AJP - Lung Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 288: L350-L358, 2005. First published October 29, 2004; doi:10.1152/ajplung.00061.2004
1040-0605/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/2/L350    most recent
00061.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maus, U. A.
Right arrow Articles by Lohmeyer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maus, U. A.
Right arrow Articles by Lohmeyer, J.

CCR2-positive monocytes recruited to inflamed lungs downregulate local CCL2 chemokine levels

Ulrich A. Maus,1 Sandra Wellmann,1 Christine Hampl,1 William A. Kuziel,2 Mrigank Srivastava,1 Matthias Mack,3 M. Brett Everhart,4 Timothy S. Blackwell,4 John W. Christman,4 Detlef Schlöndorff,3 Rainer M. Bohle,5 Werner Seeger,1 and Jürgen Lohmeyer1

1Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and Infectious Diseases, and 5Department of Pathology, Justus Liebig University, Giessen; 3Medical Policlinic, University of Munich, Munich, Germany; 2Department of Microbiology, University of Texas at Austin, Austin, Texas; and 4Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 24 February 2004 ; accepted in final form 13 October 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The CC chemokine ligand-2 (CCL2) and its receptor CCR2 are essential for monocyte trafficking under inflammatory conditions. However, the mechanisms that determine the intensity and duration of alveolar monocyte accumulation in response to CCL2 gradients in inflamed lungs have not been resolved. To determine the potential role of CCR2-expressing monocytes in regulating alveolar CCL2 levels, we compared leukocyte recruitment kinetics and alveolar CCL2 levels in wild-type and CCR2-deficient mice in response to intratracheal LPS challenge. In wild-type mice, LPS elicited a dose- and time-dependent alveolar monocyte accumulation accompanied by low CCL2 levels in bronchoalveolar lavage fluid (BALF). In contrast, LPS-treated CCR2-deficient mice lacked alveolar monocyte accumulation, which was accompanied by relatively high CCL2 levels in BALF. Similarly, wild-type mice that were treated systemically with the blocking anti-CCR2 antibody MC21 completely lacked LPS-induced alveolar monocyte trafficking that was associated with high CCL2 levels in BALF. Intratracheal application of anti-CCR2 antibody MC21 to locally block CCR2 on both resident and recruited cells did not affect LPS-induced alveolar monocyte trafficking but led to significantly increased BALF CCL2 levels. Reciprocally bone marrow-transplanted, LPS-treated wild-type and CCR2-deficient mice showed a strict inverse relationship between alveolar monocyte recruitment and BALF CCL2 levels. In addition, freshly isolated human and mouse monocytes were capable of integrating CCL2 in vitro. LPS-induced alveolar monocyte accumulation is accompanied by monocytic CCR2-dependent consumption of CCL2 levels in the lung. This feedback loop may limit the intensity of monocyte recruitment to inflamed lungs and play a role in the maintenance of homeostasis.

macrophage; neutrophil; chemokine regulation; leukocyte traffic

ACUTE PULMONARY INFLAMMATION is characterized by successive waves of early alveolar neutrophil and relatively delayed alveolar monocyte recruitment. The monocyte chemoattractant CCL2 [JE, monocyte chemoattractant protein (MCP)-1] and its receptor CCR2 are critical determinants for recruitment of monocytes to inflamed lungs. Recently, reports have identified monocytes as an active participant in the pathogenesis of acute lung inflammation and may contribute to respiratory failure (14). We have shown that bone marrow-derived blood-borne monocytes play a role in amplification of neutrophilic alveolitis in response to treatment with endotoxin (9, 10, 16). Also, CCL2-driven monocyte recruitment has been suggested to contribute to development of other inflammatory lung diseases, including idiopathic pneumonia syndrome (2).

Previous studies have characterized the sequence of recruited leukocyte populations during inflammatory responses, and a mechanism has been proposed that regulates the switch from neutrophilic to monocytic recruitment in response to treatment with endotoxin (13, 19). Although some recent studies have addressed the mechanism by which recruited macrophages are being cleared from inflamed sites (1), the mechanism(s) that determine the intensity and duration of monocyte recruitment to the lung along CCL2 gradients is not yet clear. A better understanding of this process seems important for developing strategies to restrict the leukocyte burden in inflamed organs without substantially interfering with host defense capacities.

In the present study, we tested the hypothesis that monocytes regulate their accumulation in the lungs by disrupting locally established chemokine CCL2 gradients. Our data show that alveolar recruited monocytes contribute to CCR2-mediated consumption of LPS-induced CCL2 gradients in inflamed lungs. This mechanism may serve as negative feedback loop, limiting the accumulation of monocytes into inflamed lungs and promoting the maintenance of homeostasis.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. CCR2-deficient mice were generated on a mixed C57BL/6 x 129/Ola genetic background by targeted disruption of the CCR2 gene as described previously (3). The disrupted CCR2 gene was backcrossed for six generations to wild-type BALB/c mice. Parent and offspring CCR2–/– mice on the BALB/c background were bred under specific pathogen-free (SPF) conditions. Wild-type control animals were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals 8–12 wk old and between 18–21 g were used for the described experiments. This animal study was approved by our local government committee of Giessen.

Reagents. Escherichia coli endotoxin (O111:B4) and ammonium chloride were purchased from Sigma. The function-blocking rat anti-murine CCR2 MAb (clone MC21, isotype IgG2b) has been described recently in detail (4) and has been shown to specifically block the alveolar monocyte traffic under inflammatory conditions (9). Control mice received isotype-matched nonimmune control antibody (Pharmingen, Heidelberg, Germany). Recombinant human and murine CCL2 and function-blocking anti-CCR2 antibody used for in vitro consumption experiments were purchased from R&D Systems (Wiesbaden, Germany). All antibodies and recombinant reagents were assured to be free of endotoxin as determined by Limulus amebocyte lysate assays (detection limit <10 pg/ml, Coatest; Chromogenix, Mölndal, Sweden).

