Journal of Clinical Immunology, Vol. 22, No. 3, May 2002 (©2002)

Induction of MCP-1 Expression in Airway Epithelial Cells: Role of CCR2 Receptor in Airway Epithelial Injury MATTHEW C. LUNDIEN,1 KAMAL A. MOHAMMED,1 NAJMUNNISA NASREEN,1 R. S. TEPPER,1 JOYCE A. HARDWICK,1 KERRY L. SANDERS,1 ROBERT D. VAN HORN,1 AND VEENA B. ANTONY1,2

Accepted: January 10, 2002

surface layer of the conducting airways is the first to come into contact and thus be damaged by inhaled agents in the external environment. A myriad of agents including infectious organisms, inhaled toxic substances, and physical forces are known to be injurious to airway cells (1–5). The bronchial epithelium is speculated to be a key regulator of airway biology (6) and must be capable of rapid cell turnover to repair itself. However, the mechanism of repair is not yet known. Chemokines are small (8 –12 kDa) inducible secreted cytokine proteins acting primarily as chemoattractants and activators of leukocytes. Chemokines have been divided into four subfamilies (C-C, C-X-C, C, and C-X-X-X-C) depending on spacing of highly characteristic cysteine residues within their amino terminal regions (7, 8). Prior to this work, it was unknown that BECs express the CCR2 receptor protein (CCR2). It has been shown that CCR2 interacts with monocyte chemoattractant protein-1 (MCP-1) on multiple cell lines (9, 10). MCP-1 is a C-C chemokine active on mononuclear phagocytes, basophils, T cells, and natural killer cells (7, 9). It is produced in response to diverse inflammatory signals, typically interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and bacterial lipopolysaccharide (LPS) (7). Our study was designed to elucidate a possible mechanism of airway cell proliferation and bronchial epithelial cell monolayer repair at the cellular level in an in vitro model of mechanical injury. MCP-1 is a known chemokine ligand for the CCR2 receptor. In fact, this receptor– chemokine interaction has been shown to induce haptotaxis (defined as the migration of cells along the gradients of substrate-bound attractant molecules) in mesothelial cell lines (7). In BECs we showed that MCP-1 is produced by injured BECs, and through its interaction with the CCR2 receptor induces cell proliferation and haptotaxis.

The repair of an injured bronchial epithelial cell (BEC) monolayer requires proliferation and migration of BECs into the injured area. We hypothesized that BEC monolayer injury results in monocyte chemoattractant protein-1 (MCP-1) production, which initiates the repair process. BECs (BEAS-2B from ATCC) were utilized in this study. MCP-1 interacts with CCR2B receptor (CCR2B), resulting in cell proliferation, haptotaxis, and healing of the monolayer. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to verify the presence of CCR2B. CCR2B was not merely present but also inducible by interleukin-2 (IL-2) and lipopolysaccharide (LPS). We demonstrated by immunohistochemistry that BECs express MCP-1 after injury and that receptor expression can be regulated by exposure to IL-2 and LPS. Haptotactic migration of cells was enhanced in the presence of MCP-1 and reduced in the presence of CCR2B antibody. This enhanced or depressed ability of the BECs to perform haptotactic migration was shown to be statistically significant (P ⬍ 0.05) when compared to controls. Finally, BECs proliferate in response to MCP-1 as proven by electric cell-substrate impedance sensing (ECIS) technology. MCP-1-specific antibodies were shown to neutralize the MCP-1-mediated BEC proliferation. This cascade of events following injury to the bronchial epithelium may provide insight into the mechanism of the repair process. KEY WORDS: Bronchial epithelial cells; MCP-1; CCR2B; lipopolysaccharide; IL-2; haptotaxis; chemokines.

INTRODUCTION

Physical and environmental forces play an important role in regulating the structure, function, and metabolism of the lung. The entire bronchial epithelial cell (BEC) 1

Division of Pulmonary and Critical Care Medicine, Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 2 To whom correspondence should be addressed. Fax: 317-554-0262; telephone: 317-554-0000 ext. 2041; email: [email protected].

