Impaired Pulmonary Host Defense in Mice Lacking Expression of the CXC Chemokine Lungkine

Gene targeting of Lungkine

Two BAC clones containing genomic copies of the mouse Lungkine gene were identified in a mouse 129/Sv-derived embryonic stem (ES)³ cell genomic library (Incyte Genomics, St. Louis, MO) using PCR primers corresponding to the mouse Lungkine cDNA: GV15 (5′-TTAGGCATCACTGCCTGTCA-3′) and GV16 (5′-AATGAAGCTTCTGTATAGT3′). Several fragments containing the Lungkine gene were identified by Southern blot analysis of BAC plasmid DNA using a 500-bp cDNA probe labeled with [γ-³²P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) by random priming (Megaprime DNA Labeling System; Amersham Pharmacia Biotech, Piscataway, NJ). Two BamHI fragments (7.6 kb each) that contained either the 5′ half or the 3′ half of the Lungkine cDNA were subcloned into pBluescript (Stratagene, La Jolla, CA) and mapped by restriction digest. The complete intron and exon sequences of these two subclones were further analyzed using an Applied Biosystems 377 DNA sequencer (Applied Biosystems, Foster City, CA). DNA fragments corresponding to the 5′ and 3′ regions of the Lungkine locus were, in turn, subcloned from this plasmid into the pOSDupDel vector (a gift from Oliver Smithies, University of North Carolina, Chapel Hill, NC) at either end of the neomycin resistance gene (neo). This targeting vector DNA was linearized using the restriction enzyme NotI before its introduction into 129/Sv-derived ES cells by electroporation. Lungkine-targeted ES clones were identified by Southern blotting. DNA was digested with BamHI, transferred to nylon membranes, and hybridized to a radiolabeled DNA probe derived from a region upstream of the 5′ region of homology. Cells whose DNAs were of the structure predicted for the targeted locus were used to generate chimeric mice using standard blastocyst injection procedures. Several correctly targeted ES cells were injected into blastocysts to generate chimeric mice. Lungkine heterozygous (+/−) offspring were identified using a PCR-based screening strategy using three oligonucleotide primers corresponding to the region of homology, the neo gene, and the deleted region of the Lungkine gene. Lungkine^−/− mice were generated by interbreeding these heterozygous mice.

Mice

For the experiments described here we used Lungkine^−/− mice generated in the 129 × BL6 background. Age- and sex-matched (129 SvEv × C57BL/6)F₂ control mice (Lungkine^+/+) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in a specific pathogen-free environment. All experiments in which animals were used were conducted in accordance with the institutional guidelines of Schering-Plough and the University of Michigan.

RNA analysis

Total RNA was extracted from lung tissues of wild-type and Lungkine^−/− mice using an ULTRASPEC RNA isolation reagent (Biotecx, Houston, TX). Twenty micrograms of RNA was electrophoresed in a 1% agarose gel and blotted onto a Duralone membrane (Stratagene). A Lungkine cDNA probe was ³²P-labeled using the Megaprime DNA labeling random primer kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Hybridization was conducted at 68°C in QuickHyb hybridization solution (Stratagene), and the blots were washed according to the manufacturer’s protocol. The membranes were then exposed overnight to x-ray film at −70°C with an intensifying screen.

Histology and immunohistochemistry

Tissues were either fresh-frozen for cryosection or perfused, inflated (for lung only), fixed in 4% paraformaldehyde, and processed for paraffin sections. Routinely, 5-μm paraffin sections were cut and stained with hematoxylin and eosin. For immunostaining, fresh-frozen sections were fixed with cold acetone and stained with anti-CD11b/Mac-1 or anti-Gr-1 Abs (PharMingen, San Diego, CA). Binding of the Ab was amplified with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and detected with a diaminobenzidine substrate kit from Vector Laboratories. Slides were counterstained with hematoxylin.

Preparation of lung single-cell suspension

Single-cell suspensions of the lungs were prepared as previously described (8). Briefly, freshly resected lungs were minced with scissors to a fine slurry and incubated at 37°C for 30 min in an RPMI 1640 solution (Sigma, St. Louis, MO) containing 1 mg/ml type A collagenase (Roche, Indiana-polis, IN) and 30 U/ml DNase (Sigma). The solutions were then drawn up and down 20 times in 10-ml syringes (Becton Dickinson, Franklin Lakes, NJ) to disperse the cells mechanically. The resulting cell suspensions were pelleted, resuspended, passed through Nitex mesh filters (Tetko, Kansas City, MO), and passed through a 20% Percoll gradient (Sigma) before cell counting under a hemocytometer. Cytospin slides of this suspension were then prepared and stained (Diff-Quik Stain set; Dade Behring, Newark, DE), and differential cell counts were determined using a high-power microscope. The absolute number of a leukocyte subtype was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in that sample.

