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Originally published online as doi:10.1189/jlb.0605337 on November 7, 2005

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(Journal of Leukocyte Biology. 2006;79:114-122.)
© 2006 by Society for Leukocyte Biology

Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue

Christoph A. Reichel*, Andrej Khandoga*,1, Hans-Joachim Anders{dagger}, Detlef Schlöndorff{dagger}, Bruno Luckow{dagger} and Fritz Krombach*

* Institute for Surgical Research and
{dagger} Medical Policlinic-Innenstadt, Arbeitsgruppe Klinische Biochemie, University of Munich, Germany

1 Correspondence: Institute for Surgical Research, University of Munich, Marchioninistr. 27, D-81377 Munich, Germany. E-mail: andrej.khandoga{at}med.uni-muenchen.de


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ABSTRACT
 
Leukocyte infiltration of reperfused tissue is a key event in the pathogenesis of ischemia-reperfusion. However, the role of chemokine receptors Ccr1, Ccr2, and Ccr5 for each single step of the postischemic recruitment process of leukocytes has not yet been characterized. Leukocyte rolling, firm adherence, transendothelial, and extravascular migration were analyzed in the cremaster muscle of anaesthetized C57BL/6 mice using near-infrared reflected light oblique transillumination microscopy. Prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min after onset of reperfusion, migration parameters were determined in wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice. Sham-operated wild-type mice without ischemia were used as controls. No differences were detected in numbers of rolling leukocytes among groups. In contrast, the number of firmly adherent leukocytes was increased significantly in wild-type mice as compared with sham-operated mice throughout the entire reperfusion phase. Already after 5 min of reperfusion, this increase was reduced significantly in Ccr1–/– and Ccr5–/– mice, whereas only in Ccr2–/– mice, was adherence attenuated significantly at 120 min after onset of reperfusion. Furthermore, after 120 min of reperfusion, the number of transmigrated leukocytes (>80% Ly-6G+ neutrophils) was elevated in wild-type mice as compared with sham-operated animals. This elevation was significantly lower in Ccr1–/–, Ccr2–/–, and Ccr5–/– mice. Leukocyte extravascular migration distances were comparable among groups. In conclusion, these in vivo data demonstrate that Ccr1, Ccr2, and Ccr5 mediate the postischemic recruitment of neutrophils through effects on intravascular adherence and subsequent transmigration.

Key Words: ischemia-reperfusion • leukocytes • transmigration • MIP-1{alpha} • MCP-1 • RANTES


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INTRODUCTION
 
Ischemia-reperfusion (I/R) is the most common cause for organ dysfunction and failure after myocardial infarction, hemorrhagic shock, and transplantation. A variety of studies in the last decades has shown that the extent of postischemic tissue damage strongly correlates with the number of leukocytes recruited to the reperfused tissue [1 2 3 ]. Consequently, inhibition of leukocyte infiltration appeared to be a promising therapeutical approach in the prevention of I/R-induced tissue damage as well as subsequent loss of organ function [3 ]. However, clinical trials of leukocyte adhesion blockade did not succeed in several I/R diseases including myocardial infarction, stroke, and shock (reviewed in ref. [4 ]). Moreover, recent studies revealed that transmigrated leukocytes might also be involved in tissue remodeling and healing, indicating a much more complex role of this cell population in the inflammatory response [5 6 7 8 ]. Leukocyte recruitment from the microvasculature to extravascular compartments is a highly regulated multistep process involving a variety of adhesion molecules, cytokines, and chemokines [9 10 11 ]. Over the past years, chemokines and their receptors have become the subject of intensive investigations. Chemokines are small molecules (8–14 kDa), which can be subclassified into C, CC, CXC, and CX3C chemokines according to the arrangement of their N-terminal cysteine residues [12 , 13 ]. In the leukocyte extravasation process, chemokine receptors on circulating leukocytes are supposed to interact with chemokines presented on the vascular endothelium as well as the extracellular matrix. These interactions initiate mechanisms, which finally lead to firm adherence, transmigration, and chemotaxis of leukocytes [11 , 14 15 16 ].

