CCR2 is thought to recruit monocytes to sites of infection. Two subpopulations of murine blood monocytes differing in Gr1 and CCR2 expression have been described. The exact role of CCR2 in migration of CCR2lowGr1low and CCR2highGr1high monocytes into nonlymphoid tissue is controversial. In this study, we have addressed this question in a murine model of bacterial urinary tract infection. Only Gr1high monocytes were recruited into the infected bladder. CCR2 deficiency reduced their frequency in this organ, indicating a requirement of this chemokine receptor. Importantly, CCR2-deficient mice also showed reduced Gr1high monocyte numbers in the blood, but not in the bone marrow (BM), indicating that CCR2 acted at the step of monocyte release into the circulation. The same was found also in noninfected mice, indicating a further involvement of CCR2 in steady-state BM egress. An additional requirement of CCR2 in monocyte recruitment from the blood into the bladder was excluded by tracking particle-labeled endogenous monocytes and by adoptive transfer of BM-derived monocyte subsets. These findings demonstrate that CCR2 governs homeostatic and infection-triggered release of Gr1high monocytes from the BM into the blood but is dispensable for recruitment into a nonlymphoid tissue.
Monocytes and macrophages are characterized by a lack of lymphocyte markers and by expression of CD11b and CD14 in humans and of CD11b, CD115 (M-CSF receptor), and F4/80 in mice (1, 2, 3, 4, 5). Their precursors are released from the bone marrow (BM)3 (1, 5, 6) into the bloodstream as nondividing monocytes. Murine monocytes can be subdivided by their expression of Gr1 and of the chemokine receptors CCR2 and CX3CR1 (1). Gr1highCCR2highCX3CR1low monocytes are relatively large, short-lived, and actively enter inflamed tissue (1, 2). Gr1lowCCR2lowCX3CR1high monocytes are smaller and less granular, patrol blood vessels (7, 8), and home also to noninfected tissues (1, 2). It has been shown that these monocytes can develop from Gr1high monocytes in the blood (9). Both subsets can give rise not only to macrophages but also to dendritic cells (DC) (2, 3, 8, 9, 10).
The role of chemokine receptors in regulating monocyte migration is under intense investigation (11). CXCR4 retains their precursors in the BM when its ligand SDF-1 is available (12, 13). CCR2 is required for monocyte recruitment into inflamed or infected tissue, as evidenced by a major reduction of infiltrating monocytes in CCR2−/− mice (14, 15, 16) and aggravated infection (17, 18). This has been particularly well studied in infection with Listeria monocytogenes, a Gram-positive rod that targets the spleen (15, 17, 18, 19). Likewise, inflammatory diseases involving aberrant monocyte recruitment were attenuated in CCR2−/− mice (20, 21, 22). Gr1high inflammatory monocytes, which are particularly well recruited to sites of infection, express high levels of CCR2 (1, 17). Thus, it has been concluded that CCR2 mediates entry of monocytes into infected tissue. This conclusion is supported by several in vitro studies demonstrating CCR2-mediated attachment of monocytes to inflamed endothelial cells (23, 24) and local up-regulation of the CCR2 ligand MCP-1 in inflammatory conditions (25). Several inhibitors of CCR2 or its ligand MCP-1 have been developed and are currently being tried in various diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis, asthma, or in graft-vs-host diseases (20, 25, 26, 27, 28).
A recent study has challenged this view by demonstrating that CCR2−/− mice infected with Listeria monocytogenes showed diminished monocyte numbers in the spleen and in the circulation (17). It was proposed that CCR2 mediates egress of BM progenitors into the circulation rather than into infected tissues (17, 29). Studies testing this hypothesis in infection models other than listeriosis are missing. In particular, immigration into nonlymphoid tissues, where the endothelium needs to be crossed by monocytes, has not been examined yet. In arteriosclerosis, a role of CCR2 in monocyte infiltration into inflamed vessel walls has been reported (29). However, it has recently been reported that CCR2lowGr1low monocytes were rapidly recruited in bacterial peritonitis, suggesting that CCR2-independent recruitment pathways can operate (7). The exact role of CCR2 in monocyte migration remains to be clarified.
