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Production in C-C Chemokine Receptor 2 (CCR2) Knockout Mice: Role of CCR2 in Linking the Innate and Adaptive Immune Responses1
,§
*
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94141; and
Cardiovascular Research Institute,
Department of Anatomy, and
§
Department of Medicine, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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after challenge
with purified protein derivative of Mycobacterium bovis.
In this study, we have investigated the mechanism of this cytokine
production defect. In vitro, direct activation of splenocytes with
CD3/CD28 Abs failed to reveal any differences in IFN- production
between CCR2+/+ and
CCR2-/- mice. However, after immunization,
the number of Ag-specific, IFN--producing cells in the draining
lymph nodes was decreased by 70% in the
CCR2-/- mice, suggesting an in vivo
trafficking defect. Direct measurement of cell trafficking with
fluorescently labeled CFA revealed a marked decrease in the number of
monocytes/macrophages migrating to the site of immunization and to the
draining lymph nodes in the CCR2-/- mice.
The data suggest that impaired trafficking of APCs in the
CCR2-/- mice contributes to the defect in
IFN- production. These data support the idea that CCR2-positive
monocytes/macrophages are critical in linking the innate and adaptive
immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the draining lymph nodes (DLNs) of
CCR2-/- mice (14). Decreased
production of this cytokine in CCR2-/-
mice infected with Leishmania donovani (22) or
Cryptococcus neoformans (23) has subsequently
been reported, suggesting that CCR2-dependent production of IFN- is
critical for host defense.
Because of the importance of IFN- in the immune response and disease
pathogenesis, we have investigated the basis of the reduced production
of this cytokine in the CCR2-/- mice. We
considered two possible mechanisms: first, that signaling by CCR2 acted
in concert with activation of the TCR to optimize cytokine production;
second, that abnormal trafficking of the T cells or APCs prevented the
production of activated T cells in
CCR2-/- mice. In this study, we show that
the diminished IFN- production by the
CCR2-/- mice is primarily due to a defect
in the trafficking of monocytes/macrophages to the site of
immunization; as a result, fewer Ag-laden APCs are present in the DLNs
of the CCR2-/- mice. These studies reveal
a novel role for CCR2-positive monocytes/macrophages as an important
link between the innate and adaptive immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemical reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated. Tissue culture reagents were from Life Technologies/BRL (Gaithersburg, MD). Cytokines and cytokine Abs were from R&D Systems (Minneapolis, MN).
Mice
CCR2-/- mice were generated as described (14) and were backcrossed nine times with C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). Littermate CCR2+/+ and CCR2-/- mice were then bred to generate the mice used in the experiments. All mice were maintained in specific pathogen-free conditions and were used between 6 and 12 wk of age.
Immunizations
For Ag-specific cytokine analysis, mice were immunized s.c. at
the base of the tail with 50 µl of keyhole limpet hemocyanin (KLH;
100 µg), emulsified with an equal volume of CFA. Inguinal DLNs were
removed 4 days after immunization. For studies of cell trafficking,
mice were immunized in the quadriceps with 25 µl of KLH (50 µg)
emulsified with an equal volume of CFA that had been fluorescently
labeled with a lipophilic tracer
chloromethylbenzamido-3,3'-dioctadecylindocarbocyanine (DiI) (1 mg/5 ml
CFA) (CellTracker; Molecular Probes, Eugene, OR) that is miscible in
adjuvant, thereby rendering the adjuvant fluorescent. The epimysium
covering the quadriceps and the inguinal DLNs were removed 48 h
after immunization. To investigate whether the Ag and adjuvant were
colocalized within the same cells, we emulsified OVA-FITC with CFA-DiI
and immunized in the muscle. The cells were isolated as described
below and analyzed by flow cytometery. After immunization with
OVA-FITC and CFA (without DiI), 
90% of the cells were positive for
FITC. After immunization with CFA-DiI and OVA (without FITC
conjugation), 90% of the cells were positive for DiI. After
immunization with CFA-DiI and OVA-FITC emulsified together, less than
7% of the cells were positive for FITC and negative for DiI. Thus, the
vast majority of cells isolated had taken up both adjuvant and
Ag.