Recently, it has been found that increased levels of chemokines and their receptors are expressed in numerous pathological conditions such as rheumatoid arthritis, asthma, and glomerulonephritis [17 18 19 ]. Inhibition of chemokine activity as well as blockade of chemokine receptors have been demonstrated to be beneficial, therapeutical approaches in various inflammatory disease models [20 21 22 ].

There is a growing body of evidence that chemokines and their receptors are also critically involved in the pathogenesis of I/R. Recent studies revealed that I/R induces simultaneous expression of CC chemokines such as CC chemokine ligand 3 (Ccl3)/macrophage-inflammatory protein-1 {alpha} (MIP-1{alpha}), Ccl4/MIP-1ß, Ccl2/monocyte chemoattractant protein-1 (MCP-1), and Ccl5/regulated on activation, normal T expressed and secreted (RANTES) [23 , 24 ]. Previous in vivo results clearly demonstrated that therapeutic strategies against CC chemokines might be useful in the prevention of postischemic organ dysfunction [24 , 25 ]. Blockade of Ccl3/MIP-1{alpha}, a major ligand of CC chemokine receptor 1 (Ccr1), has been shown to be associated with reduced leukocyte infiltration of the reperfused lung [24 ]. In mouse models of renal I/R and focal cerebral ischemia, blockade of chemokine receptors Ccr2 and Ccr5 was effective in the reduction of leukocyte infiltration of the reperfused tissue and in the amelioration of subsequent postischemic organ failure [26 , 27 ].

Neutrophils, monocytes, and lymphocytes, which represent the major leukocyte subpopulations recruited to postischemic tissue, have been found to express Ccr1, Ccr2, as well as Ccr5 [28 29 30 31 32 ]. The functional relevance of these chemokine receptors for extravasation of neutrophils remains disputed [26 , 27 ]. Moreover, the role of chemokine receptors Ccr2 and Ccr5 for each single step in the sequential process of leukocyte recruitment as well as for leukocyte interstitial migration behavior during I/R is not understood. Finally, the impact of Ccr1 on the extravasation process of leukocytes during I/R has not yet been analyzed.

Therefore, the objective of our study was to elucidate the role of Ccr1, Ccr2, and Ccr5 for postischemic leukocyte rolling, firm adherence, and transmigration; to characterize the effects of Ccr1, Ccr2, and Ccr5 deficiency on the recruitment of different leukocyte subpopulations during I/R; and to analyze the functional relevance of these chemokine receptors for interstitial migration of extravasated leukocytes.


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MATERIALS AND METHODS
 
Animals
C57BL/6NCrl mice were purchased from Charles River (Sulzfeld, Germany). Male Ccr1-, Ccr2-, and Ccr5-deficient mice were generated as described (refs. [33, 34] and Guillermo Perez de Lema, H. Maier, T. Franz, M. Escribese, S. Chilla, S. Segerer, N. Camarasa, H. Schmid, B. Banas, S. Kalaydjiev, D. H. Busch, K. Pfeffer, F. Mampaso, D. Schlöndorff, and B. Luckow, manuscript submitted) and backcrossed for 10 generations to the C57BL/6NCrl background. All experiments were performed with male mice at the age of 30 ± 5 weeks. Animals were raised in a specific pathogen-free environment and later housed under conventional conditions with free access to food and water. All experiments were performed according to German legislation for the protection of animals.

Surgical procedure
The surgical preparation was performed as originally described by Baez [35] with minor modifications. Mice were anesthetized using a ketamine/xylazine mixture (100 mg/kg ketamine and 10 mg/kg xylazine) administrated by intraperitoneal injection. The left femoral artery was cannulated in a retrograde manner for the administration of microspheres to the cremasteric vasculature (see below). The right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally in a relatively avascular zone, using careful electrocautery to stop any bleeding, and spread over the transparent pedestal of a custom-made microscopic stage. Epididymis and testicle were detached from the cremaster muscle and placed into the abdominal cavity. Throughout the procedure as well as after surgical preparation during intravital microscopy, the muscle was superfused with warm buffered saline.

Ischemia of the cremaster muscle was induced by clamping all supplying vessels at the basis of the cremaster muscle using a vascular clamp (Martin, Tuttlingen, Germany). Stagnancy of blood flow was then verified by intravital microscopy. After 30 min of ischemia, the vascular clamp was removed, and reperfusion was restored for 130 min.

