulating and emigrated pools were compared during experimental pneumonias. Similar percentages were observed in the blood and bronchoalveolar lavage fluid 6 or 24 h after intratracheal instillation of Streptococcus pneumoniae, demonstrating that no site on the CD18 molecule was required for either its adhesive or its signaling functions during neutrophil emigration. However, 6 h after instillation of Escherichia coli LPS or Pseudomonas aeruginosa, the percentage of CD18-deficient neutrophils in the bronchoalveolar lavage fluid was only about one-fourth of that observed in the blood. This difference persisted for at least 24 h after instillation of E. coli LPS. Thus, neutrophil emigration elicited by the Gram-negative stimuli E. coli LPS or P. aeruginosa was compromised by deficiency of CD18. These data, based on comparing WT and gene-targeted CD18-deficient neutrophils within the same animals, provide evidence for molecular pathways regulating neutrophil emigration, which could not be appreciated in previous studies with pharmacological blockade or genetic deficiency of CD18.

The β2 integrins are expressed by all leukocytes and, among other functions, mediate adhesion to endothelial cells. They are heterodimeric transmembrane glycoproteins composed of one invariant CD18 chain and one of four possible CD11 chains (CD11a, -b, -c, or -d). The spontaneous genetic deficiency of CD18 results in the loss of all immunologically recognizable CD11 and CD18 and the absence of all CD11/CD18 function (see Refs. 1 and 2 for review). Infected tissues from human or veterinary CD18-deficient patients are typically devoid of extravascular neutrophils, suggesting that CD11/CD18 complexes are essential to emigration from the blood vessels (1, 2, 3, 4, 5, 6, 7). Confirming this, blocking Abs against CD11/CD18 significantly inhibit neutrophil emigration from the systemic circulation during diverse inflammatory processes in various tissues (8, 9, 10, 11, 12, 13).

In contrast, pneumonic lungs from CD18-deficient human or veterinary patients display abundant emigrated neutrophils (6, 7, 14), demonstrating that CD18-independent pathways can be used for neutrophil emigration from the pulmonary circulation. Blocking Ab studies suggest that neutrophils use CD18-dependent or CD18-independent pathways in the lungs, depending on the stimulus inducing pneumonia. Blocking Abs against CD11/CD18 prevent neutrophil emigration by ∼70% during 4–6 h of pneumonia induced by Escherichia coli LPS, E. coli, Pseudomonas aeruginosa, IgG immune complexes, IL-1α, and phorbol esters (12, 15, 16, 17, 18), but blocking Abs against CD11/CD18 do not affect neutrophil emigration during 4–6 h of pneumonia induced by S. pneumoniae, group B Streptococcus, Staphylococcus aureus, complement fragment C5a, hyperoxia, or hydrochloric acid (12, 16, 18, 19, 20).

Consistent with clinical observations of neutrophils in the lungs of CD18-deficient patients, but contrasting with predictions based on blocking Ab studies, mice rendered CD18-deficient by gene targeting show no defect in neutrophil emigration compared with wild-type (WT)5 mice during experimental pneumonias induced by either E. coli or S. pneumoniae (21). Furthermore, experimental pneumonias induced by Pasteurella haemolytica in cows with spontaneous deficiencies of CD18 show no defect in neutrophil emigration compared with normal (CD18-expressing) cows (22). Thus, the results from all studies to date suggest that CD18 deficiency does not compromise the emigration of neutrophils from the pulmonary circulation. However, the genetic deficiency of CD18 results in systemic phenotypic alterations (1, 7, 23), including soft tissue infections, neutrophilia, splenomegaly, and lymphadenopathy, which may affect neutrophil emigration and confound comparisons of CD18-deficient and control animals. To directly examine the roles of CD11/CD18 in neutrophil emigration in the lungs, we endeavored to compare the emigration of WT and CD18-deficient neutrophils within the same physiological environment. To accomplish this, mice with both types of neutrophils in their blood were generated after reconstitution of the hemopoietic systems of lethally irradiated mice with mixtures of fetal liver cells from WT and CD18-deficient mice.

