American Journal of Respiratory and Critical Care Medicine

The CC chemokine ligand 2 (CCL2) (JE, monocyte chemotactic protein-1 [MCP-1]) and its CC chemokine receptor 2 (CCR2) are critical regulators of monocyte/macrophage trafficking. Recently, we demonstrated that application of exogenous CCL2 in the lungs of mice induced monocyte accumulation in the airspace, whereas combined bronchoalveolar instillation of CCL2 and Escherichia coli endotoxin provoked both enhanced monocyte accumulation and extensive neutrophil influx associated with loss of pulmonary endothelial/epithelial barrier function. In this study, we investigated the role of the CCL2 receptor CCR2 in alveolar leukocyte traffic. In CCR2 knockout mice or wild-type mice treated with the anti-CCR2–blocking monoclonal antibody MC21, monocyte accumulation in response to alveolar CCL2 or CCL2 plus endotoxin was inhibited by more than 90%. Unexpectedly, alveolar neutrophil accumulation in the CCL2/lipopolysaccharide (LPS) model was also drastically reduced by both approaches of CCR2 function interference. When wild-type mice treated with anti–Gr-1 monoclonal antibody to deplete neutrophils selectively or treated with antileukinate, a CXC receptor inhibitor, were challenged with alveolar CCL2 plus LPS, alveolar monocyte accumulation was markedly decreased. Wild-type mice treated with MC21 to block CCR2 function or with anti–Gr-1 to deplete neutrophils did not exhibit the vascular leakage that typically accompanies inflammation triggered by CCL2 and LPS in wild-type mice. These findings confirm a central role for CCR2 in the process of alveolar monocyte recruitment in response to CCL2 alone and combined CCL2 plus LPS and reveal a previously unobserved interdependence between monocyte and neutrophil trafficking that has important implications for the concomitant increase in vascular permeability.

The contribution of monocytes to acute lung inflammation is largely unknown, mainly because of difficulties in discriminating newly recruited monocytes from resident alveolar macrophages (1). Recently, we described a novel fluorescence-activated cell sorter (FACS)-based technique permitting discrimination of alveolar recruited monocytes from other cell populations in the alveolar compartment, including neutrophils and resident alveolar macrophages (2, 3). We showed that deposition of the monocyte chemoattractant CCL2 (JE, MCP-1) in the lungs of intact mice provoked monocyte accumulation in the alveoli in the absence of inflammation (2). In contrast, intratracheal (IT) administration of CCL2 along with a low dose of Escherichia coli endotoxin induced a strong yet self-limiting pulmonary inflammatory response characterized by tightly controlled waves of neutrophil and monocyte accumulation in the airspace (4). In parallel, enhanced permeability of the capillary endothelial/alveolar epithelial barrier was noted, with enhanced protein leakage into the alveolar compartment. These characteristics of the model resemble the key pathophysiologic features associated with the acute respiratory distress syndrome in humans (4, 5).

In mice, an abundance of evidence indicates that CCL2-induced signaling occurs exclusively through binding to CC chemokine receptor 2 (CCR2), which belongs to the family of G-protein–coupled, seven-transmembrane–spanning cell surface receptors (6). Recently, it was demonstrated that murine CCR2 is expressed at highest levels on circulating monocytes (7), and CCR2-bearing monocytes but not neutrophils are the primary cells recruited into the alveolar airspace by exogenous CCL2 (2, 4). In this study, the role of CCR2-mediated signaling in alveolar leukocyte traffic in mouse lung inflammation induced by the combination of CCL2 and lipopolysaccharide (LPS) was investigated in more detail. Studies were performed in wild-type mice, mice deficient in CCR2, wild-type mice pretreated with blocking anti-CCR2 monoclonal antibody (mAb) MC21, and mice rendered selectively neutropenic or treated with antileukinate, a CXC receptor inhibitor (8). We found that CCR2 was necessary not only for monocyte influx but also for the earlier accumulation of neutrophils. We also found that neutropenic mice or mice in which the chemotaxis of circulating neutrophils was blocked by antileukinate treatment would not support monocyte migration into the airspace in response to CCL2 and LPS. Both types of intervention inhibited the vascular leakage response to lung CCL2/LPS deposition.

