Lungkine Lacking Expression of the CXC Chemokine Impaired

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

This information is current as Shu-Cheng Chen, Borna Mehrad, Jane C. Deng, Galya of September 27, 2021. Vassileva, Denise J. Manfra, Donald N. Cook, Maria T. Wiekowski, Albert Zlotnik, Theodore J. Standiford and Sergio A. Lira J Immunol 2001; 166:3362-3368; ;

doi: 10.4049/jimmunol.166.5.3362 Downloaded from http://www.jimmunol.org/content/166/5/3362

References This article cites 22 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/166/5/3362.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Impaired Pulmonary Host Defense in Mice Lacking Expression of the CXC Chemokine Lungkine1

Shu-Cheng Chen,* Borna Mehrad,† Jane C. Deng,‡ Galya Vassileva,* Denise J. Manfra,* Donald N. Cook,* Maria T. Wiekowski,* Albert Zlotnik,§ Theodore J. Standiford,‡ and Sergio A. Lira2*

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but Downloaded from reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these ﬁndings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace. The Journal of Im- munology, 2001, 166: 3362–3368.

hemokines are a large family of 8- to 10-kDa polypeptide hypothesized that this chemokine may have a distinct biological http://www.jimmunol.org/ molecules that regulate the immune system in many as- role in pulmonary physiology, including host response to infection. C pects, including chemotaxis of leukocytes, hemopoiesis, Recently, a novel hemopoietic regulatory factor, named and angiogenesis (1–4). Based on the composition of a cysteine WECHE, was isolated from an endothelial cell line using a dif- motif located near the N terminus of these molecules, they are ferential gene expression method and was shown to be identical classiﬁed into four subgroups: C, CC, CXC, and CX3C. The CXC with Lungkine (7). During development, the expression of either chemokines are characterized by four cysteine residues at the N the transcript or the protein of Lungkine/WECHE was found in terminus, the ﬁrst two of which are separated by a nonconserved sites of hemopoietic stem and progenitor cell production and main-

amino acid. They are further classified by the presence or the ab- tenance, including day 9.5 dorsal aorta, day 10.5 yolk sac, aorta- by guest on September 27, 2021 sence of the amino acid sequence glutamic acid-leucine-arginine gonad-mesonephrons, and fetal liver. In vitro, Lungkine inhibits (the ELR motif) immediately preceding the CXC sequence. Nearly the development of erythroid (BFU-E) progenitor cells obtained all of the known neutrophil-targeted chemokines belong to the from either mouse fetal liver or bone marrow and is chemotactic ELRϩCXC chemokine family. for bone marrow cells in Transwell assays (7). Lungkine is an ELRϩCXC chemokine that is highly expressed In an attempt to determine the biological roles of Lungkine in by lung airway epithelial cells (5). It has recently been designated vivo, we have generated Lungkine null (Ϫ/Ϫ) mice. In this study, Ϫ/Ϫ CXCL15, according to the new nomenclature system (6). The ex- we report that Lungkine mice have defects in pulmonary host pression of Lungkine is up-regulated in response to a variety of defense. inflammatory stimuli, including intratracheal challenge with LPS, Nippostronglyus, and Asperigillus (5). Because Lungkine induces Materials and Methods neutrophil recruitment in vivo and in vitro (5) and because it is Gene targeting of Lungkine highly expressed in the lung in response to pathogens, it has been 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 (5Ј-TTAGGCAT CACTGCCTGTCA-3Ј) and GV16 (5Ј-AATGAAGCTTCTGTATAGT *Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 3Ј). Several fragments containing the Lungkine gene were identified by 07033; †Division of Pulmonary and Critical Care Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; ‡Department of Medicine, Division Southern blot analysis of BAC plasmid DNA using a 500-bp cDNA probe ␥ 32 of Pulmonary and Critical Care Medicine, University of Michigan Medical School, labeled with [ - P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, Ann Arbor, MI 48109; and §DNAX Research Institute, Palo Alto, CA 94304 IL) by random priming (Megaprime DNA Labeling System; Amersham Pharmacia Biotech, Piscataway, NJ). Two BamHI fragments (7.6 kb each) Received for publication October 12, 2000. Accepted for publication December Ј Ј 14, 2000. 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 re- The costs of publication of this article were defrayed in part by the payment of page striction digest. The complete intron and exon sequences of these two charges. This article must therefore be hereby marked advertisement in accordance subclones were further analyzed using an Applied Biosystems 377 DNA with 18 U.S.C. Section 1734 solely to indicate this fact. sequencer (Applied Biosystems, Foster City, CA). DNA fragments corre- 1 This work was supported in part by National Institutes of Health Grants sponding to the 5Ј and 3Ј regions of the Lungkine locus were, in turn, RO1HL56402 (to T.J.S.), RO1HL57243 (to T.J.S.), P50HL60289 (to T.J.S.), and subcloned from this plasmid into the pOSDupDel vector (a gift from Oliver K08HL04220 (to B.M.) and American Lung Association Grant RG-005-N (to B.M.). 2 Address correspondence and reprint requests to Dr. Sergio A. Lira, Department of Immunology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Ken- 3 Abbreviations used in this paper: ES, embryonic stem; p.i., postinoculation; BAL, ilworth, NJ 07033. E-mail address: [email protected] bronchoalveolar lavage; MIP-2, macrophage-inflammatory protein-2.

