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Center for Immunology and Inflammatory Disease, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital,
Pulmonary and Critical Care Divisions, Brigham and Women’s Hospital, and
Department of Medicine, Harvard Medical School, Boston, MA 02114;
Department of Pathology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada; and
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Gladstone Institute, University of California, San Francisco, CA 94141
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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were also
similar in WT and CCR2-/- mice. Finally, OVA/OVA
CCR2-/- mice developed increased airway
hyper-responsiveness to a degree similar to that in WT mice. We
conclude that following repeated airway challenges with Ag in
sensitized mice, the development of Th2 responses (elevated IgE,
pulmonary eosinophilia, and lung cytokine levels of IL-4 and IL5) and
the development of airway hyper-responsiveness are not diminished by a
deficiency in CCR2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of CCR2 in inflammatory and infectious diseases has been
investigated using mice with a targeted deletion of this receptor
(16, 17, 18, 19, 20). CCR2-/- mice developed
normally and exhibited no obvious phenotypic abnormalities in the
unchallenged state. However, following antigenic challenge and in
models of infection, CCR2-/- mice demonstrated
significant defects in monocyte recruitment and impaired IFN-
production, with resultant defects in granuloma formation and host
defense against certain intracellular pathogens (e.g., Listeria
monocytogenes, Leishmania, and Cryptococcus
neoformans) (16, 17, 18, 19, 21, 22). Taken together these
data support the hypothesis that CCR2 plays an important role in
monocyte/macrophage recruitment and in Th1-type inflammatory
responses.
The role of CCR2 in models of Th2 inflammation is less well studied. Murine models of asthma have been used to study Th2 inflammatory responses in the lung. These models are characterized by the development of Ag-induced 1) airway hyper-responsiveness (AHR), 2) recruitment of T cells and eosinophils to the airway lumen and lung, 3) elevated levels of Ag-specific IgE in the serum, 4) elevated Th2 cytokines (IL-4, IL-5) in bronchoalveolar lavage (BAL) and lung, and 5) enhanced expression of chemokines with specificities for T lymphocytes and eosinophils in the lung (23, 24, 25, 26, 27, 28, 29). We and others have shown that some of the ligands of CCR2 are up-regulated in the murine model of asthma (i.e., MCP-1, MCP-3, and MCP-5) (8, 30, 31, 32). Neutralization of MCPs with mAbs has been reported to modulate some aspects of the phenotype induced by allergen exposure. For example, neutralization of either MCP-1 or MCP-5 diminished AHR in Ag-challenged mice (31). Anti-MCP-1 also diminished leukocyte recruitment to BAL and lung interstitium, while anti-MCP-5 diminished lung interstitial, but not BAL, leukocyte recruitment. To further examine the specific role of CCR2 in an established murine model of asthma, we compared the responses of CCR2-/- mice with those of wild-type (WT) littermates following immunization and challenge with OVA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CCR2-/- mice were generated as previously described (16). CCR2-/- and CCR2+/+ (WT) littermate controls were generated from matings between heterozygous (CCR2+/-) mice of similar genetic background (Sv129xC57BL/6 hybrids). Mice were studied between 6 and 10 wk of age. The mice were bred and housed in a pathogen-free animal facility and were given food and water ad libitum.
Immunization and challenge protocol
The sensitization and challenge protocol was previously described (32). Mice were immunized with 10 µg of OVA (Sigma, St. Louis, MO) and 1 mg of aluminum hydroxide i.p. on days 0 and 7. Mice underwent aerosol challenge with either PBS or OVA (5% in PBS) for 20 min/day for 4 days, 7 days after the final immunization. Aerosol challenge was performed by placing mice in a Plexiglas box (dimensions: 22 x 23 x 14 cm) and aerosolizing OVA using a DeVilbiss nebulizer, driven by compressed air. Mice were studied 24 h after the last aerosol challenge.
