RAPID COMMUNICATION
Cloning of rat eotaxin: ozone inhalation increases mRNA and protein expression in lungs of Brown Norway rats

Yukio Ishii1, Manabu Shirato1, Akihiro Nomura1, Tohru Sakamoto1, Yoshiyuki Uchida1, Morio Ohtsuka1, Masaru Sagai2, and Shizuo Hasegawa1

1 Department of Respiratory Medicine, Institute of Clinical Medical Sciences, University of Tsukuba, and 2 National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The C-C chemokine eotaxin is thought to be important in the selective recruitment of eosinophils to the site of inflammation in guinea pigs, mice, and humans. We isolated the rat eotaxin gene to determine whether a similar molecule might play a role in the pulmonary infiltration of eosinophils during acute inflammation in the rat. The cDNA for rat eotaxin encoded a 97-amino acid protein containing a 74-amino acid mature eotaxin protein with 97.3% identity to mouse eotaxin. The recombinant protein encoded by this gene displayed specific chemotactic activity for eosinophils when analyzed with a microchemotactic chamber. The expression of eotaxin mRNA increased ~1.6-fold immediately after exposure to ozone and was 4-fold higher after 20 h. The number of lavageable eosinophils at the same time points were 3- and 15-fold greater, respectively, than control eosinophils. Immunocytochemistry revealed that alveolar macrophages and bronchial epithelial cells were positive for eotaxin. These results suggest that eotaxin may be involved in the recruitment of eosinophils into the air spaces during certain inflammatory conditions in rats.

messenger ribonucleic acid; chemokine; cytokine; inflammation; eosinophils

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE INFILTRATION of inflammatory cells into the airways is a pathological characteristic of pulmonary inflammation. Among these cells are the eosinophils, which are activated at the site of inflammation to release preformed cationic proteins, oxygen radicals, and lipid mediators (7, 26). This leads to hyperresponsiveness of the airway to certain stimuli and to damage to the airway epithelium (3, 25). Although eosinophils constitute only a minority of the circulating leukocytes, they are recruited in large numbers into tissue sites during inflammation, suggesting that specific chemotactic factors are involved in their recruitment.

The mechanism underlying the migration of eosinophils into tissue sites is not completely understood. It has been reported that the chemotactic cytokines, especially the C-C chemokines, are important (1, 5, 20). Eotaxin, a novel member of the C-C chemokine family, was recently identified, and the eotaxin genes from guinea pigs (13, 22), mice (8, 21), and humans (6, 18) have been cloned. Eotaxin displays potent and specific chemotactic activity for eosinophils in these three species both in vivo and in vitro. The expression of eotaxin mRNA was shown to be induced in the lungs during allergic inflammation. Moreover, the levels of eotaxin mRNA paralleled the kinetics of eosinophil accumulation during such inflammation (8).

To determine whether a similar molecule might be involved in the infiltration of eosinophils into the lungs during acute inflammation in rats, we molecularly cloned the rat homologue of the eotaxin gene and analyzed the expression of its mRNA and protein products during eosinophilic inflammation of the lungs in Brown Norway (BN) rats exposed to ozone. The role of eotaxin in the recruitment of eosinophils into the lung was investigated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals and exposure to ozone. Male BN rats (Charles River Laboratories, Kanagawa, Japan), 200-250 g, were exposed for 6 h to 1.2 parts/million (ppm) ozone in a chamber 1.68 m3. Ozone was generated from pure oxygen by an Arc generator and was continuously monitored with an Ox analyzer (model 806, Kimoto-Denshi, Tokyo, Japan).

Bronchoalveolar lavage. The lungs of anesthetized rats (50 mg/kg body wt ip of pentobarbital sodium) were lavaged with a total volume of 48 ml of phosphate-buffered saline (PBS) containing 3 mM EDTA before, immediately after, and 20 h after exposure to ozone. At each time point, lavaged cells were combined and resuspended in 5 ml of PBS. Cell viability was determined by trypan blue exclusion. Differential cell counts were performed by standard light-microscopic techniques based on staining with Diff-Quik (American Scientific Products, McGaw Park, IL). Results are expressed as the total number of cells recovered per lung.

