Division of Pulmonary and Critical Care Medicine, Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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Pleural injury results in the death of mesothelial cells and denudation of the mesothelial basement membrane. Repair of the mesothelium without fibrosis requires proliferation and migration of mesothelial cells into the injured area. We hypothesized that monocyte chemoattractant protein-1 (MCP-1) induces proliferative and haptotactic responses in pleural mesothelial cells (PMCs) and that the MCP-1 binding receptor CCR2 mediates the pleural repair process. We demonstrate that PMCs exhibited MCP-1-specific immunostaining on injury. MCP-1 induced proliferative and haptotactic responses in PMCs. PMCs express CCR2 in a time-dependent manner. Fluorescence-activated cell sorting analysis demonstrated that interleukin (IL)-2 upregulated CCR2 protein expression in PMCs, whereas lipopolysaccharide (LPS) downregulated the response at the initial period compared with that in resting PMCs. However, the inhibitory potential of LPS was lost after 12 h and showed a similar response at 24 and 48 h. Haptotactic migration was upregulated in PMCs that were cultured in the presence of IL-2. The increased haptotactic capacity of mesothelial cells in the presence of IL-2 correlated with increased CCR2 mRNA expression. PMCs cultured in the presence of LPS showed decreased haptotactic activity to MCP-1. Blocking the CCR2 with neutralizing antibodies decreased the haptotactic response of PMCs to MCP-1. These results suggest that the haptotactic migration of mesothelial cells in response to MCP-1 are mediated through CCR2, which may play a crucial role in reepithelialization of the denuded basement membrane at the site of pleural injury and may thus contribute to the regeneration of the mesothelium during the process of pleural repair.
monocyte chemoattractant protein-1; interleukin-2; lipopolysaccharide
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INTRODUCTION |
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INJURY TO THE PLEURA and the repair process that follows play an important role in the outcome of many diseases of the lung. The process of repair without fibrosis of the exfoliated or injured mesothelium requires the migration and proliferation of mesothelial cells for the reestablishment of a normal mesothelial monolayer. Local factors released at the site of injury are responsible for the repair process. Pleural mesothelial cells (PMCs) migrate in response to thrombin (17). The migration of cells along the gradients of substrate-bound attractant molecules is defined as haptotaxis. Migration of circulating leukocytes to the site of infection or tissue injury consists of a multistep series of adhesive and signaling events (37). The ordered influx of leukocytes to the site of inflammation is controlled by different regulatory chemokines (14, 31). Chemokines are small proteins with typical subunits of 8-12 kDa that 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. Chemokines exert their effect via a distinct group of structurally related seven-transmembrane-domain protein receptors that are within a superfamily of receptors that range in size from 339 to 372 amino acids that signal through heterodimeric GTP-binding proteins (5). Chemokine receptors are differentially and constitutively expressed, allowing leukocytes to immediately respond differentially to chemokine signals at local sites (4).
On stimulation, PMCs release C-C chemokines (24). However, it is not
known whether they themselves may be responsive to a microenvironment
containing C-C chemokines. Response to the chemoattractant would
indicate that mesothelial cells express receptors to the specific
chemokine. Monocyte chemoattractant protein-1 (MCP-1) is a C-C
chemokine active on mononuclear phagocytes, basophils, T cells, and
natural killer (NK) cells (23, 35). MCP-1 is produced by a
variety of cell types, including mesothelial cells (2, 25), human
fibroblasts, and endothelial cells (36), in response to diverse
inflammatory signals, typically interleukin (IL)-1, tumor necrosis
factor-, and bacterial lipopolysaccharide (LPS). MCP-1 levels were
significantly increased in wounds (13, 15). MCP-1 interacts with the
MCP-1 binding receptor CCR2, of which two isoforms have been cloned and
termed CCR2A and CCR2B (9). In monocytes and NK cells, the CCR2 is
expressed predominantly as the B isoform, with low levels of the A
isoform (26). In CCR2 knockout mice, monocytes failed to migrate in
response to MCP-1 (8). Although the regulation of chemokine production has been extensively investigated, little is known about the
microenvironmental signals that may affect the chemokine system by
modulating receptor expression. Chemokines such as IL-8 and regulated
on activation normal T cell expressed and presumably secreted (RANTES)
mediate haptotactic migration of neutrophils and monocytes (30, 40). Whether MCP-1 causes haptotactic migration of PMCs when surface bound
is not known. The present investigation demonstrates the role of MCP-1
in pleural injury and the role of its CCR2 in the haptotactic migration
of PMCs.