Cell culture and activation
Single-cell suspensions were prepared for Ag-specific cytokine analysis. Inguinal DLN cells (2.5 x 106/ml) were cultured in complete medium consisting of RPMI 1640 supplemented with FCS (10%, v/v), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (10 mM), nonessential amino acids (100 µM), sodium pyruvate (1 mM), and 2-ME (50 µM). The cells were restimulated in vitro with KLH (100 µg/ml), and supernatants were collected 48 h later and stored at -80°C until assayed. For nonspecific cell stimulation, spleens from naive mice were removed, and single-cell suspensions were prepared. Red cells were removed by hypertonic lysis at room temperature. Splenocytes (2.5 x 106 cells/ml) were cultured in complete medium and stimulated in vitro with anti-CD3 (10 µg/ml; PharMingen, San Diego, CA) and anti-CD28 (10 µg/ml; PharMingen), or with Con A (5 µg/ml; Sigma). Supernatants were collected 48 h later and stored at -80°C until assayed.
APC isolation
Mononuclear cells were isolated from the epimysium of the
quadriceps and from the inguinal DLNs by enzymatic digestion with 1
mg/ml collagenase D (Boehringer Mannheim, Indianapolis, IN) in RPMI
1640 containing 2% FCS for 30 min at 37°C. After homogenization in a
Dounce homogenizer, the tissue digest was centrifuged for 5 min at
900 x g, resuspended in 2 ml HBSS, and layered onto a
metrizamide gradient (1.077 g/cm3; Accurate
Chemical and Scientific, Westbury, NY). Mononuclear cells were isolated
by collecting cells in the light density fraction after centrifugation
for 20 min at 900 x g and counted with a
hemocytometer. Anti-CD11b FITC (0.25 µg/ml; PharMingen) was added to
stain macrophages, and anti-CD11c FITC (0.25 µg/ml; PharMingen)
was added to stain DCs. A FACScan (Becton Dickinson, Mountain View,
CA) was used to quantitate cells that had taken up the Ag. After flow
cytometry analysis, the CCR2+/+ and
CCR2-/- CD11b-positive cells were both found to
phagocytose the labeled adjuvant and Ag mixture equally well with
90% of both being positive for DiI.
ELISA cytokine assays
Supernatants from stimulated cells were assayed in sandwich
ELISAs, as previously described (14), except that primary
and secondary Abs were from R&D Systems. All Abs were used at the
dilutions recommended by the manufacturer. Enzyme-linked immunospot
(ELISPOT) assays for determination of IFN- production by individual
cells were performed as previously described (24).
Briefly, 96-well Immulon IV plates (Dynatech Laboratories, Chantilly,
VA) were coated overnight by incubation at 4°C with the IFN-
capture Ab. The following day, the plates were washed with PBS, and
cells were added to the first well of a row in duplicate (2 x
106 cells). Serial dilutions were conducted
across the plate, and cells were cultured overnight in complete medium
plus KLH (100 µg/ml). The next day, the cells were washed away, and
biotinylated anti-IFN- secondary Ab was added for 1 h at
room temperature. The plate was washed, and streptavidin-alkaline
phosphatase (Boehringer Mannheim) was added for 30 min at room
temperature. The plate was washed again, and the substrate,
5-bromo-4-chloro-3-indolyl phosphate (1 mg/ml; Sigma), was dissolved in
2-amino-2-methyl-1-propanol buffer (0.1 M; Sigma) mixed with 0.6%
low-melt agarose (SeaPlaque; FMC Bioproducts, Rockland, ME). After
solidification, the blue spots (each spot corresponding to an
IFN--producing cell) were counted with an inverted microscope.