Intravital microscopy
The set-up for intravital microscopy was centered around an Olympus BX 50 upright microscope (Olympus Microscopy, Hamburg, Germany), equipped for stroboscopic fluorescence epi-illumination microscopy. Light from a 75-W xenon source was narrowed to a near monochromatic beam of a wavelength of 700 nm by a galvanometric scanner (Polychrome II, TILL Photonics, Gräfelfing, Germany) and directed onto the specimen via a fluorescein isothiocyanate (FITC) filter cube equipped with dichroic and emission filters (DCLP 500, LP515, Olympus Microscopy). Microscopic images were obtained with Olympus water immersion lenses [20x/numerical aperture (NA) 0.5 and 40x/NA 0.8] and recorded with an analog black and white charged-coupled device video camera (Cohu 4920, Cohu, San Diego, CA) and an analog video recorder (AG-7350-E, Panasonic, Tokyo, Japan). Oblique illumination was obtained by positioning a mirroring surface (reflector) directly below the specimen and tilting its angle relative to the horizontal plane. The reflector consisted of a round coverglass (thickness 0.19–0.22 mm, diameter 11.8 mm), which was coated with aluminum vapor (Freichel, Kaufbeuren, Germany) and brought into direct contact with the overlying specimen. For measurement of centerline blood flow velocity, green fluorescent microspheres (0.96 µm diameter, Molecular Probes, Leiden, The Netherlands) were injected via an arterial catheter, and their passage through the vessels of interest was recorded using the FITC filter cube under appropriate stroboscopic illumination (exposure 1 ms, cycle time 10 ms, {lambda}=488 nm), integrating video images for sufficient time (>80 ms) to allow for the recording of several images of the same bead on one frame. Beads that were flowing freely along the vessels’ centerline were used to determine blood flow velocity (see below).

Quantification of leukocyte kinetics and microhemodynamic parameters
For off-line analysis of parameters describing the sequential steps of leukocyte extravasation, we used the Cap-Image image analysis software (Dr. Zeintl, Heidelberg, Germany). Rolling leukocytes were defined as those moving slower than the associated blood flow and quantified for 30 s. Firmly adherent cells were determined as those resting in the associated blood flow for more than 30 s and related to the luminal surface per 100 µm vessel length. Transmigrated cells were counted in regions of interest (ROIs) covering 75 µm on both sides of a vessel over 100 µm vessel length. For the analysis of leukocyte interstitial migration distances, ROIs were subdivided into a segment adjacent to the vessel covering 25 x 100 µm and a segment distant to the vessel covering 50 x 100 µm. Leukocyte accumulation in each segment was quantified and shown as a proportion of the number of transmigrated leukocytes in the total area. By measuring the distance between several images of one fluorescent bead under stroboscopic illumination, centerline blood flow velocity was determined. From measured vessel diameters and centerline blood flow velocity, apparent wall shear stress was calculated, assuming a parabolic flow velocity profile over the vessel cross-section [36 ].

Experimental groups
Animals were assigned randomly to the following groups: sham-operated wild-type mice without ischemia as well as wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice (n=7 each group) undergoing I/R (30/130 min).

Experimental protocol
At the beginning of each experiment, three postcapillary vessel segments in a central area of the spread-out cremaster muscle were chosen randomly by the observer among those that were at least 150 µm away from neighboring postcapillary venules and did not branch over a distance of at least 150 µm. After having obtained baseline recordings of leukocyte rolling, firm adhesion, and transmigration in all three vessel segments, ischemia was induced for 30 min. Measurements, which took ~5 min, respectively, were repeated at 5, 30, 60, 90, and 120 min after onset of reperfusion in the identical postcapillary vessel segments, and blood flow velocity was determined at 125 min after reperfusion as described above. Finally, after 130 min of reperfusion, tissue samples of the cremaster muscle were taken for immunohistological analysis. Blood samples were collected by cardiac puncture for the determination of systemic leukocyte counts using a Coulter AcT counter (Coulter Corp., Miami, FL). Animals were then killed by bleeding to death.