Urinary tract infections (UTI), such as cystitis and pyelonephritis, are among the most prevalent infections and account for considerable morbidity and medical costs (30). Most of these infections are caused by invasive Gram-negative uropathogenic Escherichia coli (UPEC) that ascend through the urinary tract (31, 32). Polymorphonuclear neutrophilic granulocytes (PMN), monocytes, and DC are rapidly recruited to the inflamed bladder (33), but only the former two cell types contribute to the early innate defense (33). Although observations in other infection models suggest that CCR2 may recruit monocytes also in UTI, we recently found that this chemokine receptor was not essential for bacterial clearance in mice (33). It is unknown whether this chemokine receptor is required for monocyte recruitment in UTI and, if so, whether it operates at the BM/blood or at the blood/bladder barrier and whether it affects Gr1low and/or Gr1high monocytes. In this study, we have addressed these open questions using a murine model of UTI.
Materials and Methods
Mice and reagents
CCR2−/− mice had been backcrossed >10 times to the C57BL/6 background (14, 33) and were bred and kept under specific pathogen-free conditions. Animal experiments had been approved by the government and local animal ethics reviewing boards. Unless indicated otherwise, all reagents were obtained from Sigma-Aldrich.
UPEC and UTI model
The UPEC strain 536 (O6:K15:H31) originating from a UTI patient was used for infections. UPEC were cultured overnight under static conditions in LB medium, harvested by centrifugation at 1200 g for 5′ and resuspended in PBS to a concentration of 1 × 1010 CFU per ml. Anesthetized female mice of 8–10 wk of age were infected by transurethral inoculation of 5 × 108 E. coli 536 (0.05 ml) into the bladder using a soft polyethylene catheter (outer diameter 0.6 mm; BD Biosciences). Expression of the virulence factor FimH on UPECs, which is crucial for invasion into urothelial cells (34), was confirmed by mannose-sensitive hemagglutination of guinea pig erythrocytes (35) and by microscopic detection of UPEC within urothelial cells (data not shown). In ∼20% of infected mice, UPEC could be grown also from kidney homogenate, but the number of CFU was ∼1000-fold lower compared with a homogenate of the extensively rinsed bladder.
Isolation and analysis of leukocytes
The digestion protocol for isolation of nonlymphoid leukocytes (36, 37) was modified to isolate leukocytes from the bladder. In brief, bladders were sliced with a scalpel and digested for 30 min at 37°C with 0.5 mg/ml collagenase and 100 μg/ml DNase I in RPMI 1640 medium (Invitrogen) containing 0.5% heat-inactivated FCS (PAA Laboratories) and 20 mM HEPES. Cell suspensions were filtered through 100-μm nylon mesh and washed with Ca2+ and Mg2+-free HBSS containing 10 mM EDTA, 0.1% BSA, and 20 mM HEPES.
Whole blood was subjected to red cell lysis buffer and washed twice in DMEM medium (Invitrogen) containing 0.02% NaN3, 2 mM EDTA, and 2% FCS. BM cells were obtained by flushing the femur with HBSS medium containing 0.1% BSA and 5 mM EDTA and then treated like blood cells.
The numbers of viable cells was determined by trypan blue staining. Fc receptors were blocked with mouse serum. Titrated amounts of the following labeled Abs from BD Pharmingen were used for staining of 1 × 106 cell samples: anti Gr-1 (RB6-8C5), anti CD11c (HL-3), anti F4/80-biotin (CI:A3-1) from Serotec, and anti CD115 (AFS98) from eBioscience. Monocytes and macrophages were identified as CD11b+, F4/80+CD115+ cells deficient in expression of the lymphoid lineage (Lin) markers CD19, NK1.1, and CD3ε (1). PMN were identified as F4/80−Gr1+SSChigh cells. Flow cytometry was performed on a LSR II cytometer (BD Biosciences) and results were analyzed using Flow Jo software (Tristar). To calculate absolute cell numbers, 10 × 104 10 μm PerCP Cy5.5-labeled micro beads (BD Biosciences) were added to the measured tubes.