Isolation of bone marrow cells and transplantation into recipient animals. Bone marrow cells were isolated under sterile conditions from the tibias and femurs of sex-matched, syngeneic CCR2+/+ and CCR2–/– donor mice (10). Briefly, single cell suspensions were carefully prepared from the bone marrow isolates and filtered through 70-µm and 40-µm nylon meshes (BD Biosciences) to remove residual cell aggregates. The cells were washed in Leibovitz L15 medium (GIBCO) before transplantation. Recipient wild-type and CCR2-deficient mice received 12 Gy of total body irradiation using a 60Co source. To reduce gastrointestinal toxicity, we applied the irradiation in two doses separated by a 3-h interval. For transplantation, the lethally irradiated recipients were sedated with ketamine and slowly infused via the lateral tail veins with donor marrow cells suspended in Leibovitz L15 medium (1 x 107 bone marrow cells/mouse). The recipient chimeric animals were then housed under SPF conditions for at least 3–4 wk with free access to autoclaved food and water. The described protocol has been shown to yield engraftment efficiencies of >97% (10).

Treatment protocols. Alveolar neutrophil and monocyte recruitment profiles were evaluated in wild-type mice and CCR2-deficient mice and, in selected experiments, also in chimeric wild-type mice (lethally irradiated wild-type mice reconstituted with bone marrow cells from CCR2-deficient mice) and chimeric CCR2-deficient mice (lethally irradiated CCR2-deficient mice reconstituted with wild-type bone marrow cells). Mice received intratracheal instillations of LPS (0.1, 1, 10, and 20 µg/mouse) for various times (0, 12, 24, 48, 72, 96, 120, and 168 h), as previously described in detail (5–10). In some inhibition experiments, wild-type mice received an injection of 250–500 µg/mouse ip of anti-CCR2 MAb MC21 6 h before inflammatory challenge. Selected mice were also treated with intratracheal instillations of E. coli LPS (20 µg/mouse) plus MC21 (100 µg/mouse) for various time points. Subsequently, mice were killed with an overdose of isoflurane. Blood samples and bronchoalveolar lavage (BAL) fluid (BALF) were collected as described earlier (5–10). Quantitation of alveolar recruited neutrophils and monocytes was done on differential cell counts of Pappenheim-stained cytocentrifuge preparations (Shandon), using overall morphological criteria, including differences in cell size and shape of nuclei and subsequent multiplication of those values with the respective absolute BAL cell counts, as recently described (8).

Determination of BALF cytokine levels. BALF cytokine levels of TNF-{alpha}, macrophage inflammatory protein (MIP)-2, CCL2 (MCP-1, JE), and CCL12 (MCP-5) collected from mice of the various treatment groups were determined with commercially available ELISA kits (R&D Systems).

Lung histology. Mice were killed with a lethal dose of isoflurane, the chest was rapidly opened, and the thoracic organs were carefully removed. We fixed lungs by instilling PBS-buffered formaldehyde solution (4.5%, pH 7.2) through the trachea at a constant pressure of 20 cmH2O, as described recently (5). Fixation was allowed to proceed overnight at room temperature; subsequently tissue samples were paraffin embedded. Sections of 5 µm from all lungs were stained with hematoxylin-eosin and evaluated for evidence of cellular infiltrates at a x40 original magnification. In selected experiments, lungs of mice were subjected to BAL (PBS, pH 7.2) before fixation in paraformaldehyde solution, as described above.

In vitro CCL2 consumption experiments. We used both human and mouse blood monocytes for CCL2 consumption experiments. Peripheral blood from healthy donors was subjected to combined Ficoll density gradient centrifugation and centrifugal elutriation to obtain highly purified monocyte and neutrophil preparations, as recently described (14). Peripheral blood monocytes (PB-Mo) of both wild-type and CCR2-deficient mice were isolated according to recently described gating and sort protocols using a DIVA-assisted FACSVantage SE flow cytometer (5). Monocytes (1 x 105 cells/ml) were cultured at 37°C in an atmosphere of 5% CO2 in RPMI 1640 supplemented with 5% FCS in the absence or presence of defined amounts of recombinant human or murine CCL2 (600 and 500 pg/ml, respectively). In selected experiments, neutrophils were tested for their ability to consume CCL2. In addition, PB-Mo collected from wild-type mice were incubated with the lysosomotropic agent ammonium chloride [20 mM, 30 min (17, 20)] before addition of recombinant murine CCL2 (500 pg/ml, 24 h) to block CCR2 receptor recycling to the cell surface. Subsequently, cell-free culture supernatants were collected and stored at –80°C until CCL2 analysis with commercial ELISA (R&D Systems).