DC differentiation from bone marrow
DCs were obtained by differentiation of bone marrow precursor cells, as previously described (25), to determine whether the absence of CCR2 affected their activation efficiency. Bone marrow plugs were isolated from the femur and tibia of the hind legs, and red cells were lysed. Cells (1.2 x 107) were cultured in 25 ml of complete medium plus GM-CSF (20 ng/ml; R&D Systems). After 48 h, an additional 12.5 ml of complete medium containing GM-CSF (40 ng/ml) was added. On day 5, 19 ml of medium was removed, and nonadherent cells were collected by centrifugation. The pelleted cells were resuspended in 19 ml of fresh medium plus GM-CSF (20 ng/ml) and added back to the culture dish. On day 8, the nonadherent cells were collected and replated in fresh medium plus GM-CSF (10 ng/ml; 2 x 105 cells/ml). On day 9, the DCs were stimulated with LPS (0111:B4; Sigma; 1 µg/ml). Supernatants were removed 24 h later and stored at -80°C until assaying for IL-12 by ELISA. The cells were also labeled with CD11c-PE (0.25 µg/ml) and MHC II-CYC (0.25 µg/ml; PharMingen) to examine DC purity by flow cytometry.
Statistical analysis
All statistical analyses were performed with the Mann-Whitney test. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by
CCR2-/- mice after immunization in a Th1
manner
We previously found that CCR2-/-
mice challenged with PPD of M. bovis in CFA produced
markedly less IFN- than CCR2+/+ mice
(14). To further investigate this finding, we immunized
CCR2-/- and
CCR2+/+ mice with KLH emulsified in CFA,
which also produces a Th1 response. Four days after immunization, cells
from the DLNs were restimulated in vitro with KLH for 48 h, and
supernatants were assayed for IFN-. As seen in Fig. 1
, and consistent with the previous data
obtained by challenging with PPD, the KLH/CFA immunization resulted in
the production of 70–90% less IFN- by the
CCR2-/- mice than by the wild-type mice.
In contrast, production of IL-5, IL-10, or IL-13 was not reduced in the
CCR2-/- mice (Fig. 1). IL-12 and IL-4
were undetectable in both CCR2 wild-type and
CCR2-/- mice (data not shown). Ab isotype
switching was also investigated, but no differences in IgG1 (induced by
IL-4) or IgG2a (induced by IFN-) (26, 27, 28) were found
(data not shown).
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To determine whether expression of CCR2 on T cells is required for
normal production of IFN-, we harvested naive splenocytes from
CCR2-/- and
CCR2+/+ mice and activated them with a
combination of anti-CD3 (10 µg/ml) and anti-CD28 (10
µg/ml). No significant differences in the amounts of IFN- produced
were found between the CCR2-/- and
CCR2+/+ mice (Fig. 2A). Because the anti-CD3
and anti-CD28 were used at saturating concentrations, additional
experiments were performed in which the concentration of anti-CD3
was varied between 2.5 and 0.00125 µg/ml, while anti-CD28 was
kept constant at 0.1 µg/ml. Once again, at all anti-CD3
concentrations examined, we failed to detect a difference in the amount
of IFN- produced by CCR2-/- and
CCR2+/+ (data not shown). Splenocyte
proliferation during this assay was also quantitated, and no
significant differences were found between the
CCR2-/- and wild-type mice (data not
shown). In contrast, when splenocytes from naive mice were activated by
Con A, significantly lower levels of IFN- were produced by the
CCR2-/- cells (Fig. 2B). These
results suggest that, at least in vitro, signaling through CCR2 is not
essential for T cells to produce optimal levels of IFN- in response
to direct activation by anti-CD3/anti-CD28, but that optimal
activation by Con A does depend upon the presence of CCR2.
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Activation of T cells to produce a Th1 response requires the
presence of the APC cytokine IL-12. To determine whether IL-12 function
was dependent upon the presence of CCR2, DCs were derived by culturing
bone marrow precursor cells of naive
CCR2-/- and
CCR2+/+ mice. After differentiation, the
DCs were activated in vitro with LPS to stimulate cytokine production.
Flow cytometry after LPS stimulation demonstrated that the cells were
90% positive for CD11c and 90% positive for MHC II (data not shown).
No differences in the production of IL-12 were found between the
CCR2-/- and
CCR2+/+ mice (Fig. 3). Thus, as was the case for T cells,
activation of CCR2 was not required for cytokine production by
DCs.