Immunohistochemistry
Tissue samples from the right cremaster muscle were taken at the end of intravital microscopy (130 min after onset of reperfusion). To determine the phenotype of transmigrated leukocytes, immunostaining of paraffin-embedded serial tissue sections from each experiment was performed. Sections were incubated with primary rat anti-mouse anti-Ly-6G, anti-CD45 (BD Biosciences, San Jose, CA), or anti-F4/80 (Serotec, Oxford, UK) immunoglobulin G (IgG) antibodies. Afterwards, the paraffin sections were stained with commercially available immunohistochemistry kits (Ly-6G, CD45, Super Sensitive Link-Label IHC detection system, BioGenex, San Ramon, CA; F4/80, Vectastain ABC kit, Vector Laboratories, Burlingame, CA), obtaining an easily detectable reddish or brownish end-product, respectively. Finally, the sections were counterstained with Mayer’s hemalaun. The number of extravascularly localized Ly-6G-, CD45-, or F4/80-positive cells was quantified by light microscopy (magnification x400) on seven sections (10 observation fields per section) from seven individual animals per experimental group in a blinded manner, respectively. The number of transmigrated Ly-6G+ cells (neutrophils) and F4/80+ cells (monocytes/macrophages) is expressed as the percentage of total CD45+ leukocytes.

Toluidine blue staining
Histochemical staining with toluidine blue (Merck, Darmstadt, Germany) was performed on paraffin-embedded tissue sections incubated with 1% toluidine blue for 10 min. Mast cells were identified easily by metachromatic staining of their granules. The number of toluidine blue-positive mast cells was counted in 10 high-power fields (=0.09766 mm2 at microscope magnification x400) per section in a blinded manner and expressed as cells/mm2.

Flow cytometry
Rat monoclonal antibodies (mAb) to detect murine Ccr2 and Ccr5 expression on native neutrophils of the peripheral blood were generated as described previously [37 ]. Anticoagulated whole blood samples were incubated with 5 µg/ml mAb MC-21 or MC-68 for 60 min on ice. The antibody MC-21 binds specifically to murine Ccr2, and the antibody MC-68 binds to murine Ccr5. As isotype control, samples were also stained with rat IgG2b (BD PharMingen, San Jose, CA). For the identification of neutrophils, samples were also incubated with a phycoerythrin-labeled CD11b (BD PharMingen) antibody. After three washing steps, cells were incubated for 60 min on ice with a FITC-labeled anti-rat polyclonal antibody (Dianova, Hamburg, Germany). After lysis of erythrocytes, stained cells were analyzed on a flow cytometer (FACSort, Becton Dickinson, San Jose, CA). Neutrophils were identified by their light-scatter properties and expression of CD11b. The cutoff to define chemokine receptor-positive cells was set according to the staining with the isotype control antibody. Approximately 20,000 gated events were collected in each analysis.

Statistics
Data analysis was performed with a statistical software package (SigmaStat for Windows, Jandel Scientific, Erkrath, Germany). The Kruskal-Wallis test followed by the Student-Newman-Keuls test was used for the estimation of stochastic probability in intergroup comparisons. Mean values and SEM are given. P values <0.05 were considered significant.


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RESULTS
 
Microhemodynamic parameters and systemic leukocyte counts
To assure intergroup comparability, quantification of inner diameters, blood flow velocities, as well as shear rates of analyzed postcapillary venules was performed. No statistically significant differences among the experimental groups were detected (Table 1 A ). Furthermore, systemic leukocyte counts were comparable among all experimental groups (Table 1B) .


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  		Table 1. Microhemodynamic Parameters and Systemic Leukocyte Counts

Leukocyte rolling
Surgical preparation induced leukocyte rolling in postcapillary venules. At baseline measurement, no significant differences were detected among experimental groups. However, after 5 min of reperfusion, there was a transient, twofold elevation in numbers of rolling leukocytes in wild-type mice as compared with sham-operated mice (38.2±5.2/30 s vs. 17.7±4.2/30 s; Fig. 1 A ). Over the time course, this elevation decreased to near-baseline levels, finally reaching a plateau after 120 min of reperfusion. Similar results were obtained in Ccr1-, Ccr2-, and Ccr5 (Fig. 1B) -deficient mice, and no statistically significant differences were detected as compared with wild-type mice.