Depletion and labeling of blood monocytes
Clodronate-liposomes and control PBS-liposomes were prepared as described earlier (38). Blood monocytes were eliminated by i.v. injection of 250 μl clodronate-liposome suspension into the lateral tail vein. Blood monocytes were labeled by injection of 0.5-μm FITC-conjugated (yellow gold) plain beads (2.5% solid microspheres; Polysciences) (39). These beads were diluted 1/25 in PBS, and 250 μl of this suspension was injected i.v. for labeling of Gr1low monocytes. To label Gr1high monocytes, 250 μl of liposomes containing clodronate were i.v. injected, followed by 250 μl of fluorescent beads i.v. 16–18 h later (39).
Recipient mice were irradiated with 9 Gy and reconstituted with 5 × 106 donor BM cells. For mixed BM chimeras, 50% of CCR2-deficient (CD45.2) and 50% of wild-type (CD45.1) BM were injected i.v.
Adoptive transfer of BM monocytes
BM cells from CCR2−/− and CCR2+/+ mice were isolated as described above. For enrichment of BM monocytes, cell suspensions were incubated with anti Ly6G (clone 1A8; BD Biosciences) labeled to biotin using a commercial kit (Pierce) followed by incubation with MACS anti-biotin, anti-CD19, anti-CD8, and anti-CD4 beads (Miltenyi Biotec). After negative selection, the cell suspension comprised 75–85% of Gr1high and 5–10% Gr1lowF4/80+CD115+ monocytes. These cells were labeled with 10 μM CMTMR (Molecular Probes), and 2 × 106 CCR2−/− and CCR2+/+ cells (ratio 1:1) were infused into infected mice. To exclude contaminations of bladder monocytes with blood cells, recipient mice were thoroughly perfused before analysis.
Bladders were homogenized in the presence of the protease inhibitors Complete Mini (Roche) in 2 ml PBS. Supernatants after centrifugation, as well as serum samples, were analyzed by a TNF-α ELISA kit (R&D Systems).
Prism (GraphPad) was used for statistical analysis. The unpaired Student’s t test was used to compare groups; a p-value of less than 0.05 was considered significant. In all figures, ∗ indicates p-value <0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
Gr1high, but not Gr1low, monocytes are recruited from the blood into the infected bladder
Blood monocytes can be subdivided into Gr1lowCCR2low and Gr1highCCR2high subsets (1). We studied the mechanisms governing migration of these subsets in bacterial infection, using a murine model of UTI, induced by transurethral instillation of UPEC (33). The BM contained considerably more Gr1high than Gr1low monocytes both in noninfected and infected mice (Fig. 1) (17). In noninfected control mice, Gr1low monocytes were more frequent in the blood and the bladder (Fig. 1). A total of 14 h after UTI induction, Gr1high monocytes had decreased in the blood and increased the bladder, whereas numbers of Gr1low monocytes were not significantly altered (Fig. 1). These findings showed that only Gr1high, but not Gr1low, monocytes were recruited to the bladder in UTI.
CCR2 deficiency reduces Gr1high monocytes in the infected and noninfected bladder and also in the blood
CCR2 has been proposed to mediate recruitment of monocytes to sites of infection (14, 15, 17, 18). Since Gr1high monocytes have been shown to express more CCR2 (1), we investigated whether their preferential recruitment to the infected bladder (Fig. 1) was mediated by CCR2. Indeed, Gr1high monocytes were less abundant in infected bladders of CCR2−/− mice (Fig. 2,C). These cells were less frequent also in noninfected CCR2−/− mice as compared with noninfected wild-type controls (Fig. 2 C), indicating an additional role for CCR2 in homeostasis.