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-producing T cells in the DLNs
We next sought to determine whether the decrease in IFN-
production represented less cytokine being produced by each
CCR2-/- T cell in the DLNs, or
alternatively, whether fewer Ag-specific IFN--producing T cells were
present in the DLNs of the CCR2-/- mice.
The ELISPOT assay was used to determine the number of Ag-specific
IFN--producing cells in the DLNs, after immunization with KLH/CFA.
As shown in Fig. 4, there were
significantly fewer Ag-specific IFN--producing cells in the DLNs of
CCR2-/- mice than in those of
CCR2+/+ littermate controls. However, we
found no differences in the total numbers of CD3-positive T cells in
the DLNs after immunization. These results were consistent with a
trafficking defect of either the T cell or the APC.
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Activation of T cells in vivo to produce IFN- requires
macrophages and/or DCs to effectively present Ag to the TCR. Because
fewer T cells appeared to be activated to produce IFN- by the
ELISPOT assay and because there did not appear to be a signaling defect
by the T cells lacking CCR2 (the CD3/CD28 activation experiments), we
considered the possibility that APC trafficking might be abnormal in
CCR2-/- mice. To test this hypothesis,
the number of infiltrating monocytes/macrophages and/or DCs at the site
was determined 48 h after immunization with fluorescently labeled
adjuvant. Resident and infiltrating mononuclear cells were isolated
from the quadriceps and quantified. As shown in Fig. 5A, there were significantly
fewer cells at the site of immunization in
CCR2-/- mice than in
CCR2+/+ mice. Further analysis revealed
that the vast majority of these cells were
CD11b+/CD11c-, suggesting
that they were macrophages (Fig. 5B). Peritoneal
macrophages, used as control cells for Ab specificity, yielded a
virtually identical staining pattern (data not shown). Using these same
techniques, the number of DiI-positive cells trafficking to the DLNs
after immunization was determined. Markedly fewer DiI-positive cells
appeared in the DLNs of CCR2-/- mice as
compared with CCR2+/+ mice (Fig. 6A), and the vast majority of
these cells were CD11b positive (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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than
CCR2+/+ mice in response to immunization with
PPD of M. bovis mice (14). In the current
study, we have confirmed these results using an alternative means of
immunization and have explored the mechanism for the selective decrease
in production of the Th1 cytokine IFN-. In this study, we report
that the basis for this defect involves impaired trafficking of APCs in
the CCR2-/- mice.
We initially speculated that the decreased Ag-specific production of
IFN- might be due to an inability of the
CCR2-/- lymphocytes to signal in response
to MCP-1 or other closely related MCP chemokines (14).
Such a mechanism would imply cross-talk between CCR2 and the TCR in the
production of IFN-. Reports from a number of groups support the
notion that in addition to acting as potent chemoattractants,
chemokines influence proliferation and cytokine production by
lymphocytes. In 1995, Bacon et al. (4) showed that the
chemokine RANTES caused proliferation of T cells in the absence of
exogenous Ag and up-regulated the cytokine IL-5. Further studies by
this group showed that RANTES induced activation and increased
expression of CD3 on T cells (29). In 1996, Taub et al.
(5) showed that RANTES, macrophage-inflammatory proteins
1
and 1ß, and MCP-1 acted in concert with anti-CD3 to activate
T cells, increasing proliferation. In another series of studies, Karpus
et al. (6) provided evidence that different chemokines
could differentially induce polarization of T cells into Th1 or Th2
cytokine-producing populations. They found that addition of MCP-1 into
T cell cultures induced IL-4 production, whereas addition of
macrophage-inflammatory protein 1 induced IFN- production
(6). These results were further supported by studies in
which neutralizing Abs to MCP-1 were added to cultures of
CD4+ T cells, and a decrease in IL-4 production
was seen along with a concomitant increase in IFN- production
(30). Taken together, these in vitro studies supported the
notion that signaling through CCR2 may play an important role in
IFN- production in vivo.