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  		Figure 1. Leukocyte rolling was quantified in postcapillary venules of the cremaster muscle at baseline conditions prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min of reperfusion. Panels show results for sham-operated and wild-type (WT) mice (A) as well as as for sham-operated wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice at representative time-points (B; mean±SEM for n=7 per group; #, P<0.05, vs. sham).

Leukocyte adherence
Prior to ischemia, only a few leukocytes were found attached to the inner vessel wall of postcapillary venules in all experimental groups. At 5 min after the onset of reperfusion, there was a marked increase in the number of firmly adherent leukocytes in wild-type animals (17.0±2.7/104 µm2) as compared with sham-operated animals (5.0±1.6/104 µm2; Fig. 2 A ), and this increase persisted throughout the entire experiment. In contrast, in Ccr1 (8.9±1.0/104 µm2)- and Ccr5 (9.1±2.0/104 µm2)-deficient animals, significantly fewer leukocytes adhered than in wild-type animals (17.0±2.7/104 µm2). At 5 min of reperfusion, Ccr2 deficiency (11.7±2.6/104 µm2; Fig. 2B ) did not significantly reduce firm leukocyte adherence but did so after 120 min (7.5±1.6/104 µm2) as compared with wild-type animals (14.4±2.1/104 µm2).



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  		Figure 2. Leukocyte firm adherence was quantified in postcapillary venules of the cremaster muscle at baseline conditions prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min of reperfusion. Panels show results for sham-operated and wild-type mice (A) as well as for sham-operated, wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice at representative time-points (B; mean±SEM for n=7 per group; *, P<0.05, vs. wild-type; #, P<0.05, vs. sham).

Leukocyte transmigration
Leukocyte transmigration was quantified in ROIs adjacent to postcapillary venules (Fig. 3 A ). At baseline conditions, the number of transmigrated leukocytes was low and did not differ between experimental groups. After onset of reperfusion, there was a progressive increase in numbers of transmigrated leukocytes in wild-type mice as compared with sham-operated mice (Fig. 3B) . At 120 min of reperfusion, this increase was significantly attenuated in Ccr1 (30.7±3.2/1.5x104 µm2)-, Ccr2 (27.4±3.1/1.5x104 µm2)-, and Ccr5 (32.1±3.0/1.5x104 µm2)-deficient mice (Fig. 3C) as compared with wild-type mice (47.1±3.7/1.5x104 µm2), resulting in an average reduction of 53.1%, 63.7%, and 48.5%, respectively.



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  		Figure 3. Leukocyte transmigration. Representative reflective light oblique transillumination (RLOT) intravital microscopic image (original objective magnification x20) of a postcapillary venule of the cremaster muscle of a wild-type animal after 120 min of reperfusion (A). Transmigrated leukocytes were quantified in the displayed ROIs at baseline conditions prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min of reperfusion. Panels show results for sham-operated and wild-type mice (B) as well as for sham-operated, wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice at representative time-points (C; mean±SEM for n=7 per group; *, P<0.05, vs. wild-type; #, P<0.05, vs. sham).

Tissue distribution of transmigrated leukocytes
As a measure of leukocyte interstitial migration behavior, distribution of transmigrated leukocytes was determined within the extravascular tissue (Fig. 4 A ). After 120 min of reperfusion in wild-type animals, over 60% (60.2±0.01%) of transmigrated leukocytes had migrated a distance of more than 25 µm (Fig. 4B) . Ccr1 (61.0±0.02%), Ccr2 (58.3±0.02%), and Ccr5 (59.4±0.01%) deficiency was not associated with alterations in the interstitial distribution of transmigrated leukocytes. Comparable results were obtained for the other observation time-points (data not shown).



