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The Journal of Immunology, 2002, 168: 4659-4666.
Copyright © 2002 by The American Association of Immunologists

Differential Roles of CC Chemokine Ligand 2/Monocyte Chemotactic Protein-1 and CCR2 in the Development of T1 Immunity1

Tim R. Traynor2,*, Amy C. Herring*, Martin E. Dorf{ddagger}, William A. Kuziel§, Galen B. Toews* and Gary B. Huffnagle3,*,{dagger}

* Pulmonary Division, Departments of Internal Medicine and {dagger} Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109; {ddagger} Department of Pathology, Harvard Medical School, Boston, MA 02115; and § Department of Molecular Genetics and Microbiology, and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-{gamma} production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-{gamma}-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII+CD11c+ and MHCII+CD11b+ cells, and produced significantly less IL-12p70 and TNF-{alpha} when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CC chemokine ligand 2 (CCL2,4 the chemokine formerly known as monocyte chemoattractant protein-1 (MCP-1); Ref. 1) and its receptor, CCR2, have been found to be involved in mediating T1/T2 polarization. CCL2 expression has been associated with T2 (Th2 and Tc2) development in both infectious (2, 3, 4, 5, 6) and allergic disease models (7, 8). In addition, CCL2 has been found to enhance IL-4 production by T cells (9, 10, 11, 12). In contrast, studies using mice that lack CCR2, the only known functional receptor for CCL2, have generally demonstrated that this receptor promotes T1 (Th1 and Tc1) development in infection models (13, 14, 15, 16, 17, 18) and attenuates the pulmonary allergic (T2) response to Asperigillus (19). The reasons for the discrepancy between the pro-T2 actions of CCL2 and the pro-T1 role of CCR2 are not known. However, in the absence of CCR2, CCL2 may promote T2 development via a second, as yet unidentified, receptor (20, 21, 22).

CCR2 is clearly important for the resolution of a pulmonary Cryptococcus neoformans infection (16). C. neoformans is an encapsulated yeast that is acquired via the respiratory tract and requires T1-type cell-mediated immunity for clearance of this opportunistic pathogen from the lung (23). T1-type cell-mediated immunity to pulmonary C. neoformans infection is characterized by IFN-{gamma} production, macrophage and lymphocyte infiltration into the lungs, and the development of Ag-specific delayed-type hypersensitivity to C. neoformans (23). The T1-type response requires CD4+ and CD8+ T cells, in addition to the production of the cytokines TNF-{alpha}, IL-12, and IFN-{gamma} (23). In contrast to CCR2+/+ mice, CCR2-/- mice produce a strong T2-type immune response to C. neoformans, and cannot clear a pulmonary C. neoformans infection (16). The T2-type response is characterized by reduced macrophage and lymphocyte recruitment, pulmonary eosinophilia, leukocyte production of IL-4 and IL-5 but not IFN-{gamma}, and increased serum IgE. These findings demonstrate that expression of CCR2 is required for the development of a T1-type response to C. neoformans infection and lack of CCR2 results in a switch to a T2-type response.

Previous studies from our laboratory demonstrated that efferent phase (days 5–14 postinfection) production of CCL2 is required for T1-driven mononuclear cell recruitment into the lungs of C. neoformans-infected mice at 2 wk postinfection (24). However, treatment of mice beginning at day 5 of infection with anti-CCL2 Ab did not produce the pulmonary eosinophilia seen in CCR2-/- mice (16, 24). It is possible that the production of CCL2 before day 5 prevented the development of pulmonary eosinophilia (T2-type response). The objective of this study was to determine the role of CCL2 in the afferent and efferent phase of T1 immunity and compare it to the role of CCR2 in T1 to T2 switching of the pulmonary response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Wild-type and CCR2-/- mice (25) on an outbred C57BL/6 x 129 genetic background were maintained at the University of Michigan Unit for Laboratory Animal Medicine Facilities (Ann Arbor, MI) under specific pathogen-free conditions in enclosed filter top cages. Clean food and water was given ad libitum. The mice were handled and maintained using microisolator techniques with daily veterinarian monitoring. Bedding from the mice was transferred weekly to cages of uninfected "sentinel" mice that were subsequently bled at weekly intervals and found to be negative for Abs to mouse hepatitis virus, Sendai virus, and Mycoplasma pulmonis. Mice were 8–12 wk of age at the time of infection and there were no age-related differences in the responses of these mice to C. neoformans infection.

C. neoformans

C. neoformans strain 52D was obtained from the American Type Culture Collection (24067; Manassas, VA). For infection, yeast were grown to stationary phase (48–72 h) at 37°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit, MI) on a shaker. The cultures were then washed in nonpyrogenic saline, counted on a hemocytometer, and diluted to 3.3 x 105 CFU/ml in sterile nonpyrogenic saline.