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MATERIALS AND METHODS |
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Cytokines and reagents. Recombinant human MCP-1, monoclonal anti-MCP-1 antibody (mouse IgG1, which does not cross-react with other C-C chemokines), monoclonal anti-human CCR2 antibody (mouse IgG1), polyclonal goat anti-human MCP-1 antibody (IgG), and mouse IgG1 isotype were purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-2 was from PeproTech (Rocky Hill, NJ). LPS, goat anti-mouse IgG-FITC antibody, and nonspecific goat IgG were purchased from Sigma (St. Louis, MO). [3H]thymidine was obtained from Amersham Life Science (Arlington Heights, IL).
Isolation and culture of human PMCs. Pleural fluid was obtained via thoracentesis from patients with transudative pleural effusions secondary to congestive heart failure according to a protocol approved by the Indiana University (Indianapolis, IN) Institutional Review Board. The majority of patients had intractable congestive heart failure and symptomatic pleural effusions. None of the subjects had evidence of an infectious etiology for the pleural effusion. The pleural fluid was removed into a heparinized container and centrifuged at 1,000 g for 10 min, and the supernatant was discarded. The cell pellet was briefly exposed to cold hypotonic solution to lyse the red blood cells. The cells were resuspended in medium 199 (GIBCO BRL, Life Technologies, Grand Island, NY) containing 15% fetal bovine serum (Harlan Bioproducts, Indianapolis, IN), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The cells were plated in 75-cm2 culture flasks (Corning Costar) and incubated overnight at 37°C in 5% CO2-95% air. The following day, the medium was changed to remove nonadherent cells. Mesothelial cells were characterized by the presence of a classic cobblestone morphology (1), absence of factor VIII antigen, and presence of cytokeratin (11). When the cells were confluent, they were seeded onto 24-well culture plates as required for different assays. All cells were utilized between the second and fourth passages.
PMC proliferative activity. Mesothelial cell proliferation was
determined with a [3H]thymidine uptake assay as
described by Hott et al. (17) and Lauber et al. (20). The
cells (2 × 104) were plated in 48-well
plates and grown to near confluence (<60%) so that there would be
enough space for the cells to proliferate. The medium was then replaced
by medium 199 and incubated overnight. Various concentrations of MCP-1
(10, 25, 50, and 100 ng/ml), polyclonal goat anti-human MCP-1-specific
antibody (IgG, 10 µg/ml), or nonspecific goat IgG as a control were
added at the same concentrations. After 24 h of incubation at 37°C
in 5% CO2-95% air, 0.5 µCi of
[3H]thymidine was added to each well, and the
plates were incubated for an additional 24 h. The cells were washed in
complete Hanks' balanced salt solution three times and removed from
the plates with 0.25% trypsin-EDTA, precipitated in 5%
trichloroacetic acid, and centrifuged at 3,000 rpm for 10 min. They
were resuspended in 0.1 N sodium hydroxide, placed in scintillation
fluid, and counted in a -scintillation counter. All experiments were
done in triplicate, and the data are expressed as percent stimulation {[counts per minute produced by the test factors/counts
per minute produced by serum-free medium (SFM)] × 100}. The selected MCP-1 concentrations had no effect on the
mesothelial cell viability as demonstrated by trypan blue exclusion.