Determination of pulmonary resistance and airway responsiveness
Airway responsiveness was measured as previously described (32, 33). In brief, dose-response curves to methacholine were obtained in anesthetized and ventilated mice 24 h after the last aerosol challenge. Methacholine was administered sequentially in increasing doses (33–1000 µg/kg i.v.). From the relationship between the dose administered and pulmonary resistance (RL), the effective dose that resulted in a doubling of RL was determined by log-linear interpolation. This dose is referred to as the effective dose required to increase RL to 200% of control values (ED200RL) and was used as a measure of airway responsiveness. Because the doses of agonist are given in geometrically increasing amounts, it is common to log-transform this index.
Bronchoalveolar lavage
BAL was performed after the physiological measurements as previously described (32). In brief, the lungs were lavaged with six 0.5-ml aliquots of PBS containing 0.6 mM EDTA. Recovered live cells (trypan blue exclusion) were enumerated in a hemocytometer, and cell differential counts were determined by enumerating macrophages, neutrophils, eosinophils, and lymphocytes on Wright-stained (Leukostat, Fisher Scientific, Pittsburgh, PA) cytocentrifuge preparations. BAL cytokine and eosinophil peroxidase levels were measured using the lavage fluid recovered from the first 1 ml of instilled PBS/EDTA to avoid overdilution of the BAL fluid components. BAL supernatants were aliquoted and frozen (-80°C) for subsequent cytokine and eosinophil peroxidase (EPO) determinations.
Histology and immunohistochemistry
Lungs were harvested after BAL and inflation-fixed to total lung capacity in 10% formalin. Formalin-preserved lung tissue was stained with hematoxylin and eosin using standard protocols. The severity of the inflammatory response was graded semiquantitatively for the following features: overall inflammation, granulomatous inflammation, giant cells, macrophages, lymphocytes, and eosinophils. Each of these features was graded on a scale of 0–3, where 0 represented none, 1 mild, 2 moderate, and 3 severe, based on the severity and extent of the features in the histological sections. The slides were randomized and blinded before grading and were read by one person (F.G.).
Flow cytometry
Flow cytometry was performed as previously described (29). In brief, BAL cells were suspended in staining buffer (PBS with 10% mouse serum) and incubated with the appropriate conjugated Abs at 4°C for 30 min. After washing with PBS, the cells were fixed with 1% paraformaldehyde. Single- and three-color flow cytometries were performed on a FACScan (Becton Dickinson, San Jose, CA) cytofluorometer, and analysis was performed using LYSYS software (Hewlett Packard, Palo Alto, CA).
Total serum IgE and OVA-specific IgE
OVA-specific IgE levels were measured by capture ELISA as previously described (32). ELISA microtiter plates were coated with a purified anti-mouse IgE mAb (PharMingen, San Diego, CA) at a concentration of 2 µg/ml and blocked with PBS-10% FCS. Serum samples were diluted in PBS-10% FCS and incubated in the wells for 2 h. After washing with PBS-Tween, biotinylated OVA (10 µg/ml) was added to the wells and incubated for 1 h. The plates were washed with PBS-Tween followed by the addition of avidin alkaline phosphatase (Sigma) for 1 h. The plates were washed with PBS-Tween and distilled water before the addition of the phosphatase substrate. The plates were allowed to develop for 30 min. The plates were read in an ELISA plate reader at OD405nm. Total serum IgE was measured by capture ELISA in a manner similar to the detection of OVA-specific IgE. A biotinylated rat anti-mouse IgE (PharMingen) was used to detect captured IgE in place of the biotinylated OVA.
BAL EPO levels
BAL EPO levels were measured as previously described (34). One hundred microliters of sample or standard (porcine EPO; ExOxEmis, San Antonio, TX) were plated on 96-well plates and incubated with 100 µl of the assay reaction mixture containing 0.05 M Tris buffer, pH 8.0 (Sigma), 0.0036% H2O2 (Fisher Scientific, Pittsburgh, PA), 0.001% Triton X (Sigma), and 0.18 mg/ml ortho-phenylenediamine (Sigma). The plates were incubated in the dark for 30 min. The reaction was terminated with 50 µl of 4 M H2SO4/well. The plates were read on an ELISA plate reader at OD490nm.