Animals

Wild-type BALB/c mice (18–21 g) were purchased from Charles River (Sulzfeld, Germany). CCR2 knockout mice were generated by targeted disruption of the CCR2 gene as recently described (9), and the disrupted CCR2 allele was backcrossed for six generations to inbred BALB/c animals. The BALB/c CCR2−/− breeders were maintained under specific pathogen-free conditions. Experimental animals were used between 8–10 weeks of age. Experimental protocols involving animals were approved by institutional and local government committees.

Reagents

The red fluorescent dye PKH26-PCL and diluent B solution were purchased from Zynaxis (Malvern, CA; Sigma, Deisenhofen, Germany). Recombinant CCL2 was purchased from R&D Systems (Wiesbaden, Germany). The hexapeptide antileukinate (Ac-RRWWCR-NH2) was synthesized and purified at Synpep Corp. (Dublin, CA). All reagents and mAbs used were ascertained to be endotoxin free by limulus amebocyte lysate (LAL) assay (COATEST; Chromogenix, Mölndal, Sweden; detection limit less than 10 pg/ml). E. coli lipopolysaccharide (O111:B4) was purchased from Sigma.

Antibodies

Azide-free preparations of rat monoclonal anti-murine Gr-1 (clone RB6-8C5, rat isotype IgG2b) were purchased from BD Biosciences (Heidelberg, Germany). The antibody is a complement-fixing isotype and is widely used for depletion of circulating polymorphonuclear neutrophils (10, 11). Phycoerythrin (PE)-conjugated anti–Gr-1 or fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 mAb preparations used for phenotypic analysis were from BD and Serotec (München, Germany), respectively. The function-blocking rat anti-murine CCR2 mAb (clone MC21, isotype IgG2b) has been described recently in detail (7). Isotype-matched rat IgG2b control antibody was obtained from BD Biosciences. For inhibition experiments, mice received an intraperitoneal injection of 250–500 μg per mouse of anti-CCR2 mAb 6 hours before IT instillation of CCL2 and LPS.

Depletion of Circulating Neutrophils and Counting Peripheral Blood Leukocytes

Dose–response experiments were done to determine the optimal amount of anti–Gr-1 that would result in successful neutrophil depletion. Groups of four mice were given an intraperitoneal injection of 1, 2, 5, 7.5, 10, 20, 100, and 200 μg of anti–Gr-1 mAb. After 24 hours, mice were killed with isoflurane (Forene; Abbott, Wiesbaden, Germany), and anticoagulated whole blood was collected. Successful neutrophil depletion (more than 90%) was verified by flow cytometry and on Pappenheim-stained blood smears (Merck, Darmstadt, Germany). Monocytes and neutrophils were gated according to their forward- and side-scatter properties. Highly Gr-1–positive, F4/80-negative neutrophils as well as F4/80-positive and either Gr-1low or Gr-1neg monocyte subpopulations were analyzed by direct dual-color immunofluorescence staining using FITC-conjugated anti-F4/80 mAbs in combination with PE-conjugated anti–Gr-1 mAbs according to protocols described elsewhere (2, 12). Optimal signal-to-noise ratios required for flow cytometric discrimination of Gr-1high neutrophils and Gr-1low and Gr-1neg monocytes were obtained with 1:100 dilutions of anti–Gr-1 mAb preparations. Peripheral blood cell counts were performed on Pappenheim-stained blood smears obtained from untreated mice and from mice treated with anti–Gr-1 mAbs.

Preparation and Implantation of Antileukinate Containing Micro-osmotic Pumps

Micro-osmotic pumps (model 1003D; Alzet Corp., distributed by Charles River Laboratories, Sulzfeld, Germany) were used for subcutaneous delivery of antileukinate. Pumps were preincubated for 4 hours at 37°C in sterile saline according to the manufacturer's instructions and were subsequently filled with 100 μl of antileukinate solution (2 μg/μl in phosphate-buffered saline, pH 7.4). After mice were anesthetized, an approximately 1-cm incision was made in the skin of the abdominal region, and a subcutaneous pocket was formed using sterile forceps. The pumps were implanted in these preformed pockets, and wounds were closed with sterile sutures. In addition to the systemic administration of antileukinate via micro-osmotic pumps (pumping rate, 1 μl/hour), mice received a single intravenous injection of antileukinate (20 μg in phosphate-buffered saline, pH 7.4, per mouse) directly before IT instillation of CCL2 and LPS at the time points analyzed. To evaluate the effect of antileukinate on alveolar monocyte recruitment under noninflammatory conditions, other groups of antileukinate-treated mice received only CCL2.