Flow cytometric analysis of CCR2. PMCs were cultured in SFM or SFM with either IL-2 (200 U/ml, based on preliminary observations) or LPS [10 µg/ml, based on previously published work (2)] for 1-48 h at 37°C in a 5% CO2 atmosphere. The PMCs were trypsinized and washed three times in PBS with 5% BSA and 5 mM sodium azide and incubated for 45 min at 4°C in the presence of either mouse anti-human CCR2 monoclonal antibody (1 µg/106 cells) or mouse IgG1 isotype. The cells were washed three times and labeled with rabbit anti-mouse IgG1-FITC conjugate to detect the antibody bound to the antigens. After incubation, the cells were washed three times and fixed in 4% paraformaldehyde. The fluorescence associated with the cells was analyzed by flow cytometry with a FACStar (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Fluorescence data were collected on a log scale, and the relative fluorescence intensity is reported by comparing the light-scatter characteristics of treated cells to those of normal cells analyzed in the same experiment.
Histochemical immunostaining of PMCs. The PMCs were immunostained with an avidin-biotin conjugate and peroxidase as previously described (24). Briefly, PMCs were cultured in glass chambered slides to confluence. To understand whether the injury could induce MCP-1 expression in PMCs, confluent PMC monolayers were injured with a sterile needle. Briefly, with a sterile 26-gauge intradermal needle, the confluent PMC monolayers were injured carefully by gently touching the glass surface without dislodging the intact monolayer. A 45-60° angle between the needle point and the surface of a glass slide allowed a perfect injury without dislodging the mesothelial monolayer. The whole process was performed in minimal time, and the slides were reincubated. Twenty-four hours after injury, the cells were fixed in methanol. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in PBS and treated with 0.1% Triton X-100. The slides were rinsed three times in PBS and blocked with 1% normal horse serum for 30 min to reduce nonspecific binding. They were incubated for 50 min in the presence of rabbit anti-human MCP-1 antibody (1:1,200). The negative controls were incubated in the presence of normal rabbit IgG. The slides were rinsed again with PBS three times and then incubated for 30 min with avidin-biotin-conjugated goat anti-rabbit IgG (VECTASTAIN Elite ABC Kit, Vector Laboratories, Burlingame, CA). The slides were washed in PBS and incubated for 5 min in peroxidase substrate (3,3'-diaminobenzidine; Vector Laboratories), rinsed in PBS, counterstained with Mayer's hematoxylin (Sigma), dehydrated in graded alcohol solutions and xylene, and then mounted in Permount (Fisher, Fair Lawn, NJ).
RT-PCR. Total cellular RNA was isolated from PMCs with TRI REAGENT (10). RT-PCR was performed as previously described (25). One microgram of total RNA was reverse transcribed into cDNA. The first strand of cDNA was synthesized in a total volume of 20 µl in the presence of 5 mmol/l of MgCl2, 50 mmol/l of KCl, 10 mmol/l of Tris · HCl, pH 8.3, 1 mmol/l of deoxynucleotide triphosphates, 1 U/ml of RNase inhibitor, 15 µM primer, and 2.5 U/ml of Moloney murine leukemia virus RT (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 (26) for human CCR2A (GenBank
accession no. U03882) and human CCR2B (GenBank accession no. U03905).
Human -actin was amplified as a positive control. The
following oligonucleotide sequences of the primers were used: CCR2A:
sense, 5'-TGCTACTCGGGAA- TCCTGAA-3' (700-719 bp),
and antisense, 5'-TTCAGGGGCTCTGCCAATT-3' (1,134-1,116
bp), product length 434 bp; CCR2B: sense,
5'-TGCTACTCGGGAATCCTGAA-3' (741-760 bp), and
antisense, 5'-ACACACACAGCCCTGAGGTTCC-3' (1,290-1,270 bp), product length 550 bp; and
-actin, sense,
5'-GCACTCTTCCAGCCTTCCTTCC-3' (819-840 bp), and
antisense, 5'-TGCTTGCTGATCCACATCTGCT-3' (1,120-1,099 bp),
product length 302 bp.