Statistics. The data are expressed as means ± SD. Significant differences between treatment groups were estimated by Mann-Whitney U-test. Differences were assumed to be significant when P values were <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Alveolar neutrophil and monocyte recruitment and cytokine production induced by intratracheal LPS treatment in wild-type and CCR2-deficient mice. In BALF of both untreated wild-type and CCR2-deficient mice, alveolar macrophages represented the main cellular component, with alveolar neutrophils rarely exceeding 1–2% of the total BAL cell counts. Instillation of endotoxin into the lungs of wild-type and CCR2-deficient mice elicited a dose- and time-dependent recruitment of alveolar neutrophils with similar kinetics between these groups peaking at ~24 to 48 h posttreatment (Fig. 1, left). In contrast, LPS treatment induced alveolar monocyte accumulation, peaking at ~48–72 h posttreatment in wild-type mice but not CCR2-deficient mice. Alveolar monocyte recruitment was highly significantly reduced in CCR2-deficient mice (Fig. 1, right), demonstrating that alveolar monocyte traffic in LPS-induced pulmonary inflammation is predominantly mediated via CCR2.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 1. Time- and dose-response effects of intratracheal (IT) application of Escherichia coli endotoxin on the alveolar neutrophil and monocyte recruitment in wild-type (WT) and CCR2-deficient (KO) mice. WT mice (solid bars) and CCR2 KO (open bars) were treated for various times with increasing concentrations of E. coli endotoxin, as indicated. Subsequently, mice were killed, and total bronchoalveolar lavage (BAL) cell numbers and differentials were analyzed as described in METHODS. Values of total BAL fluid (BALF) leukocyte subpopulations are shown as means ± SD (n = 5–9 animals each). *P <0.01 vs. WT mice.

 
We additionally performed lung histology to exclude the possibility that differences in monocyte numbers observed in BALF of LPS-challenged wild-type vs. CCR2-deficient mice were due to a selective, LPS-induced retention of monocytes in the lungs of CCR2-deficient mice. As shown in Fig. 2, instillation of LPS into the lungs of wild-type (Fig. 2C) and CCR2-deficient mice (Fig. 2D) induced a similar pattern of interstitial and alveolar accumulating leukocytes (48 h posttreatment), associated with a thickening of the alveolar septae, compared with untreated controls (Fig. 2, A and B, respectively). However, when the lungs of LPS-treated wild-type and CCR2-deficient mice were subjected to BAL before preparation for lung histology, post-BAL histological examination revealed no selective retention of alveolar recruited monocytes in either of the investigated treatment groups (Fig. 2, E and F), demonstrating that different numbers of alveolar monocytes observed in BALF of the two treatment groups were not due to a selective monocytic sticking in the lungs of CCR2-deficient mice.



View larger version (132K):
[in this window]
[in a new window]
  		Fig. 2. Effect of IT LPS application on lung leukocyte recruitment in WT compared with CCR2 KO mice. Groups of WT mice and CCR2 KO mice (n = 3 each) were left untreated (A, B) or received IT instillations of LPS (20 µg/mouse, 48 h; C, D) or received IT instillations of LPS for 48 h followed by BAL before lung histological examination (E, F). Subsequently, mice were killed, and lungs were prepared for lung histology as described in METHODS.

 
We next compared BALF CCL2, CCL12, TNF-{alpha}, and MIP-2 concentrations in wild-type and CCR2-deficient mice challenged with intratracheal application of LPS. TNF-{alpha} and MIP-2 levels were similar in wild-type mice and CCR2-deficient mice under baseline conditions and showed similar time- and dose-dependent induction patterns with peak values observed at ~12 h postchallenge (Fig. 3, left and middle). In contrast, CCR2-deficient mice showed baseline BALF CCL2 levels in the same range as wild-type mice but demonstrated ~10-fold elevated LPS-induced BALF CCL2 levels with peak values observed at ~48–72 h posttreatment (Fig. 3, right). In contrast to the strongly different BALF CCL2 levels observed in LPS-challenged wild-type vs. CCR2-deficient mice, BALF CCL12 (MCP-5) levels of the same mice did not show significant differences between both treatment groups [wild type (24 h), 5 ± 8 pg/ml vs. CCR2 knockout (24 h), 19 ± 14 pg/ml; wild type (48 h), 109 ± 42 pg/ml vs. CCR2 knockout (48 h), 150 ± 30 pg/ml; wild type (72 h), 114 ± 37 pg/ml vs. CCR2 knockout (72 h), 148 ± 67 pg/ml; mean ± SD, n = 5 each]. These data again support the role of CCR2 as primary receptor involved in CCL2 turnover in vivo.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 3. Time- and dose-response effects of IT application of E. coli endotoxin on alveolar TNF-{alpha}, macrophage inflammatory protein (MIP)-2, and CCL2 levels in WT and CCR2 KO mice. WT mice (solid bars) and CCR2 KO mice (open bars) were treated for various times with increasing concentrations of E. coli endotoxin, as indicated. Subsequently, mice were killed, and BALF TNF-{alpha}, MIP-2, and CCL2 levels were measured by ELISA. Values are shown as means ± SD (n = 5–9 animals each). *P < 0.01 vs. WT mice.

 
Effect of systemic or intra-alveolar administration of neutralizing CCR2 antibodies on LPS-induced alveolar CCL2 levels in wild-type mice. The inverse relationship between alveolar monocyte accumulation and BALF CCL2 levels observed in wild-type mice vs. CCR2-deficient mice prompted us to evaluate whether systemic treatment of wild-type mice with the anti-CCR2 antibody MC21 would influence alveolar monocyte trafficking and BALF CCL2 levels in response to LPS. We observed that wild-type mice pretreated with the anti-CCR2 antibody MC21 before intratracheal LPS application showed a selectively reduced alveolar monocyte but not neutrophil accumulation, which was associated with increased BALF CCL2 levels peaking at 48–72 h posttreatment (Fig. 4, A and C). Interestingly, this systemic (MC21-mediated) blockade of CCR2, resulting in decreased LPS-induced alveolar monocyte recruitment, significantly decreased alveolar TNF-{alpha} levels at 24 h posttreatment, indicating that alveolar recruited monocytes contribute to LPS-induced intra-alveolar liberation of proinflammatory mediators such as TNF-{alpha} (Fig. 4D).