Surgical intratracheal inoculation

Mice were anesthetized by i.p. injection of pentobarbital (0.074 mg/g weight of mouse) and restrained on a small surgical board. A small incision was made through the skin over the trachea and the underlying tissue was separated. A 30-gauge needle was bent and attached to a tuberculin syringe filled with diluted C. neoformans culture. The needle was inserted into the trachea and 30 µl of inoculum (104 CFU) was dispensed into the lungs. The needle was removed and the skin closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.

Bronchoalveolar lavage

Mice were lavaged after cannulation of the trachea with polyethylene tubing (PE50) that was attached to a 25-gauge needle on a tuberculin syringe. The lungs were lavaged twice with 0.8 ml of PBS containing 5 mM EDTA. The recovered fluid (1.3–1.4 ml total) was spun at 1500 rpm and the supernatant was removed and stored at -20°C until analyzed for eotaxin (Quantikine M; R&D Systems, Minneapolis, MN) or IFN-{gamma} (OptEIA; BD PharMingen, San Diego, CA) by sandwich ELISA using the manufacturer’s instructions supplied with the cytokine-specific kits.

CCL2 neutralizing Ab

Monoclonal anti-CCL2 Ab (2H5) (26) was purified from hybridoma supernatants. Supernatants were passed through a protein G column (Amersham Pharmacia Biotech, Piscataway, NJ) and bound Ab was eluted with 0.1 M glycine-HCl buffer (pH = 2.7). The glycine-HCl buffer was exchanged for PBS using a Centriplus filter (Millipore, Bedford, MA), and the Ab concentration was determined by OD. Anti-CCL2 activity was confirmed by ELISA. For in vivo CCL2 neutralization, animals were injected i.p. with either PBS as a control or 100 µg of Ab at days 0, 4, 8, and 12 post C. neoformans infection.

Lung leukocyte isolation

At day 14 postinfection, individual lungs were excised, minced, and enzymatically digested for 30 min in 15 ml of digestion buffer (RPMI, 5% FCS, antibiotics, 1 mg/ml collagenase, and 30 µg/ml DNase). The cell suspension and undigested fragments were further dispersed by triturating with a 10-ml syringe. The total cell suspension was then pelleted and the erythrocytes were lysed by resuspending them in ice-cold NH4Cl buffer (0.83% NH4Cl, 0.1% KHCO3, and 0.037% Na2EDTA, pH 7.4). A total of 10-fold excess of media was added to return the solution to isotonicity. The isolated leukocytes were repelleted and resuspended in complete media. Total cell numbers were assessed in the presence of trypan blue (viability >85%) using a hemocytometer. Subsequent flow cytometric analysis (described below) was used to determine the percentage of total leukocytes (CD45CLA+) within the lung cell suspension for correction of hemocytometer counts. Subsets of isolated leukocytes (neutrophils, eosinophils, macrophages, and total lymphocytes) were determined by Wright-Giemsa staining of samples cytospun onto slides. As reported previously (16, 27), recruited leukocyte numbers were calculated by subtracting uninfected values, as determined in a parallel cohort of uninfected mice (the values for uninfected wild-type and uninfected CCR2-/- mice were not significantly different), from the values measured in infected lungs.

Flow cytometric analysis

Leukocytes (5 x 105) were incubated for 30 min on ice with staining buffer (FA buffer, Difco; 0.1% NaN3, 1% FCS). Each sample was incubated with: 1) 0.12 µg of CyChrome-labeled anti-CD45 (30-F11, BD PharMingen); and either 2) 0.25 µg each of FITC-labeled anti-CD4 (RM4-5) and PE-labeled anti-CD8 (53-6.7); or 3) 0.25 µg of FITC-labeled anti-B220 (RA3-6B2). In other experiments, samples of lung-associated lymph node (LALN) cell suspensions were stained with PE-labeled anti-I-Ad/I-Ed (2G9) and either FITC-labeled anti-CD11b (M1/70) or FITC-labeled anti-CD11c (HL3). The samples were washed in staining buffer and fixed in 2.5% paraformaldehyde in buffered saline. Stained samples were stored in the dark at 4°C until analyzed by flow cytometry (Coulter Elite ESP; Coulter, Hialeah, FL). Samples were gated for CD45+ cells and then analyzed for staining by the specific FITC- and PE-labeled anti-lymphocyte markers. Recruited lymphocyte numbers in the lungs were calculated by subtracting resident values, as determined in a parallel cohort of uninfected mice (the values for uninfected wild-type and uninfected CCR2-/- mice were not significantly different) from the values measured in infected lungs.

Lung leukocyte culture and cytokine production

Isolated leukocytes (15 x 106) were cultured in 6-well plates with 3 ml of compete medium at 37°C and 5% CO2 with no additional stimulus. Supernatants were harvested at 24 h and assayed for IFN-{gamma}, IL-4, and IL-5 production by ELISA (OptEIA, BD PharMingen).