PCR was performed with 5 µl of RT product in a reaction mixture
containing 2 mmol/l of MgCl2, 50 mmol/l of KCl, 10 mmol/l of Tris · HCl, pH 8.30, 15 µM specific
oligonucleotide primer, and 2.5 U of Taq DNA polymerase
(Perkin-Elmer Cetus). The samples were amplified in a thermal cycler
(GeneAmp PCR System 9600, Perkin-Elmer Cetus), preheated for 1.30 min
at 95°C followed by 30 cycles, with each cycle composed of
denaturation at 95°C for 15 s, primer annealing at 60°C for 30 s for CCR2A and 66°C for 30 s for CCR2B, and extension at 72°C
for 30 s. The amplified product obtained was subjected to
electrophoresis analysis in 2% agarose gel with a 21 g/l of Tris, 11 g/l of boric acid, and 0.002 M EDTA, pH 8.00, running buffer. The gels
were stained with ethidium bromide and transferred onto a nylon
membrane (Zeta-Probe, Bio-Rad, Hercules, CA). The membranes were baked
at 80°C for 30 min under vacuum. Hybridization was conducted
according to standard protocols (20). The following oligonucleotide
sequences to hybridize CCR2A, CCR2B, and -actin were used: CCR2A:
5'-GGAGTCCTTGTGTAGTCACT-3', probe length 20 bp, 1,090-1,071 bp;
CCR2B: 5'-TGCTTTCGGAAGAACACCGAG-3', probe length 21 bp,
1,048-1,028 bp; and
-actin:
5'-CCAGACAGCACTGTGTTGGCGTACAGGTCT-3', probe length 30 bp,
943-914 bp.
Fifty nanograms of oligonucleotide were labeled with
[-32P]ATP (3,000 Ci/mmol) in the presence of
T4 kinase (Boehringer Mannheim, Mannheim, Germany) in 50 µl of kinase
reaction buffer and then passed through Bio-Spin chromatography columns
(Bio-Rad). The membranes were hybridized with
[
-32P]ATP-labeled probe, and the blots were
exposed and autoradiographed.
PMC haptotaxis assay. Haptotaxis was assayed in triplicate by using Boyden chambers (19). Before the motility assay, the uncoated polycarbonate filters (8-µm pore size; Nucleopore, Millipore) were placed in the chambers, the dull side of the filter facing the upper side of the wells. The upper wells were filled with chemokine MCP-1 (50 ng/ml) in serum-free medium 199. Control wells were filled with 1 mg/ml of BSA. The lower wells remained empty. The chambers were left overnight at 37°C in humidified air in the presence of 5% CO2. The filters were then removed, rinsed in PBS, and air-dried. Some of the filters treated with MCP-1 were carefully rehydrated with PBS and incubated for 1 h with monoclonal anti-MCP-1 antibody (mouse IgG1, 10 µg/ml) or with nonspecific mouse IgG1 as a control before the onset of assay. Similarly, to see the effect of CCR2 in PMC haptotaxis, the activated PMCs were incubated with mouse anti-human CCR2 antibody for 1 h, washed in serum-free medium 199, 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 that were filled with Hanks' balanced salt solution. Cells (1 × 105) stimulated with SFM, IL-2 (200 U/ml), or LPS (10 µg/ml) were seeded into each upper chamber and incubated for 3 h at 37°C. At the end of the incubation, the medium in the upper wells was discarded by suction. The filters were removed and washed with PBS, fixed in Formalin, and stained with Diff-Quik (Baxter, Rome, Italy). The stained filters were then mounted on glass slides, and cell migration was quantitated by counting the number of cells on the distal surface of the filter under an optical microscope. Ten high-power oil-immersion fields (×100) were counted. The results are expressed as haptotactic index in which the number of cells per 10 high-power fields is visualized.
Statistical analysis. Data were analyzed with the SigmaStat statistical software package (Apple Computer, Cupertino, CA) and are expressed as means ± SE. The difference between the group means was analyzed by analysis of variance (ANOVA) with the use of the Student-Newman-Keuls test. The data were considered significant if P values were <0.05.
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RESULTS |
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Isolated pleural fluid cells were characterized as PMCs. In general, ~200 ml of centrifuged transudative pleural fluid yielded a 75-cm2 confluent flask of mesothelial cells within 7-14 days when cultured as described in Isolation and culture of human PMCs. The mesothelial origin of cells in the second passage was confirmed by positive staining for vimentin, cytokeratin, and hyaluronic acid mucin (23). The morphology was documented to be a cobblestone pattern by phase-contrast microscopy, and numerous microvilli were noted on transmission electron microscopy. All cells were utilized between the second and fourth passages in 24-well plates.
