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The Role of CC Chemokine Receptor 2 in Alveolar Monocyte and Neutrophil Immigration in Intact Mice

Department of Internal Medicine, Justus-Liebig-University, Giessen; Medical Policlinic, University of Munich, Munich; Institute for Animal Physiology, University of Munich, Munich, Germany; Department of Microbiology, University of Texas at Austin, Austin, Texas; and Department of Surgery, North Shore University Hospital, Manhasset, New York

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: ulrich.a.maus{at}med.uni-giessen.de

	ABSTRACT

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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.

Key Words: lung • monocyte • CC chemokine receptor 2 • CC chemokine ligand 2 • inflammation

	INTRODUCTION

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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.

	METHODS

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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-1^low or Gr-1^neg 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-1^high neutrophils and Gr-1^low and Gr-1^neg 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 (2–4). 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 (2–4).

Flow Cytometry
A FACStar^PLUS 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.

	RESULTS

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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).

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of CCR2 ablation or inhibition on alveolar leukocyte trafficking in response to CCL2 and LPS. Groups of wild-type mice (black bars), CCR2-deficient mice (white bars), and anti-CCR2–treated mice (hatched bars) were left untreated (0 hours) or received an IT application of CCL2 and LPS. Mice were killed, and bronchoalveolar lavage was performed on the untreated mice at 3, 6, 12, 24, 48, or 72 hours after instillation of CCL2 and LPS. Quantitation of alveolar recruited monocytes (A) and neutrophils (B) was performed by the FACS technique. Values are presented as mean ± SEM (n = 5 for all time points except at 72 hours, where n = 3). Alv-Mo, alveolar-recruited monocytes; PMN, polymorphonuclear neutrophils; *p < 0.05 for comparison with wild-type mice.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of function-blocking anti-CCR2 mAbs on alveolar monocyte recruitment. Wild-type mice received an intravenous injection of the red fluorescent intravital dye PKH26 for labeling of the resident alveolar macrophage pool. PKH26 was used to increase the red fluorescence emission properties of these cells (designated as rAM in the upper right quadrant of the dot plots A–C). After 24 hours, some mice received an intra-alveolar instillation of CCL2 (A, 50 µg/mouse) or an intraperitoneal injection of anti-CCR2 mAb (500 µg per mouse) followed by IT instillation of CCL2 alone (B) or with LPS (10 ng per mouse) (C) (n = 3 for each group). After an additional 24 hours, mice were killed, intra-alveolar leukocytes were harvested by bronchoalveolar lavage, and leukocyte differentials were determined by FACS analysis. A representative FACS profile is shown for each treatment protocol. Alv-Mo, alveolar recruited monocytes; rAM, resident alveolar macrophages; PMN, polymorphonuclear neutrophils; Ly, lymphocytes.

View larger version (79K): [in this window] [in a new window]   Figure 3. (A–D) Immunophenotyping of circulating and alveolar recruited neutrophils and monocytes from wild-type mice. Peripheral blood was harvested from untreated wild-type mice and was analyzed by dual-color flow cytometry for distribution of Gr-1+ and F4/80+ cells. (A) Negative control. (B) Specific F4/80-FITC (x axis) and Gr-1–PE (y axis) staining indicates that circulating neutrophils are Gr-1 positive (Gr-1+ PMN, arrow) and F4/80 negative. Circulating F4/80+ monocytes exhibit bimodal Gr-1 staining with Gr-1neg (B, Gr-1- Mo, arrow) and Gr-1low monocytes (B, Gr-1+ Mo, arrow). The dot plots in C and D show a corresponding FACS profile of F4/80 and Gr-1 expression by alveolar-recruited neutrophils and monocytes recovered from PKH26-treated mice challenged with CCL2 and LPS. (C) Negative control, showing the lack of PKH26 fluorescence by recruited neutrophils (C, PMN) and slightly increased PKH26 emission by recruited monocytes (C, Alv-Mo) and highly increased PKH26 fluorescence by resident alveolar macrophages (C, rAM). (D) Immunophenotyping of Gr-1 expression by alveolar recruited neutrophils and monocytes and resident alveolar macrophages, respectively. rAM lack Gr-1 expression (D, rAM, arrow), whereas alveolar recruited neutrophils are strongly Gr-1+ (D, Gr-1+ PMN, arrow). Alveolar recruited monocytes show a similar bimodal Gr-1 staining pattern as circulating monocytes (D, Gr-1+ Alv-Mo, arrow versus Gr-1- Alv-Mo, arrow). (E) Depletion of neutrophils in wild-type mice with anti–Gr-1. Mice were given an intraperitoneal injection of control antibody (CL) or increasing amounts of anti–Gr-1 (clone RB6-8C5) (5, 10, and 20 µg per mouse). After 24 hours, peripheral blood was collected, and neutrophils were gated according to their forward scatter and Gr-1 surface expression profile in the fluorescence 2 channel for PE. Monocytes were gated according to their FSC and F4/80 FITC expression, and Gr-1 coexpression was analyzed in the PE channel. Note that treatment of mice with 5 µg or more of anti–Gr-1 mAb effectively depleted circulating neutrophils (second row, histogram on the left), whereas 10 µg or more per mouse of anti-Gr-1 mAb additionally strongly depleted Gr-1+ monocytes (third row, histogram on the right). A representative FACS profile is shown. Mo, monocyte; PMN, polymorphonuclear neutrophil.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of transient neutropenia on alveolar monocyte accumulation in wild-type mice. (A) Groups of mice were left untreated (black bars) or were made transiently neutropenic by pretreatment with anti–Gr-1 mAb. After 24 hours, neutropenic mice received an IT instillation of CCL2 plus LPS (white bars). At the indicated time points, cells were harvested by bronchoalveolar lavage, and the alveolar monocyte subpopulations were quantitated. Values are presented as mean ± SEM (n = 5 mice per group). *p < 0.05 for comparison between the neutropenic and normal mouse groups. (B) Representative histogram of the distribution profile of Gr-1 expression by residual alveolar recruited monocytes collected from transiently neutropenic mice challenged for 48 hours with CCL2 and LPS.

