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* Pulmonary Division, Departments of Internal Medicine and
Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109;
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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--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-
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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- 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-, IL-12, and IFN-
(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-, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- (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-, 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- and
IL-5 production by ELISA. To measure TNF- 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). 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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). Similarly, total
lymphocyte recruitment was reduced by 67 and 70% in CCL2-neutralized
and CCR2-deficient mice, respectively. Fig. 2 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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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. 3). 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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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. 3), 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
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- as described for IL-5 (Fig. 3). Production of IL-4 by lung leukocytes from
CCR2-/- mice and CCL2-neutralized mice was
significantly greater than for leukocytes from
CCR2+/+ mice (Fig. 4). In contrast, IFN- production by
lung leukocytes from CCR2-/- mice and
CCL2-neutralized mice was significantly reduced compared with
leukocytes from CCR2+/+ mice. Although IFN-
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- levels by
>90% compared with CCR2+/+ mice (Fig. 4). These
data, together with the IL-5 data presented in Fig. 3, 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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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. 5). 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. 5). 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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). 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. 6). Neutralization of CCL2 did not alter
the percentage or total number of both of these types of cells (Figs. 5 and 6). 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. 6). LALN cells from
CCR2-/- mice also produced significantly less
IL-12p70 and TNF- following stimulation with heat-killed
cryptococci or C. albicans than LALN cells from
CCR2+/+ mice or CCL2-neutralized
CCR2+/+ mice (Fig. 7). The lack of IL-12 and TNF-
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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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- or IL-5 (Fig. 8). LALN
T cells from CCR2+/+ and anti-CCL2-treated
CCR2+/+ mice produced significant levels of
IFN- 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- 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. 8). 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--producing T cells in LALN. In contrast, CCL2/MCP-1
is not required for the development of Ag-specific IFN--producing T
cells in the draining lymph nodes.
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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. 9a). Cytokine production by
isolated pulmonary leukocytes was also not affected by in vivo
neutralization of CCL2 (Fig. 9b). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1 and 2).
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. 3). 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. 3). 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. 3) 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- 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. 6). Consistent with the
ELISA data, there also appear to be fewer IFN--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- 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- 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. 5–7). 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. 8),
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- 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. 8). 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. 3 and 4), but plays no role in polarization if lymph node responses alone are
assayed (Fig. 6). 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.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University College of Veterinary Medicine, Pullman, WA 99164.
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
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.
Received for publication May 17, 2001. Accepted for publication March 4, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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