MCP-1 induced mesothelial cell proliferation. To determine the
effect of MCP-1 in regulating human mesothelial cell proliferation, various concentrations (10, 25, 50, and 100 ng/ml) of MCP-1, polyclonal goat anti-MCP-1 antibody (10 µg/ml), and nonspecific goat IgG (control) were added to the cell cultures grown in SFM. MCP-1, in a
significant and dose-dependent manner, stimulated mesothelial cell
proliferation as demonstrated by [3H]thymidine
incorporation (Fig. 1A). Maximal
[3H]thymidine uptake occurred in response to 50 ng/ml of MCP-1. Incubation with 100 ng/ml of MCP-1 did not increase
[3H]thymidine incorporation above the level
with 50 ng/ml of MCP-1. Furthermore, administration of specific
polyclonal anti-human MCP-1 antibody resulted in a significant
inhibition of mesothelial cell proliferation compared with that in
MCP-1-stimulated cultures (P < 0.001; Fig. 1B).
Nonspecific goat IgG was not effective in neutralizing the
MCP-1-induced proliferation in PMCs.
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PMCs expressed CCR2. CCR2 expression on mesothelial cells was
tested by fluorescence-activated cell-sorting analysis at various time
intervals (1, 3, 6, 12, 24, and 48 h). Resting PMCs expressed CCR2.
CCR2 expression in resting PMCs was found to be 36.94% at 24 h (Fig.
2). IL-2 upregulated CCR2
expression in PMCs in a time-dependent manner. IL-2 showed a maximum
response (55.20%) after 24 h. The difference between SFM- and
IL-2-induced responses was significant (P < 0.05).
IL-2-stimulated PMCs were 55.20 and 53.80% positive for CCR2 at 24 and
48 h, respectively. The difference between 24- and 48-h IL-2-stimulated
cultures was not significant. In PMCs that were cultured in the
presence of LPS, CCR2 expression was downregulated until 6 h (3.51%).
The inhibitory effect of LPS was gradually restored after 12 h
(9.02%), and after 24 h (27.57%), the response plateaued. The
difference between 24- and 48-h LPS-stimulated cultures was not
significant.
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Injured PMCs expressed MCP-1. When confluent PMC monolayers
were subjected to mechanical injury in vitro, the injured PMCs densely
stained positive for MCP-1 (Fig. 3),
whereas uninjured PMCs did not stain positive for MCP-1.
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IL-2 and LPS modulated CCR2 mRNA expression in PMCs. PMCs
expressed CCR2A and CCR2B isoform receptor mRNAs (Fig.
4). We amplified the CCR2A and
CCR2B transcripts of 434 and 550 bp, respectively. When CCR2A
amplification was performed with RNA isolated from IL-2-activated PMCs
and from the human myeloid leukemia THP-1 cell line as a positive
control, we also amplified a 1,649-bp-long sequence with the expected
434-bp sequence. We hybridized the product with a 20-bp CCR2-specific
probe to rule out nonspecific amplification. Both bands (1,649- and
434-bp-long sequences) strongly hybridized with this probe. PMCs
cultured in SFM showed increased CCR2A and CCR2B mRNA expression
compared with cells cultured in the presence of LPS (Fig. 4C).
IL-2 induced a time-dependent expression of CCR2A and CCR2B mRNAs in
PMCs (Fig. 4B). Maximum expression was noticed at the end of 24 h. The message intensity of both the 1,649- and 434-bp-long sequences
was higher in IL-2-stimulated PMCs compared with SFM-stimulated cells.
To evaluate the intensity of the message, the CCR2A receptor-specific
bands from SFM- and IL-2-stimulated cultures at a 24-h time period were
analyzed by laser densitometry and normalized with the housekeeping
gene -actin. The densitometric analysis of the CCR2A-specific mRNA
expressed in the IL-2-stimulated cultures was found to be 24- and
2.8-fold higher than the mRNAs from SFM cultures for the 1,649- and
434-bp bands, respectively. However, in resting and LPS-stimulated
PMCs, the signal for the 1,649-bp sequence was low. LPS inhibited CCR2A and CCR2B mRNA expression in PMCs (Fig. 4C). The LPS-mediated inhibition persisted until 6 h. The inhibitory effect of LPS was not
due to toxicity. The LPS concentration used in this study was not toxic
to PMCs as tested by trypan blue dye exclusion. However, after 12 h,
the inhibitory effect of LPS was decreased.