Smithies, University of North Carolina, Chapel Hill, NC) at either end of characteristics and then were examined for FL1 and FL2 fluorescence ex- the neomycin resistance gene (neo). This targeting vector DNA was lin- pression. The absolute number of a leukocyte type was determined by earized using the restriction enzyme NotI before its introduction into 129/ multiplication of the percentage of that cell type by the total number of Sv-derived ES cells by electroporation. Lungkine-targeted ES clones were lung leukocytes in that sample. For analyses of bone marrow and blood, identified by Southern blotting. DNA was digested with BamHI, trans- single-cell suspensions were prepared from individual tissues by passage ferred to nylon membranes, and hybridized to a radiolabeled DNA probe through a 100-␮m pore size nylon cell strainer (BD Biosciences Labware, derived from a region upstream of the 5Ј region of homology. Cells whose Bedford, MA) in RPMI 1640 medium containing 10% FCS. DNAs were of the structure predicted for the targeted locus were used to generate chimeric mice using standard blastocyst injection procedures. Hematology Several correctly targeted ES cells were injected into blastocysts to gen- Blood samples were collected from the infraorbital sinus into sterile evac- erate chimeric mice. Lungkine heterozygous (ϩ/Ϫ) offspring were identi- uated tubes with added EDTA (Vacutainer Systems; Becton Dickinson, fied using a PCR-based screening strategy using three oligonucleotide Rutherford, NJ). Hematologic values were determined with an automated primers corresponding to the region of homology, the neo gene, and the Ϫ Ϫ system (Cell-Dyn 3500; Abbott, Chicago, IL). Platelet counts were per- deleted region of the Lungkine gene. Lungkine / mice were generated by formed manually when the instrument was unable to provide accurate interbreeding these heterozygous mice. platelet counts due to excessive clumping or excessively large platelets. Mice Klebsiella pneumoniae inoculation For the experiments described here we used LungkineϪ/Ϫ mice generated in the 129 ϫ BL6 background. Age- and sex-matched (129 SvEv ϫ We chose to use K. pneumoniae strain 43816, serotype 2 (American Type ϩ/ϩ Culture Collection, Manassas, VA) in our studies because a murine model C57BL/6)F2 control mice (Lungkine ) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in a specific patho- of pneumonia has been well characterized using this strain (9, 10). K. gen-free environment. All experiments in which animals were used were pneumoniae was grown in tryptic soy broth (Difco, Detroit, MI) for 18 h conducted in accordance with the institutional guidelines of Schering- at 37°C. The concentration of bacteria in broth was determined by mea- Downloaded from Plough and the University of Michigan. suring the amount of absorbance at 600 nm. A standard of absorbencies based on known CFU was used to calculate the inoculum concentration. An RNA analysis inoculum of 1 ϫ 102 organisms was chosen, as this dose allowed for the development of substantial inflammation by 36–48 h without excessive Ϫ/Ϫ Total RNA was extracted from lung tissues of wild-type and Lungkine mortality in wild-type animals. Mice were anesthetized with ϳ1.8–2 mg of mice using an ULTRASPEC RNA isolation reagent (Biotecx, Houston, pentobarbital/animal i.p. The trachea was exposed, and 30 ␮l of inoculum TX). Twenty micrograms of RNA was electrophoresed in a 1% agarose gel or saline was administered via a sterile 26-gauge needle. The skin incision and blotted onto a Duralone membrane (Stratagene). A Lungkine cDNA was closed with surgical staples. http://www.jimmunol.org/ probe was 32P-labeled using the Megaprime DNA labeling random primer kit (Amersham Pharmacia Biotech) according to the manufacturer’s in- Determination of lung K. pneumoniae CFU structions. Hybridization was conducted at 68°C in QuickHyb hybridization solution (Stratagene), and the blots were washed according to the At the time of sacrifice, the right ventricle was perfused with 1 ml of PBS, manufacturer’s protocol. The membranes were then exposed overnight to the lungs were removed aseptically and placed in 3 ml of sterile saline, and x-ray film at Ϫ70°C with an intensifying screen. the tissues were homogenized with a tissue homogenizer under a vented hood. The lung homogenates were placed on ice, and serial 1/5 dilutions Histology and immunohistochemistry 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 Tissues were either fresh-frozen for cryosection or perfused, inflated (for were counted. lung only), fixed in 4% paraformaldehyde, and processed for paraffin sec- by guest on September 27, 2021 tions. Routinely, 5-␮m paraffin sections were cut and stained with hema- Bronchoalveolar lavage (BAL) toxylin and eosin. For immunostaining, fresh-frozen sections were fixed with cold acetone and stained with anti-CD11b/Mac-1 or anti-Gr-1 Abs BAL was performed to obtain airspace cells as previously described (11). (PharMingen, San Diego, CA). Binding of the Ab was amplified with a The trachea was exposed and intubated using a 0.97-mm outside diameter Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and de- polyethylene catheter. BAL was performed by instilling PBS containing tected with a diaminobenzidine substrate kit from Vector Laboratories. 5 mM EDTA in 1-ml aliquots. Approximately 5 ml of lavage fluid was Slides were counterstained with hematoxylin. retrieved per mouse. Cytospins were then prepared from BAL cells and stained with Diff-Quik (Dade Behring, Newark, DE), and differential Preparation of lung single-cell suspension counts were determined. Single-cell suspensions of the lungs were prepared as previously described Lung harvesting for cytokine analysis (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, At designated time points, mice were sacrificed by carbon dioxide inhala- St. Louis, MO) containing 1 mg/ml type A collagenase (Roche, Indiana- tion, and blood was collected by direct cardiac puncture. The pulmonary polis, IN) and 30 U/ml DNase (Sigma). The solutions were then drawn up vasculature was perfused with 1 ml of PBS containing 5 mM EDTA via the and down 20 times in 10-ml syringes (Becton Dickinson, Franklin Lakes, right ventricle, and whole lungs were harvested for assessment of the var- NJ) to disperse the cells mechanically. The resulting cell suspensions were ious cytokine protein levels. Lungs were homogenized in 1.5 ml of com- pelleted, resuspended, passed through Nitex mesh filters (Tetko, Kansas plete protease inhibitor lysis buffer (Roche). Homogenates were incubated City, MO), and passed through a 20% Percoll gradient (Sigma) before cell on ice for 30 min, then centrifuged at 2500 rpm for 10 min. Supernatants counting under a hemocytometer. Cytospin slides of this suspension were were collected, passed through a 0.45-␮m pore size filter (Gelman Sci- then prepared and stained (Diff-Quik Stain set; Dade Behring, Newark, ences, Ann Arbor, MI), then stored at Ϫ20°C for assessment of cytokine DE), and differential cell counts were determined using a high-power mi- levels. croscope. The absolute number of a leukocyte subtype was determined by multiplication of the percentage of that cell type by the total number of Murine cytokine ELISA lung leukocytes in that sample. Murine TNF-␣, macrophage-inflammatory protein-2 (MIP-2), and KC Flow cytometry were measured using a modification of a double-ligand method as described previously (12). Briefly, flat-bottom 96-well microtiter plates (Im- Total lung leukocytes were isolated from animals as described above. For muno-Plate I 96-F; Nunc, Copenhagen, Denmark) were coated with 50 identification of lung leukocyte subsets, FITC- or PE-conjugated CD4, ␮l/well rabbit Ab against the various cytokines (1 ␮g/ml in 0.6 M NaCl,