The hemopoietic systems of lethally irradiated C57BL/6 host mice were reconstituted after injection of fetal liver cells as described (24). In short, CD18-deficient mice (23), provided by Dr. Arthur L. Beaudet, and WT mice of similar randomly mixed C57BL/6 × 129/Sv background were mated with the like genotype, and fetuses were collected after 14 days of gestation. Single-cell suspensions were prepared from WT and CD18-deficient fetal livers, and mixtures of 1–2 × 106 total cells were injected i.v. into host mice that had received radiation doses of 800 and 400 rad, separated by 3 h, from a 137Cs source. To minimize possible systemic physiological effects caused by CD18 deficiency, these studies were performed using mice reconstituted with mixtures of fetal liver cells in which a minority of the inoculum was from CD18-deficient animals (≤10% in the E. coli LPS and S. pneumoniae experiments and 25% in the P. aeruginosa experiments). After transplantation, mice received trimethoprim-sulfamethoxazole in their drinking water and were maintained under barrier conditions. Animals were analyzed a minimum of 4 wk after irradiation to allow for reconstitution. Several mice reconstituted with mixed WT and CD18-deficient fetal liver cells had their lungs lavaged (see below) and then fixed by instillation of 10% formalin at 22 cm H2O. Lavage fluids were analyzed for neutrophil content, and histological sections from the fixed lungs were examined to determine whether irradiation and reconstitution resulted in pulmonary inflammation.

Pneumonias were induced by intratracheal (i.t.) instillations (21). Mice were anesthetized by i.m. injection of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (5 mg/kg). The tracheas were surgically exposed, and 50 μl of E. coli LPS serotype O55:B5 (Sigma, St. Louis, MO) at 2 mg/ml, of P. aeruginosa at 1 × 108 CFU/ml, or of S. pneumoniae at 5 × 109 CFU/ml were instilled i.t. At the indicated times, mice were killed by inhalation of a lethal overdose of halothane. Blood was collected from the inferior vena cava, and erythrocytes were hypotonically lysed. Bronchoalveolar lavage (BAL) was performed after cannulating the trachea. A syringe containing 1 ml of ice-cold PBS containing 0.6 mM EDTA was inserted into the cannula, 0.5 ml were injected while the chest was massaged, and then as much volume as possible was recovered. The lavage steps were twice repeated without changing the syringe, after which the syringe was removed from the cannula and the contents were evacuated into a test tube and kept on ice. The entire procedure was repeated twice more, so that the lungs received a total of 9 lavages of 0.5 ml pooled in ≤3 ml final volume. Blood and BAL leukocytes were washed with fresh PBS/EDTA, and WT and CD18-deficient cells were differentiated using mAb against CD11a and CD11b.

For flow cytometric analyses, blood and BAL cells were stained with saturating concentrations of a FITC-conjugated mAb against murine CD11a (M17/4, PharMingen, San Diego, CA) and a PE-conjugated mAb against the murine granulocyte marker Gr-1 (RB6-8C5, PharMingen). Overlapping spectra of FITC and PE were accounted for by adjusting compensation using cells stained with single Abs. Gr-1 bright cells had forward and right angle scatter characteristics consistent with granulocytes. The percentage of neutrophils that were CD18 deficient was assessed for each sample by scoring 5000 Gr-1 bright cells as either positive or negative for CD11a/CD18.

For immunohistochemical analyses, blood and BAL cells were cytocentrifuged onto glass slides, allowed to air dry, and then fixed with acetone-methanol (1:1). Slides were treated with a rat mAb against murine CD11b (M1/70, PharMingen), and M1/70 was visualized using biotinylated goat anti-rat IgG and a streptavidin-alkaline phosphatase detection system (Kirkegaard and Perry, Gaithersburg, MD). Control slides demonstrated no staining of WT or CD18-deficient cells when nonspecific rat IgG replaced M1/70 and no staining of cells from CD18-deficient mice by M1/70. Slides were counterstained with hematoxylin, and the percentage of neutrophils which were CD18-deficient was assessed for each sample by scoring 300 polymorphonuclear cells as either positive or negative for CD11b/CD18.