Polymerase chain reaction cloning. Total RNA was extracted by the guanidinium thiocyanate method (4) from the lungs of BN rats 20 h after exposure to ozone. RNA (1 µg) was reverse transcribed with oligo(dT), and 5 µl of the resulting cDNA were amplified with a DNA thermal cycler (Perkin-Elmer Cetus, Branchburg, NJ) with the oligonucleotide primers 5'-CCTCCACCATGCAGAGCTCC and 5'-AGGCTCTGGGTTAGTGTCAA, corresponding to base pairs (bp) 40-59 and 355-374, respectively, of mouse eotaxin cDNA (8). Polymerase chain reaction (PCR) conditions were 30 cycles of denaturation at 95°C for 45 s, annealing at 45°C for 60 s, and extension at 72°C for 90 s. The amplified PCR products were subcloned directly into a pGEM-T vector (TA vector, Promega, Madison, WI) and sequenced by the dideoxy chain termination method with an autosequencer (ABI PRISM-310, Perkin-Elmer).

5' Rapid amplification of cDNA ends. With the use of total RNA from the lungs of BN rats 20 h after exposure to ozone, first-strand cDNA was synthesized with a Marathon cDNA amplification kit (Clontech Laboratories, Palo Alto, CA). After second-strand synthesis, the cDNA adaptor was ligated with T4 DNA ligase, and rapid amplification of cDNA ends (RACE) PCR was performed with the oligonucleotide primers 5'-CCATCCTAATACGACTCACTATAGGGC and 5'-AGGCTCTGGGTTAGTGTCAA. PCR conditions were 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 4 min. The amplified PCR products were subcloned directly into a pCR3.1-Uni expression vector (TA vector, Invitrogen, San Diego, CA) and sequenced.

Production of recombinant eotaxin. Eotaxin cDNA subcloned into a pCR3.1-Uni expression vector was transfected into COS cells with LipofectAMINE (GIBCO BRL, Gaithersburg, MD). After 72 h of culture, the supernatants of eotaxin-transfected or mock-transfected (vector alone) COS cells were concentrated by ultrafiltration in a Centricon-3 (Amicon, Danvers, MA). The recombinant protein was characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions on 15% gels and stained with Coomassie blue or was transferred to polyvinylidene difluoride membranes (Nippon Bio-Rad Laboratories, Tokyo, Japan) for Western blot analysis with anti-murine eotaxin antibody (Pepro Tech, London, UK).

Chemotaxis. Normal rat granulocytes were obtained from peripheral blood via the abdominal aorta with two-step Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) density gradient centrifugation. Isolated cells were washed two times with Hanks' balanced salt solution (HBSS), and the residual red blood cells were removed by hypotonic lysis. The chemotactic assay was performed with a 48-well modified Boyden chamber (Neuro Probe, Cabin John, MD). The cells were resuspended to a concentration of 1.5 × 106 cells/ml in HBSS containing 2% bovine serum albumin, and 50 µl (7.5 × 104 cells) were placed in the top of the chamber. The lower wells of the chambers were filled with various concentrations of COS cell supernatants. The chambers were incubated at 37°C for 30 min; cells that crossed the polycarbonate filter (3-µm pore; Neuro Probe) and adhered to the bottom were stained with Diff-Quik and counted in a high-performance field (×1,000). The results are presented as the total number of cells per 5 high-performance field.

RNA analysis. Total RNA was extracted from the lungs of anesthetized rats that were removed at the same time as bronchoalveolar lavage (BAL) was performed. RNA (20 µg/lane) was electrophoresed on formaldehyde-agarose gels and transferred to nylon membranes (Hybond-N+, Amersham). Blots were prehybridized at 68°C for 30 min in ExpressHyb hybridization solution (Clontech) and hybridized with a [alpha -32P]dCTP-labeled 0.3-kilobase (kb) rat eotaxin cDNA PCR fragment at 68°C for 60 min in the same solution. After removal of the probe, the blots were hybridized with a [alpha -32P]dCTP-labeled 0.47-kb rat beta -actin cDNA fragment (17). The blots were washed with 2× saline-sodium citrate (SSC)-0.05% SDS at room temperature for 30 min, followed by 0.1× SSC-0.1% SDS at 50°C for 30 min. Autoradiograms were made with a bioimaging analyzer (BAS5000, Fuji Photo Film, Tokyo, Japan).