Importantly, CCR2 deficiency reduced Gr1high monocytes also in the blood (Fig. 2,B), whereas no significant differences were seen in the BM of CCR2-competent and -deficient mice (Fig. 2,A). These observations raised the possibility that CCR2 acted at the step of Gr1high monocyte release from the BM, as recently suggested (17, 29). This interpretation was further corroborated by lower levels of the effector cytokine TNF-α in the serum and bladder homogenate of CCR2-deficient mice (Fig. 2 D), consistent with a reduction of monocyte functionality.
Homeostatic release of Gr1high monocytes into the blood is also controlled by CCR2
To further examine the effect of CCR2 on Gr1high monocyte release from the BM in the steady state (Fig. 2), we reconstituted lethally irradiated CCR2+/+ mice with a 1:1 mixture of BM from CCR2+/+ CD45.1 and from CCR2−/− CD45.2 mice. After 6 wk, we determined the numbers of Gr1high monocytes and those of PMN, which served as a CCR2-independent control cell population. In the BM from one femur, CCR2 deficiency neither affected the absolute numbers of monocytes and PMN, nor the ratio between CCR2+/+ (CD45.1+) and CCR2−/− (CD45.2) cells, indicating that entry of the injected cells into the recipient BM was independent of CCR2 (Fig. 3,A). In contrast, Gr1high monocytes in the blood were significantly reduced when they lacked CCR2, as compared with wild-type cells (Fig. 3,B). The numbers of PMN in the blood were not affected by CCR2 (Fig. 3 B). These data confirmed that not only inflammatory but also homeostatic release of Gr1high monocytes into the blood stream depended on CCR2.
Applicability of latex (Lx) bead labeling for in vivo tracking of Gr1low and Gr1high monocytes in CCR2−/− mice
The requirement of CCR2 for egress of monocytes from the BM (Figs. 2 and 3) did not exclude an additional role in mediating entry into the bladder. To investigate this possibility, we used a recently developed technique that allows selective in vivo labeling of either Gr1high or Gr1low monocytes. Gr1low monocytes in the blood can be labeled by i.v. injection of FITC fluorescent Lx beads, which are spontaneously phagocytosed by these cells (39). Such phagocytosis was not affected by CCR2 expression, since Gr1low blood monocytes of CCR2+/+ and CCR2−/− mice showed comparable uptake of one or, less frequently, several beads, resulting in labeling of 6–10% of these cells for 1–5 days (Fig. 4,A). Preferential labeling of Gr1high monocytes was conducted by injection of Lx beads in the absence of Gr1low monocytes. This could be achieved by i.v. injection of clodronate-loaded liposomes (clo-lip) (39), which efficiently depleted both monocyte subsets in the blood within less than 10 h (Fig. 4,B). The Gr1high monocyte subset re-emerged after 24 h, whereas Gr1low monocytes remained absent for 5–7 days (Fig. 4,B). Gr1high monocytes could be labeled by injecting Lx beads 18 h after depletion with clo-lip (Fig. 4,B), resulting in efficient labeling of 25–30% of Gr1high monocyte at 66 h after depletion, both in CCR2+/+ and CCR2−/− mice (Fig. 4,B). At this time point, Gr1low monocytes in fact had not yet re-appeared (Fig. 4 B). These findings demonstrated the applicability of the monocyte-subset-specific Lx bead labeling technique (39) in CCR2−/− and CCR2+/+ mice.