In each group, the percentages of CD18-deficient neutrophils in the blood and in the BAL fluid were compared by paired t tests, and differences were considered significant when p < 0.05. Results were expressed as mean and SEM. Each group consisted of five to eight mice.

Whereas peripheral blood neutrophils from WT mice are uniformly positive for CD11a, CD11b, and CD18, neutrophils from CD18-deficient mice are uniformly negative for CD11a and CD11b as well as CD18 (21, 23), similar to patients with spontaneous deficiencies of CD18 (1, 2). Abs against CD11a and CD11b were used to differentiate WT and CD18-deficient neutrophils in the present studies. When lethally irradiated C57BL/6 mice were reconstituted with WT fetal liver cells, their peripheral blood neutrophils expressed CD11a/CD18 (Fig. 1,A). When lethally irradiated C57BL/6 mice were reconstituted with CD18-deficient fetal liver cells, their peripheral blood neutrophils did not express CD11a/CD18 (Fig. 1,B). When lethally irradiated C57BL/6 mice were reconstituted with mixtures of WT and CD18-deficient fetal liver cells, anti-CD11a/CD18 staining revealed both WT and CD18-deficient neutrophils in the circulating blood (Fig. 1 C). Similar results were observed when WT and CD18-deficient neutrophils were differentiated using immunohistochemistry for CD11b/CD18 (data not shown).

FIGURE 1.

Circulating WT and CD18-deficient neutrophils after hemopoietic reconstitution. Neutrophils were identified by bright staining with a PE-conjugated mAb against murine Gr-1 (RB6-8C5). WT and CD18-deficient cells were differentiated using a FITC-conjugated mAb against CD11a/CD18 (M17/4). The y-axes depict relative numbers of peripheral blood neutrophils that express the level of CD11a/CD18 designated on the x-axes. A, Neutrophils from peripheral blood of mice reconstituted with WT fetal liver cells; B, neutrophils from peripheral blood of mice reconstituted with CD18-deficient fetal liver cells; C, neutrophils from peripheral blood of mice reconstituted with mixtures of WT and CD18-deficient fetal liver cells.

FIGURE 1.

Circulating WT and CD18-deficient neutrophils after hemopoietic reconstitution. Neutrophils were identified by bright staining with a PE-conjugated mAb against murine Gr-1 (RB6-8C5). WT and CD18-deficient cells were differentiated using a FITC-conjugated mAb against CD11a/CD18 (M17/4). The y-axes depict relative numbers of peripheral blood neutrophils that express the level of CD11a/CD18 designated on the x-axes. A, Neutrophils from peripheral blood of mice reconstituted with WT fetal liver cells; B, neutrophils from peripheral blood of mice reconstituted with CD18-deficient fetal liver cells; C, neutrophils from peripheral blood of mice reconstituted with mixtures of WT and CD18-deficient fetal liver cells.

Close modal

The lungs of mice which had been irradiated and reconstituted with mixed WT and CD18-deficient fetal liver cells were examined for signs of inflammation. In the absence of experimentally induced pneumonia, neutrophils were not recovered by BAL. Fixed sections from the lungs of these mice did not reveal evidence of infection, radiation-induced pneumonitis, or neutrophilic infiltration of the interstitium or air spaces.