In the current study, we tested this hypothesis but found that
CCR2-/- T cells produced as much IFN-
as CCR2+/+ T cells after direct activation
of CD3/CD28. These results are in contrast to the recent report of Sato
et al. (22), who found that T cells derived from
CCR2-/- mice produced significantly less
IFN- than cells from CCR2+/+ mice in
response to CD3/CD28 activation. Because Sato et al. used lower
concentrations of the CD3/CD28 Abs, we repeated the experiments using a
wide range of CD3 Ab concentrations, but again failed to detect any
differences between the CCR2+/+ and
CCR2-/- mice. Nor did we find any
difference in the proliferation of CCR2+/+
and CCR2-/- splenocytes in response to
CD3/CD28. The reason for the difference between our results and those
of Sato et al. (22) is not apparent at this time.
Because in our hands the presence of CCR2 did not play a role in
IFN- production in response to direct activation of the TCR, we
turned our attention to the other major CCR2-positive cell type, the
APCs, and asked whether they were functioning abnormally in the
CCR2-/- mice. Although T cells can be
directly activated in vitro by the addition of
anti-CD3/anti-CD28, activation by Ags requires APCs to present
Ag to the TCR to provide costimulatory signals and, in the case of Th1
responses, to provide IL-12 (31). We therefore next
examined the ability of LPS-activated DCs to produce IL-12, and found
that the CCR2-/- cells produced as much
IL-12 as wild-type cells, ruling out the possibility that the Th1
defect was due to a problem with IL-12 production. We concluded from
these experiments that CCR2 was not required for cytokine production by
either T cells or APCs, if these cells were directly activated in
vitro.
The lack of detectable cytokine defects in isolated cells led us to suspect that impaired trafficking of leukocytes to the site of immunization and/or to the DLNs was the underlying abnormality in the CCR2-/- mice. Indeed, macrophage trafficking defects in the CCR2-/- mice have been well documented. Fewer macrophages enter the peritoneal cavity after thioglycolate administration (14, 15, 16), and fewer macrophages are recruited to the aortic wall (18, 21) in CCR2-/- than in CCR2+/+ mice. In addition, Kurihara et al. (15) found that CCR2-/- mice were susceptible to lethal infection with Listeria monocytogenes, which they attributed to a lack of macrophage trafficking into infected tissues. None of these studies, however, considered macrophage recruitment in the context of Ag presentation. In mice, CCR2 is expressed on both immature DCs (17) and monocytes/macrophages. We therefore hypothesized that trafficking of these cells to the site of immunization, and subsequently to the DLNs, might be impaired.
We began the analysis of cell trafficking by determining the number of
Ag-specific IFN--producing cells in the DLNs after immunization,
using the ELISPOT assay. These studies revealed that there were indeed
fewer Ag-specific IFN--producing cells in the DLNs of the
CCR2-/- mice, even though the total
number of CD3+ cells was equivalent in the
CCR2-/- and
CCR2+/+ mice. It was still not clear,
however, whether the primary defect was at the level of the T cell or
the APC, because both are required for efficient cytokine production. A
clue came from a result in our earlier studies (14), and
confirmed in this study, that splenocytes from
CCR2-/- mice produced significantly less
IFN- than cells from CCR2+/+ mice after
activation with Con A. Unlike anti-CD3/anti-CD28, optimal
activation of T cells by Con A requires the presence of APCs. It is of
interest in this regard that we found decreased Con A-induced IFN-
production in naive mice as well as in KLH/CFA-immunized
CCR2-/- mice, implying that CCR2 may play
a role in the constitutive trafficking of APCs as well as in activated
APC trafficking, even though the total number of CD11b-positive cells
is the same in the CCR2+/+ and
CCR2-/- mice (14).
Consistent with this hypothesis are the reports of constitutive MCP-5
expression in naive LNs (32) and the recent findings of Gu
et al. (33), who have noted the presence of MCP-1 in both
the spleen and lymph nodes of naive mice. It is also possible that
MCP-1 production by APCs serves to increase the efficiency of Ag
presentation by attracting CCR2-positive T cells within the LN. In this
regard, Sallusto et al. (34) have found that mature DCs
express MCP-1 and MCP-2.
Taken together, these data implicated the APC as the culprit and
suggested that a defect in monocyte/macrophage and/or DC trafficking
might be the primary cause of the IFN- production defect in
CCR2-/- mice. That equivalent levels of
IFN- were produced by CCR2+/+ and
CCR2-/- splenocytes in response to
CD3/CD28 further suggested that T cell trafficking was not the problem.