BAL cytokine ELISAs
BAL cytokine determinations were performed by capture ELISA
according to the protocols provided by the manufacturers (IL-4 and
IFN-, PharMingen; IL-5, Endogen, Cambridge, MA). The limit of
detection of the IL-4 and IFN- assays was 10 pg/ml and for the IL-5
assay 5 pg/ml.
RNA isolation and ribonuclease protection assay (RPA)
Lung RNA was isolated as previously described (32).
In brief, lungs were homogenized in 8 ml of 4 M guanidine thiocyanate,
25 mM sodium acetate (pH 7.0), and 0.1 M 2-ME using a Polytron
(Brinkmann Instruments, Westbury, NY). RNA was isolated by gradient
density centrifugation over 5.7 M CsCl/25 mM sodium acetate (pH 5.0;
32,000 rpm, 25°C, for 18 h). For the RPA analysis, 5 µg of
total RNA was analyzed for cytokine expression using the mCK-1 Th1/Th2
RPA template set (PharMingen). Radiolabeled RNA probes for IL-4, IL-5,
IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, IFN-, L-32, and GAPDH were
prepared according to the manufacturer’s instructions by in vitro
transcription using 100 µCi of 3000 Ci/mmol
[
-32P]UTP and T7 RNA polymerase. Following
in vitro transcription, the reaction mixture was treated with DNase and
the radiolabeled probes were isolated by phenol-chloroform extraction
and ethanol precipitation. The radio-labeled probes were dissolved in
hybridization buffer and added to each RNA sample at a final
concentration of 6 xx104 cpm/µl. Following an overnight incubation
at 56°C, single-stranded RNA was digested with RNase and
double-stranded protected RNA was purified by phenol-chloroform
extraction and ethanol precipitation. Samples were analyzed on a 5%
acrylamide/8 M urea gel. After drying, the gel was exposed to film for
18 days.
Statistical analysis
Student’s t test (unpaired, two-tailed) was used to calculate significance levels between treatment groups for BAL cell counts and differentials, EPO levels, and IgE levels. A Kruskal-Wallis test was used to compare the methacholine-induced airway responsiveness. Lung histology scores were compared using the Fisher’s exact test. p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recovery of cells from the BAL of OVA/PBS WT and
CCR2-/- control mice revealed a predominance of
alveolar macrophages in both groups, without any significant difference
between WT and CCR2-/- mice (Fig. 1
). In OVA/OVA WT and
CCR2-/- mice, a significant increase in total
cells, neutrophils, eosinophils, and lymphocytes was seen compared with
their respective OVA/PBS controls (all p < 0.05);
alveolar macrophage numbers were not significantly different between
OVA/OVA- and OVA/PBS-treated mice (Fig. 1). There was no significant
difference in BAL cell recoveries (total or specific cell types)
between OVA/OVA WT and CCR2-/- mice (Fig. 1).
These findings indicate that mice deficient in CCR2 had similar numbers
of resident BAL cells as WT mice (i.e., OVA/PBS group). In addition,
CCR2-/- mice were able to recruit significant
numbers of inflammatory cells into the airway lumen following Ag
challenge (OVA/OVA) in a manner comparable to WT mice.
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The level of EPO activity in the BAL supernatants of OVA/PBS and
OVA/OVA WT and CCR2-/- mice was determined by
colorimetric assay (Fig. 3). The levels
of EPO activity were significantly higher in OVA/OVA WT and
CCR2-/- mice compared with their respective
OVA/PBS controls (p < 0.01 and
p < 0.003, respectively), in agreement with the
increased numbers of eosinophils seen in the OVA/OVA groups. There was
no difference, however, between the WT and
CCR2-/- mice with respect to EPO activity when
comparing similarly treated mice.