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MATERIALS AND METHODS

Bronchial Epithelial Cell Culture BEAS-2B human BECs, purchased from American Type Culture Collection (ATCC; Manassas, VA), had been isolated by ATCC from normal human bronchial epithelium obtained from the autopsy of noncancerous individuals. These cells were immortalized by infection with adenovirus 12-SV40 virus hybrid and cloned. The cells were, upon arrival, flash thawed and cultured in bronchial epithelial cell growth medium (BEGM) with “bullet kit” additives. The bullet kit additives were single aliquots of bovine pituitary extract, insulin, hydrocortisone, human recombinant epidermal growth factor, retinoic acid, transferrin, epinephrine, triiodothyronine, and gentamycin/amphotericin B. Streptomycin/penicillin and amphotericin B were added to decrease the risk of bacterial or fungal contamination. Bovine serum albumin (BSA) was added to reach a 5% solution. Media and bullet kit were purchased from Clonetics division of BioWhittaker (Walkersville, MD). Cytokines and Reagents Cytokines and reagents were purchased from various companies. Human IL-2, human recombinant monocyte chemoattractant protein-1 (rMCP-1), and mouse antihuman MCP-1 were purchased from Peprotech, Inc. (Rocky Hill, NJ). LPS from Salmonella typhimurium and mouse IgG isotype were purchased from Sigma (St Louis, MO). Mouse IgG was purchased from Calbiochem (LaJolla, CA). Mouse monoclonal anti-human CCR2 IgG2b was purchased from R&D Systems (Minneapolis, MN). Isolation of RNA and Reverse Transcriptase-Mediated Polymerase Chain Reaction (RT-PCR) and Southern Analysis Bronchial epithelial cells were cultured in serum-free media (SFM), SFM with IL-2 (200 U/ml), or SFM with LPS. Total cellular RNA was isolated from BEAS-2B BECs using RNeasy Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s recommendations. RT-PCR was performed as previously described (7, 11). One microgram of total RNA was reverse transcribed into cDNA. The first strand of cDNA was synthesized in a total volume of 20 ␮l in the presence of 5 mmole/liter MgCl2, 50 mmole/liter of KCL, 10 mmole/liter of Tris-HCl (pH 8.3), 1 mm/Liter deoxynucleotide triphosphates, 1 U/ml RNase inhibitor, 15 ␮M primer, and 2.5

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U/ml Maloney murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus; Norwalk, CT). The reverse transcription was conducted at 42°C for 15 min and the reaction was stopped by incubation at 99°C for 5 min. cDNA was amplified with specific primers (7, 12) for human CCR2A (GeneBank accession No. U03905) and CCR2B (GeneBank accession No. U03905). Human ␤-actin was amplified as a positive control. The oligonucleotide sequence of the primers used for CCR2A was sense, 5⬘TGCTACTCGGGAATCCTGAA-3⬘ (700 –719 bp), and antisense, 5⬘-TTCAGGGGCTCTGCCAATT-3⬘ (1134 –1116 bp). The product length for CCR2A was 434 bp. The oligonucleotide sequence of the primers used for CCR2B was sense, 5⬘-TGCTACTCGGGAATCCTGAA-3⬘ (741–760 bp), and antisense, 5⬘ACACACACAGCCCTGAGGTTCC-3⬘ (1290 –1270 bp). The product length for CCR2B was 550 bp. The ␤-actin sequence used was sense, 5⬘-GCACTCTTCCAGCCTTCCTTCC-3⬘ (819 – 840 bp), and antisense, 5⬘-TCGTTGCTGATCCACATCTGCT-3⬘ (1120 –1099 bp). The product length for ␤-actin was 302 bp. PCR was performed with 5 ␮l of RT product in a reaction mixture containing 2 mmole/liter MgCl2, 50 mmole/liter KCL, 10 mmole/liter Tris-HCL (pH 8.30), 15 ␮M specific oligonucleotide primer, and 2.5 U Taq DNA polymerase (Perkin-Elmer Cetus). The samples were amplified in a thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer Cetus) and preheated for 90 min at 95°C followed by 30 cycles. Each cycle was composed of denaturation at 95°C for 15 sec, primer annealing at 66°C for 30 sec for CCR2B, and extension at 72°C for 30 sec. The amplified product obtained was subjected to electrophoresis analysis in 2% agarose gel with 89 mM Tris, 89 mM boric acid, and 2 mM EDTA (pH 8.00) running buffer. The gels were stained with ethidium bromide and the bands were visualized under ultraviolet (UV) light and photographed on Polaroid film. The PCR products, run on an agarose gel, were transferred to a Nylon filter by capillary blotting for Southern hybridization (13) and were detected with ␥-32P d-CTPlabeled oligonucleotide probe 5⬘-CCAGACAGCACTGTGTTGGCGTACAGGTCT-3⬘ (␤-actin), 5⬘-TGCTTTCGGAAGAACACCGAG-3⬘ (CCR2B). The expected sizes of PCR products are 302 bp for ␤-actin and 550 bp for CCR2B. Immunohistochemical Staining of BECs The BECs were immunostained with an avidin– biotin conjugate and peroxidase as previously described (14). Briefly, BECs were cultured on glass four-chambered slides to confluence. The monolayer was then injured