We therefore sought to directly quantitate the number of cells that
entered the site of immunization and to track them to the DLNs in
CCR2-/- and
CCR2+/+ mice. To directly visualize cells
that interacted with the Ag, we immunized mice with KLH and
fluorescently labeled CFA (35). Thus, any cell that
phagocytosed the Ag could be detected by fluorescence analysis. Using
this technique, Dupuis et al. (35) showed that DiI-labeled
APCs trafficked to the T cell areas of the DLNs, where they could
present Ags. In our studies, 70% fewer fluorescently labeled cells
were found at the site of immunization in the quadriceps in the
CCR2-/- mice than in the wild-type mice.
Analysis of these cells for expression of CD11b and CD11c revealed that
greater than 80% of the fluorescent cells were macrophages, and we
failed to detect any cells that were strongly positive for CD11c,
suggesting the absence of DCs. This result is also consistent with
recent work by Randolph et al. (36), who found that after
s.c. injection with FITC-latex beads, the majority of cells that
arrived at the site were CD11b+
monocytes/macrophages. We further found a marked reduction in the
number of fluorescent CD11b+ cells in the DLNs of
CCR2-/- mice, as compared with the
CCR2+/+ mice.
These findings suggest that impaired trafficking of the
monocytes/macrophages to the site results in fewer Ag-bearing APCs in
the DLNs and contributes to reduced production of IFN- in
CCR2-/- mice after immunization. However,
we have not ruled out a trafficking defect in the T cells themselves,
although this would have to be limited to Ag-specific T cells because
we did not detect differences in the number of total CD3-positive cells
in LNs from CCR2+/+ vs
CCR2-/- mice. Adoptive transfer
experiments will be required to address this question directly.
Our results immediately raise the question of why wild-type levels of IL-5, IL-10, and IL-13 are produced in the CCR2-/- mice. One possibility is that CCR2 may be expressed on only a subpopulation of APCs that tend to induce Th1 responses. In this regard, recent reports suggest the presence of at least two populations of DCs that can differentially polarize T cells to produce either Th1 or Th2 cytokines (37, 38). There have also been reports suggesting that distinct populations of macrophages exist. Stein et al. (39) suggested the presence of two separate populations of macrophages: APC1s that promoted inflammatory responses and APC2s that were designated noninflammatory inducers. It is possible, therefore, that CCR2 is preferentially expressed on the APC1-type macrophages that predominate at sites of inflammation. This would be consistent with the presence of MCP-1 at sites of inflammation (40, 41, 42, 43) and would extend to the APC the recent paradigm that different chemokine receptors are present on Th1 vs Th2 lymphocytes. A second possibility is that we did not detect a defect in Th2 cytokines because the genetic background of the CCR2-/- mice (C57BL/6) and the immunization protocol (KLH/CFA) favored the production of Th1 cytokines. In this regard, Warmington et al. (44) found reduced levels of both Th1 and Th2 cytokines when CCR2-/- mice were immunized in a more Th2-like manner with Schistosoma mansoni egg Ags. To provide a more rigorous test of the role of the genetic background, we are crossing the CCR2 null mice onto a pure BALB/c background, and we will then test their ability to synthesize and secrete Th2 cytokines after immunization in a Th2 manner.
Gu et al. (33) have recently reported that
MCP-1-/- mice have a defect in the production
of Th2 cytokines, but make normal levels of IFN-. The disparity in
cytokine production between the ligand (MCP-1) and receptor (CCR2)
knockouts is intriguing, and suggests several plausible explanations
for reconciling the data. First, it is possible that there are
additional functional receptors for MCP-1. Thus, Nibbs et al.