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Histopathologic examination of lung tissue from OVA/PBS WT and
CCR2-/- mice revealed normal lung histology in
both groups (Fig. 4, A and
B, respectively). Low power views of lung sections from both
OVA/OVA WT (Fig. 4C) showed a pleomorphic peribronchial and
perivascular inflammation, as has been previously described with this
model (32). The lungs from OVA/OVA
CCR2-/- (Fig. 4D) mice showed a
similar perivascular and peribronchial inflammation. High power views
of lungs from OVA/OVA WT (Fig. 4E) and
CCR2-/- (Fig. 4F) mice revealed the
presence of eosinophils in the inflammatory infiltrates in both groups
(arrows). Semiquantitative grading of the slides from the OVA/OVA WT
and CCR2-/- mice revealed that the numbers of
eosinophils, lymphocytes, macrophages, giant cells, and overall
inflammation were similar between the two groups (Table I). Insome OVA/OVA WT and
CCR2-/- mice, areas of inflammation in the lung
periphery consisting primarily of focal collections of macrophages and
lymphocytes (granulomatous-like inflammation) were observed (Table I).
Granulomatous-like inflammation was absent or mild in 16 of 16 WT mice
and 10 of 18 CCR2-/- mice. More severe grades
of granulomatous-like inflammation (grades 2 and 3) were seen in 8 of
18 CCR2-/- mice compared with WT mice (Table I;
p < 0.001, by Fisher’s exact test).
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Immunization and aerosol challenge with OVA was associated with
the development of increased titers of both total and OVA-specific IgE
in both WT and CCR2-/- mice, compared with
their respective OVA/PBS controls (p < 0.05
for both groups; Fig. 5). There was no
significant difference in the levels of total and OVA-specific IgE in
similarly treated WT and CCR2-/- mice. These
data indicate that a deficiency in CCR2 did not affect the ability to
induce a Th2-dependent humoral response.
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Levels of the Th2 cytokines IL-4 and IL-5 were assessed in BAL
fluid samples of OVA/PBS and OVA/OVA WT and
CCR2-/- mice (Table II). Levels of IL-4 and IL-5 were below
the limit of detection in the OVA/PBS-treated mice of either genotype.
Both WT and CCR2-/- OVA/OVA-treated mice showed
elevated IL-4 and IL-5 levels in the BAL fluid compared with their
respective OVA/PBS controls. There was no difference in BAL IL-4 levels
between OVA/OVA WT and CCR2-/- mice
(p = 0.85). There was a trend toward greater
levels of IL-5 in the BAL of CCR2-/- mice
compared with WT, but this difference did not reach statistical
significance (p = 0.09). IFN- levels were
assessed in the BAL fluid of OVA/OVA WT and
CCR2-/- mice. Four of seven BAL samples from WT
OVA/OVA mice and three of seven samples from
CCR2-/- OVA/OVA mice had undetectable levels of
IFN-. The remaining WT samples had IFN- levels that ranged from
16–51 pg/ml, while the remaining CCR2-/-
samples had a range of 13–41 pg/ml. There was no difference in the
mean BAL IFN- levels between WT and
CCR2-/- mice.
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To assess the levels of Th1 and Th2 cytokines in the lung tissue,
RPA analysis was performed on whole lung RNA isolated from OVA/PBS and
OVA/OVA, WT and CCR2-/- mice (Fig. 6). Constitutive expression of IL-15 mRNA
was seen in all mice. There was increased expression of IL-5, IL-13,
and IFN- in the OVA/OVA WT and CCR2-/-mice
compared with their respective OVA/PBS controls. However, no
differences were seen between the OVA/OVA WT and
CCR2-/- mice with respect to the induction of
any of the Th1 or Th2 cytokines. There was no induction of IL-2, IL-4,
IL-6, IL-9, or IL-10 in any group.
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OVA/OVA WT mice had significantly increased methacholine
responsiveness (as reflected by a lower log
ED200RL) compared with
OVA/PBS WT controls (p < 0.020; Fig. 7). Similarly, OVA/OVA
CCR2-/- mice developed significantly enhanced
methacholine responsiveness compared with their OVA/PBS controls
(p < 0.0006; Fig. 7). There was no significant
difference in airway responsiveness between similarly treated WT and
CCR2-/- mice. These findings indicate that
Ag-induced AHR develops in CCR2 cell-deficient mice and that the
increased responsiveness was not significantly different from that of
similarly treated WT animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in CCR2-/- mice have demonstrated diminished macrophage/monocyte recruitment in murine models of Th1-type granulomatous inflammation (16, 17, 18). The lack of any differential recruitment of macrophages in CCR2-/- mice in our model is probably due to the nature of the inflammatory response in this model (i.e., Th2-type inflammation), which may be less dependent on recruited monocytes than a Th1 response.