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using the point of a 26-gauge intradermal needle to produce a scratch across the surface of the slide. The slides were reincubated for 24 hr, then fixed in methanol. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in PBS. PBS was subsequently used to rehydrate the cells. After the PBS was removed, the cells were blocked with 1% normal horse serum for 20 min to reduce nonspecific binding and treated with 0.1% Triton X-100 for 10 min to permeabilize the cells. The slides were washed three times with PBS then incubated for 40 min in the presence of mouse anti-human MCP-1/CCR2 antibody. The negative controls were incubated in the presence of monoclonal mouse IgG isotype. The slides were rinsed three times with PBS, then incubated for 30 min with avidin– biotin conjugated rabbit anti-mouse IgG (Vectastain Elite ABC kit, Vector Laboratories, Inc, Burlingame, CA). The slides were washed in PBS, incubated for 5 min in peroxidase substrate (3,3-diaminobenzidine; Vector Laboratories), rinsed with deionized water, counterstained with Mayer’s hematoxylin (Sigma), rinsed with water, dehydrated in graded alcohol solutions and xylene, and finally mounted in Cytoseal XYL (Stephens Scientific, Riverdale, NJ). Bronchial Epithelial Cell Haptotaxis Assay Haptotaxis assays were performed in triplicate using Boyden chambers (15). Before the motility assay, the uncoated polycarbonate filters (8 ␮m pore size; Nucleopore, Millipore) were placed in the chambers with the dull side of the filter facing the upper side of the wells. The upper wells were filled with MCP-1 (20 ng/ml) in SFM (BEBM plus additives). Control wells were filled with 1% BSA in BEBM plus additives media. The upper wells were incubated with MCP-1 (20 ng/ml) in SFM or 1% BSA in SFM overnight at 37°C in humidified air in the presence of 5% CO2. The filters were then removed and air dried. Some of the filters treated with MCP-1 were rehydrated with PBS and incubated for 1 hr with monoclonal antibody to MCP-1 (10 ␮g/ml) or with nonspecific mouse IgG (isotype). Similarly, to see the effect of CCR2B in BEC haptotaxis, the activated BECs were incubated with mouse anti-human CCR2B antibody for 1 hr, washed in SFM, and used for the haptotaxis assay. Finally, the filters were placed in 48-well Boyden chambers with the coated surface of the filter facing toward the lower wells, which were filled with Hanks’ Balanced Salt Solution (HBSS). Cells stimulated with SFM, IL-2 (200 U/ml), or LPS (10 ␮g/ml) were seeded into each upper chamber and incubated for 3 hr at 37°C. At the end of the incubation, the media in the upper wells was discarded by suction. The filters were removed and