The intratracheal instillation of E. coli LPS to mice with hemopoietic systems reconstituted with mixed WT and CD18-deficient fetal liver cells resulted in neutrophil emigration by 6 h, as measured by the recovery of neutrophils by BAL (1.8 ± 0.6 × 105 neutrophils/ml). To determine whether the WT and CD18-deficient neutrophils were equally capable of emigrating 6 h after E. coli LPS instillation, the percentage of CD18-deficient neutrophils in the circulating pool (peripheral blood) and in the emigrated pool (BAL fluid) were compared. In these animals, 9.3% of the circulating neutrophils were CD18-deficient, but only 2.8% of the emigrated neutrophils were CD18-deficient (Fig. 2,A), indicating that CD18-deficient neutrophils had a defect in emigration compared with neutrophils expressing CD11/CD18. The BAL fluid contained a smaller fraction of CD18-deficient neutrophils than the circulating blood in every mouse, whether the cells were differentiated by anti-CD11b staining of polymorphonuclear cells examined by immunohistochemistry (Fig. 2 A) or by anti-CD11a staining of Gr-1-positive cells examined by flow cytometry (data not shown).

FIGURE 2.

Percentages of blood and BAL neutrophils which were CD18-deficient during E. coli LPS pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. A, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 6 h after instillation of E. coli LPS. B, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 24 h after instillation of E. coli LPS. ∗, Significant differences between blood and BAL fluid.

FIGURE 2.

Percentages of blood and BAL neutrophils which were CD18-deficient during E. coli LPS pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. A, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 6 h after instillation of E. coli LPS. B, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 24 h after instillation of E. coli LPS. ∗, Significant differences between blood and BAL fluid.

Close modal

After 24 h of E. coli LPS pneumonia in mice with hemopoietic systems reconstituted from mixtures of WT and CD18-deficient stem cells, 2.7 ± 0.5 × 106 neutrophils/ml were recovered in the BAL fluid. Cytospins immunohistochemically stained for CD11b/CD18 revealed that only 1.4% of these emigrated neutrophils were CD18-deficient, whereas 14.1% of the circulating neutrophils were CD18-deficient (Fig. 2 B). Similar results were observed when WT and CD18-deficient cells were differentiated by anti-CD11a staining of Gr-1-positive cells examined by flow cytometry (data not shown).

To determine whether CD18-deficient neutrophils were compromised in emigration during pneumonia induced by living Gram-negative organisms, P. aeruginosa were instilled i.t. into mice with CD18-negative and -positive neutrophils in their blood. After 6 h, 3.4 ± 0.6 × 105 neutrophils/ml were recovered in the BAL fluid. Immunohistochemical staining for CD11b/CD18 revealed that 46% of the neutrophils were CD18-deficient in the peripheral blood, whereas only 11% of the neutrophils were CD18-deficient in the BAL fluid (Fig. 3).

FIGURE 3.

Percentages of blood and BAL neutrophils that were CD18-deficient during P. aeruginosa pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. ∗, Significant difference between blood and BAL fluid.

FIGURE 3.

Percentages of blood and BAL neutrophils that were CD18-deficient during P. aeruginosa pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. ∗, Significant difference between blood and BAL fluid.

Close modal

To determine whether CD18-deficient neutrophils were compromised in emigration during streptococcal pneumonia, S. pneumoniae were instilled i.t. into mice with hemopoietic systems reconstituted from mixtures of WT and CD18-deficient stem cells. After 6 h, 1.4 ± 0.6 × 106 neutrophils/ml were recovered in the BAL fluid. At this time, 12.7% of the circulating neutrophils were CD18-deficient and 12.8% of the emigrated neutrophils were CD18-deficient (Fig. 4A), indicating that CD18-deficient neutrophils did not have a defect in emigration compared with CD18-positive neutrophils.

FIGURE 4.

Percentages of blood and BAL neutrophils that were CD18-deficient during S. pneumoniae pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. A, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 6 h after instillation of S. pneumoniae. B, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 24 h after instillation of S. pneumoniae. Blood and BAL fluid did not differ significantly.

FIGURE 4.

Percentages of blood and BAL neutrophils that were CD18-deficient during S. pneumoniae pneumonia. Mice had hemopoietic systems reconstituted from mixtures of WT and CD18-deficient fetal liver cells, and WT and CD18-deficient neutrophils from blood and BAL fluid were differentiated using immunohistochemical staining for CD11b/CD18 in cytospin preparations. A, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 6 h after instillation of S. pneumoniae. B, Percentages of CD18-deficient neutrophils in blood and BAL fluid collected 24 h after instillation of S. pneumoniae. Blood and BAL fluid did not differ significantly.