CCR2 is expressed on activated T lymphocytes, and many of the ligands for CCR2 (e.g., MCP-1, -2, and -3) are chemotactic for these cells (35). As such, one might have expected a differential recruitment of lymphocytes to the BAL in CCR2-/- mice. However, the BAL differentials and the flow cytometric analysis of BAL lymphocytes demonstrated no difference in the recruitment of T or B lymphocytes in Ag-challenged CCR2-/- mice compared with WT mice. These data suggest that alternate chemoattractants are recruiting lymphocytes to the lung in this model. Likewise, eosinophil recruitment to the airways was not affected by a deficiency in the CCR2 receptor. These data demonstrate that the recruitment of eosinophils to the airways is not dependent on the presence of CCR2 in this model and suggest that the signals required to recruit eosinophils to the lung and airway of these mice are intact.
AHR was assessed in CCR2-/- and WT mice to determine the role of CCR2 in regulating this physiological response in the murine model. We found that CCR2-/- mice had baseline airway responsiveness (i.e., OVA/PBS group) similar to WT mice. Following Ag challenge, CCR2-/- mice developed increased airway responsiveness compared with their own OVA/PBS controls; however, their responsiveness was not significantly different from that in OVA/OVA WT mice. These data demonstrate that CCR2 is not critical for the development of increased airway responsiveness following repeated aerosol Ag challenge in sensitized mice.
BAL cytokine levels and lung cytokine mRNA expression were analyzed to
assess whether differences existed between WT and
CCR2-/- mice in an allergic model of
inflammation. Cytokine responses have been reported to be altered in
CCR2-/- mice in models of Th1-type inflammation
and infection (16, 17, 18, 19, 21, 22). Diminished production of
IFN- has been reported and has been associated with defects in
delayed-type hypersensitivity responses, granuloma formation, and
defective host immune response to intracellular pathogens. Further
studies have suggested that the expression of CCR2 plays an important
role in determining the polarization of T cell responses to
intracellular pathogens, favoring a Th2 response when CCR2 was lacking
(19, 22). If the predominant cytokine defect in
CCR2-/- mice is a diminished production of
IFN-, we might have expected to see a Th2-permissive effect in our
model. We noted a trend toward higher levels of IL-5, but not IL-4,
cytokines in the BAL fluid; however, this did not reach statistical
significance, and there was no difference in the pulmonary
eosinophilia, total and OVA-specific IgE production, lung cytokine mRNA
expression, or AHR.
In a model of Th2-granulomatous inflammation (schistosomal
Ag-elicited), CCR2-/- mice displayed diminished
expression of some Th1 (IFN-) and Th2 (IL-4) cytokines in the lungs
and lymphoid tissue, but granuloma formation was not inhibited
(36). In our studies both OVA/OVA WT and
CCR2-/- mice developed the characteristic
peribronchial and perivascular inflammation with lymphocytes and
eosinophils that characterizes this model. The protocol used in our
studies also led to the development of small areas of
granulomatous-like inflammation in the lungs of both OVA/OVA WT and
CCR2-/- mice. These areas were characterized by
focal collections of lymphocytes and macrophages. Interestingly,
OVA/OVA CCR2-/- mice developed a greater degree
of granulomatous-like inflammation in the lung compared with WT mice
following OVA inhalation. The difference in inflammatory response in
the lungs of CCR2-/- mice warrants further
investigation.