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washed with PBS, fixed in formalin, and stained with Diff-Quik (Baxter, Rome, Italy). The stained filters were then mounted on glass slides. Cell migration was quantitated by counting the number of cells on the distal surface of the filter under an optical microscope. Ten high-power fields were counted per slide. The results are expressed as the number of cells migrated per 10 high-power fields. ECIS Proliferation Assay Proliferation data were taken in duplicate using electrical cell-substrate impedance sensing (ECIS) technology. ECIS is a device formulated to measure the activity of cells electronically. Cells are cultured on thin film electrodes evaporated onto the bottom of tissue culture dishes (16, 17). When cells attach and spread on these electrodes, the measured impedance will increase, since the current cannot pass through the insulating cell membrane but instead must pass around the cells. One volt of electricity at 4 KHz is applied to the sample through a 1 M⌺ resistor (18, 19). The final average resistance gives a convenient measure of when cells have reached confluence (20). An eight-electrode culture dish with one electrode/ well was precoated with 400 ␮l of media and experimental additives and then allowed to incubate for 2 hr. The additives consisted of the following: rMCP-1 (20 ng/ml), rMCP-1 plus antibody to MCP-1 (10 ␮g/ml), rMCP-1 plus mouse IgG isotype (10 ␮g/ml), and media only (control). BEAS-2B cells were trypsinized, pelleted, examined for viability, and counted. Two hundred microliters of media and additives were then gently removed from each well and 200 ␮l of cell suspension was added. There were a total of 1.25 ⫻ 105 BEAS-2B cells per 400 ␮l well volume. ECIS collected resistance values every 10 min for a total run time of 48 hr. In order to monitor BEC proliferation, we also evaluated direct total cell counts as described earlier (21). Briefly, 5 ⫻ 104 BECs were cultured in a 24-well plate in SFM. In some of the wells MCP-1 (20 ng/ml), either with MCP-1neutralizing antibodies or isotype antibodies, was included. After 48 hr of incubation, the cultures were trypsinized and the total cells were counted on a hemacytometer. STATISTICAL ANALYSIS

Data were analyzed with the Sigma Stat statistical software package (Apple Computer, Cupertino, CA) and are expressed as means ⫾ standard error (SE). The difference between the group means was analyzed by analysis of variance (ANOVA) with the use of the

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Fig. 1. Bronchial epithelial cells express CCR2B. BECs were incubated with SFM, IL-2, or LPS for 1, 3, 6, 12, and 24 hr. The expression of CCR2B mRNA was assessed by RT-PCR. The left panel indicates mRNA expression of CCR2B by resting cells in SFM. The middle panel indicates mRNA expression of CCR2B by cells stimulated with IL-2. The right panel indicates mRNA expression of CCR2B by cells stimulated with LPS. ␤-actin served as the positive control. This is a representative of three independent experiments.

Student–Newman–Keuls test. The data were considered significant if P values were ⬍ 0.05. RESULTS

CCR2B Exists on BECs Cultured in SFM and Its Expression Is Modulated by IL-2 and LPS in a Time-Dependent Manner Resting BECs were tested for the presence of CCR2A and CCR2B by RT-PCR at various time intervals (1, 3,

6, 12, and 24 hr). The product lengths selected were 434 bp for CCR2A, 550 bp for CCR2B, and 302 bp as a positive control. BECs were also stimulated with IL-2 (200 U/ml) and LPS (10 ␮g/ml) in SFM for 24 hr, then tested for CCR2. CCR2A was not expressed (data not shown). Figure 1 shows that resting BECs express CCR2B as evidenced by RT-PCR. CCR2B expression is modulated by the presence of IL-2 and LPS. IL-2 up-regulates CCR2B expression in a time-dependent manner and shows a maximum response at 12 hr in BECs cultured in the presence of LPS, CCR2B expres-

Fig. 2. CCR2B receptor expression by bronchial epithelial cells. BECs were incubated with SFM, IL-2, or LPS for 1, 3, 6, 12, and 24 hr. The CCR2B receptor-specific RT-PCR product was assessed by Southern analysis. Lanes 1, 2, 3, 4, and 5 represent 1, 3, 6, 12, and 24 hr, respectively. The OD values represent the mean of three independent experiments. ␤-Actin served as the positive control.