CD8a, CD3, CD11c, B220, pan NK, Mac-1, F4/80, and Gr-1 were used (all 0.26 M H3BO4, and 0.08 M NaOH, pH 9.6) for 16 h at 4°C and then reagents from PharMingen). In addition, cells were stained with anti-CD45 washed with PBS (pH 7.5) and 0.05% Tween 20 (wash buffer). Nonspecific (PharMingen), allowing discrimination of leukocytes from nonleukocytes binding sites on the microtiter plates were blocked with 2% BSA in PBS and thus eliminating any nonspecific binding of cell surface markers on and incubated for 90 min at 37°C. After rinsing four times with wash nonleukocytes. Stained samples were kept in the dark at 4°C until analyzed buffer, the plates were added with diluted (undiluted and 1/10) cell-free on a FACScan cytometer (Becton Dickinson, San Jose, CA) using supernatants (50 ␮l) in duplicate, followed by incubation for 1 h at 37°C. CellQuest software (Becton Dickinson). Leukocyte populations were ana- Plates were washed four times, followed by the addition of 50 ␮l/well lyzed by gating on CD45-positive cells of the appropriate light scatter biotinylated rabbit Abs against the specific cytokines (3.5 ␮g/ml in PBS 3364 LUNGKINE IN HOST DEFENSE Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Generation of LungkineϪ/Ϫ 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 5Ј 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 (ϩ/ϩ), Lungkine heterozygous (ϩ/Ϫ), and Lungkine null (Ϫ/Ϫ) mice is shown. C, Northern blot analysis of LungkineϪ/Ϫ mice. Total RNA was isolated from lungs of two separate LungkineϪ/Ϫ lines (L #25 and L #69) and from wild-type mice. A total of 20 ␮g 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 LungkineϪ/Ϫ mice.