Flow cytometry

Total lung leukocytes were isolated from animals as described above. For identification of lung leukocyte subsets, FITC- or PE-conjugated CD4, CD8a, CD3, CD11c, B220, pan NK, Mac-1, F4/80, and Gr-1 were used (all reagents from PharMingen). In addition, cells were stained with anti-CD45 (PharMingen), allowing discrimination of leukocytes from nonleukocytes and thus eliminating any nonspecific binding of cell surface markers on nonleukocytes. Stained samples were kept in the dark at 4°C until analyzed on a FACScan cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson). Leukocyte populations were analyzed by gating on CD45-positive cells of the appropriate light scatter characteristics and then were examined for FL1 and FL2 fluorescence expression. The absolute number of a leukocyte type was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in that sample. For analyses of bone marrow and blood, single-cell suspensions were prepared from individual tissues by passage through a 100-μm pore size nylon cell strainer (BD Biosciences Labware, Bedford, MA) in RPMI 1640 medium containing 10% FCS.

Hematology

Blood samples were collected from the infraorbital sinus into sterile evacuated tubes with added EDTA (Vacutainer Systems; Becton Dickinson, Rutherford, NJ). Hematologic values were determined with an automated system (Cell-Dyn 3500; Abbott, Chicago, IL). Platelet counts were performed manually when the instrument was unable to provide accurate platelet counts due to excessive clumping or excessively large platelets.

Klebsiella pneumoniae inoculation

We chose to use K. pneumoniae strain 43816, serotype 2 (American Type Culture Collection, Manassas, VA) in our studies because a murine model of pneumonia has been well characterized using this strain (9, 10). K. pneumoniae was grown in tryptic soy broth (Difco, Detroit, MI) for 18 h at 37°C. The concentration of bacteria in broth was determined by measuring the amount of absorbance at 600 nm. A standard of absorbencies based on known CFU was used to calculate the inoculum concentration. An inoculum of 1 × 10² organisms was chosen, as this dose allowed for the development of substantial inflammation by 36–48 h without excessive mortality in wild-type animals. Mice were anesthetized with ∼1.8–2 mg of pentobarbital/animal i.p. The trachea was exposed, and 30 μl of inoculum or saline was administered via a sterile 26-gauge needle. The skin incision was closed with surgical staples.

Determination of lung K. pneumoniae CFU

At the time of sacrifice, the right ventricle was perfused with 1 ml of PBS, the lungs were removed aseptically and placed in 3 ml of sterile saline, and the tissues were homogenized with a tissue homogenizer under a vented hood. The lung homogenates were placed on ice, and serial 1/5 dilutions were made. Ten microliters of each dilution was plated on soy-base blood agar plates (Difco) and incubated for 18 h at 37°C, and then the colonies were counted.

Bronchoalveolar lavage (BAL)

BAL was performed to obtain airspace cells as previously described (11). The trachea was exposed and intubated using a 0.97-mm outside diameter polyethylene catheter. BAL was performed by instilling PBS containing 5 mM EDTA in 1-ml aliquots. Approximately 5 ml of lavage fluid was retrieved per mouse. Cytospins were then prepared from BAL cells and stained with Diff-Quik (Dade Behring, Newark, DE), and differential counts were determined.

Lung harvesting for cytokine analysis

At designated time points, mice were sacrificed by carbon dioxide inhalation, and blood was collected by direct cardiac puncture. The pulmonary vasculature was perfused with 1 ml of PBS containing 5 mM EDTA via the right ventricle, and whole lungs were harvested for assessment of the various cytokine protein levels. Lungs were homogenized in 1.5 ml of complete protease inhibitor lysis buffer (Roche). Homogenates were incubated on ice for 30 min, then centrifuged at 2500 rpm for 10 min. Supernatants were collected, passed through a 0.45-μm pore size filter (Gelman Sciences, Ann Arbor, MI), then stored at −20°C for assessment of cytokine levels.