Immunocytochemistry. At the same time as BAL, the rats were anesthetized and perfusion fixed first with saline and then with 4% paraformaldehyde-PBS via the pulmonary artery. The lungs were removed and immersed in the same fixative for 2 h at 4°C, washed with PBS three times for 5 min each, transferred to PBS containing 30% sucrose for 18 h at 4°C, and embedded in optimum cutting temperature compound (Miles, Elkhart, IN). Cryostat sections (8 µm) were cut and mounted on poly-L-lysine-coated glass slides. After incubation with 2% normal goat serum in 0.01 M phosphate buffer containing 0.5 M NaCl and 0.1% Tween 20 (Sigma Chemical) for 20 min at room temperature, the sections were incubated for 1 h at room temperature with a 1:125 dilution of rabbit anti-mouse eotaxin antibody (Pepro Tech); as a control, nonimmune rabbit serum was used. After the sections were washed, they were incubated with biotinylated goat anti-rabbit immunoglobulin G and then with avidin-biotinylated peroxidase complex (ABC Kit, Vector Laboratories, Burlingame, CA). Reactions were visualized with 3,3'-diaminobenzidine tetrahydrochloride in the presence of H2O2.

Statistics. Data are presented as means ± SD and were analyzed by standard one-way analysis of variance in combination with Duncan's multiple comparison test. A level of P < 0.05 was accepted as statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning of rat eotaxin cDNA. To identify the rat homologue of the eotaxin gene, we utilized PCR primers corresponding to highly conserved regions of the mouse and human eotaxin genes (8). After amplification, a single PCR product of ~330 bp was obtained; its size resembled that of the mouse eotaxin cDNA fragment (334 bp) obtained with these primers. The 5' sequence of this cDNA fragment was extended by RACE, and the product was cloned and sequenced.

The coding region of cDNA consisted of an open reading frame of 291 bases encoding a 97-amino acid protein that contained a 23-amino acid signal peptide (Fig. 1A). By homology with the NH2 terminus of the mature mouse protein, signal peptide cleavage was predicted to occur between alanine and histidine, resulting in a 74-amino acid mature protein. It is a member of the C-C chemokine family as indicated by the cysteine pair at amino acids 9 and 10 (Fig. 1A). As with other eotaxins, rat eotaxin is characterized by the presence of a two-amino acid gap located before the second proline of the putative mature protein as well as a highly conserved domain near the carboxy terminus (ICADPKKKWVQD; Fig. 1B). Comparison of the predicted amino acid sequence of rat eotaxin with those of other eotaxins revealed a high degree of homology to the mouse protein (97.3%) but a lesser homology to guinea pig (63.5%) and human (62.2%) eotaxins (Fig. 1B). Rat eotaxin showed lower amino acid homology to other rat C-C chemokines such as regulated on activation normal T cell expressed and secreted (RANTES) (31.8%), macrophage inflammatory protein (MIP)-1alpha (31.9%), and MIP-1beta (39.1%).


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Fig. 1.   A: nucleotide sequence and deduced amino acid sequence of rat eotaxin cDNA coding region. Underlined amino acids correspond to predicted signal sequence. Arrowhead, signal peptidase cleavage site predicted by homology with NH2 terminus of mature mouse eotaxin. B: amino acid sequence alignment of rat eotaxin (Eot) with mouse, guinea pig (GP), and human eotaxins and other rat C-C chemokines. Amino acids are numbered from predicted mature NH2 terminus after signal peptidase cleavage. Asterisks indicate 4 cysteine residues characteristic of C-C chemokine family. RANTES, regulated on activation normal T cell expressed and secreted; MIP, macrophage inflammatory protein. These sequence data are available from GenBank under accession no. U96637.