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 4. Effect of systemic anti-CCR2 antibody application on alveolar CCL2 and TNF-{alpha} levels and alveolar leukocyte traffic in LPS-treated WT mice. WT mice either received IT application of E. coli LPS (20 µg/mouse, solid bars) for various time points or were pretreated systemically with the blocking anti-CCR2 MAb MC21 (crosshatched bars) before LPS instillation procedures. At 24, 48, or 72 h post-LPS treatment, mice were killed and subjected to BAL for determination of differential BAL cell counts and BALF cytokine levels. Data are presented as means ± SD (n = 6 animals per time point). *P < 0.01 vs. LPS-treated animals (A, D) and P < 0.05 vs. LPS-treated animals (C).

 
To further specify whether CCR2 expressed by recruited or resident cells within the alveolar compartment is directly involved in regulating alveolar space CCL2 levels, wild-type mice received intratracheal coapplication of LPS (20 µg/mouse) and anti-CCR2 antibody MC21 (100 µg/mouse). In fact, this coapplication resulted in a marked increase of BALF CCL2 levels compared with control antibody-treated mice (Fig. 5A). At the same time, BALF monocyte counts were not significantly different from those observed in wild-type mice treated with LPS alone (data not shown). In contrast to CCL2, wild-type mice cochallenged with LPS plus MC21 had similar BALF TNF-{alpha} levels as in wild-type controls. (Fig. 5B).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. Effect of IT anti-CCR2 antibody application on LPS-induced alveolar CCL2 and TNF-{alpha} levels. Mice received co-instillations of either control antibodies plus E. coli LPS (20 µg/mouse, solid bars) or blocking anti-CCR2 Ab MC21 (100 µg/mouse) plus E. coli LPS (20 µg/mouse, hatched bars) for various time points. Subsequently, mice were killed and subjected to BAL for determination of differential BAL cell counts and BALF cytokine levels. Data are presented as means ± SD (n = 5 animals/time point). *P < 0.01 vs. animals treated with coapplications of control Ab plus LPS.

 
Effect of intratracheal LPS application on alveolar monocyte recruitment and alveolar CCL2 levels in chimeric wild-type and chimeric CCR2-deficient mice. To further distinguish the respective role of CCR2 expressed on recruited monocytes vs. resident alveolar cells in regulating alveolar space CCL2 levels, we compared LPS-induced CCL2 levels in reciprocally transplanted chimeric wild-type and chimeric CCR2-deficient mice. Chimeric wild-type mice treated with intratracheal application of LPS showed a significantly reduced alveolar monocyte accumulation together with drastically increased BALF CCL2 levels, comparable to those observed in CCR2-deficient mice (Fig. 6, A and B). On the other hand, LPS instillation into the lungs of chimeric CCR2-deficient mice provoked a significantly increased alveolar monocyte traffic compared with CCR2-deficient mice, with peak values observed at ~48 h posttreatment. In parallel, chimeric CCR2-deficient mice exhibited significantly reduced intra-alveolar CCL2 levels (Fig. 6, C and D). These data suggest a major role of CCR2-positive alveolar recruited monocytes in regulating alveolar space CCL2 levels in lung inflammation.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 6. Effect of CCR2 chimerism in WT and CCR2 KO mice on E. coli LPS-induced alveolar monocyte accumulation and alveolar CCL2 levels. WT mice (solid bars in A and B) and chimeric WT mice (open bars in A and B) or CCR2 KO mice (solid bars in C and D) and chimeric CCR2 KO mice (open bars in C and D) were treated with IT application of E. coli LPS for various time points, as indicated. Subsequently, mice were killed and subjected to BAL for determination of BAL cell differentials and BALF cytokine levels. Values are shown as means ± SD (n = 5–9 animals/time point). A and B: *P < 0.05 compared with WT mice; C and D: *P < 0.05 compared with CCR2 KO mice.