Close modal

After 24 h of S. pneumoniae pneumonia in mice reconstituted with mixtures of WT and CD18-deficient stem cells, 1.0 ± 0.2 × 106 neutrophils/ml were recovered by BAL. Similar percentages of neutrophils were CD18-deficient in the blood and the BAL fluid (Fig. 4 B), suggesting that neutrophil emigration over this time frame remained free of requirements for CD11/CD18 adhesion complexes.

Blocking Abs against CD11/CD18 prevent neutrophil emigration by ∼70% during 4–6 h of pneumonia induced by E. coli LPS, E. coli, or P. aeruginosa (12, 17, 18). However, no defect in emigration is observed in neutrophil emigration in CD18-deficient mice during 6 h of E. coli pneumonia (21). The reason for these discrepant results is unclear but may reflect confounding effects of either Ab treatments or targeted gene deletions. Blocking Abs against CD11/CD18 may have effects other than simply the prevention of ligand binding. For example, they may cross-link CD11/CD18 molecules or interact with Fc receptors. The targeted deletion of CD18 induces systemic phenotypic changes (23), including peripheral blood neutrophilia and chronic inflammatory lesions, which also may confound comparisons of neutrophil emigration in CD18-deficient and WT mice.

The present results compared the emigration of WT and CD18-deficient neutrophils within the same animal and are free from these confounding factors. Pneumonias were induced in hemopoietically reconstituted mice with both WT and CD18-deficient neutrophils circulating in their peripheral blood, to directly compare the emigrating abilities of these neutrophils. The percentage of neutrophils that were CD18-deficient was significantly smaller in the BAL fluid than in the blood 6 h after i.t. instillation of E. coli LPS to mice with ∼10% of their circulating cells CD18-deficient. Similar results were observed 6 h after the i.t. instillation of living P. aeruginosa to mice with ∼50% of their circulating cells CD18-deficient. These data suggest that CD18 deficiency compromises neutrophil emigration during acute pneumonia elicited by either of these two Gram-negative stimuli.

Blocking Ab studies and comparisons of WT and CD18-deficient animals investigate the effects of inhibiting CD11/CD18 function on all hemopoietic cells. Accordingly, any observed differences might be the result of CD11/CD18 expressed by either neutrophils or other cells. For example, macrophages express CD11/CD18 molecules. Because CD11/CD18 can mediate uptake or signaling in response to complement-opsonized particles, LPS, and other stimuli (23, 25, 26, 27, 28, 29, 30), the expression of inflammatory mediators produced by macrophages could be affected by blocking CD11/CD18 function, leading to decreased emigration through mechanisms other than blockade of neutrophil CD18. In the present studies, CD11/CD18-positive and CD11/CD18-negative neutrophils were directly compared within the same environment, while exposed to the same chemoattractants, cytokines, and lipid mediators. Thus, the compromised emigration of CD18-deficient neutrophils under these conditions demonstrates a critical role for the CD11/CD18 molecules expressed by neutrophils per se.

Although the present studies were free from several limitations discussed above inherent to comparisons of Ab-treated or gene-targeted mice with controls, limitations inherent to the present techniques also bear consideration. First, neutrophil emigration was studied by analysis of lavaged cells. It is conceivable that CD18 deficiency affects the adhesion of emigrated neutrophils to the alveolar epithelium, and such differential adhesion could affect the relative recovery by lavage of WT and mutant cells. Second, the mice in these studies were lethally irradiated and then hemopoietically reconstituted before the studies of pneumonia. These experimental manipulations could potentially affect the regulation of acute inflammatory responses in the lungs in as yet unrecognized ways. Blocking Ab studies (12, 17) and the studies reported in this article are subject to different experimental limitations, but the results collected with either approach suggest that CD11/CD18 is critical to neutrophil emigration during 4–6 h of pneumonia elicited by LPS or P. aeruginosa.