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).

View larger version (26K): [in this window] [in a new window]   Figure 5. Effect of antileukinate on alveolar leukocyte accumulation in response to CCL2 and LPS. Untreated wild-type mice (black bars) or animals systemically pretreated with the CXC receptor inhibitor antileukinate (AL) (white bars) were challenged with CCL2 and LPS. After the indicated times, the mice were killed, and pulmonary cells were harvested with quantitation of alveolar recruited polymorphonuclear neutrophils (A, PMN), and monocytes (B, Alv-Mo). Values are expressed as mean ± SEM (n = 5 mice per group). *p < 0.05 for comparison between the two groups.

View larger version (23K): [in this window] [in a new window]   Figure 6. Pulmonary leakage associated with inflammation induced by CCL2 and LPS. Wild-type mice were treated as indicated. At 1 hour before the mice were killed, they received an intravenous injection of FITC-labeled albumin (1.5 mg per mouse). Serum and lavage fluids were collected, and lung permeability index was determined as described (4). Values are presented as mean ± SEM (n = 5 mice per group). *p < 0.05 versus the 0-hour time point. +p < 0.05 versus the respective time points shown in A.

	DISCUSSION

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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 ß2 integrin -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.

	Acknowledgments

 
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."

Received in original form December 4, 2001; accepted in final form April 26, 2002

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				L. Ziegler-Heitbrock The CD14+ CD16+ blood monocytes: their role in infection and inflammation J. Leukoc. Biol., March 1, 2007; 81(3): 584 - 592. [Abstract] [Full Text] [PDF]

				L. Landsman, C. Varol, and S. Jung Distinct Differentiation Potential of Blood Monocyte Subsets in the Lung J. Immunol., February 15, 2007; 178(4): 2000 - 2007. [Abstract] [Full Text] [PDF]

				C. Varol, L. Landsman, D. K. Fogg, L. Greenshtein, B. Gildor, R. Margalit, V. Kalchenko, F. Geissmann, and S. Jung Monocytes give rise to mucosal, but not splenic, conventional dendritic cells J. Exp. Med., January 22, 2007; 204(1): 171 - 180. [Abstract] [Full Text] [PDF]

				A. Zarbock, M. Schmolke, T. Spieker, K. Jurk, H. Van Aken, and K. Singbartl Acute Uremia but Not Renal Inflammation Attenuates Aseptic Acute Lung Injury: A Critical Role for Uremic Neutrophils J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3124 - 3131. [Abstract] [Full Text] [PDF]

				U. A. Maus, S. Janzen, G. Wall, M. Srivastava, T. S. Blackwell, J. W. Christman, W. Seeger, T. Welte, and J. Lohmeyer Resident Alveolar Macrophages Are Replaced by Recruited Monocytes in Response to Endotoxin-Induced Lung Inflammation Am. J. Respir. Cell Mol. Biol., August 1, 2006; 35(2): 227 - 235. [Abstract] [Full Text] [PDF]

				S. Herold, W. von Wulffen, M. Steinmueller, S. Pleschka, W. A. Kuziel, M. Mack, M. Srivastava, W. Seeger, U. A. Maus, and J. Lohmeyer Alveolar Epithelial Cells Direct Monocyte Transepithelial Migration upon Influenza Virus Infection: Impact of Chemokines and Adhesion Molecules J. Immunol., August 1, 2006; 177(3): 1817 - 1824. [Abstract] [Full Text] [PDF]