Lymph node cell isolation and culture

At 14 days postinfection, LALN (or superior mediastinal nodes) were excised and cells dispersed by mashing in media (RPMI, 5% FCS, antibiotics). Isolated LALN cells (2 x 106) were cultured in 24-well plates with 1 ml of compete medium at 37°C and 5% CO2 with no additional stimulus or one of the following: heat-killed C. neoformans (HKC) at a ratio of 2:1 (HKC:node cells) or both phorbol 12 myristate 13-acetetate (50 ng/ml) and ionomycin (50 ng/ml). These suboptimal concentrations of PMA and ionomycin were shown to have little effect on uninfected cells. Supernatants were harvested at 24 h and assayed for IFN-{gamma} and IL-5 production by ELISA. To measure TNF-{alpha} and IL-12p70 production by ELISA (OptEIA, BD PharMingen), LALN were digested with collagenase as described for the lung cultures and cultured as described above for 24 h with no additional stimulus, HKC, or with heat-killed Candida albicans at a 2:1 ratio of heat-killed yeast:node cells.

Statistics

Data (mean ± SEM) for each experimental group were derived from three separate experiments and analyzed by two-way ANOVA. For individual comparisons of multiple groups, post hoc test for simple main effects was used to calculate p values. Means with p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CCL2 neutralization on pulmonary mononuclear cell recruitment

Our first aim was to compare the effects of CCL2 neutralization (beginning at day 0) to CCR2 deficiency on C. neoformans-induced lung leukocyte recruitment. Total lung leukocytes were isolated from enzymatically digested lungs and pulmonary leukocyte recruitment was determined as outlined in Materials and Methods. Following infection, the lungs of wild-type mice showed a vigorous inflammatory response with 76 million leukocytes recruited at 14 days postinfection (Fig. 1Go). Leukocyte recruitment was reduced 55% by neutralization of CCL2 during the first 14 days of infection. A similar reduction (46%) in leukocyte recruitment was apparent in infected mice lacking CCR2. Thus, neutralization of CCL2 and deletion of CCR2 both result in similar reductions in total leukocyte recruitment into the lungs.



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  		FIGURE 1. Effects of CCL2 neutralization on C. neoformans-induced pulmonary leukocyte recruitment in resistant wild-type mice. Wild-type and CCR2-/- mice were inoculated intratracheally with 104 CFU of C. neoformans. For in vivo neutralization of CCL2 in wild-type mice, Ab ({alpha}-CCL2) was administered i.p. at days 0, 4, 8, and 12 postinfection. Lungs were harvested at day 14 for isolation of lung leukocytes (see Materials and Methods). Samples of individual lung leukocyte suspensions were cytospun onto slides and stained with Wright-Giemsa stain for visual quantification of macrophages and lymphocytes. *, p < 0.05 in comparison with CCR2+/+; n = 10 for each group; values are means ± SEM.

 
Cytological and flow cytometric analysis was used to determine which leukocyte subsets could account for the reduction in total leukocyte recruitment in CCL2-neutralized mice at 14 days postinfection. Both CCL2 neutralization and CCR2 deficiency reduced pulmonary macrophage recruitment by 54 and 65%, respectively (Fig. 1Go). Similarly, total lymphocyte recruitment was reduced by 67 and 70% in CCL2-neutralized and CCR2-deficient mice, respectively. Fig. 2Go illustrates that a pulmonary C. neoformans infection in wild-type mice induces vigorous recruitment of both T and B lymphocytes by 14 days postinfection. CCL2 neutralization and CCR2 deficiency resulted in comparable decreases in both CD4+ T cell recruitment (reductions of 77 and 82%, respectively) and CD8+ T cell recruitment (reductions of 82 and 90%, respectively). Recruitment of B220+ cells was not significantly affected by either CCL2 neutralization or by deletion of CCR2. These results demonstrate that macrophage and T cell recruitment are similarly reduced by either CCL2 neutralization or deletion of the CCL2 receptor CCR2.



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  		FIGURE 2. Effects of CCL2 neutralization ({alpha}-CCL2) on C. neoformans-induced recruitment of lymphocyte subsets in wild-type mice. Samples of lung leukocyte suspensions from 14-day infected mice were stained with fluorochrome-labeled Abs specific for lymphocyte subsets (CD4+, CD8+, and B220+) and analyzed by flow cytometry (described in Materials and Methods). *, p < 0.05 in comparison with CCR2+/+; n = 10 for each group; values are means ± SEM.