Treatment Protocol

Fluorescence labeling of resident alveolar macrophages in vivo was performed as recently described in detail (24). Four treatment groups were evaluated: (1) CCR2-deficient mice receiving IT instillation of CCL2 (50 μg per mouse) in the presence of E. coli LPS (10 ng per mouse), (2) wild-type mice receiving intraperitoneal injections of the anti-CCR2 mAb MC21 followed by IT instillation of CCL2 and LPS, (3) transiently neutropenic wild-type mice receiving IT instillation of CCL2 with or without LPS, and (4) antileukinate-treated wild-type mice receiving an IT instillation of CCL2 with or without LPS.

Lung Permeability Assay

Analysis of lung barrier function was monitored by leakage of FITC–albumin into the alveolar space as described in detail elsewhere (4, 13).

Collection of Blood Samples and Bronchoalveolar Lavage

Mice in the various experimental groups were killed with an overdose of isoflurane at 3, 6, 12, 24, 48, and 72 hours after treatment with CCL or CCL2 plus LPS. Blood samples and bronchoalveolar lavage fluids were collected as described earlier (24).

Flow Cytometry

A FACStarPLUS flow cytometer was used throughout this study and was operated as recently detailed (14). Identification of leukocyte populations in the lungs was performed as described elsewhere (2, 3).

Statistics

All data are given as mean ± SEM. Statistical significance between treatment groups was estimated by the Mann-Whitney U test. Differences were considered to be statistically significant when p values were less than 0.05.

Effect of CCR2 Deficiency on Alveolar Leukocyte Recruitment in Response to CCL2 and LPS

Neutrophil and monocyte trafficking in wild-type mice in response to combined intrapulmonary CCL2 and LPS challenge is characterized by early alveolar neutrophil influx, which peaks at approximately 12 hours after challenge, followed by monocyte accumulation, which peaks at approximately 48 hours after challenge (Figures 1A and 1B)

. Pretreatment of wild-type mice with the anti-CCR2 mAb MC21 resulted in a more than 90% reduction in alveolar monocyte recruitment in response to CCL2 alone (Figures 2A and 2B) or CCL2 plus LPS (Figures 1A and 2C). CCR2 knockout mice also exhibited a severe defect in alveolar monocyte recruitment in response to CCL2 and LPS (Figure 1A). Surprisingly, in addition to the expected drop in monocyte recruitment, the alveolar neutrophil accumulation in CCR2 knockout mice was also reduced by more than 90% (Figure 1B). Similarly, pretreatment of wild-type mice with the anti-CCR2 mAb MC21 significantly reduced the peak alveolar neutrophil accumulation observed at approximately 12 hours after CCL2 and LPS treatment (Figure 1B).

Effect of Neutrophil Depletion or Antileukinate Treatment on Alveolar Monocyte Trafficking in Response to CCL2 and LPS

The finding that disruption of the CCR2 gene or blocking CCR2 protein with MC21 suppressed alveolar monocyte and neutrophil accumulation in the lungs led us to hypothesize that alveolar monocyte and neutrophil recruitment are interdependent in our model of acute lung inflammation. To evaluate whether neutrophil depletion impaired alveolar monocyte accumulation in response to CCL2 and LPS, wild-type mice were rendered transiently neutropenic by treatment with anti–Gr-1. In earlier experiments on wild-type mice to determine the optimal concentration of anti–Gr-1 to deplete neutrophils, dual-color flow cytometry revealed homogeneous Gr-1 cell surface antigen expression on neutrophils and absence of staining for the monocyte marker F4/80. Circulating monocytes exhibited homogeneous staining for F4/80 and a biphasic Gr-1 cell surface distribution (Figures 3A and 3B)

, extending previous reports (12). Importantly, the Gr-1 expression profile detected on circulating neutrophils and monocytes was similar to the profile detected on neutrophils and monocytes recovered from the bronchoalveolar lavage fluid obtained from wild-type mice treated with CCL2 and LPS (Figures 3C and 3D). This demonstrates that both Gr-1–positive and Gr-1–negative monocyte subpopulations were nonselectively recruited into the lungs. Careful titration experiments showed that intraperitoneal injection of approximately 7.5 μg of anti–Gr-1 was sufficient to obtain a more than 90% depletion of highly Gr-1–positive neutrophils (Figure 3E) while avoiding depletion of weakly Gr-1–positive monocytes. This finding was confirmed by light microscopic examination of Pappenheim-stained blood smears obtained from mice treated with 7.5 μg anti–Gr-1. Control mice showed 84 ± 2% lymphocytes, 6 ± 1% monocytes, and 9 ± 2% polymorphonuclear neutrophils, whereas anti-Gr-1–treated animals showed 89 ± 3% lymphocytes, 10 ± 2% monocytes, and 1 ± 0.5% polymorphonuclear neutrophils (mean ± SEM, n = 4).