(45) have described a murine chemokine receptor, known as
D6, which binds MCPs and other chemokines, but does not signal. More
recently, Schweickart et al. (46) cloned a human chemokine
receptor (CCR11) that binds and signals to MCPs, although at relatively
high concentrations. It is not yet known if the murine form of CCR11 is
activated by murine MCPs. Second, we know that in the mouse, MCP-3 and
MCP-5 are also potent CCR2 agonists (32, 47). No
information is available on how these chemokines might influence the
balance of Th1 vs Th2 responses, and it will be of interest to examine
cytokine profiles in MCP-3- and MCP-5-deficient mice, when they become
available. Finally, there are technical differences in the experimental
protocols used to immunize the MCP-1-/- and
CCR2-/- mice. Thus, Gu et al.
(33) immunized MCP-1-/- mice in a
Th2 manner, whereas we immunized CCR2-/-
mice in a Th1 manner. Because both strains of mice are on the C57BL/6
background, a side-by-side comparison using identical immunization
protocols should resolve many of these questions.
In summary, we have found that
CCR2-/- mice have a profound and
selective defect in IFN- production when immunized in a Th1 manner
and that this defect is due, at least in part, to impaired trafficking
of monocytes/macrophages to the immunization site and APCs to the DLNs.
These studies reveal an important function for CCR2 in the modulation
of the immune response. It is interesting to speculate that the
importance of CCR2 in this regard might depend upon whether or not
MCP-1 (or other CCR2 agonists) are generated at the site of
inflammation as part of an immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Israel F. Charo, Gladstone Institute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA 94141-9100.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; DC, dendritic cell; DiI, 3,3'dioctadecylindocarbocyanine; DLN, draining lymph node; ELISPOT, enzyme-linked immunospot; KLH, keyhole limpet hemocyanin; PPD, purified protein derivative.
Received for publication March 21, 2000. Accepted for publication September 13, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. K. Henke, C. G. Pearce, D. M. Moaveni, A. J. Moore, E. M. Lynch, C. Longo, M. Varma, N. A. Dewyer, K. B. Deatrick, G. R. Upchurch Jr, et al. Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model. J. Immunol., September 1, 2006; 177(5): 3388 - 3397. [Abstract] [Full Text] [PDF]
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 P. Fiorina, M. J. Ansari, M. Jurewicz, M. Barry, V. Ricchiuti, R. N. Smith, S. Shea, T. K. Means, H. Auchincloss Jr., A. D. Luster, et al. Role of CXC Chemokine Receptor 3 Pathway in Renal Ischemic Injury J. Am. Soc. Nephrol., March 1, 2006; 17(3): 716 - 723. [Abstract] [Full Text] [PDF]
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G. P. de Lema, H. Maier, T. J. Franz, M. Escribese, S. Chilla, S. Segerer, N. Camarasa, H. Schmid, B. Banas, S. Kalaydjiev, et al. Chemokine Receptor Ccr2 Deficiency Reduces Renal Disease and Prolongs Survival in MRL/lpr Lupus-Prone Mice J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3592 - 3601. [Abstract] [Full Text] [PDF]
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 T. H. Terwey, T. D. Kim, A. A. Kochman, V. M. Hubbard, S. Lu, J. L. Zakrzewski, T. Ramirez-Montagut, J. M. Eng, S. J. Muriglan, G. Heller, et al. CCR2 is required for CD8-induced graft-versus-host disease Blood, November 1, 2005; 106(9): 3322 - 3330. [Abstract] [Full Text] [PDF]
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C. Qu, E. W. Edwards, F. Tacke, V. Angeli, J. Llodra, G. Sanchez-Schmitz, A. Garin, N. S. Haque, W. Peters, N. van Rooijen, et al. Role of CCR8 and Other Chemokine Pathways in the Migration of Monocyte-derived Dendritic Cells to Lymph Nodes J. Exp. Med., November 15, 2004; 200(10): 1231 - 1241. [Abstract] [Full Text] [PDF]
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 B.-C. Chiu, C. M. Freeman, V. R. Stolberg, J. S. Hu, K. Zeibecoglou, B. Lu, C. Gerard, I. F. Charo, S. A. Lira, and S. W. Chensue Impaired Lung Dendritic Cell Activation in CCR2 Knockout Mice Am. J. Pathol., October 1, 2004; 165(4): 1199 - 1209. [Abstract] [Full Text] [PDF]