Expression and eosinophil chemotactic activity of recombinant eotaxin. To evaluate the molecular size and functional chemotactic properties of the protein encoded by cloned rat eotaxin cDNA, the latter was subcloned into an expression vector and used for transient transfection of COS cells. The supernatant from these cells was concentrated 10-fold and analyzed by Western blotting with an anti-mouse eotaxin antibody. A single of ~8.5 kDa reacted with this antibody (Fig. 2A). This molecular mass was consistent with that calculated from the deduced amino acid sequence (~8.4 kDa).


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Fig. 2.   A: Western blot analysis of supernatants from COS cells transfected with expression vector alone (lane 1) or with rat eotaxin cDNA in expression vector (lane 2). Both samples were stained with anti-mouse eotaxin antibody. Nos. on left, molecular mass markers in kDa. B: chemotaxis of rat eosinophils (solid bars) and neutrophils (open bars) to a fivefold concentrated supernatant (sup) from COS cells transfected with expression vector alone (moc/sup ×5) and unconcentrated (eot/sup) or fivefold concentrated (eot/sup ×5) supernatant from eotaxin-transfected COS cells. Results are total number of cells in 5 high-performance field (hpf; ×1,000). C: proportion of eosinophils and neutrophils in cell suspension medium after density gradient centrifugation (upper chamber) and in cells attached to filter as a result of chemotactic assay (migrated).

The chemotactic activity of the recombinant protein toward rat granulocytes was assayed in vitro in a microchemotaxis chamber. The granulocytes were obtained from the peripheral blood of normal rats, with a ratio of eosinophils to neutrophils of ~1:9 (Fig. 2C). The recombinant protein demonstrated chemotactic activity toward eosinophils, whereas it lacked activity toward neutrophils (Fig. 2B); 90.2% of the cells that had migrated toward a fivefold concentrate of eotaxin cDNA supernatant were eosinophils (Fig. 2C). No chemotactic activity on eosinophils was observed in supernatant from cells transfected with vector alone (Fig. 2B).

Expression of eotaxin mRNA and protein in lung tissues. We next determined by Northern blot analysis whether the expression of rat eotaxin mRNA was correlated with the degree of eosinophil infiltration in the acute pulmonary inflammation induced by exposure to ozone. The size of the rat eotaxin message (~900 bp) was found to be consistent with those of mature mouse (8, 21) and human (6, 18) eotaxin mRNAs. Although a small amount of eotaxin mRNA was constitutively expressed in rat lungs before exposure to ozone, its expression was increased after ozone exposure (Fig. 3). Analysis of the density ratios of the eotaxin and beta -actin messages demonstrated that the expression of eotaxin mRNA was enhanced ~1.6-fold immediately after exposure to ozone and 4-fold 20 h later. The number of eosinophils in BAL fluid at the same time points was 3-fold and 15-fold greater, respectively, than the control number (Fig. 3).


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Fig. 3.   Top: expression of rat eotaxin mRNA in lungs of Brown Norway rats and hybridization of this blot with a beta -actin probe. Lane 1, before exposure to ozone; lane 2, immediately after 6-h exposure to 1.2 parts/million ozone; lane 3, 20 h after exposure to ozone. Blots are representative of each group. Bottom: no. of eosinophils recovered by bronchoalveolar lavage in ozone-exposed Brown Norway rats. Results are means ± SD per lung for 4 animals. * Significantly different compared with rats before exposure to ozone (air), P < 0.01.

We assayed protein expression and tissue localization of eotaxin by immunocytochemistry. Eotaxin staining was localized most strongly in alveolar macrophages 20 h after exposure to ozone (Fig. 4C) as well as in a fraction of the bronchial epithelial cells (Fig. 4D). We observed only weak immunoreactivity to eotaxin on alveolar macrophages immediately after exposure to ozone (Fig. 4B) but none before ozone exposure (Fig. 4A). No immunoreactivity to any cell type was detected with nonimmune rabbit serum at any time.