 
Monocytes are capable of lowering CCL2 levels in vitro. We additionally performed cell culture experiments to confirm the proposed mechanism of monocyte-dependent CCL2 consumption in vitro. We used both highly purified human (Fig. 7A) and mouse PB-Mo (Fig. 7B) (purities >95%) for CCL2 consumption assays. In supernatants of unstimulated monocytes, CCL2 protein was nearly undetectable (Fig. 7, A and B, bar 2). Also, no spontaneous degradation of recombinant CCL2 protein was observed during the 24-h time period in the absence of monocytes (Fig. 7, A and B, bars 3 and 4). Addition of human monocytes (1 x 105 cells/ml) to culture media containing defined concentrations of recombinant CCL2 led to a time-dependent decrease of CCL2 protein, peaking at 24 h postinduction (Fig. 7A, bars 5–7). Similarly, highly purified PB-Mo collected from wild-type mice (1 x 105 cells/ml) also consumed CCL2 protein levels within the 24-h observation period (P < 0.001, Fig. 7B, bar 5). Addition of function-blocking anti-CCR2 antibodies to the monocyte cultures strongly inhibited this CCL2 consumption process (Fig. 7A, bars 8–10; Fig. 7B, bar 7). Similarly, blockade of receptor recycling by the lysosomotropic agent ammonium chloride significantly inhibited CCL2 consumption by wild-type PB-Mo (Fig. 7B, bar 6). Lack of CCL2 consumption was also observed in cultures of PB-MO collected from CCR2-deficient mice incubated with CCL2 for 24 h (Fig. 7B, bar 8). Notably, addition of neutrophils instead of monocytes to cell cultures did not induce any detectable CCL2 consumption (data not shown). These data strongly support the concept that the consumption of CCL2 protein observed in vivo is monocyte and CCR2 dependent.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 7. Monocytes downregulate CCL2 protein levels in vitro. Human (A) or murine peripheral blood monocytes (PB-Mo, B) were incubated in the absence or presence of defined concentrations of CCL2 protein in vitro. After defined time points, culture supernatants were collected and analyzed for CCL2 protein content by ELISA. Bar 1 (A, B), CCL2 content of medium alone; bar 2 (A, B), CCL2 release by unstimulated monocytes after 24 h of culture; bars 3 and 4 (A, B), CCL2 levels in culture media at 0 and 24 h after addition of recombinant CCL2 protein (600 pg/ml, A; 500 pg/ml, B). Bars 5–7 (A), monocyte-dependent consumption of CCL2 protein (600 pg/ml) in culture media at 1 h (bar 5), 4 h (bar 6), and 24 h (bar 7). Bars 8–10 (A), function-blocking anti-CCR2 Ab (5 µg/ml) to monocyte cultures inhibited CCR2-dependent CCL2 turnover by monocytes at 1, 4, and 24 h. Bar 5 (B), WT monocyte-dependent consumption of CCL2 protein (500 pg/ml) in culture media at 24 h. Bars 6, 7 (B), CCL2 consumption in cultures (24 h) of WT PB-Mo after pretreatment with the lysosomotropic agent ammonium chloride (20 mM, 30 min; bar 6) or in the presence of function-blocking anti-CCR2 antibody MC21 (bar 7). Bar 8 (B), CCL2 consumption in cultures of monocytes collected from CCR2 KO mice at 24 h. The data are shown as means ± SE (n = 3–6 independent experiments). A: *P < 0.01 bar 5 vs. 4; **P < 0.003 bar 6 vs. 4; ***P < 0.0004 bar 7 vs. 4. B: ***P < 0.0004 bar 5 vs. 4; ++bars6–8 vs. 5.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the current study, we tested the hypothesis that CCR2-positive alveolar recruited monocytes limit their further accumulation in LPS-challenged mouse lungs by downregulating the alveolar levels of CCL2. We found that LPS-treated wild-type mice with intact CCR2 expression showed a robust CCR2-dependent alveolar monocyte accumulation, associated with relatively low BALF CCL2 levels. In contrast, CCR2-deficient and chimeric wild-type mice reconstituted with the hematopoietic system of CCR2-deficient mice lacking monocytic CCR2 expression demonstrated a heavily reduced alveolar monocyte traffic in response to LPS together with highly elevated alveolar CCL2 levels. Conversely, reconstitution of CCR2-deficient mice with the hematopoietic system of wild-type mice raised their LPS-induced alveolar monocyte accumulation to wild-type values, again accompanied with a sharp drop in BALF CCL2 levels. Furthermore, systemic anti-CCR2 antibodies to block LPS-induced alveolar monocyte traffic in wild-type mice significantly increased alveolar CCL2 levels. Moreover, intra-alveolar blockade of CCR2 receptor function by intratracheal antibody administration in LPS-challenged mice did not affect the alveolar monocyte accumulation but again significantly increased alveolar CCL2 levels. Finally, addition of human or mouse monocytes to culture media containing defined amounts of recombinant CCL2 protein caused a sharp and time-dependent drop of CCL2 levels in vitro, which was not observed upon addition of anti-CCR2 antibodies to the cultures or when highly purified CCR2-deficient monocytes were employed. Together, the data suggest that CCR2-positive monocytes regulate alveolar CCL2 levels in inflamed lungs. As alveolar CCL2 generation represents the main driving force of monocyte accumulation in this compartment, the consumption of alveolar CCL2 by CCR2-positive monocytes appearing in the alveolar space may represent an important negative feedback mechanism, limiting excess monocyte accumulation in this compartment and promoting a return to homeostasis.

It has been proposed that early recruited neutrophils elicit subsequent alveolar monocyte recruitment by local release of CCL2 (21). In addition, resident alveolar epithelial cells, due to their polarized CCL2 release upon inflammatory challenge, also appear to be involved (14). Both mechanisms may be operative in the described system, given that neutrophil accumulation preceded alveolar monocyte accumulation in LPS-inflamed lungs of both wild-type and CCR2-deficient mice. The strikingly different BALF CCL2 levels noted in LPS-treated wild-type and CCR2-deficient mice, however, were not due to different neutrophil recruitment profiles in these two treatment groups. Moreover, we found that primary isolates of type II epithelial cells collected from wild-type and CCR2-deficient mice responded with the same amount of CCL2 release upon LPS stimulation in vitro, thus making differential CCL2 releasing capacities of wild-type vs. CCR2 knockout epithelial cells unlikely (M. Srivastava, unpublished observation). In addition, because neutrophils were recently shown to lack CCR2 expression, it is rather unlikely that neutrophils are directly involved in local CCL2 depletion processes (9, 10). Accordingly, neutrophils failed to lower recombinant CCL2 levels in vitro (data not shown). Thus the significant differences in BALF CCL2 levels between wild-type and CCR2-deficient mice support the hypothesis that CCR2-positive monocytes themselves are actively involved in regulating local CCL2 levels within inflamed lungs. Indeed, in addition to CCR2 deletion, blockade of CCR2 function by systemic MC21 administration in wild-type mice attenuated the alveolar monocyte but not neutrophil traffic with a concomitant increase in alveolar CCL2 levels. From the presented data, we cannot exclude the notion that other mechanisms may also contribute to CCL2 removal from inflamed lungs such as proteolytic degradation in alveolar lining fluids. In this regard, Tylaska et al. (18) recently reported that CCL2 levels in allogeneic sponges implanted subcutaneously in CCR2-deficient mice remain elevated for up to 6–8 days after implant compared with wild-type controls, which was prolonged compared with the CCL2 kinetics (up to 3 days) reported to occur in LPS-challenged CCR2-deficient mice in the current study. However, both different experimental models (acute vs. chronic) and different body compartments (lung vs. systemic) investigated may contribute to the differences in CCL2 removal kinetics reported in this study compared with that of Tylaska et al. (18).