Murine cytokine ELISA

Murine TNF-α, macrophage-inflammatory protein-2 (MIP-2), and KC were measured using a modification of a double-ligand method as described previously (12). Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F; Nunc, Copenhagen, Denmark) were coated with 50 μl/well rabbit Ab against the various cytokines (1 μg/ml in 0.6 M NaCl, 0.26 M H₃BO₄, and 0.08 M NaOH, pH 9.6) for 16 h at 4°C and then washed with PBS (pH 7.5) and 0.05% Tween 20 (wash buffer). Nonspecific binding sites on the microtiter plates were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. After rinsing four times with wash buffer, the plates were added with diluted (undiluted and 1/10) cell-free supernatants (50 μl) in duplicate, followed by incubation for 1 h at 37°C. Plates were washed four times, followed by the addition of 50 μl/well biotinylated rabbit Abs against the specific cytokines (3.5 μg/ml in PBS (pH 7.5), 0.05% Tween 20, and 2% FCS), and incubated for 30 min at 37°C. Streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was then added after washing the plates, and incubation proceeded for 30 min at 37°C. Plates were washed again four times, and chromogen substrate (Bio-Rad) was added. The plates were incubated at room temperature to the desired extinction, and the reaction was terminated with 50 μl/well 3 M H₂SO₄ solution. Plates were read at 490 nm in an ELISA plate reader. Standards were half-log dilutions of recombinant murine cytokines from 1 pg/ml to 100 ng/ml. This ELISA method consistently detected murine cytokine concentrations >25 pg/ml. The ELISA did not cross-react with IL-1, IL-2, IL-4, or IL-6. In addition, the ELISA did not cross-react with other members of the murine chemokine family, including murine MIP-1, JE, RANTES, or ENA-78.

In vivo neutrophil chemotaxis

The mouse air-pouch model for in vivo chemotaxis has been described in detail previously (13). Briefly, sex- and age-matched Lungkine^−/− and wild-type mice were anesthetized with ether. On experimental day 0, 5 ml of sterile air was injected s.c. under the dorsal skin; the resultant space was reinjected with 3 ml of sterile air on day 3. On day 5, 0.2 μg of Escherichia coli LPS (Sigma) in 1 ml of carboxymethylcellulose (0.5% in saline; Fluka, Buchs, Switzerland) was injected into the pouches. The animals were sacrificed 4 h later, and the air-pouchs were lavaged with 2 ml of sterile PBS. The resulting cell suspensions were pelleted, resuspended, and counted under a hemocytometer. Cytospin slides were prepared and stained (Diff-Quik Stain set; Dade Behring), and differential cell counts were determined using a high-power microscope. The absolute number of a leukocyte type was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in that sample.

Statistical analysis

Data were analyzed using the InStat version 2.01 statistical package (GraphPad software; GraphPad, San Diego, CA). Survival data were compared using Fisher’s exact test. All other data were expressed as the mean ± SEM and compared using an unpaired two-tailed Mann-Whitney U (nonparametric) test. A p < 0.05 was considered to be statistically significant.

Generation of Lungkine^−/− mice

DNA sequence analysis of the Lungkine genomic locus revealed that it contains three introns and four exons. The translation start site is located in exon 1, and the open reading frame spans all four exons (Fig. 1⇓A). A targeting strategy was designed to delete a 3-kb sequence of Lungkine genomic DNA that includes the entire first and second exons and part of the third exon (Fig. 1⇓A). The targeting vector shown in Fig. 1⇓ was used to transfect ES cells. Two independent ES cell clones containing the targeted loci were identified by Southern blot analysis and injected into mouse blastocysts. Chimeras obtained from both clones were bred with C57BL/6J mice to generate heterozygotes (Lungkine^+/−). PCR analysis of tail DNA from these founders confirmed germline transmission of the targeted allele (Fig. 1⇓B). Further intercrossing of these Lungkine^+/− mice yielded Lungkine^−/− mice within the expected Mendelian ratios, demonstrating that Lungkine expression is not essential for embryonic development.

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FIGURE 1.

Generation of Lungkine^−/− mice. A, Targeting strategy. The DNA structures of the targeting vector, the Lungkine locus, and the targeted locus are shown. Arrows indicate the transcriptional direction of each gene. Open rectangles, exons. A DNA probe (filled rectangle) from a 5′ upstream homologous region of Lungkine gene was used to screen the ES cells. A 7.6- and a 5-kb BamHI fragment were expected for the wild-type Lungkine locus and for the targeted locus, respectively. Restriction enzymes shown are: H, HindIII; H/P, HindIII/PlmI; B, BamHI; and E, EcoRI. B, An analysis of mouse genomic DNA by PCR. An example of PCR products of wild-type (+/+), Lungkine heterozygous (+/−), and Lungkine null (−/−) mice is shown. C, Northern blot analysis of Lungkine^−/− mice. Total RNA was isolated from lungs of two separate Lungkine^−/− lines (L #25 and L #69) and from wild-type mice. A total of 20 μg of total RNA was loaded on each lane. Lungkine cDNA was used as a probe for the hybridization. No Lungkine mRNA was detected in lungs from either line of Lungkine^−/− mice.