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sion is down-regulated until 6 hr. The inhibitory effect of LPS is restored after 12 hr. Southern blot analysis of CCR2B RT-PCR product also confirmed similar results (Fig. 2). Densitometry studies of Southern blots indicate a significant inhibition of CCR2B receptor in presence of LPS, while in the presence of IL-2, a marked increase in CCR2B receptor expression was noted. Injured Bronchial Epithelial Cells Express MCP-1 and CCR2B Receptor When confluent monolayers of BECs are subjected to mechanical injury, the BECs stain positive for MCP-1 in the cytoplasm evidenced by immunohistochemical staining (Fig. 3B). BECs positioned in areas away from sites of injury do not stain positive for MCP-1. Injured BECs demonstrated dark positive staining with CCR2B antibody. Cells that were distant from the site of injury were not positive for CCR2B receptor (Fig. 3C). CCR2B and MCP-1 Mediate Haptotactic Activity in Bronchial Epithelial Cells BECs demonstrate a haptotactic response in a concentration-dependent manner. Maximum activity was noticed at 20 ng/ml of MCP-1. To assess the effect of cytokines and endotoxin, the cells were pretreated with IL-2 or LPS and tested for haptotactic activity against MCP-1 treated cells. Antibody to CCR2B was added to all conditions to assess receptor influence over the haptotactic response. Also, a group of filters were pretreated with antibody to MCP-1 to assess the haptotactic influence of this cytokine. Table I shows that cells with CCR2B blocked by antibody had a significant (P ⬍ 0.05) decrease in haptotactic activity when compared to MCP-1 and controls (BSA). The MCP-1 antibody group showed a significant decrease in migration compared to cells stimulated with MCP-1 in SFM and IL-2, but not MCP-1-stimulated cells in the presence of LPS. The MCP-1 group had a significant increase in activity over the BSA (control) group in the presence of IL-2 and LPS, but not in the resting cells. Recombinant MCP-1-Induced Bronchial Epithelial Cell Proliferation In order to determine the effect of MCP-1 on airway cell proliferation, a concentration response curve was done (data not shown). Based on optimal response, 20 ng/ml of MCP-1 was selected. Recombinant MCP-1 (20 ng/ml), antibody to MCP-1 (10 ␮g/ml), and mouse IgG

Fig. 3. Immunohistochemical staining for detection of MCP-1 in human bronchial epithelial cells after injury. (A) Incubation in the presence of nonspecific mouse IgG antibody (isotype). (B) Incubation in the presence of mouse anti-human MCP-1-specific antibody. (C) Incubation in the presence of mouse anti-human CCR2 specific antibody. Arrows represent areas of MCP-1/CCR2 specific dense positive staining. Cells near the injured area have more intense staining. This is a single representative of four different slides stained.

isotype (10 ␮g/ml) were added to cell cultures of BEAS-2B and incubated for 24 hr in SFM. As seen in Fig. 4A, MCP-1 stimulated the proliferation of cells as evidenced by a rise in resistance over time. Resistance values were gathered in real time by the ECIS equipment. The resistance was markedly increased compared

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Table I. Neutralizing Antibodies to MCP-1 and CCR2 Receptor Inhibit MCP-1-Mediated Haptotactic Migration of Bronchial Epithelial Cellsa Number of cells migrated against samples BECs activation

BSA (1 mg/ml)

Recombinant MCP-1 (20 ng/ml)

Isotype antibody (5 ␮g/ml)

CCR2 antibody (5 ␮g/106 cells)

MCP-1 antibody (5 ␮g/ml)

SFM IL-2 (200 U/ml) LPS (10 ␮g/ml)

41.0 ⫾ 5.57 48.3 ⫾ 3.06 39.6 ⫾ 4.04

53.6 ⫾ 4.16 69.6 ⫾ 4.73b 48.67 ⫾ 3.06b

43.6 ⫾ 3.32 47.3 ⫾ 2.89 38.0 ⫾ 2.01

29.6 ⫾ 1.84b,c 27.3 ⫾ 3.79b,c 24.6 ⫾ 5.03b,c

32.3 ⫾ 4.73c 33.3 ⫾ 5.69c 43.6 ⫾ 1.15

Values are means ⫾ SE of number of cells migrated per 10 high-power fields. Cells were cultured in the presence of serum-free medium (SFM), interleukin-2 (IL-2), and lipopolysaccharide (LPS) overnight as described in Materials and Methods. The data represented are the means of three independent experiments. b Significantly different from BSA response with P value ⬍ 0.05. c Significantly different from monocyte chemoattractant protein-1 (MCP-1) response with P value ⬍ 0.05. a

to BECs alone in culture and also was increased over rMCP-1 in the presence of isotype. When an antibody to MCP-1 was added, the cells did not proliferate. Similar results were obtained with direct cell count analysis (Fig. 4B). MCP-1 induced an 80% increase in BEC proliferation. Inclusion of MCP-1 neutralizing antibodies significantly reduced BEC proliferation compared to isotype antibody. DISCUSSION