(pH 7.5), 0.05% Tween 20, and 2% FCS), and incubated for 30 min at of sterile air was injected s.c. under the dorsal skin; the resultant space was 37°C. Streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was reinjected with 3 ml of sterile air on day 3. On day 5, 0.2 ␮gofEscherichia then added after washing the plates, and incubation proceeded for 30 min coli LPS (Sigma) in 1 ml of carboxymethylcellulose (0.5% in saline; Fluka, at 37°C. Plates were washed again four times, and chromogen substrate Buchs, Switzerland) was injected into the pouches. The animals were sac- (Bio-Rad) was added. The plates were incubated at room temperature to the riﬁced 4 h later, and the air-pouchs were lavaged with 2 ml of sterile PBS. desired extinction, and the reaction was terminated with 50 ␮l/well 3 M The resulting cell suspensions were pelleted, resuspended, and counted

H2SO4 solution. Plates were read at 490 nm in an ELISA plate reader. under a hemocytometer. Cytospin slides were prepared and stained (Diff- Standards were half-log dilutions of recombinant murine cytokines from 1 Quik Stain set; Dade Behring), and differential cell counts were determined pg/ml to 100 ng/ml. This ELISA method consistently detected murine cy- using a high-power microscope. The absolute number of a leukocyte type tokine concentrations Ͼ25 pg/ml. The ELISA did not cross-react with was determined by multiplication of the percentage of that cell type by the IL-1, IL-2, IL-4, or IL-6. In addition, the ELISA did not cross-react with total number of lung leukocytes in that sample. other members of the murine chemokine family, including murine MIP-1, JE, RANTES, or ENA-78. Statistical analysis In vivo neutrophil chemotaxis Data were analyzed using the InStat version 2.01 statistical package The mouse air-pouch model for in vivo chemotaxis has been described in (GraphPad software; GraphPad, San Diego, CA). Survival data were com- detail previously (13). Brieﬂy, sex- and age-matched LungkineϪ/Ϫ and pared using Fisher’s exact test. All other data were expressed as the wild-type mice were anesthetized with ether. On experimental day 0, 5 ml mean Ϯ SEM and compared using an unpaired two-tailed Mann-Whitney The Journal of Immunology 3365

LungkineϪ/Ϫ 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, LungkineϪ/Ϫ 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 ϭ 14 for bone marrow and n ϭ 10 for blood; data not shown). Similarly, hematocrits and total white blood cell counts of LungkineϪ/Ϫ mice did not differ significantly from those of wild- type mice (n ϭ 15; data not shown).