Northern blot analysis was used next to examine Lungkine mRNA expression in the lung tissues of Lungkine^−/− mice. As shown in Fig. 1⇑C, a Lungkine mRNA of the predicted size was detected in the wild-type mouse, but not in either line of Lungkine^−/− mice.

Lungkine^−/− mice develop normally and have a normal complement of leukocytes in the lung

The Lungkine^−/− mice developed normally and were fertile. Routine histologic analysis of all organs was unremarkable. Given Lungkine’s expression pattern and its chemotactic properties, we focused our initial analysis on the lung using immunohistochemistry and flow cytometry. Anti-Mac-1 and anti-Gr-1 Abs were used in immunohistochemical staining to examine the resident macrophage and neutrophil populations in the lung tissues of Lungkine^−/− animals. No significant differences from wild-type mice were seen in the number or localization of these cell types in the lungs of Lungkine^−/− mice (data not shown). The resident leukocyte population in lung tissue was further analyzed by flow cytometry. We did not detect significant differences in the absolute or relative numbers of distinct leukocytes (CD3-, CD4-, CD8a-, CD11c-, B220-, pan NK-, Mac-1-, F4/80-, and Gr-1-expressing subsets) between Lungkine^−/− and wild-type animals (n = 10; data not shown).

Lungkine^−/− mice have normal leukocyte subpopulations

To evaluate a possible role for Lungkine in hemopoiesis, leukocyte subpopulations in peripheral blood and bone marrow were analyzed by flow cytometry. Compared with wild-type mice, Lungkine^−/− mice had no significant changes in the absolute or relative numbers of distinct cell types (CD11c-, B220-, pan NK-, Mac-1-, F4/80-, Gr-1-, CD4-, CD8a-, and CD3-expressing subsets; n = 14 for bone marrow and n = 10 for blood; data not shown). Similarly, hematocrits and total white blood cell counts of Lungkine^−/− mice did not differ significantly from those of wild-type mice (n = 15; data not shown).

Lungkine^−/− mice are more susceptible to pneumonia induced by Klebsiella

Given the known chemotactic properties of this molecule for neutrophils and its high levels of expression in the lung (5), we next examined the susceptibility of Lungkine^−/− mice to Klebsiella pneumonia. Both Lungkine^−/− and wild-type animals developed lethargy and ruffled fur 48 h postinoculation (p.i.) with a sublethal dose. However, only 56% of Lungkine^−/− mice survived for longer than 10 days compared with an 85% survival rate for wild-type mice (Fig. 2⇓).

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FIGURE 2.

Survival following K. pneumoniae (1 × 10² CFU) infection. Data represent the average values of two separate experiments. There were 13 or 14 mice in each group. Values of p were <0.05 compared with wild-type mice.

To investigate the cause of the observed increased mortality in Lungkine^−/− mice, we measured the lung bacterial burden of Lungkine^−/− and wild-type control mice. Lungs of animals challenged with 1 × 10² CFU K. pneumoniae were harvested 48 h p.i. As shown in Fig. 3⇓, there was a 73-fold increase in lung bacterial burden in Lungkine^−/− mice compared with wild-type controls. This observation indicated that the absence of Lungkine resulted in significant impairment of the clearance of K. pneumoniae from the lung.

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FIGURE 3.

Quantification of lung K. pneumoniae CFU. Mice were challenged i.t. with K. pneumoniae (1 × 10² CFU), and their lungs were harvested 48 h later to determine K. pneumoniae CFU(log₁₀). Data represent the average values of two separate experiments. There were 12 mice/group. ∗, p = 0.025 compared with wild-type mice challenged with K. pneumoniae.

Characterization of inflammatory cell influx in lungs of K. pneumoniae-infected mice

