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*
Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033;
Division of Pulmonary and Critical Care Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lungkine is an ELR+CXC chemokine that is highly expressed by lung airway epithelial cells (5). It has recently been designated CXCL15, according to the new nomenclature system (6). The expression of Lungkine is up-regulated in response to a variety of inflammatory stimuli, including intratracheal challenge with LPS, Nippostronglyus, and Asperigillus (5). Because Lungkine induces neutrophil recruitment in vivo and in vitro (5) and because it is highly expressed in the lung in response to pathogens, it has been hypothesized that this chemokine may have a distinct biological role in pulmonary physiology, including host response to infection.
Recently, a novel hemopoietic regulatory factor, named WECHE, was isolated from an endothelial cell line using a differential gene expression method and was shown to be identical with Lungkine (7). During development, the expression of either the transcript or the protein of Lungkine/WECHE was found in sites of hemopoietic stem and progenitor cell production and maintenance, including day 9.5 dorsal aorta, day 10.5 yolk sac, aorta-gonad-mesonephrons, and fetal liver. In vitro, Lungkine inhibits the development of erythroid (BFU-E) progenitor cells obtained from either mouse fetal liver or bone marrow and is chemotactic for bone marrow cells in Transwell assays (7).
In an attempt to determine the biological roles of Lungkine in vivo, we have generated Lungkine null (-/-) mice. In this study, we report that Lungkine-/- mice have defects in pulmonary host defense.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 [
-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 x BL6 background. Age- and sex-matched (129 SvEv x 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 x 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-
, 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A). 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. 1B). 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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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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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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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. 5A). 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. 5B).
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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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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-, 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 x
107 ± 2.9 x 106 and
8.6 x 106 ± 2.7 x
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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rapid clearance of bacterial pathogens from the respiratory tract is mediated by resident alveolar macrophages and neutrophils that are recruited from the blood into the airspace (15, 16, 17). This neutrophil recruitment is mediated by the production in the lung of chemotactic cytokines (16). ELR+ CXC chemokines, including MIP-2 and KC, contribute to antibacterial host defense by affecting neutrophil trafficking and activation (9, 11, 18).
The increased mortality in Lungkine-/- mice following infection with K. pneumonia demonstrates that Lungkine is a chemokine that has a central role in pulmonary host defense. Our finding that the numbers of neutrophils are decreased in the BAL of infected Lungkine-/- mice at 24 h p.i. (but not in the parenchyma) suggests that one function of Lungkine might be to direct neutrophils from the parenchyma into the airspace. This reduction may be of notable functional importance, given the key role of neutrophils in innate defense against bacteria in the respiratory tract that have not been killed by resident alveolar macrophages (15). In fact, at 48 h p.i., the burden of bacteria was considerably greater in Lungkine-/- mice than in wild-type control animals. The increased bacterial load may lead to tissue invasion and dissemination that is not adequately controlled despite the impressive influx of neutrophils seen in Lungkine-/- mice at this time. A similar increase in both neutrophils and bacteria was seen in mice lacking the receptor for neutrophil chemoattractant C5a following infection with Pseudomonas aeruginosa, although the C5a-deficient mice did not display the early deficit in airway neutrophils seen in the Lungkine-/- mice (19). This increased neutrophilic content found in the airways of both these mouse strains is probably secondary to an increased bacterial load. However, as in all genetically engineered, loss-of-function mice, we cannot exclude the possibility that other unidentified, compensatory neutrophil attractants are up-regulated in Lungkine-/- mice. Lungkine protein has been shown by others to be secreted into the alveolar space in response to inflammatory stimuli (5). This pattern of expression and secretion may favor directed migration of leukocytes from the vascular and/or interstitial spaces into the airway lumen and alveolar spaces. If that is the case, it is unclear why so few neutrophils are normally present within the lung airspace and airway despite the fact that Lungkine is expressed at high levels in these structures. Apparent paradoxes of this sort are not unique to Lungkine. Other chemokines, such as eotaxin (20, 21), have potent chemoattractant roles in vitro and are expressed at high levels in tissues that are not normally infiltrated by eosinophils, the target cells of eotaxin. We propose that Lungkine may act as a permissive factor, facilitating the transmigration of neutrophils into the airways during the early phases of inflammation. For example, Lungkine may act specifically on activated neutrophils or may work in concert with undefined molecules that are present only on the surface of inflamed endothelium. It is also possible that Lungkine, like other ELR+ CXC chemokines, may affect polymorphonuclear leukocyte activities, including respiratory burst and antimicrobial activities (1, 2, 22). Finally, the site of production (distal airway epithelial cells) and the magnitude of expression show similarities to the expression of defensin proteins (23), and this raises the possibility that Lungkine may have direct antimicrobial properties that are independent of its effects on neutrophils. Exploring these mechanistic possibilities will probably contribute to a better understanding of the role of Lungkine in host defenses and to insights into the biology of a subset of chemokines expressed constitutively in specific organs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sergio A. Lira, Department of Immunology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033.
3 Abbreviations used in this paper: ES, embryonic stem; p.i., postinoculation; BAL, bronchoalveolar lavage; MIP-2, macrophage-inflammatory protein-2.
Received for publication October 12, 2000. Accepted for publication December 14, 2000.
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