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MCP-1 mediated haptotactic activity in PMCs. PMCs demonstrated
haptotactic response to MCP-1 in a concentration-dependent manner.
Maximum activity was noticed at 50 ng/ml of MCP-1 (Table 1); the activity plateaued thereafter. 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.
Pretreatment of PMCs with IL-2 resulted in an increased haptotactic
response (P < 0.005). However, PMCs cultured in the presence
of LPS showed significantly decreased haptotactic migration compared
with PMCs cultured in SFM or IL-2 (P < 0.001). The treatment
of MCP-1-coated filters with monoclonal anti-MCP-1 antibody
significantly reduced the haptotactic migration of PMCs (P < 0.001). The pretreatment of PMCs with mouse anti-human CCR2 antibody
for 1 h resulted in significant inhibition of haptotactic migration of
PMCs (P < 0.001).
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DISCUSSION |
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Pathogenic organisms or natural fibers such as asbestos that enter the pleural space may cause pleural injury and subsequent denudation of the pleural mesothelium. The factors responsible for the reepithelialization of the pleura in vivo are largely unknown. A multitude of factors such as chemokines or cytokines present in pleural space at the site of injury are potentially chemotactic and/or mitogenic to PMCs. However, the role of chemokines in pleural injury and possible mesothelial repair has not been reported. In this study, we investigated the role of MCP-1 (a C-C chemokine) in the process of repair of the denuded mesothelium. We report that MCP-1 induces PMC proliferation in a concentration-dependent manner. MCP-1 also mediates haptotactic activity in PMCs. PMCs cultured in the presence of IL-2 demonstrated a higher haptotactic response to MCP-1 compared with resting cells and cells cultured in the presence of LPS.
The present findings are interesting because little work has been done to define the mechanisms of repair of an injured pleura. Mesothelial cells are metabolically active cells capable of producing their own basement membrane components (29). Several studies have indicated that after various insults, regeneration of a new mesothelium is rapid (28), with association of mitotic activity and shortening of the S phase (3, 39). In an earlier study, Hott et al. (17) demonstrated that thrombin induced proliferation and chemotactic activity of mesothelial cells. The present results indicate that injury caused MCP-1 expression in PMCs. MCP-1 induced PMC proliferation in a concentration-dependent manner. The MCP-1-mediated proliferation was significantly inhibited when MCP-1-specific antiserum was added to PMC cultures. Abundant expression of MCP-1 in wounds indicates that injury causes the expression of MCP-1 in local tissues (13, 15). Therefore, we speculate that in the case of pleural injury, PMCs release MCP-1, and this released MCP-1 induces PMC proliferation.
Ligand-receptor interactions are essential to mount a cellular
migratory response. It is likely that CCR2 (MCP-1 binding receptor) mediates the haptotactic responses in PMCs to MCP-1. Flow cytometric analysis demonstrated that resting PMCs express CCR2 and that IL-2
augmented CCR2 expression in PMCs. CCR2 facilitated haptotactic activity in the resting PMC cells, and IL-2 potentiates the PMC haptotactic activity by inducing CCR2 expression. Inhibition of the PMC
haptotactic response to MCP-1 with the neutralizing antibody to CCR2
suggests that PMC haptotactic activity to MCP-1 is mediated through the
CCR2. In CCR2 knockout [CCR2(/
)] mice,
monocytes failed to migrate in response to MCP-1 (8). The CCR2 has two isoforms, CCR2A and CCR2B, and expression of the particular isoform varies from cell to cell depending on disease conditions. PMCs expressed both the A and B isoforms of CCR2 mRNA in a time-dependent manner. It has been reported that activated monocytes, NK cells, and
dendritic cells express abundant amounts of the CCR2B isoform and, to a
lesser extent, the CCR2A isoform (9, 26). Our results with RT-PCR and
Southern analysis studies demonstrate that PMCs abundantly express the
CCR2B isoform compared with the CCR2A isoform. Mesothelial cells
respond to laminin and fibronectin by haptotaxis (16). Davila and
Crouch (12) demonstrated that mesothelial cells produce extracellular
matrix during pleural injury. Another study (38) demonstrated that
ultrastructurally, mesothelial cells at an area of regeneration take on
a macrophage-like morphology, implying an ability to migrate. Taken
together, these findings demonstrated that in the case of pleural
injury, PMCs proliferate and migrate laterally by haptotaxis, resulting
in pleural repair without fibrosis.