When neutropenic wild-type mice were subsequently challenged with an IT instillation of CCL2 and LPS, alveolar monocyte recruitment was strongly reduced (Figure 4A)

, suggesting that the extensive alveolar monocyte accumulation observed in the response of wild-type mice to CCL2 and LPS required the presence of neutrophils. Because the residual alveolar recruited monocytes in neutropenic mice demonstrated a similar distribution pattern of Gr-1neg to Gr-1low cells as observed for circulating monocytes, it is unlikely that anti–Gr-1 treatment depleted the population of Gr-1low monocytes (Figure 4B).

Interestingly, there was no difference between peak monocyte recruitment in wild-type or transiently neutropenic mice 48 hours after IT instillation of CCL2 alone into the lungs (data not shown), indicating that neutrophils were not required for this response.

To investigate whether blockade of neutrophil chemotaxis would affect alveolar monocyte recruitment in response to CCL2 plus LPS, wild-type mice were pretreated systemically with the CXC receptor inhibitor antileukinate. Treatment of mice with antileukinate was highly effective in blocking early alveolar neutrophil accumulation in response to CCL2 and LPS (Figure 5A)

. Importantly, the strongly reduced alveolar neutrophil trafficking was associated with strongly reduced alveolar monocyte accumulation (Figure 5B). In contrast, systemic pretreatment of mice with antileukinate did not affect peak alveolar monocyte accumulation induced by CCL2 alone (data not shown).

Effect of CCR2 Blockade or Transient Neutropenia on Pulmonary Barrier Function

Loss of lung barrier function has a major impact on pulmonary gas exchange in inflamed lungs. Therefore, we examined whether reduced leukocyte trafficking across the pulmonary endothelial/epithelial barrier observed in mice treated with anti-CCR2 mAb as well as neutropenic wild-type mice affected the leakage response that occurs in wild-type mice in response to CCL2 and LPS (Figure 6A)

(4). Mice pretreated with the anti-CCR2 mAb MC21 and subsequently challenged with CCL2 plus LPS did not exhibit a major leakage response during the 24-hour observation period (Figure 6B; p < 0.05). Similarly, neutropenic wild-type mice also showed a significant reduction in protein permeability relative to the leakage observed in wild-type mice treated with CCL2 and LPS (Figure 6C; p < 0.05).

Alveolar monocyte migration in response to lung deposition of CCL2 or CCL2 and LPS was virtually fully blocked in both CCR2 knockout mice and wild-type mice pretreated with a blocking anti-CCR2 mAb. Surprisingly, neutrophil accumulation, which normally constitutes the first wave of inflammatory leukocytes to accumulate in the lungs in response to CCL2 and LPS in wild-type mice, was also largely suppressed when functional CCR2 was absent. On the other hand, alveolar monocyte accumulation in response to CCL2 and LPS was markedly reduced when either selective depletion or blockade of the chemotactic response of circulating neutrophils was achieved in wild-type mice. Both interference with CCR2/monocytes (function-blocking anti-CCR2 mAb) and neutrophil depletion also largely attenuated the vascular leakage response induced by combined CCL2 and LPS treatment in wild-type mice.

We evaluated the role of CCR2-mediated signaling in alveolar leukocyte traffic using a recently established mouse model of CCL2 plus LPS-induced acute pulmonary inflammation (4). We hypothesized that employment of CCR2 knockout mice would lead to a largely impaired alveolar influx of monocytes. In fact, CCR2 knockout mice demonstrated a more than 90% blockade of alveolar monocyte traffic in response to CCL2 alone and to combined CCL2/LPS challenge. Furthermore, suppressed monocyte migration in response to CCL2/LPS challenge was also achieved when wild-type mice were pretreated with a novel anti-CCR2–blocking mAb (7). It is well established that CCL2 is a critical chemoattractant for monocytes and that CCR2 is the primary (if only) receptor on monocytes for CCL2. Indeed, CCL2 is known to promote monocyte firm adhesion to the vascular endothelium under dynamic flow conditions in vitro (15). This process is suppressed by CCL2 antagonists and is presumably operative via CCR2-mediated monocyte β2 integrin α-chain activation (U. Maus, unpublished observation). Similarly, CCR2 knockout mice are defective in monocyte firm adhesion to the vascular endothelium in response to the application of exogenous CCL2 in vivo (9). Thus, it is likely that monocyte accumulation in inflammation induced by CCL2 and LPS is mediated by CCR2.