Type II alveolar epithelial cells were recently shown to express CCR2 mRNA and respond to CCL2 stimulation in vitro, presumably via CCR2 (11). In addition, resident alveolar macrophages were also demonstrated to exhibit some CCR2 expression, albeit at much lower levels than on monocytic precursors (8). Tylaska et al. (18) recently demonstrated that peritoneal macrophages from wild-type mice are capable of integrating CCL2 gradients in a CCR2-dependent manner in vitro, and this CCR2-dependent control of locally released CCL2 levels at inflammatory sites was also reported to occur in response to allogeneic sponge implants in wild-type but not CCR2-deficient mice. However, in the present study, a major role of resident alveolar macrophages in consuming locally released CCL2 levels within the alveolar compartment is unlikely due to the findings in the chimeric mice. Chimeric wild-type mice, recently shown to exhibit a CCR2-negative circulating monocyte but CCR2-positive alveolar macrophage phenotype (10), demonstrated similar alveolar CCL2 levels as did CCR2-deficient mice, strongly supporting the predominant role of the mononuclear cells in regulating alveolar CCL2 levels. This view is further supported by the fact that consumption of alveolar CCL2 along with strong monocyte traffic into the alveolar space was noted in chimeric CCR2-deficient mice. In this line, highly elevated BALF CCL2 levels observed in LPS-treated wild-type mice upon intratracheal MC21 application are considered to primarily reflect MC21 interference with CCR2 function on newly recruited monocytes rather than resident lung cells, including alveolar macrophages and epithelial cells. However, the MC21-induced increase in alveolar CCL2 levels noted in wild-type mice was not associated with increased numbers of alveolar recruited monocytes. The most probable explanation for this observation may be that diffusion of the antibody toward the interstitial compartment has hampered additional monocytes from further accumulating within the alveolar compartment of MC21-treated mice.

We and others recently showed that monocyte recruitment to the lungs in response to CCL2 or CCL2 plus LPS (5, 7) or during idiopathic pneumonia syndrome (2) and septic acute respiratory distress syndrome (14) was accompanied by increased monocytic CD14 cell surface expression. These CD14-positive alveolar monocytes exhibited increased TNF-{alpha} mRNA levels and responded with greater TNF-{alpha} release upon LPS stimulation in vitro than circulating CD14-negative monocytes, suggesting that transmigration across the highly specialized endothelial/alveolar epithelial barrier primes but not fully activates monocytes, irrespective of the event eliciting the migratory response (5). Similar to these reported results, here we report that reduced BALF TNF-{alpha} levels were observed in wild-type mice pretreated with MC21 to effectively block the alveolar monocyte but not neutrophil appearance in response to LPS. Another explanation for the significantly reduced BALF TNF-{alpha} levels observed in MC21-pretreated, LPS-challenged wild-type mice has been offered by a recent report, showing that CCL2 elicited a TNF-{alpha} response in monocytes/macrophages in vitro (12), which would not be observed in mice pretreated with MC21 to block CCR2-mediated monocyte recruitment. However, two aspects argue against the idea that this mechanism plays also a significant role in the described mouse system in vivo: First, we found that LPS-treated wild-type mice receiving intratracheal applications of MC21 to block local CCR2 receptor function on newly recruited monocytes showed heavily increased BALF CCL2 levels without a notable decrease in BALF TNF-{alpha} levels compared with LPS-treated controls, suggesting that the contribution of alveolar recruited monocytes to intra-alveolar liberated TNF-{alpha} levels is independent from the CCR2/CCL2 axis but may involve other signaling pathways such as CD14. Second, LPS-treated CCR2-deficient mice not capable of responding to CCL2 showed similar BALF TNF-{alpha} levels as detected in LPS-treated wild-type mice, again indicating that the CCR2/CCL2 axis appears to play a minor role in the local TNF-{alpha} release in the described mouse model.

Our data suggest that consumption of alveolar CCL2 levels by monocyte CCR2 receptors is a possible mechanism that accounts for the reciprocal relationship between monocytic recruitment and CCL2 concentrations in BALF, although a role of monocytes in cell-cell signaling events initiated by the CCL2-CCR2 axis to downregulate CCL2 in the major cellular sources of this chemokine in the alveolar compartment (presumably alveolar epithelial cells) cannot be fully excluded. The concept of CCL2 scavenging by CCR2-positive monocytes is further supported by the CCL2 consumption assays in vitro: a sharp drop in CCL2 levels was observed in cultures employing human or wild-type mouse monocytes, but not in cultures of wild-type monocytes with blocked CCR2 receptor function (i.e., impaired receptor recycling using ammonium chloride or antibody blockade of CCR2) or CCR2-deficient monocytes. Notably, similar to these findings regarding the role of monocytes in CC chemokine consumption, a recent report demonstrated that G-CSF-enhanced neutrophil recruitment to LPS-inflamed rat lungs was associated with strongly reduced BALF MIP-2 and cytokine-induced neutrophil chemoattractant levels due to scavenging of soluble CXC chemoattractants by recruited neutrophils as a mechanism of lowering chemokine gradients in the surrounding alveolar space (22). It is important to add that similar to the chemokine receptors’ lowering local chemokine levels, local chemokine levels may in turn affect chemokine receptor levels, reflecting the complex regulatory interplay of receptor-ligand interactions. In addition, other parameters are known to regulate chemokine receptor expression levels at inflammatory sites, such as the pathogen-associated molecular patterns themselves (17a) and leukocytic differentiation processes taking place during leukocyte migration towards inflamed tissues, as recently demonstrated for the CCR2 expression profiles on circulating monocytes vs. alveolar macrophages (8).