				V. H. Rao, D. T. Meehan, D. Delimont, M. Nakajima, T. Wada, M. A. Gratton, and D. Cosgrove Role for Macrophage Metalloelastase in Glomerular Basement Membrane Damage Associated with Alport Syndrome Am. J. Pathol., July 1, 2006; 169(1): 32 - 46. [Abstract] [Full Text] [PDF]

				X. Lin, H. Yang, T. Sakuragi, M. Hu, L. L. Mantell, S. Hayashi, Y. Al-Abed, K. J. Tracey, L. Ulloa, and E. J. Miller {alpha}-Chemokine receptor blockade reduces high mobility group box 1 protein-induced lung inflammation and injury and improves survival in sepsis Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L583 - L590. [Abstract] [Full Text] [PDF]

				M. Srivastava, S. Jung, J. Wilhelm, L. Fink, F. Buhling, T. Welte, R. M. Bohle, W. Seeger, J. Lohmeyer, and U. A. Maus The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression Profiles J. Immunol., August 1, 2005; 175(3): 1884 - 1893. [Abstract] [Full Text] [PDF]

				J. J. Osterholzer, T. Ames, T. Polak, J. Sonstein, B. B. Moore, S. W. Chensue, G. B. Toews, and J. L. Curtis CCR2 and CCR6, but Not Endothelial Selectins, Mediate the Accumulation of Immature Dendritic Cells within the Lungs of Mice in Response to Particulate Antigen J. Immunol., July 15, 2005; 175(2): 874 - 883. [Abstract] [Full Text] [PDF]

				M. Aurrand-Lions, C. Lamagna, J. P. Dangerfield, S. Wang, P. Herrera, S. Nourshargh, and B. A. Imhof Junctional Adhesion Molecule-C Regulates the Early Influx of Leukocytes into Tissues during Inflammation J. Immunol., May 15, 2005; 174(10): 6406 - 6415. [Abstract] [Full Text] [PDF]

				O. Dewald, P. Zymek, K. Winkelmann, A. Koerting, G. Ren, T. Abou-Khamis, L. H. Michael, B. J. Rollins, M. L. Entman, and N. G. Frangogiannis CCL2/Monocyte Chemoattractant Protein-1 Regulates Inflammatory Responses Critical to Healing Myocardial Infarcts Circ. Res., April 29, 2005; 96(8): 881 - 889. [Abstract] [Full Text] [PDF]

				J. H. Jennings, D. J. Linderman, B. Hu, J. Sonstein, and J. L. Curtis Monocytes Recruited to the Lungs of Mice during Immune Inflammation Ingest Apoptotic Cells Poorly Am. J. Respir. Cell Mol. Biol., February 1, 2005; 32(2): 108 - 117. [Abstract] [Full Text] [PDF]

				U. A. Maus, S. Wellmann, C. Hampl, W. A. Kuziel, M. Srivastava, M. Mack, M. B. Everhart, T. S. Blackwell, J. W. Christman, D. Schlondorff, et al. CCR2-positive monocytes recruited to inflamed lungs downregulate local CCL2 chemokine levels Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L350 - L358. [Abstract] [Full Text] [PDF]

				R. W. Saeed, S. Varma, T. Peng, K. J. Tracey, B. Sherry, and C. N. Metz Ethanol Blocks Leukocyte Recruitment and Endothelial Cell Activation In Vivo and In Vitro J. Immunol., November 15, 2004; 173(10): 6376 - 6383. [Abstract] [Full Text] [PDF]

				I. E. Konstantinov, S. Arab, R. K. Kharbanda, J. Li, M. M. H. Cheung, V. Cherepanov, G. P. Downey, P. P. Liu, E. Cukerman, J. G. Coles, et al. The remote ischemic preconditioning stimulus modifies inflammatory gene expression in humans Physiol Genomics, September 16, 2004; 19(1): 143 - 150. [Abstract] [Full Text] [PDF]

				H. M. Razavi, L. F. Wang, S. Weicker, M. Rohan, C. Law, D. G. McCormack, and S. Mehta Pulmonary Neutrophil Infiltration in Murine Sepsis: Role of Inducible Nitric Oxide Synthase Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 227 - 233. [Abstract] [Full Text] [PDF]

				U. A. Maus, M. Srivastava, J. C. Paton, M. Mack, M. B. Everhart, T. S. Blackwell, J. W. Christman, D. Schlondorff, W. Seeger, and J. Lohmeyer Pneumolysin-Induced Lung Injury Is Independent of Leukocyte Trafficking into the Alveolar Space J. Immunol., July 15, 2004; 173(2): 1307 - 1312. [Abstract] [Full Text] [PDF]

				J. L. Lomas-Neira, C.-S. Chung, P. S. Grutkoski, E. J. Miller, and A. Ayala CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice J. Leukoc. Biol., July 1, 2004; 76(1): 58 - 64. [Abstract] [Full Text] [PDF]