An important and unexpected finding of this study was the marked reduction in the neutrophil response in both CCR2 knockout mice and anti-CCR2–treated wild-type mice after lung challenge with CCL2 and LPS. Of note, the anti-CCR2 mAb exhibited no unspecific leukocyte binding, either in wild-type or in CCR2 knockout mice, as analyzed by flow cytometry (data not shown). Because circulating neutrophils do not express CCR2 (U. M. and colleagues, unpublished observations) (7) and are not attracted into the airspaces in response to CCL2 alone (2), we hypothesized that reduced alveolar neutrophil recruitment observed in wild-type mice pretreated with blocking anti-CCR2 mAb and CCR2 knockout mice might be secondary to impaired (CCR2-dependent) cross-talk between monocytes and neutrophils. Interestingly, both experimental approaches involving depletion of circulating neutrophils or selective blockade of neutrophil chemotaxis (8, 16) suggest that alveolar monocyte accumulation in response to CCL2 alone is largely neutrophil independent, whereas obviously an interdependence between monocytes and neutrophils is essential for increased leukocyte immigration in response to CCL2 and LPS. Interestingly, in the systemic circulation, normal numbers of neutrophils that accumulate during thioglycollate-induced peritonitis in CCR2 knockout mice were observed even though the subsequent wave of monocyte influx was totally abrogated (9). The basis for this difference is currently not clear. It might be related to the different stimuli used. Alternatively, the pulmonary endothelial/epithelial barrier may represent a specialized site with respect to leukocyte cooperativity or interdependence of inflammatory cell recruitment.

An explanation for the relationship between monocyte accumulation and previous neutrophil influx has been offered previously. It has been proposed that early neutrophil recruitment regulates subsequent monocyte accumulation during acute lung inflammatory processes through the local release of CCL2 (17). As most of the neutrophil influx precedes the monocyte accumulation in the current CCL2/LPS model, this mechanism may be operative. The reduced monocyte recruitment that we observed in the lungs of neutropenic mice or mice blocked for neutrophil chemotaxis with antileukinate is also consistent with this idea. However, such “back signaling” from already recruited leukocytes from the alveolar toward the vascular compartment may not explain the strong impact of CCR2/monocyte blockade on the neutrophil immigration when considering the kinetics of these processes. Thus, additional cross-talk between monocytes and neutrophils, particularly during acute lung inflammatory conditions, seems to occur at the onset or during the process of transendothelial/epithelial migration, and resolution of this issue is the focus of ongoing experiments.

Alveolar leukocyte recruitment may occur while the capillary endothelial/alveolar epithelial barrier properties are fully maintained, as demonstrated for the CCL2-mediated monocyte influx in wild-type mice. Leukocyte immigration may, however, be linked with severe loss of barrier properties, as observed in wild-type mice in response to alveolar CCL2 plus endotoxin challenge. This study suggests that it is not the stimulus per se (CCL2, LPS) but the related leukocyte influx that is largely responsible for the increased endothelial/epithelial permeability. The vascular leakage was largely suppressed in neutropenic mice or mice treated with anti-CCR2 antibodies. Because of reciprocal dependence of monocyte and neutrophil recruitment, the question of which of these two leukocyte subpopulations primarily affects loss of endothelial/epithelial barrier integrity in this model deserves further investigation.

In conclusion, the studies reported here have uncovered a novel interdependence of the neutrophil and monocyte responses in a model of acute pulmonary inflammation. These results suggest that in certain inflammatory situations, anti-CCR2 blocking agents may have enhanced antiinflammatory effects and support vascular integrity.

The authors are grateful for the excellent technical assistance of M. Lohmeyer and G. Mansouri.

Supported by the Deutsche Forschungsgemeinschaft, grant SFB 547 “Cardiopulmonary Vascular System.”

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Correspondence and requests for reprints should be addressed to Ulrich A. Maus, Ph.D., Department of Internal Medicine, Justus-Liebig-University, Klinikstrasse 36, Giessen 35392, Germany. E-mail:

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