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  		Figure 4. Leukocyte interstitial migration. Exemplary RLOT microscopic image (original objective magnification x25) of a postcapillary venule of the cremaster muscle of a wild-type animal after 120 min of reperfusion (A). Tissue distribution of transmigrated leukocytes was quantified in the displayed, subdivided ROIs at baseline conditions prior to 30 min of ischemia as well as at 5, 30, 60, 90, and 120 min of reperfusion. Panel shows results of quantitative analysis for sham-operated, wild-type, Ccr1–/–, Ccr2–/–, and Ccr5–/– mice at representative time-points. No significant differences were detected among experimental groups (B; mean±SEM for n=7 per group).

Phenotyping transmigrated leukocytes
To identify the phenotype of transmigrated leukocytes, immunostaining for CD45 (common leukocyte antigen), Ly-6G (neutrophils), and F4/80 (monocytes/macrophages) of cremasteric tissue samples was performed. In all experimental groups undergoing I/R, 80–90% of transmigrated leukocytes were Ly-6G+ neutrophils, and 5–10% were F4/80+ monocytes/macrophages. No statistically significant differences were detected among experimental groups.

Chemokine receptor expression on murine neutrophils
To determine membrane expression of Ccr2 and Ccr5 on blood-borne neutrophils of wild-type mice, fluorescein-activated cell sorter analysis was performed. As shown in Figure 5 , chemokine receptor Ccr2 was detected on 97.3 ± 0.9% of neutrophils, and Ccr5 was expressed on 94.5 ± 2.4% of neutrophils isolated from peripheral blood (mean±SEM; n=3).



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  		Figure 5. Membrane expression of Ccr2 and Ccr5 on neutrophils. Flow cytometric analysis of Ccr2 (A) and Ccr5 (B) expression on murine neutrophils isolated from the peripheral blood. Representative results for shifts of the mean fluorescence from Ccr2 and Ccr5 on neutrophils (solid histograms) as compared with isotype-matched control IgG (open histograms) are shown. Values given are mean percentage of respective Ccr expression (n=3).

Cremasteric mast cell density
To assess the number of mast cells in the cremaster muscle of wild-type as well as chemokine receptor-deficient mice, histochemical staining with toluidine blue was performed on paraffin sections. No significant differences were observed in the number of cremasteric mast cells among wild-type (5.7±1.1/mm2) as well as Ccr1 (7.8±1.1/mm2)-, Ccr2- (5.9±1.1/mm2)-, and Ccr5 (6.7±1.4/mm2)-deficient mice.


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DISCUSSION
 
I/R induces tissue damage, which is closely associated with leukocyte infiltration of the postischemic tissue [1 2 3 ]. The infiltrating leukocytes might also contribute to tissue remodeling and healing, pointing to a more differentiated role of this cell population during inflammatory conditions [5 6 7 8 ]. Recently, it has been shown that the chemokine receptors Ccr2 and Ccr5 as well as Ccl3/MIP-1{alpha} (an important Ccr1 ligand) are critically involved in the recruitment of leukocytes to reperfused tissue [24 , 26 , 27 ]. However, the role of Ccr1, Ccr2, and Ccr5 for each single step in the leukocyte extravasation process during I/R has not been characterized before. We used the technical approach of RLOT intravital microscopy in chemokine receptor-deficient mice to analyze leukocyte rolling and firm adherence simultaneously as well as transendothelial and interstitial migration of leukocytes dynamically over the time course.

Leukocyte rolling in postcapillary venules was strongly enhanced within the first minutes after onset of reperfusion and was not affected by Ccr1, Ccr2, and Ccr5 deficiency. Our data are in agreement with recent in vivo findings that lack of Ccr1 or Ccr2 does not alter leukocyte rolling on the endothelium of cremasteric venules after stimulation with Ccl3/MIP-1{alpha} and Ccl2/MCP-1, respectively [38 , 39 ].

Firm adherence to the endothelium is the second step in the sequential process of leukocyte recruitment as well as a prerequisite for transendothelial and interstitial migration of leukocytes.

Our findings are the first to demonstrate in vivo that Ccr1, Ccr2, and Ccr5 play major roles for firm adherence of leukocytes under postischemic conditions. Previously, it has been shown that Ccl5/RANTES-induced firm arrest of human leukocytes in a flow chamber model was reduced significantly after blockade of Ccr1 [40 ]. Moreover, lack of Ccr1 and Ccr2 was reported to attenuate Ccl3/MIP-1{alpha}- and Ccl2/MCP-1-induced firm adherence of leukocytes in the in vivo cremaster model [38 , 39 ].