CCR2 is dispensable for recruitment of in vivo-labeled monocyte subsets into the bladder
To investigate the role of CCR2 in monocyte immigration into the infected bladder, we infected CCR2+/+ and CCR2−/− mice with UPEC 2 days after labeling Gr1high (Fig. 5,B) or, as a control, Gr1low (Fig. 5,A) monocytes as described above. Another day thereafter, Gr1low monocytes had been efficiently labeled in the blood (Fig. 5,A), but very few labeled cells could be recovered from bladders of infected and noninfected animals (Fig. 5,A), confirming that this monocyte subset was recruited very slowly to the infected and noninfected bladder. This result also implied that cells recovered from the bladder were not contaminating blood cells because, in this case, their abundance should have been similar to that in the blood. Loss of CCR2 did not affect this low-level recruitment (Fig. 5 A), as expected for this CCR2low Gr1low monocyte subset.
Labeled Gr1high monocytes efficiently entered the infected bladder and reached proportions comparable to the blood (Fig. 5,B). Importantly, CCR2 deficiency reduced their immigration into the bladder neither in the steady state, nor in infection (Fig. 5 B), indicating that this chemokine receptor was dispensable for recruitment of Gr1high monocytes in both situations. Furthermore, if CCR2 would mediate Gr1high monocyte recruitment into the bladder, then blood levels of CCR2-competent cells should have been lower than those of CCR2-deficient ones.
CCR2 is dispensable for recruitment of adoptively transferred monocytes into the bladder
To confirm the interpretations above with a different approach, we cotransferred CCR2+/+ and CCR2−/− BM monocytes into recipient mice with UTI, and subsequently determined the ratio between these subsets in BM, blood, and bladder. To this end, a 1:1 mixture of CD45.1 CCR2+/+ and CD45.2 CCR2−/− monocytes was labeled with CMTMR and then transferred. Fig. 6 A shows an analysis of this mixture.
One hour after transfer, the ratio between CCR2+/+ and CCR2−/−monocytes in the BM, the blood, and the bladder was determined (Fig. 6,B; analysis of the bladder shown as example in Fig. 6,C). Some transferred Gr1low and Gr1high monocytes were detected at this early time point in the BM at 1:1 ratio (Fig. 6,B), verifying our previous finding that entry of monocytes into the BM was CCR2 independent. Although the proportion of Gr1high monocytes in the BM and blood was somewhat lower than before transfer (79 or 80 vs 84% before transfer), their proportion in the bladder was increased (87 vs 84%), consistent with selective and rapid recruitment of Gr1high monocytes to the site of infection. Importantly, CCR2+/+ and CCR2−/− monocytes were similarly frequent in the infected bladder, and this was true for both Gr1high and Gr1low monocytes (Fig. 6, B and C), confirming that monocyte entry into this organ did not require CCR2.
The traditional view of CCR2 function has recently been challenged by experiments suggesting that this chemokine receptor may affect monocyte entry into infected tissues only indirectly by promoting monocyte release from the BM (17). These findings were obtained in splenic listeriosis and, therefore, did not exclude an additional role of CCR2 in monocyte entry into nonlymphoid tissues, where these cells have to cross other endothelial barriers to leave the blood. A role of CCR2 in such recruitment is supported by in vitro studies showing CCR2-dependent monocyte attachment to inflamed endothelium (23, 24). We addressed this question in UTI, one of the most prevalent bacterial infections. We found that only Gr1high monocytes, which express high levels of CCR2, accumulated in the infected organs, consistent with the proposed role of this subset in inflammation (1). The absolute numbers of Gr1high monocytes that disappeared from the blood were lower than their increase in the bladder, which may be due to their recruitment into other tissues, to their death in the bladder, or to their urinary loss by leukocyturia. Gr1low monocytes are usually not assumed to show CCR2 dependency, since they express little CCR2 levels in the blood (1). Consistently, these cells did not accumulate in the infected bladder. This result somewhat differs from observations in Listeria monocytogenes peritonitis, where Gr1low monocytes recently were reported to be recruited into the peritoneal cavity, although at a much lower frequency than Gr1high monocytes (7). The absence of recruited Gr1low monocytes in our UTI model may be due to differences between the immune responses against Gram-negative UPEC and Gram-positive Listeria. Alternatively, it may relate to the distinct infection sites studied.