 
Effects of CCL2 neutralization on pulmonary granulocyte recruitment

The effects of CCL2 neutralization on lung granulocyte recruitment during infection were also assessed, given that eosinophilia is one of the hallmark characteristics of the T2-type immune responses in the lungs of C. neoformans-infected CCR2-deficient mice (16). There were no significant differences in neutrophil recruitment between any of the three treatment groups (5.3 ± 1.6, 2.1 ± 1.2, and 2.6 ± 1.2 million for CCR2+/+, anti-CCL2 treated CCR2+/+, and CCR2-/-, respectively; data not shown). As previously reported, CCR2-/- mice developed a marked eosinophilia at 2 wk postinfection (>90% increase; Fig. 3Go). However, this eosinophilia was not reproduced by CCL2 neutralization as both control-infected wild-type and anti-CCL2-treated wild-type mice had similar numbers of lung eosinophils at 2 wk postinfection. These results demonstrate that CCL2 neutralization in wild-type mice does not produce the eosinophilia seen in CCR2-deficient mice following C. neoformans infection.



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  		FIGURE 3. Effects of CCL2 neutralization ({alpha}-CCL2) on eosinophil recruitment, lung leukocyte production of IL-5, and BALF eotaxin levels. a, Samples of individual lung leukocyte suspensions from day 14 infected mice (described in Materials and Methods) were cytospun onto slides and stained with Wright-Giemsa stain for visual quantification of eosinophils. b, Total lung leukocytes were isolated from individual lungs at day 14 postinfection and cultured for 24 h without any additional stimulus. Culture supernatants were then harvested and analyzed for IL-5 production by ELISA. c, BALF samples were obtained at day 14 postinfection and analyzed for eotaxin by ELISA. The dotted lines represent IL-5 or eotaxin levels in a parallel cohort of uninfected mice (the values for uninfected wild-type and uninfected CCR2-/- mice were not significantly different). {dagger}, p < 0.05 for CCR2-/- vs all other groups; *, p < 0.05 in comparison with CCR2+/+; n = 10 for each group; values are means ± SEM.

 
Lung leukocyte IL-5 production was determined based on previous findings demonstrating that this cytokine is required for pulmonary eosinophilia during C. neoformans infection (28). Leukocytes were isolated from infected lungs at 2 wk postinfection and cultured for 24 h with no additional stimulus. Culture supernatants were harvested and assayed for IL-5 production by ELISA. Fig. 3Go shows that production of IL-5 by leukocytes from control-infected wild-type mice was similar to that for uninfected mouse leukocytes. CCL2 neutralization in wild-type mice resulted in a 3-fold increase in IL-5 production to a level similar to that of leukocytes from infected CCR2-deficient mice. These results show that either CCL2 neutralization or CCR2 deletion results in equivalent increases in production of IL-5 by lung leukocytes.

Because CCL2 neutralization in wild-type mice fails to produce eosinophilia despite an increase in pulmonary IL-5, pulmonary eotaxin levels were assayed to determine the role of this chemokine in eosinophil recruitment. Eotaxin is an eosinophil-selective chemoattractant whose actions are potentiated by IL-5 (29). At 2 wk postinfection, CCR2-/- mice showed significantly elevated bronchoalveolar lavage fluid (BALF) levels of eotaxin compared with infected wild-type mice (Fig. 3Go), whereas CCL2 neutralization in wild-type mice did not result in increased pulmonary eotaxin levels. These results demonstrate that the development of eosinophilia in Cryptococcus-infected CCR2-/- mice correlates with both elevated eotaxin levels and increased IL-5 production. In contrast, CCL2 neutralization in wild-type mice results in increased IL-5 production without an increase in eotaxin or eosinophil recruitment.

Effects of CCL2 neutralization on pulmonary IL-4 and IFN{gamma} production

The effects of CCL2 neutralization on production of T1- and T2-type cytokines were determined. Lung leukocytes were isolated from C. neoformans-infected mice, cultured without additional stimulus, and assayed for IL-4 and IFN-{gamma} as described for IL-5 (Fig. 3Go). Production of IL-4 by lung leukocytes from CCR2-/- mice and CCL2-neutralized mice was significantly greater than for leukocytes from CCR2+/+ mice (Fig. 4Go). In contrast, IFN-{gamma} production by lung leukocytes from CCR2-/- mice and CCL2-neutralized mice was significantly reduced compared with leukocytes from CCR2+/+ mice. Although IFN-{gamma} production by cultured lung leukocytes from CCL2-neutralized mice was greater than that from CCR2-/- mice, both CCL2 neutralization and CCR2 deletion decreased BALF IFN-{gamma} levels by >90% compared with CCR2+/+ mice (Fig. 4Go). These data, together with the IL-5 data presented in Fig. 3Go, demonstrate that both CCL2 neutralization and CCR2 deficiency result in a switch from T1- to T2-type cytokine production within the lungs.