We now show that Ccr1, Ccr2, and Ccr5 are differentially involved in mechanisms mediating firm adherence of leukocytes during the initial reperfusion phase. Whereas Ccr1 and Ccr5 predominantly regulate leukocyte firm adherence initially after onset of reperfusion, Ccr2 appears to play a role at later time-points. An explanation for these observations might give the involvement of monocytes/macrophages into the pathogenesis of I/R. Whereas the relevance of monocytes/macrophages for the healing phase after I/R injury has been well documented [41 ], only little was known about the role of this cell population during the early reperfusion phase. However, there is an enlarging body of evidence that in addition to neutrophils, monocytes infiltrate the postischemic tissue early after onset of reperfusion [42 , 43 ] and release a number of proinflammatory substances [44 ]. Moreover, it has been demonstrated in a recent study that Ccr2+ mononuclear leukocytes are potent facilitators of neutrophil recruitment during inflammatory conditions [45 ]. Thereby, a secondary generation of proinflammatory mediators by activated Ccr2+ monocytes and T cells is suggested to subsequently promote extravasation of neutrophils. Consequently, Ccr2-dependent, firm adherence of leukocytes to the postischemic endothelium might require an interplay between different leukocyte subpopulations such as neutrophils and Ccr2+ mononuclear cells and could therefore be delayed during the time course. In addition, recent findings proposing that expression of the Ccr2 ligand Ccl2/MCP-1 is regulated by expression of the CXC chemokine MIP-2 could give another explanation for these delayed effects of Ccr2 [46 ]. Therefore, our observations might as well be the consequence of time-dependent expression profiles of chemokines during the initial reperfusion phase, indicating a certain hierarchy of chemokines and their respective receptors during I/R.

To enter the extravascular compartment, leukocytes have to pass the endothelial barrier as well as the basal lamina. In our experiments, we were able to demonstrate that Ccr1, Ccr2, and Ccr5 contribute to the recruitment of leukocytes to the reperfused tissue. Furthermore, immunophenotyping revealed that 80–90% of transmigrated leukocytes were Ly-6G+ neutrophils, and 5–10% were F4/80+ monocytes/macrophages. Deficiency of Ccr1, Ccr2, as well as Ccr5 was not associated with alterations in the percentage of Ly6G+ and F4/80+ to total CD45+ leukocytes. Consequently, we conclude that Ccr1, Ccr2, and Ccr5 are critically involved in the regulation of neutrophil and monocyte extravasation during I/R. These results are in agreement with previous observations in different organs. Lack of Ccr2 in the reperfused kidney [26 ] as well as blockade of Ccl3/MIP-1{alpha} in the postischemic lung were associated with diminished leukocyte infiltration of the respective tissue [24 ]. In the present study, the functional relevance of Ccr1 for neutrophil recruitment during I/R has been elucidated for the first time and is supported by previous studies under different inflammatory conditions [39 , 47 ]. In striking contrast, blockade of Ccr5 after focal cerebral ischemia in mice did not affect recruitment of neutrophils, whereas in the same study, Ccr5 appeared to regulate macrophage infiltration of the postischemic brain [27 ]. An explanation for this discrepancy could be organ-specific effects, assuming that in addition to circulating leukocytes and endothelial/epithelial cells, also resident cells, which determine the characteristics of a definite tissue, play major roles in the inflammatory response [48 , 49 ].