To determine whether the increased bacterial CFU in Lungkine^−/− mice was associated with impaired cellular infiltration into the lung, we first examined the lungs of infected mice by histologic analysis at 24 and 48 h following Klebsiella inoculation. These time points were chosen to examine both early and maximum influxes of leukocytes, respectively (9). As shown in Fig. 4⇓, a dense neutrophilic infiltrate was observed in both the interstitial and alveolar compartments of wild-type lung. In contrast, neutrophils were strikingly absent from the alveoli of Lungkine^−/− lung sections at 24 h p.i. In addition, compared with wild-type mice, an increase in basophilically stained bacterial particles was seen in lungs of Lungkine^−/− mice at 48 h p.i. (Fig. 4⇓, C and D). To further characterize the apparent absence of airspace neutrophils in Lungkine^−/− lung at 24 h p.i., we quantified the cell numbers in the BAL. In uninfected mice no differences in the relative or absolute numbers of total BAL cells, neutrophils, or mononuclear cells were noted between wild-type and Lungkine^−/− mice. However, at 24 h p.i., an average 10-fold increase in the numbers of BAL neutrophils was observed in wild-type mice compared with baseline, whereas there was no appreciable increase in the numbers of airspace neutrophils in Lungkine^−/− mice (Fig. 5⇓A). In contrast, at 48 h the numbers of BAL neutrophils in Lungkine^−/− mice was even greater than that in wild-type animals, although this difference was not significant (p = 0.14). No significant differences in numbers of BAL mononuclear cells were observed at either 24 or 48 h following Klebsiella challenge (Fig. 5⇓B).

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FIGURE 4.

Lung histopathology following K. pneumoniae inoculation. Hematoxylin- and eosin-stained sections of lung harvested 24 h (A and B) and 48 h (C and D) after K. pneumoniae inoculation. A and C, Wild-type mice; B and D, Lungkine^−/− mice. In Lungkine^−/− mice, bacteria particles are visible under higher magnification at 48 h p.i. (arrows in inset of D; magnification, ×300). One of two representative experiments is shown.

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FIGURE 5.

Cell numbers in BAL of wild-type and Lungkine^−/− mice 24 and 48 h following K. pneumoniae inoculation. Panels depict BAL neutrophil and mononuclear cell numbers at baseline and 24 and 48 h after i.t. K. pneumoniae administration, respectively. Data represent the average values of four separate experiments for neutrophils and two separate experiments for mononuclear cells. There were 4 mice/group for uninfected animals and 10–22/group for Klebsiella-challenged animals. ∗, p = 0.0012 compared with wild-type mice challenged with K. pneumoniae.

Lungkine is produced primarily by airway epithelial cells and is secreted into the airway (5). To determine whether Lungkine preferentially affects infiltration of neutrophils into the airway or into the parenchyma, we infected mice with K. pneumonia and prepared cells from both the lung airspace (BAL) and the parenchyma 24 h after bacterial challenge. As shown above, a reduction in the numbers of infiltrating neutrophils was observed in the airspace compartment of Lungkine^−/− mice compared with that in wild-type mice. In contrast, higher numbers of neutrophils were observed in the parenchyma of Lungkine^−/− mice compared with that of wild-type mice, although no statistically significant difference was seen between the two groups (Fig. 6⇓). This result suggests that a major function of Lungkine is to facilitate migration of neutrophils from the parenchyma into the airway.

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FIGURE 6.

Parenchymal neutrophil cell numbers in the lungs of wild-type and Lungkine^−/− mice 24 h after K. pneumoniae inoculation. Data represent the average values of two separate experiments. There were 12 mice/group. p = 0.29 compared with wild-type mice challenged with K. pneumoniae.

Analysis of lung neutrophil-chemotactic mediators and extrapulmonary neutrophil chemotaxis in Lungkine^−/− mice

We next addressed whether a reduction in airway neutrophils of Lungkine-deficient mice is secondary to impaired production of other neutrophil chemotactic mediators. Levels of TNF-α, MIP-2, and KC in the lung were measured 24 h p.i. with K. pneumoniae. No significant differences in the levels of these cytokines were seen between lungs of wild-type and Lungkine-deficient animals (data not shown), indicating that the reduced neutrophil influx into the airway of Lungkine^−/− mice is independent of other neutrophil chemoattractants.

To assess whether the Lungkine deficiency results in a general defect in neutrophil mobilization, we measured neutrophil chemotaxis at an extrapulmonary site. We used a well-characterized model in which LPS is injected into an s.c. air-pouch, (13, 14). As shown in Fig. 7⇓, at 4 h postinjection, an early time point at which the accumulation of neutrophils can be reliably assessed (14), there was no significant difference in the number of neutrophils between wild-type and Lungkine^−/− animals (1.03 × 10⁷ ± 2.9 × 10⁶ and 8.6 × 10⁶ ± 2.7 × 10⁶ neutrophils, respectively). This indicates that Lungkine is not generally required for neutrophil trafficking and suggests that it is specific to the airspace of the lung.

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FIGURE 7.

Number of neutrophils in the s.c. air-pouch in wild-type and Lungkine^−/− mice after LPS instillation. LPS was injected into the air-pouches in wild-type and Lungkine^−/− mice, and the infiltrating cells were lavaged at 4 h after injection as described in Materials and Methods. There were six mice per group. p = 0.65 compared with wild-type mice challenged with LPS.