Previous studies have suggested an important role for the CCR2 ligands MCP-1 and MCP-5 in murine models of allergic pulmonary inflammation (31). Neutralization of MCP-1 by mAb diminished the recruitment of all leukocyte subsets in the lung interstitium and BAL in sensitized mice following repeated airway challenge. The diminished leukocyte recruitment was associated with a 70% decrease in AHR. Neutralization of MCP-5 resulted in a significant decrease in the interstitial inflammatory infiltrate, but not in BAL cell recruitment, and was also associated with a decrease in AHR. These data suggest that both MCP-1 and MCP-5 are involved in leukocyte trafficking in the lung in allergic pulmonary inflammation and also influence the development of AHR. Our experiments suggest that in the absence of the receptor for MCP-1 and MCP-5 (i.e., CCR2), airway inflammation and AHR are not diminished. The apparent differences between our findings and those of Gonzalo may be explained by the fact that neutralization of the ligand produces effects different from those resulting from a life-long absence of the receptor. Alternatively, ligand neutralization may simply result in a phenotype distinct from that occurring from deletion of the receptor. This is suggested by the recent observation that MCP-1-deficient mice respond differently to immunologic challenges compared with CCR2-/- mice. MCP-1-deficient mice have been reported to have a defect in the development of Th2 responses (37). This is in contrast to CCR2-/- mice, which favor a polarization toward a Th2 phenotype in models of infection with intracellular parasites (19, 22).
The role of CCR2-/- in the development of AHR has been reported in a separate model of allergic pulmonary inflammation (38). Campbell et al. found partially diminished airway responsiveness in CCR2-/- mice following sensitization and challenge with cockroach allergen. The diminished airway resistance in CCR2-/- mice was accompanied by diminished BAL histamine levels, suggesting a role for pulmonary mast cells in this process. Interestingly, as in our studies, pulmonary eosinophilia did not differ between WT and CCR2-/- mice. The authors concluded that the diminished AHR was not dependent on pulmonary eosinophilia, but, rather, on a mast cell-dependent pathway (38). The differences in AHR in CCR2 mice seen in our experiments compared with those reported by Campbell et al. may be a consequence of methodologic differences in the models. In murine models of asthma the end points of AHR and pulmonary eosinophilia can develop by at least two distinct pathways: 1) a mast cell-dependent pathway and 2) a CD4+ T cell-dependent pathway (39, 40). The protocol used for sensitization and challenge may determine the relative importance of mast cell- vs T cell-mediated processes in regulating tissue eosinophilia and AHR. Mast cells and IgE are believed to play an important role in models where sensitization and challenge are relatively attenuated (40, 41, 42). In contrast, in protocols that use repeated airway challenges in sensitized mice, AHR and pulmonary eosinophilia develop independently of IgE, B cells, and mast cells, suggesting a primary role for T cells in these models (32, 42, 43, 44, 45). This may explain the differences seen between our studies (multiple challenge model) and those of Campbell et al. (single-challenge model) (38).
In summary, previous studies have shown that CCR2 plays an important role in Th1-type inflammatory responses and may dictate the polarization of T cell responses toward a Th2 phenotype during infection with intracellular parasites. In a murine model of allergic pulmonary inflammation, the development of Th2 responses (elevated IgE, pulmonary eosinophilia, and cytokine levels of IL-4 and IL-5) and the development of AHR were not significantly affected by the absence of CCR2, suggesting that this receptor is not essential for the development of these responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James A. MacLean, Bulfinch 422, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; AHR, airway hyper-responsiveness; BAL, bronchoalveolar lavage; ED200RL, effective dose of methacholine required to increase pulmonary resistance to 200% of the control value; EPO, eosinophil peroxidase; RL, pulmonary resistance; RPA, RNase protection assay; WT, wild type.
Received for publication February 22, 2000. Accepted for publication September 8, 2000.
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References |
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are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1-, or CCR2-deficient mice. J. Immunol. 163:5519.This article has been cited by other articles:
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I. Goya, R. Villares, A. Zaballos, J. Gutierrez, L. Kremer, J.-A. Gonzalo, R. Varona, L. Carramolino, A. Serrano, P. Pallares, et al. Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease J. Immunol., February 15, 2003; 170(4): 2138 - 2146. [Abstract] [Full Text] [PDF]
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