In this context, tissue mast cells have been documented to release a number of substances inducing extravasation of neutrophils immediately after onset of reperfusion [49 ]. Recent findings revealed that mast cells express Ccr1, Ccr2, as well as Ccr5 [50 ], and it has been shown that this cell population plays a critical role in CC chemokine-induced neutrophil recruitment in vivo [51 ]. Moreover, Ccl3/MIP-1{alpha} has been shown to mediate extravasation of neutrophils in vivo through a sequential release of tumor necrosis factor {alpha} and leukotriene B4 [47 ]. Consequently, CC chemokines might trigger neutrophil recruitment during the postischemic inflammatory response indirectly via intermediary activation of resident cells such as mast cells and subsequent generation of further proinflammatory mediators. In the present study, we were able to verify that lack of Ccr1, Ccr2, and Ccr5 is not associated with alterations in the number of cremasteric mast cells, suggesting that reduced postischemic neutrophil recruitment in chemokine receptor-deficient mice might rather be a result of a lower activation of mast cells than the consequence of a lower mast cell density in the cremaster muscle. It is interesting enough that we were also able to demonstrate that chemokine receptors Ccr2 and Ccr5 are expressed on the surface of native murine neutrophils. Therefore, it seems plausible that Ccr2- and Ccr5-dependent firm adherence and (subsequent) transmigration of neutrophils to postischemic tissue might as well be mediated by direct interactions between these chemokine receptors on neutrophils and their respective chemokine ligands.

The question of whether Ccr1, Ccr2, and Ccr5 regulate transendothelial migration directly or if the observed effects are the consequence of diminished leukocyte firm adherence cannot be answered clearly in this in vivo study. However, previous in vitro data revealed that in addition to leukocyte firm adherence, CC chemokines and their respective receptors also mediate leukocyte transmigration via modulation of the activation state of leukocyte integrins [40 , 52 , 53 ]. Thereby, interactions between activated integrins and respective binding partners, such as junctional adhesion molecule-A, are supposed to regulate transendothelial migration of leukocytes [11 ]. Therefore, the reduced postischemic recruitment of leukocytes in animals, being deficient for the respective receptors, might be the consequence of reduced leukocyte firm adherence and effects on mechanisms mediating the leukocyte transmigration process directly. However, Ccr1, Ccr2, and Ccr5 deficiency did not block transmigration of leukocytes completely during I/R, suggesting participation of additional mechanisms. In this context, adhesion molecules expressed on the surface of endothelial cells and leukocytes, such as CD31, CD99, junctional adhesion molecule-A, and vascular endothelial cadherin [54 ], or leukocyte proteases [55 ] may facilitate this process independently of Ccr1, Ccr2, and Ccr5, respectively.

Finally, after their transendothelial migration, leukocytes are suggested to migrate along a chemokine gradient to the sites of inflammation [56 ]. During I/R, various cell types, including fibroblasts, tissue macrophages, as well as mast cells, could be the source of substances mediating locomotion of leukocytes within the interstitial tissue. In the present study, we have shown that I/R induces leukocyte migration within the perivascular space. It is interesting that lack of Ccr1, Ccr2, as well as Ccr5 was not associated with alterations in the tissue distribution of transmigrated leukocytes. These results are supported by previous observations under different inflammatory conditions. In a recent study, we were able to show that Ccr1 deficiency has no effect on interstitial migration behavior of transmigrated leukocytes in the mouse cremaster muscle after stimulation with Ccl3/MIP-1{alpha} [39 ].

In summary, our in vivo data demonstrate that Ccr1, Ccr2, and Ccr5 contribute to the recruitment of neutrophils during the early reperfusion phase through effects on intravascular adherence and subsequent transendothelial migration. Furthermore, we found that Ccr1, Ccr2, and Ccr5 have no impact on interstitial migration behavior of transmigrated leukocytes. Finally, our intravital microscopic findings provide insights into a dynamic regulation of leukocyte extravasation through chemokine receptors uncovering a hierarchy of Ccr1, Ccr2, and Ccr5 in the early postischemic inflammatory response.


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ACKNOWLEDGEMENTS
 
This study was supported by EU Network of Excellence "MAIN" (LSHG-CT-2003-502935, H-J. A., D. S., and F. K.) and Deutsche Forschungsgemeinschaft (Graduate Program 438 "Vascular Biology in Medicine", B. L., D. S., and F. K.). The data presented in this manuscript are part of the doctoral thesis of C. A. R. B. L. and F. K. shared senior authorship. The authors thank Phil Murphy and Ji-Liang Gao for providing the Ccr1-deficient mice as well as Anne-Marie Allmeling, Silvia Münzing, and Alke Schropp for technical assistance.

Received June 24, 2005; revised August 19, 2005; accepted August 24, 2005.


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