Chronic inflammation, triggered by infectious and toxic agents, is thought to be necessary and sufficient to account for the nature of asthma pathology (22). Inflammation subsequent to airway injury has been widely studied (1–5). Bronchial alveolar lavage and biopsy studies have demonstrated that a wide variety of inflammatory cells and mediators are involved in the process (11, 23–25). We present evidence that injurious forces alter the BEC monolayer microenvironment and speculate that an examination of the normal healing mechanism will provide a link in the understanding of stress adaptation of the surrounding tissue in the mechanically dynamic environment of the lung. In this study, BECs (BEAS-2B) from ATCC were utilized. BEAS-2B cells may not be typical of primary bronchial epithelial cells. Airway remodeling is a conceptual term applied to describe the dynamic processes that lead to structural changes in the airways of asthmatics, which result in an irreversible component of obstruction (26). Many believe the cause of this structural morphogenesis to be related to airway inflammation, but the mechanism is not yet clear. Although the concept of airway remodeling and its role in chronic persistent airflow obstruction is widely accepted, it should be recognized that airway remodeling is still just a concept (26). The underlying causes of airway remodeling are not well understood. There is now a growing appreciation that a significant degree of airflow obstruction may

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persist in some asthmatic patients who have never smoked and who have received aggressive therapy (26 – 28). It has been speculated that underlying structural changes in the airways cause persistent obstruction. Asthma is characterized by inflammation, and it is reasonable to assume that airway remodeling is an injury–repair response driven by inflammatory process (26). How inflammation leads to remodeling and why it occurs in some settings but not others are not understood. Also, forces that inhibit remodeling in the normal lung, even in the presence of epithelial injury, have not been studied. Subepithelial fibrosis has been implicated as the underlying cause of a thickened basement membrane. Deposition of collagen types III and V as well as fibronectin has been shown to account for the thickening (29). The airway epithelium may play a role in subepithelial fibrosis and perhaps other aspects of airway remodeling as well. The airway epithelium is a rich source of growth factors, cytokines, chemokines, and other mediators (4, 5, 30). An important target for these products appears to be the myofibroblasts, which are considered responsible for the synthesis and deposition of many of the matrix components found in the enlarged subepithelial layer (31). The bronchial epithelial cell layer of the airways is subject to recurrent environmental injury under physiological circumstances throughout the life of the individual. These stressors may be transient but may be severe enough to cause exfoliation of the airway. Therefore, BECs must be capable of rapid turnover and migration to quickly repair the epithelium. Daily damage and repair does not lead to fibrosis and remodeling. So, we must ask the following questions: Why does normal, daily injury not lead to fibrosis? How is repair without fibrosis accomplished? One can certainly speculate that duration, extent, and/or chronicity of injury may play a critical role. However, factors surrounding the injury/healing mechanism are largely unknown. Inflammation involving a myriad of inflammatory mediators certainly plays a

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Fig. 4. MCP-1-induced BEC proliferation. (A) BEC proliferation as evidenced by increased resistance over time using the ECIS electrode array. rMCP-1 (20 ng/ml) added to BECs promotes proliferation (open circles) as compared to control (closed squares) and nonspecific mouse IgG isotype (open squares). Antibody to MCP-1 (open triangles) led to a decrease in proliferative activity. (B) BEC proliferation as evidenced by direct cell counts. The data represent mean ⫾ SE four independent experiments run in triplicate.

role in response to injury and may cause further damage in an acute or chronic setting. The mechanism by which the epithelium repairs itself in the absence of fibrosis has not been reported. In this study, we investigated a possible mechanism of BEC monolayer repair after injury.