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  		FIGURE 4. Effects of CCL2 neutralization ({alpha}-CCL2) on C. neoformans-induced production of IL-4 and IFN-{gamma} in the lungs of wild-type mice. Total lung leukocytes were isolated from individual lungs at day 14 postinfection and cultured for 24 h without any additional stimulus. Culture supernatants were then harvested and analyzed for a, IL-4; or b, IFN-{gamma} production by ELISA. c, BALF samples were collected at 14 days postinfection and analyzed for IFN-{gamma} by ELISA. The dotted lines represent IL-4 or IFN-{gamma} in a parallel cohort of uninfected mice (the values for uninfected wild-type and uninfected CCR2-/- mice were not significantly different). *, p < 0.05 in comparison with CCR2+/+; {dagger}, p < 0.05 for CCR2-/- vs all other groups; n = 10 for each group; values are means ± SEM.

 
Comparison of CCL2 neutralization and CCR2 deletion on LALN expansion

The effect of CCL2 neutralization on LALN expansion was determined by measuring total node cell numbers and by determining the B:T cell ratio (Fig. 5Go). Analysis of LALN from uninfected mice was not possible because mice maintained under specific pathogen-free conditions in microisolator cages do not have macroscopically visible LALN before infection. Therefore, LALN expand solely in response to inoculation with C. neoformans. By 2 wk postinfection, LALN had expanded dramatically and cell suspensions could be made from the harvested nodes (Fig. 5Go). The total cell yield for LALN isolated from control-infected wild-type mice was 24.0 million/mouse. CCL2 neutralization did not significantly reduce total LALN cell numbers (20.0 million/mouse). However, there were significantly fewer cells in the LALN of CCR2-/- mice compared with LALN of CCR2+/+ mice (14.6 million/mouse, p < 0.05). Thus, CCR2 expression is required for maximal expansion of LALN following pulmonary C. neoformans infection.



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  		FIGURE 5. Comparison of the T and B cell composition of LALN in CCR2+/+ mice, anti-CCL2-treated CCR2+/+ mice, and CCR2-/- mice. Cell suspensions were made from draining lymph nodes harvested from mice at 2 wk postinfection (described in Materials and Methods). Samples of LALN cell suspensions were stained with fluorochrome-labeled Abs specific for lymphocyte subsets (CD4+, CD8+, and B220+) and analyzed by flow cytometry (described in Materials and Methods). Total lymph node cells and lymphocyte subsets were determined on a per mouse basis. Positive staining cells accounted for >90% of the cells in suspension for all groups. *, p < 0.05 in comparison with corresponding CCR2+/+ group; {dagger}, p < 0.05 for CCR2-/- vs both corresponding CCR2+/+ and Ab-treated CCR2+/+ groups; n = 10 for each group; values are means ± SEM.

 
Flow cytometry was used to determine the numbers of CD4+, CD8+, and B220+ cells within the LALN (Fig. 5Go). There were no significant differences in LALN B cell numbers between the three groups. The CD4+ and CD8+ T cell numbers in the LALN of CCR2-/- mice were less than in the LALN of either CCR2+/+ mice or anti-CCL2-treated CCR2+/+ mice. The reduction in T cells resulted in a greater LALN B:T cell ratio for CCR2-/- mice (1.82) when compared with B:T ratios of CCR2+/+ mice (1.36) or anti-CCL2-treated CCR2+/+ mice (1.33). These results demonstrate that deficient CCR2 expression also results in altered lymphocyte expansion within the LALN compared with wild-type and anti-CCL2-treated mice.

Comparison of CCL2 neutralization and CCR2 deletion on APC numbers and function in the LALN

We also analyzed changes in the expression of MHCII, CD11b, and CD11c on LALN leukocytes to determine whether there was a dichotomy in the roles of CCR2 and CCL2 on the recruitment of potential APCs into the LALN. At 2 wk postinfection, the LALN of CCR2+/+ mice contained readily detectable numbers of MHCII+CD11b+ and MHCII+CD11c+ cells (Fig. 6Go). Neutralization of CCL2 did not alter the percentage or total number of both of these types of cells (Figs. 5Go and 6Go). In contrast, the percentage and total number of both MHCII+CD11b+ and MHCII+CD11c+ cells in the LALN was lower in CCR2-/- mice compared with CCR2+/+ mice (Fig. 6Go). LALN cells from CCR2-/- mice also produced significantly less IL-12p70 and TNF-{alpha} following stimulation with heat-killed cryptococci or C. albicans than LALN cells from CCR2+/+ mice or CCL2-neutralized CCR2+/+ mice (Fig. 7Go). The lack of IL-12 and TNF-{alpha} production is consistent with a defect in the number of potential T1-promoting APCs in the LALN of CCR2-/- mice. Altogether, these results indicate that CCR2, but not CCL2, plays a role in the recruitment of potential T1-promoting APCs to the draining lymph nodes.