LungkineϪ/Ϫ mice are more susceptible to pneumonia induced by Klebsiella FIGURE 2. Survival following K. pneumoniae (1 ϫ 102 CFU) infection. Data represent the average values of two separate experiments. Given the known chemotactic properties of this molecule for neu- There were 13 or 14 mice in each group. Values of p were Ͻ0.05 trophils and its high levels of expression in the lung (5), we next compared with wild-type mice. examined the susceptibility of LungkineϪ/Ϫ mice to Klebsiella pneumonia. Both LungkineϪ/Ϫ and wild-type animals developed Downloaded from Ͻ lethargy and rufﬂed fur 48 h postinoculation (p.i.) with a sublethal U (nonparametric) test. A p 0.05 was considered to be statistically Ϫ/Ϫ signiﬁcant. dose. However, only 56% of Lungkine mice survived for longer than 10 days compared with an 85% survival rate for wild- Results type mice (Fig. 2). Generation of LungkineϪ/Ϫ mice To investigate the cause of the observed increased mortality in LungkineϪ/Ϫ mice, we measured the lung bacterial burden of

DNA sequence analysis of the Lungkine genomic locus revealed http://www.jimmunol.org/ LungkineϪ/Ϫ and wild-type control mice. Lungs of animals chal- that it contains three introns and four exons. The translation start lenged with 1 ϫ 102 CFU K. pneumoniae were harvested 48 h p.i. site is located in exon 1, and the open reading frame spans all four As shown in Fig. 3, there was a 73-fold increase in lung bacterial exons (Fig. 1A). A targeting strategy was designed to delete a 3-kb burden in LungkineϪ/Ϫ mice compared with wild-type controls. sequence of Lungkine genomic DNA that includes the entire first This observation indicated that the absence of Lungkine resulted in and second exons and part of the third exon (Fig. 1A). The target- significant impairment of the clearance of K. pneumoniae from ing vector shown in Fig. 1 was used to transfect ES cells. Two the lung. independent ES cell clones containing the targeted loci were identified by Southern blot analysis and injected into mouse blasto- Characterization of inflammatory cell influx in lungs of K. cysts. Chimeras obtained from both clones were bred with pneumoniae-infected mice by guest on September 27, 2021 ϩ/Ϫ C57BL/6J mice to generate heterozygotes (Lungkine ). PCR Ϫ/Ϫ analysis of tail DNA from these founders confirmed germline To determine whether the increased bacterial CFU in Lungkine transmission of the targeted allele (Fig. 1B). Further intercrossing mice was associated with impaired cellular infiltration into the of these Lungkineϩ/Ϫ mice yielded LungkineϪ/Ϫ mice within the lung, we first examined the lungs of infected mice by histologic expected Mendelian ratios, demonstrating that Lungkine expres- analysis at 24 and 48 h following Klebsiella inoculation. These sion is not essential for embryonic development. time points were chosen to examine both early and maximum in- Northern blot analysis was used next to examine Lungkine fluxes of leukocytes, respectively (9). As shown in Fig. 4, a dense mRNA expression in the lung tissues of LungkineϪ/Ϫ mice. As neutrophilic infiltrate was observed in both the interstitial and alveolar compartments of wild-type lung. In contrast, neutrophils shown in Fig. 1C, a Lungkine mRNA of the predicted size was Ϫ/Ϫ detected in the wild-type mouse, but not in either line of were strikingly absent from the alveoli of Lungkine lung sec- LungkineϪ/Ϫ mice. tions at 24 h p.i. In addition, compared with wild-type mice, an

LungkineϪ/Ϫ mice develop normally and have a normal complement of leukocytes in the lung The LungkineϪ/Ϫ mice developed normally and were fertile. Rou- tine 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 LungkineϪ/Ϫ animals. No significant differences from wild-type mice were seen in the number or localization of these cell types in the lungs of LungkineϪ/Ϫ mice (data not shown). The resident leukocyte population in lung tissue was further analyzed by flow FIGURE 3. Quantification of lung K. pneumoniae CFU. Mice were cytometry. We did not detect significant differences in the absolute challenged i.t. with K. pneumoniae (1 ϫ 102 CFU), and their lungs were or relative numbers of distinct leukocytes (CD3-, CD4-, CD8a-, harvested 48 h later to determine K. pneumoniae CFU(log10). Data repre- CD11c-, B220-, pan NK-, Mac-1-, F4/80-, and Gr-1-expressing sent the average values of two separate experiments. There were 12 mice/ Ϫ/Ϫ .p ϭ 0.025 compared with wild-type mice challenged with K ,ء .subsets) between Lungkine and wild-type animals (n ϭ 10; group data not shown). pneumoniae. 3366 LUNGKINE IN HOST DEFENSE