The present findings are interesting because CCR2B was previously not known to exist on BECs. It is our contention that in addition to its existence, it may play a critical and complex role in BEC function. CCR2 has a well-known interaction with the chemokine MCP-1. This CCR2/MCP-1 interaction causes an inflammatory re-

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sponse, most notably the chemotactic recruitment of monocytes. We tested for the presence of CCR2A by RT-PCR but found no expression (data not shown). The CCR2B subtype was present and is described in the results section. Chemokines, in general, have been thought of historically simply as chemoattractants. However, recent work indicates that chemokines may be involved in more diverse phases of the immune response (32). Growing evidence implicates chemokines in a complex network of immune response signaling and regulation. To add to the growing list of discovered chemokine–receptor functions, this article suggests an autocrine role of the BEC for monolayer repair in the absence of fibrosis. Cells in the damaged microenvironment act in an autocrine manner, producing MCP-1, which then interacts with CCR2B on the cell surface, thus stimulating migration, proliferation, and finally repair of the injured area. The increased MCP-1 in the injured monolayer implies a functional relationship between MCP-1 and CCR2B. In the monolayer microenvironment, repair occurs by cell proliferation and migration. Proliferation was enhanced in our study when cells were placed in the presence of higher than normal levels of MCP-1. This implies that the MCP-1/CCR2B interaction functions to excite cells to grow and divide. A significant inhibition of BEC proliferation in presence of MCP-1 neutralizing antibodies suggests that MCP-1 is implicated in BEC proliferation. The inhibition of BEC migration in the presence of CCR2B antibody suggests that haptotactic activity is mediated through CCR2B. While increased CCR2B expression in the presence of IL-2 suggests a traditional inflammatory role, the finding that LPS downregulates receptor expression implies a response to pathogens. Bacteria may use LPS as a method to promote cell infection and damage, thus disabling the autocrine proliferative response of the cell monolayer to injury. It was expected that IL-2 and LPS would influence cell haptotaxis as was shown in work with pleural mesothelial cells (7). However, we did not find this to be reproducible in BECs. We did find that blocking CCR2B significantly decreased the haptotactic response. Further studies designed to greatly increase receptor concentration while monitoring haptotactic migration may explain our findings. The significance of the present work is that it demonstrates an autocrine mechanism that BECs may use to perform local repair of injury without fibrosis and subsequent airway remodeling. BECs express CCR2B and have inhibited haptotactic movement in its absence. MCP-1 is expressed in cell injury and stimulates cell

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proliferation, and therefore may be a critical component in local repair.

ACKNOWLEDGMENT

This work was supported in part by NIH-R01-AI 45338-02.

REFERENCES 1. Riley DJ, Rannels DE, Low RB, Jensen, Jacobs TP: NHLBI Workshop Summary. Effect of physical forces on lung structure, function, and metabolism. Am Rev Respir Dis 142(4):910 –914, 1990 2. Salvi SS, Nordenhall C, Blomberg A, et al: Acute exposure to diesel exhaust increases IL-8 and GRO-alpha production in healthy human airways. Am J Respir Crit Care Med 161(2 Pt 1):550 –557, 2000 3. Felix JA, Chaban VV, Woodruff ML, Dirksen ER: Mechanical stimulation initiates intercellular Ca2⫹ signaling in intact tracheal epithelium maintained under normal gravity and simulated microgravity. Am J Respir Cell Mol Biol 18(5):602– 610, 1998 4. Polito AJ, Proud D: Epithelia cells as regulators of airway inflammation. J Allergy Clin Immunol 102(5):714 –718, 1998 5. Raeburn D, Webber SE: Proinflammatory potential of the airway epithelium in bronchial asthma. Eur Respir J 7(12):2226 –2233, 1994 6. Holgate ST, Lackie P, Wilson S, et al: Bronchial epithelium as a key regulator of airway allergen sensitization and remodeling in asthma. Am J Respir Crit Care Med 162(3 Pt 2):S113–117, 2000 7. Nasreen N, Mohammed KA, Galffy G, et al: MCP-1 in pleural injury: CCR2 mediates haptotaxis of pleural mesothelial cells. Am J Physiol Lung Cell Mol Physiol 278(3):L591–598, 2000 8. Baggiolini M, Dewald B, Moser B: Human chemokines: An update. Annu Rev Immunol 15:675–705, 1997 9. Sozzani S, Locati M, Zhou D, et al: Receptors, signal transduction, and spectrum of action of monocyte chemotactic protein-1 and related chemokines. J Leukoc Biol 57(5):788 –794, 1995 10. Antony VB, Hott JW, Kunkel SL, et al: Pleural mesothelial cell expression of C-C (monocyte chemotactic peptide) and C-X-C (interleukin 8) chemokines. Am J Respir Cell Mol Biol 12(6):581– 588, 1995 11. Nasreen N, Hartman DL, Mohammed KA, Antony VB: Talcinduced expression of C-C and C-X-C chemokines and intercellular adhesion molecule-1 in mesothelial cells. Am J Respir Crit Care Med 158(3):971–978, 1998 12. Polentarutti N, Allavena P, Bianchi G, et al: IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: Characterization of a predominant 3.4-kilobase transcript containing CCR2B and CCR2A sequences. J Immunol 158(6):2689 –2694, 1997 13. Wegener J, Keese CR, Giaever I: Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 259(1):158 –166, 2000 14. Mohammed KA, Nasreen N, Ward MJ, et al: Mycobacteriummediated chemokine expression in pleural mesothelial cells: role of C-C chemokines in tuberculous pleurisy. J Infect Dis 178(5): 1450 – 456, 1998