In summary, the current study presents data that support the role of a mechanism shaping the intensity and duration of alveolar monocyte accumulation in acute lung inflammation. CCR2 expression by circulating monocytes is a prerequisite to mount an appropriate alveolar monocyte accumulation in LPS-challenged wild-type mice. The appearance of CCR2-positive monocytes in the alveolar space is the main determinant causing consumption of alveolar CCL2 levels. This negative feedback mechanism may limit excess alveolar monocyte accumulation and promote a return to homeostasis. It is possible that this mechanism of self-regulating alveolar monocyte traffic may offer new perspectives for developing effective therapeutic interventions to selectively modulate monocyte recruitment to the lung in critically ill patients.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The current study has been supported by German Research Foundation Grant 547 "Cardiopulmonary Vascular System" and by the national network on community-acquired pneumonia CAPNETZ.


   ACKNOWLEDGMENTS
 
We are very grateful to the excellent technical support of Marlena Stein in lung histology experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. A. Maus, Dept. of Internal Medicine, Div. of Pulmonary and Critical Care Medicine, Justus-Liebig-Univ., Klinikstr. 36, Giessen 35392, Germany (E-mail: Ulrich.A.Maus{at}med.uni-giessen.de)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bellingan GJ, Xu P, Cooksley H, Cauldwell H, Shock A, Bottoms S, Haslett C, Mutsaers SE, and Laurent GJ. Adhesion molecule-dependent mechanisms regulate the rate of macrophage clearance during the resolution of peritoneal inflammation. J Exp Med 196: 1515–1521, 2002.[Abstract/Free Full Text]
  2. Bhalla KS and Folz RJ. Idiopathic pneumonia syndrome after syngeneic bone marrow transplant in mice. Am J Respir Crit Care Med 166: 1579–1589, 2002.[Abstract/Free Full Text]
  3. Kuziel WA, Morgan SJ, Dawson TC, Griffin S, Smithies O, Ley K, and Maeda N. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc Natl Acad Sci USA 94: 12053–12059, 1997.[Abstract/Free Full Text]
  4. Mack M, Cihak J, Simonis C, Luckow B, Proudfoot AEI, Plachy J, Brühl H, Frink M, Anders HJ, Vielhauer V, Pfirstinger J, Stangassinger M, and Schlöndorff D. Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice. J Immunol 166: 4697–4703, 2001.[Abstract/Free Full Text]
  5. Maus U, Herold S, Muth H, Maus R, Ermert L, Ermert M, Weissmann N, Rosseau S, Seeger W, Grimminger F, and Lohmeyer J. Monocytes recruited into the alveolar air space of mice show a monocytic phenotype, but upregulate CD14. Am J Physiol Lung Cell Mol Physiol 280: L58–L68, 2001.[Abstract/Free Full Text]
  6. Maus U, Huwe J, Ermert L, Ermert M, Seeger W, and Lohmeyer J. Molecular pathways of monocyte emigration into the alveolar air space of intact mice. Am J Respir Crit Care Med 165: 95–100, 2002.[Abstract/Free Full Text]
  7. Maus U, Huwe J, Maus R, Seeger W, and Lohmeyer J. Alveolar JE/MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, sequential neutrophil and monocyte influx and vascular leakage in mice. Am J Respir Crit Care Med 164: 406–411, 2001.[Abstract/Free Full Text]
  8. Maus U, Koay MA, Delbeck T, Mack M, Ermert M, Ermert M, Blackwell TS, Christman JW, Schlöndorff D, Seeger W, and Lohmeyer J. Role of resident alveolar macrophages in leukocyte traffic into the bronchoalveolar space under inflammatory versus noninflammatory conditions. Am J Physiol Lung Cell Mol Physiol 282: L1245–L1252, 2002.[Abstract/Free Full Text]
  9. Maus U, von Grote K, Kuziel WA, Mack M, Miller EJ, Schlöndorff D, Seeger W, and Lohmeyer J. The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice. Am J Respir Crit Care Med 166: 268–273, 2002.[Abstract/Free Full Text]
  10. Maus U, Waelsch K, Kuziel WA, Mack M, Christman JW, Blackwell TS, Schlöndorff D, Seeger W, and Lohmeyer J. Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis. J Immunol 170: 3273–3278, 2003.[Abstract/Free Full Text]
  11. Moore BB, Peters-Golden M, Christensen PJ, Lama V, Kuziel WA, Paine R III, and Toews GB. Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2. Am J Physiol Lung Cell Mol Physiol 284: L342–L349, 2003.[Abstract/Free Full Text]
  12. Robinson SC, Scott KA, and Balkwill FR. Chemokine stimulation of monocyte matrix metalloproteinase-9 requires endogenous TNF-{alpha}. Eur J Immunol 32: 404–412, 2002.[CrossRef][Web of Science][Medline]
  13. Robson RL, McLoughlin RM, Witowski J, Loetscher P, Wilkinson TS, Jones SA, and Topley N. Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-{gamma} controls neutrophil migration across the mesothelium in vitro and in vivo. J Immunol 167: 1028–1038, 2001.[Abstract/Free Full Text]
  14. Rosseau S, Hammerl P, Maus U, Walmrath HD, Schütte H, Grimminger F, Seeger W, and Lohmeyer J. Phenotypic characterization of alveolar monocyte recruitment in the adult respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 279: L25–L35, 2000.[Abstract/Free Full Text]
  15. Rosseau S, Selhorst J, Wiechmann K, Leissner K, Maus U, Mayer K, Grimminger F, Seeger W, and Lohmeyer J. Monocyte migration through the alveolar epithelial barrier: adhesion molecule mechanisms and impact of chemokines. J Immunol 164: 427–435, 2000.[Abstract/Free Full Text]