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, LungkineϪ/Ϫ mice. In LungkineϪ/Ϫ mice, bacteria particles are visible under higher magnification at 48 h p.i. (arrows in inset of D; magnification, ϫ300). One of two representative experiments is shown. Downloaded from http://www.jimmunol.org/ increase in basophilically stained bacterial particles was seen in the parenchyma, we infected mice with K. pneumonia and pre- lungs of LungkineϪ/Ϫ mice at 48 h p.i. (Fig. 4, C and D). To pared cells from both the lung airspace (BAL) and the parenchyma further characterize the apparent absence of airspace neutrophils in 24 h after bacterial challenge. As shown above, a reduction in the LungkineϪ/Ϫ lung at 24 h p.i., we quantified the cell numbers in numbers of infiltrating neutrophils was observed in the airspace the BAL. In uninfected mice no differences in the relative or ab- compartment of LungkineϪ/Ϫ mice compared with that in wild- solute numbers of total BAL cells, neutrophils, or mononuclear type mice. In contrast, higher numbers of neutrophils were ob- Ϫ Ϫ Ϫ Ϫ cells were noted between wild-type and Lungkine / mice. How- served in the parenchyma of Lungkine / mice compared with by guest on September 27, 2021 ever, at 24 h p.i., an average 10-fold increase in the numbers of that of wild-type mice, although no statistically significant differ- BAL neutrophils was observed in wild-type mice compared with ence was seen between the two groups (Fig. 6). This result sug- baseline, whereas there was no appreciable increase in the numbers gests that a major function of Lungkine is to facilitate migration of of airspace neutrophils in LungkineϪ/Ϫ mice (Fig. 5A). In contrast, neutrophils from the parenchyma into the airway. at 48 h the numbers of BAL neutrophils in LungkineϪ/Ϫ mice was Analysis of lung neutrophil-chemotactic mediators and even greater than that in wild-type animals, although this differ- Ϫ/Ϫ ence was not significant ( p ϭ 0.14). No significant differences in extrapulmonary neutrophil chemotaxis in Lungkine mice numbers of BAL mononuclear cells were observed at either 24 or We next addressed whether a reduction in airway neutrophils of 48 h following Klebsiella challenge (Fig. 5B). Lungkine-deficient mice is secondary to impaired production of Lungkine is produced primarily by airway epithelial cells and is other neutrophil chemotactic mediators. Levels of TNF-␣, MIP-2, secreted into the airway (5). To determine whether Lungkine pref- and KC in the lung were measured 24 h p.i. with K. pneumoniae. erentially affects infiltration of neutrophils into the airway or into No significant differences in the levels of these cytokines were

FIGURE 5. Cell numbers in BAL of wild- type and LungkineϪ/Ϫ 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/ p ϭ ,ء .group for Klebsiella-challenged animals 0.0012 compared with wild-type mice challenged with K. pneumoniae. The Journal of Immunology 3367