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  		FIGURE 6. Comparison of APC populations of LALN in CCR2+/+ mice, anti-CCL2-treated CCR2+/+ mice, and CCR2-/- mice at day 7 postinfection. Cell suspensions were made from draining lymph nodes harvested from mice at week 1 postinfection. Samples of LALN cell suspensions were stained with PE-labeled anti-I-Ad/I-Ed (2G9) and either FITC-labeled anti-CD11b (M1/70) or FITC-labeled anti-CD11c (HL3) and analyzed by flow cytometry. Lymph nodes from five animals/group were pooled for analysis. Plot shown is representative of two experiments.

 


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  		FIGURE 7. IL-12p70 and TNF-{alpha} production by LALN cells from C. neoformans-infected CCR2+/+ mice, anti-CCL2-treated CCR2+/+ mice, and CCR2-/- mice. Cell suspensions made from LALN of infected mice (week 1 postinfection) were cultured for 24 h with no additional stimulus, HKC, or with heat-killed C. albicans. Culture supernatants were then harvested and analyzed for a, IL-12 p70; and b, TNF-{alpha} by ELISA. *, p < 0.05 in comparison with corresponding no stimulus group; n = 2 experiments for each group (nodes from five animals were pooled for each experiment); values are the mean + SE.

 
Comparison of CCL2 neutralization and CCR2 deletion on T cell polarization within the LALN

Cytokine production by LALN T cells was determined to further assess the roles of CCL2 and CCR2 on T1/T2 polarization. Isolated LALN cells from the three groups of mice were cultured 24 h with either no additional stimulus, C. neoformans Ag, or suboptimal concentrations of PMA/ionomycin to stimulate recently activated T cells. Because complement depletion studies have demonstrated that CD4+ and CD8+ T cells are the predominate cell type responsible for cytokine production in our culture system (data not shown), cytokine production was normalized to the number of T cells in culture to eliminate any biasing which may occur due to the reduced number of T cells in cultures of LALN from CCR2-/- mice. Without stimulation, LALN cells from any of the three groups of mice did not produce appreciable amounts of IFN-{gamma} or IL-5 (Fig. 8Go). LALN T cells from CCR2+/+ and anti-CCL2-treated CCR2+/+ mice produced significant levels of IFN-{gamma} in response to either C. neoformans Ag or low-dose PMA/ionomycin. In contrast, LALN T cells from infected CCR2-/- mice failed to produce significant amounts of IFN-{gamma} following stimulation with either C. neoformans Ag or low-dose PMA/ionomycin. The LALN of all groups produced some IL-5 in response to low-dose PMA/ionomycin; however, node cells from CCR2-/- mice tended to produce more of this cytokine (Fig. 8Go). LALN cell production of IL-4 could not be detected for any of the groups of mice (data not shown). These results demonstrate that expression of CCR2 is required for the development of Ag-specific IFN-{gamma}-producing T cells in LALN. In contrast, CCL2/MCP-1 is not required for the development of Ag-specific IFN-{gamma}-producing T cells in the draining lymph nodes.



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  		FIGURE 8. IFN-{gamma} and IL-5 production by LALN cells from C. neoformans-infected CCR2+/+ mice, anti-CCL2-treated CCR2+/+ mice, and CCR2-/- mice. Cell suspensions made from LALN of infected mice were cultured for 24 h with no additional stimulus, HKC, or PMA/ionomycin (both at 50 ng/ml). Culture supernatants were then harvested and analyzed for, IFN-{gamma} (a) and IL-5 (b) by ELISA. Cytokine production was normalized to 106 cultured T cells (CD4+ and CD8+) as determined by flow cytometry. *, p < 0.05 in comparison with corresponding no stimulus group; n = 3 experiments for each group (nodes from two or four animals were pooled for each experiment); values are means ± SEM.

 
Effects of CCL2 neutralization on induction of T2-type cytokines in CCR2-/- mice

In our initial study of pulmonary C. neoformans infection in CCR2-deficient mice, one model proposed was that CCL2 may interact with a receptor other than CCR2 to promote T2-type immunity in these mice (16). This hypothesis was tested by neutralizing CCL2 during the first 2 wk of C. neoformans infection in CCR2-/- mice. CCL2 neutralization had no effect on lymphocyte recruitment compared with control infected CCR2-/- mice (Fig. 9Goa). Cytokine production by isolated pulmonary leukocytes was also not affected by in vivo neutralization of CCL2 (Fig. 9Gob). Furthermore, neither LALN development nor cytokine production by LALN cells from infected CCR2-/- mice was affected by neutralization of CCL2 (data not shown). These results demonstrate that the induction of T2-type cytokine-producing lung leukocytes in infected CCR2-/- mice is not dependent on CCL2.