Importantly, we found that CCR2−/− mice displayed reduced abundance of Gr1high monocytes in the steady-state, both in the blood and in noninfected tissue (17). This finding is consistent with previous observations in CCR2−/− mice, and provides formal proof of a homeostatic role of CCR2. In infected CCR2-deficient mice, this reduction was far more pronounced in the bladder. Importantly, this was not only true at the site of infection in the bladder, but also in the blood, and was detectable also on the functional level of a monocyte/macrophage effector molecule, TNF-α. Furthermore, several experimental approaches did not reveal any evidence that Gr1high monocytes use CCR2 to enter the inflamed bladder. Instead, Gr1high monocyte homeostasis and “availability” in cases of infection was primarily regulated on the level of egress from the BM compartment. Therefore, the CCR2 ligand, MCP-1 most likely reached the BM via the blood stream, which is supported by the finding that MCP-1 levels were increased not only in infected organs, but also in the blood stream (40). An alternative, not mutually exclusive explanation for CCR2 signaling in the BM could be local MCP-1 production in this site, as recently suggested (41). Such local production might facilitate monocytes egress from the BM also in noninfected mice. In contrast to previous observations (17), we did not observe accumulation of Gr1high monocytes in the BM of CCR2-deficient mice. Possibly, the BM niche could not accommodate more monocyte precursors when CCR2 was lacking.
Of note, there is experimental evidence for a role of CCR2 in Gr1high monocyte recruitment at the level of egress from the BM compartment in other noninfectious inflammatory conditions, namely atherosclerosis. By dissecting effects of different chemokines for monocyte subset recruitment, several studies independently highlighted regulation of atherosclerosis-associated Gr1high monocytosis via CCR2 and its ligand MCP-1 (29, 42, 43). The degree of Gr1high monocyte levels in the blood directly correlated with the accumulation of macrophages in plaques (29, 42, 43). However, especially in atherosclerosis, Gr1high monocytes may also additionally use CCR2 at the level of transendothelial migration into the intima of chronically inflamed blood vessels (43).
Our finding of CCR2-dependent monocyte egress of the BM does not exclude that CCR2 may play a role in monocyte entry into arteriosclerotic large arteries, nor into tissues other than the bladder. Nevertheless, the present results warrant reassessment of previous in vivo infection studies and of in vitro studies that support CCR2-dependent endothelial attachment. Such studies usually used human umbilical vein cells or ex vivo cultured endothelial cells isolated from large vessels, which may not generally reflect endothelial functions in the entire body or in capillaries. Also, isolated BM monocytes from wild-type or CCR2−/− mice might differ in maturation or activation, either related to intrinsic differentiation properties or to ex vivo isolation procedures.
In summary, our study demonstrates that the chemokine receptor CCR2 is required for release of Gr1high monocytes into the blood, both in homeostasis and infection, but is dispensable for recruitment into the infected bladder. Although the mechanisms underlying such recruitment remain to be elucidated, these findings question therapeutic attempts to treat inflammatory diseases in nonlymphoid tissues by local CCR2 blockade (20, 25, 26, 27, 28). Systemic administration that reaches the BM may be effective, but the concomitant general reduction of blood monocytes might result in increased susceptibility to circulating pathogens.
We thank W. A. Kuziel for CCR2−/− mice and Steffen Jung for reading the paper. We acknowledge support by the Flow Cytometry Core Facility of the Institute of Molecular Medicine and Experimental Immunology and by the House for Experimental Therapy.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by the Deutsche Forschungsgemeinschaft (Grants Ku1063/4, Ku1063/5, Ku1063/6, and Ta434/2-1).
Abbreviations used in this paper: BM, bone marrow; UPEC, uropathogenic Escherichia coli; UTI, urinary tract infection; PMN, polymorphonuclear neutrophilic granulocyte; DC, dendritic cell; clo-lip, clodronate-loaded liposome; Lx, latex.