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15. Klominek J, Robert KH, Sundqvist KG: Chemotaxis and haptotaxis of human malignant mesothelioma cells: effects of fibronectin, laminin, type IV collagen, and an autocrine motility factor-like substance. Cancer Res 53(18):4376 – 4382, 1993 16. Keese CR, Giaever I: Substrate mechanics and cell spreading. Exp Cell Res 195(2):528 –532, 1991 17. Giaever I, Keese CR: Use of electric fields to monitor the dynamical aspect of cell behavior in tissue culture. IEEE Trans Biomed Eng 33(2):242–247, 1986 18. Giaever I, Keese CR: A morphological biosensor for mammalian cells. Nature 366(6455):591–592, 1986 19. Wegener J, Keese CR, Giaever I: Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 259(1):158 –166, 2000 20. Keese CR, Giaever I: A biosensor that monitors cell morphology with electrical fields. IEEE Eng Med Biol 402– 408, 1994 21. Galffy G, Mohammed KA, Ward MJ, et al: Interleukin-8, an autocrine growth factor for malignant mesothelioma. Cancer Res 59:367–371, 1999 22. Elias JA, Zhu Z, Chupp G, Homer RJ: Airway remodeling in asthma. J Clin Invest 104(8):1001–1006, 1999 23. Azzawi M, Bradley B, Jeffery PK, et al: Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am Rev Respir Dis 142(6 Pt 1):1407– 413, 1990 24. Bradley BL, Azzawi M, Jacobson M, et al: Eosinophils, T lymphocytes, mast cells, neutrophils, and macrophages in bron-

LUNDIEN ET AL.

25.

26.

27. 28.

29.

30.

31.

32.

chial biopsy specimens from atopic subjects with asthma: Comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol 88(4):661– 674, 1991 Broide DH, Gleich GJ, Cuomo AJ, et al: Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J Allergy Clin Immunol 88(4):637– 648, 1991 Fish JE, Peters SP: Airway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol 104(3 Pt 1):509 – 516, 1999 Peat JK, Woolcock AJ, Cullen K: Rate of decline of lung function in subjects with asthma. Eur J Respir Dis 70(3):171–179, 1987 Lange P, Parner J, Vestbo J, et al: A 15-year follow-up study of ventilatory function in adults with asthma. N Engl J Med 339(17): 1194 –1200, 1998 Roche WR, Beasley R, Williams JH, Holgate ST: Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1(8637):520 –524, 1989 Vignola AM, Chanez P, Chiappara G, et al: Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis. Am J Respir Crit Care Med 156(2 Pt 1):591–599, 1997 Sousa AR, Poston RN, Lane SJ, et al: Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids. Am Rev Respir Dis 147(6 Pt 1):1557–1561, 1993 Marquez G, Martinez AC: Chemokines: The times they are a-changin’. J Clin Invest 107(7):791–792, 2001

Journal of Clinical Immunology, Vol. 22, No. 3, May 2002 (©2002)