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  		FIGURE 9. Effects of CCL2 neutralization ({alpha}-CCL2) on immune response development in mice lacking CCR2. CCR2-/- mice were infected with C. neoformans, treated with CCL2 Ab, and harvested at day 14 as described in Figs. 1–4GoGoGoGo for neutralization of CCL2 in wild-type mice. a, Effects of CCL2 neutralization on lymphocyte recruitment in infected CCR2-/- mice. b, Effects of in vivo neutralization of CCL2 on cytokine production by isolated lung leukocytes. n = 10 for each group; values are means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objective of this study was to determine whether CCL2 is the primary ligand involved in CCR2-dependent development of T1-type immunity. Using a pulmonary C. neoformans infection model, neutralization of CCL2 or deletion of CCR2 result in comparable macrophage and T cell recruitment deficits as well as a similar switch from T1- to T2-type cytokine production within the infected lung. However, CCL2 neutralization does not result in pulmonary eosinophilia nor does it produce an IFN-{gamma} defect within the draining lymph nodes, as does CCR2 deletion. These results suggest that the efferent phase of cell-mediated immunity (mononuclear cell recruitment) uses the CCL2/CCR2 signaling axis, while the afferent phase (T1 polarization) involves a CCL2-independent, CCR2 signaling pathway.

The results presented in this study demonstrate that the CCR2/CCL2 signaling pathway is required for the efferent phase recruitment of macrophages and T cells during infection. Neutralization of CCL2 in wild-type mice produced decreases in macrophage and T cell recruitment comparable to that observed in CCR2-deficient mice (Figs. 1Go and 2Go). These results are also consistent with our previous report that CCL2 neutralization after the development of immunity (post day 5) dramatically reduces macrophage and T cell recruitment during a pulmonary C. neoformans infection (24). This is consistent with the fact that both monocytes and activated T cells are known to express CCR2 (30) and CCL2 is chemotactic for these cells (31, 32). In addition, both CCL2- (2) and CCR2-deficient mice have monocyte recruitment defects (13, 14, 25). Thus, macrophage and T cell recruitment is largely dependent on the CCR2/CCL2 signaling pathway during the efferent phase of the immune response to pulmonary C. neoformans infection.

In contrast to the equivalent effects on mononuclear cell recruitment, CCL2 neutralization does not produce the increased eosinophilia seen following deletion of CCR2 (Fig. 3Go). A similar discrepancy has been observed for CCL2 and CCR2 in producing eosinophilia during allergic pulmonary inflammation in mice. CCL2 neutralization has been shown to abolish eosinophilia associated with the pulmonary allergic response (7), while CCR2 deletion does not diminish (33) or even increases eosinophilia (19, 34). Increased eosinophilia following CCL2 neutralization in C. neoformans infection does not occur despite the fact that lung leukocytes isolated from these mice produce high levels of IL-5 (Fig. 3Go). Eosinophilia in C. neoformans-infected mice (16, 27) is a T2-driven process that is dependent on the production of IL-5 (28). The finding that CCR2 deficiency, but not CCL2 neutralization, up-regulated eotaxin levels in the airways (Fig. 3Go) suggests the reduced eosinophilia in CCL2-neutalized mice results from a trafficking deficit and not to a lack of a pro-eosinophilic environment provided by IL-5 in the lungs of infected mice.

Compared with CCL2 neutralization, CCR2 deletion produced dramatically differing effects within the lymph nodes of infected mice. In contrast to LALN cells from either wild-type or CCL2-neutralized wild-type mice, LALN cells isolated from infected CCR2-deficient mice failed to produce IFN-{gamma} in response to specific Ag stimulation or in response to concentrations of PMA and ionomycin that induce cytokine production only from recently activated T cells (Fig. 6Go). Consistent with the ELISA data, there also appear to be fewer IFN-{gamma}-producing CD4+ and CD8+ T cells in the lungs of CCR2-/- compared with CCR2+/+ mice (measured by intracellular flow cytometry, data not shown). These findings are similar to previous studies using CCR2-deficient mice which demonstrated a defect in Ag-specific IFN-{gamma} production within the draining lymph nodes following either challenge with purified protein derivative of Mycobacterium bovis (13), Leishmania major infection (15), Mycobacterium tuberculosis (18), or immunization with keyhole limpet hemocyanin (17). The combination of the Ag-specific IFN-{gamma} defect and increased IL-5 production suggest that, unlike either wild-type or CCL2-neutralized wild-type mice, LALN development in C. neoformans-infected CCR2-/- mice is characterized by the lack of T1 polarization.