  16. Sabroe I, Jones EC, Usher LR, Whyte MKB, and Dower SK. Toll-like receptor (TLR) 2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol 168: 4701–4710, 2002.[Abstract/Free Full Text]
  17. Samanta AK, Oppenheim JJ, and Matsushima K. Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils. J Biol Chem 265: 183–189, 1990.[Abstract/Free Full Text]
  18. Sica A, Saccani A, Borsatti A, Power CA, Wells TNC, Luini W, Polentarutti N, Sozzoni S, and Mantovani A. Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes. J Exp Med 185: 969–974, 1997.[Abstract/Free Full Text]
  19. Tylaska LA, Boring L, Weng W, Aiello R, Charo IF, Rollins BJ, and Gladue RP. CCR2 regulates the level of MCP-1/CCL2 in vitro and at inflammatory sites and controls T cell activation in response to alloantigen. Cytokine 18: 184–190, 2002.[CrossRef][Web of Science][Medline]
  20. Ulich TR, Watson LR, Yin SM, Guo KZ, Wang P, Thang H, and del Castillo J. The intratracheal instillation of endotoxin and cytokines. I. Characterization of LPS-induced IL-1 and TNF mRNA expression and the LPS-, IL-1-, and TNF-induced inflammatory infiltrate. Am J Pathol 138: 1485–1496, 1991.[Abstract]
  21. Wang JM, Hishinuma A, Oppenheim JJ, and Matsushima K. Studies of binding and internalization of human recombinant monocyte chemotactic and activating factor (MCAF) by monocytic cells. Cytokine 5: 264–275, 1993.[CrossRef][Web of Science][Medline]
  22. Yamamoto T, Kajikawa O, Martin TR, Sharar SR, Harlan JM, and Winn RK. The role of leukocyte emigration and IL-8 on the development of lipopolysaccharide-induced lung injury in rabbits. J Immunol 161: 5704–5709, 1998.[Abstract/Free Full Text]
  23. Zhang P, Bagby GJ, Kolls JK, Welsh DA, Summer WR, Andresen J, and Nelson S. The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin. J Immunol 166: 458–465, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
J. L. Martin, L. Koodie, A. G. Krishnan, R. Charboneau, R. A. Barke, and S. Roy
Chronic Morphine Administration Delays Wound Healing by Inhibiting Immune Cell Recruitment to the Wound Site
Am. J. Pathol., February 1, 2010; 176(2): 786 - 799.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Wygrecka, L. M. Marsh, R. E. Morty, I. Henneke, A. Guenther, J. Lohmeyer, P. Markart, and K. T. Preissner
Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung
Blood, May 28, 2009; 113(22): 5588 - 5598.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
T. Ueno, K. Tanaka, M. Jurewicz, T. Murayama, I. Guleria, P. Fiorina, J. C. Paez, A. Augello, A. Vergani, M. Wong, et al.
Divergent Role of Donor Dendritic Cells in Rejection versus Tolerance of Allografts
J. Am. Soc. Nephrol., March 1, 2009; 20(3): 535 - 544.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
D. Sun, C. O. Martinez, O. Ochoa, L. Ruiz-Willhite, J. R. Bonilla, V. E. Centonze, L. L. Waite, J. E. Michalek, L. M. McManus, and P. K. Shireman
Bone marrow-derived cell regulation of skeletal muscle regeneration
FASEB J, February 1, 2009; 23(2): 382 - 395.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. L. Lin, Y. Suzuki, H. Nakano, E. Ramsburg, and M. D. Gunn
CCR2+ Monocyte-Derived Dendritic Cells and Exudate Macrophages Produce Influenza-Induced Pulmonary Immune Pathology and Mortality
J. Immunol., February 15, 2008; 180(4): 2562 - 2572.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. M. Kelly, K. McNagny, D. L. Williams, N. van Rooijen, L. Maxwell, C. Gwozd, C. H. Mody, and P. Kubes
The Lung Responds to Zymosan in a Unique Manner Independent of Toll-Like Receptors, Complement, and Dectin-1
Am. J. Respir. Cell Mol. Biol., February 1, 2008; 38(2): 227 - 238.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
C. Penido, M. F. S. Costa, M. C. Souza, K. A. Costa, A. L. P. Candea, C. F. Benjamim, and M. d. G. M. O. Henriques
Involvement of CC chemokines in {gamma}{delta} T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway
Int. Immunol., January 1, 2008; 20(1): 129 - 139.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
O. Ochoa, D. Sun, S. M. Reyes-Reyna, L. L. Waite, J. E. Michalek, L. M. McManus, and P. K. Shireman
Delayed angiogenesis and VEGF production in CCR2 / mice during impaired skeletal muscle regeneration
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2007; 293(2): R651 - R661.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
C. Hansell and R. Nibbs
Professional and Part-Time Chemokine Decoys in the Resolution of Inflammation
Sci. Signal., May 1, 2007; 2007(384): pe18 - pe18.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
K. L. Hokeness, E. S. Deweerd, M. W. Munks, C. A. Lewis, R. P. Gladue, and T. P. Salazar-Mather
CXCR3-Dependent Recruitment of Antigen-Specific T Lymphocytes to the Liver during Murine Cytomegalovirus Infection
J. Virol., February 1, 2007; 81(3): 1241 - 1250.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/2/L350    most recent
00061.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (15)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maus, U. A.
Right arrow Articles by Lohmeyer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maus, U. A.
Right arrow Articles by Lohmeyer, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.