Discussion In this report, we describe the generation and preliminary characterization of mice lacking the novel CXC chemokine Lungkine. In the adult mouse, Lungkine is produced at appreciable levels only by lung epithelial cells and collects in the lung airspace, suggesting that it might function in pulmonary host defense. We now show that deletion of the Lungkine gene is associated with diminished host defense against the pulmonary pathogen K. pneumoniae. 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 (15–17). This neutrophil recruitment is mediated by the production in the lung of chemotactic cytokines (16). ELRϩ CXC chemokines, including MIP-2 and KC, contribute to antibacterial host defense by affecting neutrophil trafficking and activation (9, 11, 18). FIGURE 6. Parenchymal neutrophil cell numbers in the lungs of wild- Ϫ/Ϫ type and LungkineϪ/Ϫ mice 24 h after K. pneumoniae inoculation. Data The increased mortality in Lungkine mice following infec- represent the average values of two separate experiments. There were 12 tion with K. pneumonia demonstrates that Lungkine is a chemo- mice/group. p ϭ 0.29 compared with wild-type mice challenged with K. kine that has a central role in pulmonary host defense. Our finding Downloaded from pneumoniae. that the numbers of neutrophils are decreased in the BAL of infected LungkineϪ/Ϫ 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 seen between lungs of wild-type and Lungkine-deficient animals be of notable functional importance, given the key role of neutro- (data not shown), indicating that the reduced neutrophil influx into phils in innate defense against bacteria in the respiratory tract that the airway of LungkineϪ/Ϫ mice is independent of other neutrophil have not been killed by resident alveolar macrophages (15). In http://www.jimmunol.org/ chemoattractants. fact, at 48 h p.i., the burden of bacteria was considerably greater in To assess whether the Lungkine deficiency results in a general LungkineϪ/Ϫ mice than in wild-type control animals. The in- defect in neutrophil mobilization, we measured neutrophil chemo- creased bacterial load may lead to tissue invasion and dissemina- taxis at an extrapulmonary site. We used a well-characterized tion that is not adequately controlled despite the impressive influx model in which LPS is injected into an s.c. air-pouch, (13, 14). As of neutrophils seen in LungkineϪ/Ϫ mice at this time. A similar shown in Fig. 7, at 4 h postinjection, an early time point at which increase in both neutrophils and bacteria was seen in mice lacking the accumulation of neutrophils can be reliably assessed (14), there the receptor for neutrophil chemoattractant C5a following infec-

was no significant difference in the number of neutrophils between tion with Pseudomonas aeruginosa, although the C5a-deficient by guest on September 27, 2021 Ϫ Ϫ wild-type and Lungkine / animals (1.03 ϫ 107 Ϯ 2.9 ϫ 106 and mice did not display the early deficit in airway neutrophils seen in 8.6 ϫ 106 Ϯ 2.7 ϫ 106 neutrophils, respectively). This indicates the LungkineϪ/Ϫ mice (19). This increased neutrophilic content that Lungkine is not generally required for neutrophil trafficking found in the airways of both these mouse strains is probably sec- and suggests that it is specific to the airspace of the lung. ondary to an increased bacterial load. However, as in all geneti- cally engineered, loss-of-function mice, we cannot exclude the possibility that other unidentified, compensatory neutrophil attract- ants are up-regulated in LungkineϪ/Ϫ 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 ELRϩ CXC chemokines, may affect polymorphonuclear leukocyte FIGURE 7. Number of neutrophils in the s.c. air-pouch in wild-type and Ϫ Ϫ activities, including respiratory burst and antimicrobial activities Lungkine / mice after LPS instillation. LPS was injected into the air- pouches in wild-type and LungkineϪ/Ϫ mice, and the infiltrating cells were (1, 2, 22). Finally, the site of production (distal airway epithelial lavaged at 4 h after injection as described in Materials and Methods. There cells) and the magnitude of expression show similarities to the were six mice per group. p ϭ 0.65 compared with wild-type mice chal- expression of defensin proteins (23), and this raises the possibility lenged with LPS. that Lungkine may have direct antimicrobial properties that are 3368 LUNGKINE IN HOST DEFENSE independent of its effects on neutrophils. Exploring these mecha- 10. Greenberger, M. J., R. M. Strieter, S. L. Kunkel, J. M. Danforth, R. E. Goodman, nistic possibilities will probably contribute to a better understand- and T. J. Standiford. 1995. Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia. J. Immunol. 155:722. ing of the role of Lungkine in host defenses and to insights into the 11. Tsai, W. C., R. M. Strieter, J. M. Wilkowski, K. A. Bucknell, M. D. Burdick, biology of a subset of chemokines expressed constitutively in spe- S. A. Lira, and T. J. Standiford. 1998. Lung-specific transgenic expression of KC enhances resistance to Klebsiella pneumoniae in mice. J. Immunol. 161:2435. cific organs. 12. Rovai, L. E., H. R. Herschman, and J. B. Smith. 1998. The murine neutrophil- chemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinet- ics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regula- Acknowledgments tion in endotoxemia. J. Leukocyte Biol. 64:494. We thank Channa Young, Petronio Zalamea, Linda Hamilton, Susan Ab- 13. Edwards, J. 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