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B.-C. Chiu, C. M. Freeman, V. R. Stolberg, J. S. Hu, E. Komuniecki, and S. W. Chensue The Innate Pulmonary Granuloma: Characterization and Demonstration of Dendritic Cell Recruitment and Function Am. J. Pathol., March 1, 2004; 164(3): 1021 - 1030. [Abstract] [Full Text] [PDF]
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H. Bruhl, J. Cihak, M. A. Schneider, J. Plachy, T. Rupp, I. Wenzel, M. Shakarami, S. Milz, J. W. Ellwart, M. Stangassinger, et al. Dual Role of CCR2 during Initiation and Progression of Collagen-Induced Arthritis: Evidence for Regulatory Activity of CCR2+ T Cells J. Immunol., January 15, 2004; 172(2): 890 - 898. [Abstract] [Full Text] [PDF]
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R. W. DePaolo, B. J. Rollins, W. Kuziel, and W. J. Karpus CC Chemokine Ligand 2 and Its Receptor Regulate Mucosal Production of IL-12 and TGF-{beta} in High Dose Oral Tolerance J. Immunol., October 1, 2003; 171(7): 3560 - 3567. [Abstract] [Full Text] [PDF]
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A. Iwasaki The Importance of CD11b+ Dendritic Cells in CD4+ T Cell Activation In Vivo: With Help from Interleukin 1 J. Exp. Med., July 21, 2003; 198(2): 185 - 190. [Full Text] [PDF]
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M. L. deSchoolmeester, M. C. Little, B. J. Rollins, and K. J. Else Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection J. Immunol., May 1, 2003; 170(9): 4693 - 4700. [Abstract] [Full Text] [PDF]
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 H. M. Scott and J. L. Flynn Mycobacterium tuberculosis in Chemokine Receptor 2-Deficient Mice: Influence of Dose on Disease Progression Infect. Immun., November 1, 2002; 70(11): 5946 - 5954. [Abstract] [Full Text] [PDF]
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N. Omata, M. Yasutomi, A. Yamada, H. Iwasaki, M. Mayumi, and Y. Ohshima Monocyte Chemoattractant Protein-1 Selectively Inhibits the Acquisition of CD40 Ligand-Dependent IL-12-Producing Capacity of Monocyte-Derived Dendritic Cells and Modulates Th1 Immune Response J. Immunol., November 1, 2002; 169(9): 4861 - 4866. [Abstract] [Full Text] [PDF]
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A. M. Krackhardt, S. Harig, M. Witzens, R. Broderick, P. Barrett, and J. G. Gribben T-cell responses against chronic lymphocytic leukemia cells: implications for immunotherapy Blood, June 17, 2002; 100(1): 167 - 173. [Abstract] [Full Text] [PDF]
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T. R. Traynor, A. C. Herring, M. E. Dorf, W. A. Kuziel, G. B. Toews, and G. B. Huffnagle Differential Roles of CC Chemokine Ligand 2/Monocyte Chemotactic Protein-1 and CCR2 in the Development of T1 Immunity J. Immunol., May 1, 2002; 168(9): 4659 - 4666. [Abstract] [Full Text] [PDF]
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R. T. Palframan, S. Jung, G. Cheng, W. Weninger, Y. Luo, M. Dorf, D. R. Littman, B. J. Rollins, H. Zweerink, A. Rot, et al. Inflammatory Chemokine Transport and Presentation in HEV: A Remote Control Mechanism for Monocyte Recruitment to Lymph Nodes in Inflamed Tissues J. Exp. Med., November 5, 2001; 194(9): 1361 - 1374. [Abstract] [Full Text] [PDF]
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B. P. Chen, W. A. Kuziel, and T. E. Lane Lack of CCR2 Results in Increased Mortality and Impaired Leukocyte Activation and Trafficking Following Infection of the Central Nervous System with a Neurotropic Coronavirus J. Immunol., October 15, 2001; 167(8): 4585 - 4592. [Abstract] [Full Text] [PDF]
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 W. Peters, H. M. Scott, H. F. Chambers, J. L. Flynn, I. F. Charo, and J. D. Ernst Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis PNAS, July 3, 2001; 98(14): 7958 - 7963. [Abstract] [Full Text] [PDF]
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