Our studies demonstrate that APC numbers and function are deficient in the LALN of CCR2-/- mice during C. neoformans infection ( Figs. 5–7GoGoGo). These results are consistent with previous reports of potentially altered APC trafficking in CCR2-deficient mice (15, 17). Different populations of dendritic cells (35, 36, 37, 38) and macrophages (39) have been reported to exist which can polarize T cell differentiation to either T1 or T2. Therefore, if APCs promoting T1 differentiation express CCR2, the loss of CCR2 may prevent T1 polarization because T1-APC do not appear in the draining lymph nodes (a recent report suggests that circulating dendritic cell precursors express CCR2; Ref. 40). Because neutralization of CCL2 does not prevent Ag-specific T1 differentiation in the LALN (Fig. 8Go), other CCR2 ligands, such as CCL7 (MCP-3) and CCL12 (MCP-5), likely provide the primary signaling for T1-promoting APC trafficking to the LALN. Therefore, the lack of Ag-specific IFN-{gamma} production in the LALN of infected CCR2-/- mice appears to be due to a defect in CCR2-mediated T1-promoting APC movement from the lungs to the LALN via a ligand other than CCL2.

Both CCR2-/- mice and CCL2-neutralized mice manifest a T2 response in the lungs, but neither show evidence of a T2 response in the LALN. Where does T2 polarization occur during the infection? Recent studies from Gajewska et al. (41), using an airway OVA model to study T2 polarization, indicate that the spleen can be a site of T2 polarization to Ag encountered in the respiratory tract. In other studies, OVA instilled in the airways could be detected on APCs in the spleen (42). Thus, one possibility is that T2 cells develop in the spleens of CCR2-/- mice and migrate to the lungs. A second possibility is that nonpolarized T cells migrate from the LALN to the lung and polarization of T2 cells occurs in situ. The expansion of the LALN in CCR2-deficient mice occurred without detectable T cell polarization (Fig. 8Go). This suggests that the T cell expansion is driven by migration of non-T1 promoting APCs or the capture of free Ag draining from the lung by resident LALN APC. The pulmonary environment appears to preferentially favor T2 differentiation in the absence of strong T1-type signals (43, 44). If CCL2 is neutralized, our data indicate that T1-promoting APCs are still capable of migrating to the LALN but trafficking of T1 cells from the LALN to the lungs is blocked. The end result is migration of T2 cells from another site (spleen?) or in situ T2 polarization of nonpolarized T cells in the lungs.

The studies presented here, along with those from other laboratories, demonstrate that CCL2/MCP-1 can promote both T1 and T2 responses. CCL2 is associated with both T1 and T2 polarization (2, 4, 6, 10, 11, 45, 46, 47, 48), and the neutralization of CCL2 can eliminate the manifestation of both T1 and T2 responses (7, 24, 49). Our studies suggest that the T1 or T2 promoting activity of CCL2 in vivo depends on additional factors that may include the timing of CCL2 induction (afferent vs efferent), type of pathogen/Ag, route of inoculation/immunization, and the tissue site. For example, CCL2 is a pro-T1 factor if pulmonary cytokine responses are measured (Figs. 3Go and 4Go), but plays no role in polarization if lymph node responses alone are assayed (Fig. 6Go). The data in this manuscript also suggest a mechanism to explain the apparent dichotomy between the role of CCL2 and its receptor CCR2 in models of T1/T2 immunity. Thus, the pleiotropic activity of CCL2 (chemotaxin, T1, T2, activation, etc.) is influenced by the inflammatory milieu, a function of the type and tissue location of an infectious agent. The pleiotropic activity of mediators such as CCL2 is critical for effective adaptive host defenses.


   Acknowledgments
 
We thank Michal A. Olszewski and Deirdra D. Williams for their technical assistance and insights.


   Footnotes
 
1 This work was supported by the following grants: National Institutes of Health Grants R01-HL65912 (to G.B.H.), R01-HL63670 (to G.B.H.), R01-HL51082 (to G.B.T.), and T32-HL07749 (to T.R.T.); Veterans Administration Merit Review Grant (to G.B.T.); Burroughs-Wellcome Fund (to G.B.H.); and an Institute for Cellular and Molecular Biology Research Grant (to W.A.K.). Additional support for flow cytometry came from the University of Michigan Comprehensive Cancer Center National Institutes of Health CA46592, the University of Michigan Multipurpose Arthritic Center National Institutes of Health AR20557, and the University of Michigan Biomedical Research Core Facilities Core Flow Cytometry Facility. T.R.T. is a Parker B. Francis Fellow in Pulmonary Research. Back

2 Current address: Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University College of Veterinary Medicine, Pullman, WA 99164. Back

3 Address correspondence and reprint requests to Dr. Gary B. Huffnagle, Pulmonary Division, Department of Internal Medicine, University of Michigan Medical Center, 6301 Medical Science Research Building III Box 0642, Ann Arbor, MI 48109-0642. E-mail address: ghuff{at}umich.edu Back

4 Abbreviations used in this paper: CCL2, CC chemokine ligand 2; BALF, bronchoalveolar lavage fluid; LALN, lung-associated lymph node; MCP-1, monocyte chemotactic protein-1; HKC, heat-killed C. neoformans. Back

Received for publication May 17, 2001. Accepted for publication March 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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