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Fig. 4.   Immunocytochemical localization of eotaxin in lungs of Brown Norway rats before (A), immediately after (B), and 20 h (C and D) after 6-h exposure to 1.2 parts/million ozone. Arrowheads, cells positive for eotaxin. Bars, 10 µm.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Eotaxin was first identified as a potent eosinophil chemoattractant in BAL fluid that was obtained after challenging sensitized guinea pigs with allergen (9, 14). Since then, several in vitro and in vivo studies have shown eotaxin to be a potent and selective eosinophil chemoattractant in humans (6, 18) and mice (8, 21). The novel rat C-C chemokine we identified here is selectively chemotactic for eosinophils. On the basis of sequence similarity and function, this chemokine can be considered a rat equivalent of eotaxins.

In agreement with the findings in guinea pigs (13, 22) and humans (6), eotaxin mRNA was expressed in rat lungs even before the exposure to ozone. It has been reported that eosinophils mainly reside in the tissue, inasmuch as there are normally several hundred times as many eosinophils in the tissue as in the blood (24). Eosinophils, like neutrophils, are often present in the walls of blood vessels or the interstitium of the lung as marginated and/or interstitial pools. Therefore, it is likely that the constitutive expression of eotaxin mRNA we observed in unexposed rat lungs was associated with baseline eosinophil homing to the marginated and/or interstitial pools. However, we were unable to detect eotaxin protein immunocytochemically in unexposed lungs. One possible explanation is that the amount of eotaxin protein in these samples may be below the level of detection. Alternatively, eotaxin mRNA may be expressed, but not translated, before the exposure to ozone.

After exposure to ozone, we observed that the number of BAL-recovered eosinophils was increased, suggesting that these cells were recruited in the airways or alveolar spaces by ozone stimulation. Interestingly, eotaxin mRNA and protein expression were also enhanced in the lungs, suggesting that the translation of eotaxin mRNA occurs within a short time. The localization of eotaxin protein at luminal sites such as alveolar macrophages and bronchial epithelial cells during ozone-induced pulmonary inflammation suggests that the additional expression of eotaxin may be involved in the migration of eosinophils from the marginated and/or interstitial pools into the air spaces. Similar results have also been observed in mice, in which eotaxin expression was found to parallel the accumulation of eosinophils during allergic inflammation (8).

Although we have shown that rat eotaxin acts as a selective chemoattractant of eosinophils in vitro and that its expression can be correlated with the number of BAL-recovered eosinophils, it is not clear whether eotaxin contributes to the ozone-induced eosinophilic inflammation in vivo. Indeed, it has been reported that several chemotactic factors such as MIP-2 (11, 12), cytokine-induced neutrophil chemoattractant (10, 12), and leukotriene (LT) B4 (15, 16, 23) are induced by exposure to ozone; among these, LTB4 is chemotactic for eosinophils (19). However, the epithelial cell response was detected only after high in vitro exposure (~4 ppm ozone), and increases in LTB4 have not been observed in vivo (15, 16, 23). Thus, among the ozone-inducible chemoattractants, only eotaxin is upregulated by near-ambient levels of ozone, suggesting a specific contribution of eotaxin to the recruitment of eosinophils into the lungs of rats exposed to ozone in vivo. Our findings that eotaxin mRNA was not expressed in the lungs of Sprague-Dawley rats 20 h after a 6-h exposure to 1.2 ppm ozone (data not shown) do not contradict this hypothesis, inasmuch as ozone exposure leads to the accumulation of neutrophils in the lung of Sprague-Dawley rats (2, 12). Although the mechanisms that underlie the influx of inflammatory cells into tissue sites are not well known, it is likely that the appearance in tissues of different types of inflammatory cells during acute inflammation is regulated, at least in part, by the differential expression of chemokines. The rat eotaxin cDNA we have cloned may be an important tool for exploring the molecular mechanisms for eosinophil traffic during inflammation.

    ACKNOWLEDGEMENTS

This study was supported in part by Ministry of Education of Japan Research Grant 08770425 and by a research grant from the Kanae Foundation of Research for New Medicine.

    FOOTNOTES

Address for reprint requests: Y. Ishii, Dept. of Respiratory Medicine, Institute of Clinical Medical Sciences, Univ. of Tsukuba, Tsukuba, Ibaraki 305, Japan.

Received 6 May 1997; accepted in final form 8 October 1997.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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