The mechanism of regulation of chemokine-receptor expression in PMCs and their role in PMC haptotaxis remain unclear. The chemokines and cytokines released at the site of injury in the milieu of the pleural space regulate the repair process. Cytokines and LPS were noticed to modulate the expression of IL-8A and IL-8B receptors in polymorphonuclear leukocytes (21). In basophils, IL-5 upregulated CCR4R expression (27). IL-2 regulates CCR2 expression and chemotactic responsiveness in T lymphocytes (22). Our findings demonstrate that the incubation of PMCs with IL-2 resulted in a dramatic increase in the expression of CCR2, and it is likely that augmented receptor levels underlie the better haptotactic migratory responsiveness of activated PMCs to MCP-1. Therefore, it is probable that under inflammatory conditions, the action of chemokines may be regulated at the level of cytokine production as well as of receptor expression.
Host defense responses to pleural infection involve the recruitment of leukocytes, which can lead to clearance of the infecting organisms (18). Several soluble factors including IL-2 are known to be released in the pleural space (6, 33). During pleural inflammation, the recruited lymphocytic population may secrete IL-2 and other cytokines (7, 32), which mount secondary responses including augmenting the PMC responses in the pleural space. IL-2 upregulates CCR2 expression in PMCs and also increases haptotactic migration. PMCs release MCP-1 in response to inflammatory stimuli (24, 25); this released MCP-1 at the site of injury may induce proliferation and haptotactic responses in PMCs in a paracrine fashion. The presence of IL-2 amplifies CCR2 expression and thus facilitates MCP-1-mediated haptotactic activity in PMCs.
In pleural infections, the invading pathogens release endotoxin in the pleural space. In an earlier study by Antony et al. (2), bacterial endotoxin (LPS) has been found to stimulate MCP-1 expression in PMCs. However, in the present study, LPS was noticed to downregulate CCR2 (CCR2A and CCR2B) expression and also to suppress the migratory capacity of PMCs. Endotoxin suppressed CCR2 expression in peripheral blood monocytes (34). LPS caused a rapid loss of steady-state levels of CCR2 message through message degradation in a monocytic (THP-1) cell line (41). LPS dramatically reduced the expression of CCR2 on the surface of peripheral blood cells in mice and completely blocked macrophage infiltration into the peritoneal cavity (42). These findings suggest that in the presence of endotoxins, chemokine and chemokine-receptor mRNA expression are regulated by different mechanisms. Inhibition of CCR2 expression in PMCs with LPS indicates that endotoxins present in the milieu of pleural injury affect the pleural repair process.
The significance of the present work is that it demonstrates a potentially important and primary role of PMCs in the process of repair without fibrosis of an injured pleura. PMCs express CCR2 and are able to respond to the paracrine release of MCP-1 by injured mesothelial cells. MCP-1, through its ability to stimulate PMC proliferation as well as haptotaxis, may also play a central role in this repair process.
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ACKNOWLEDGEMENTS |
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We acknowledge the help of Diana L. Baxter (Medical Media, Veterans Affairs Medical Center, Indianapolis, IN) for photographic work.
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FOOTNOTES |
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This work was supported in part by National Institute of Allergy and Infectious Diseases Grants R01-AI-37454-03 and R01-AI-41877-01.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. B. Antony, Veterans Affairs Medical Center, 1481 West 10th St., 111-P, Indianapolis, IN 46202 (E-mail: vantony{at}iupui.edu).
Received 29 June 1999; accepted in final form 13 September 1999.
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