To determine whether the requirements for CD11/CD18 changed as the pneumonia progressed, the percentages of CD18-deficient neutrophils were compared in the blood and BAL fluid 24 h after the i.t. instillation of E. coli LPS. Similar to observations at the earlier time points, a significantly smaller percentage of neutrophils were CD18-deficient in the BAL fluid compared with the blood 24 h after instillation of E. coli LPS. In previous studies of peritonitis induced in rabbits by the injection of E. coli or protease peptone, blocking Abs against CD18 compromised neutrophil emigration during the first several hours, but emigration during 24 h of peritonitis was no longer affected by blocking Abs (31). These results indicate that CD18-independent pathways become available over this time frame of peritonitis. The present data suggest that neutrophil emigration during E. coli LPS pneumonia remains dependent on CD18 for at least 24 h. Although these studies used different animal species and inflammatory stimuli, the present results suggest that the temporal regulation of CD18 dependence in mediating neutrophil emigration may differ in the vascular beds of the lungs and of the peritoneum.

In contrast to the results with E. coli LPS or P. aeruginosa pneumonias, similar percentages of CD18-deficient neutrophils were present in the blood and BAL fluid 6 h after the instillation of S. pneumoniae. Previous studies demonstrate that blocking Abs against CD11/CD18 do not affect neutrophil emigration during 4–6 h of pulmonary inflammation induced by S. pneumoniae, group B Streptococcus, Staphylococcus aureus, complement fragment C5a, hyperoxia, or hydrochloric acid (12, 16, 18, 19, 20). Several factors have been suggested as responsible for the inability of blocking Abs to inhibit neutrophil emigration during such pneumonias, including: 1) failure to obtain sufficient Ab concentrations to the required anatomic or cellular locations to completely prevent ligand binding; 2) alternative epitopes on CD11/CD18 that mediate neutrophil emigration and are not blocked by the Abs; or 3) a distinct pathway for neutrophil emigration that does not require CD11/CD18. The present data, indicating that the emigration of CD18-deficient neutrophils is not compromised compared with WT during streptococcal pneumonia, suggest that S. pneumoniae elicits a distinct pathway for neutrophil emigration in the lungs that is truly CD18 independent, not utilizing any region of CD11/CD18 complexes for either adhesion or signaling events essential for emigration. Furthermore, the present results demonstrate that neutrophil emigration remains CD18 independent throughout 24 h of pneumonia induced by S. pneumoniae.

These roles for CD11/CD18 in mediating neutrophil emigration during pneumonias could not be appreciated when comparing neutrophil emigration in CD18-deficient and WT mice (21), likely due to the diverse physiological effects resulting from the genetic deficiency of CD18 by all leukocytes. However, these roles for CD11/CD18 became clear in mice with reconstituted hemopoietic systems in which the behaviors of cells of different genotypes were studied within the same physiological environment. To our knowledge, these data are the first to demonstrate that the deficiency of an adhesion molecule compromises neutrophil emigration during pneumonia. Furthermore, these studies identified CD11/CD18 expressed by neutrophils per se as critical to mediating emigration during E. coli LPS and P. aeruginosa pneumonias. These requirements for CD11/CD18 persisted for at least 24 h of E. coli LPS pneumonia. Finally, the data conclusively demonstrate that in contrast to E. coli LPS or to P. aeruginosa, S. pneumoniae induces neutrophil emigration in the lungs that does not require CD11/CD18.

We thank Dr. Arthur L. Beaudet for providing the WT and CD18-deficient mice and Kathryn E. Marino, Sabrina D. Bhagwan, and Amy Imrich for technical assistance.

1

This work was supported by U.S. Public Health Service Grant HL 52466 and by a Research Grant from the Cystic Fibrosis Foundation. B.H.H. was supported by a fellowship from the Cancer Research Fund of the Damon-Runyon-Walter Winchell Foundation.

5

Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; i.t., intratracheal.

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