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* Tularik, Inc., South San Francisco, CA 94080; and
Department of Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The exact mechanisms that drive recruitment of these pathogenic Th2 cells to the sites of inflammation are not completely understood. Several chemokine receptors are selectively expressed in Th2 cells under in vitro and in vivo stimulation conditions, and frequently induce migration of these cells when stimulated with their specific ligands. These include the family of receptors that bind to the CC chemokine family, namely, CCR3, CCR4, and CCR8 (2, 3, 4). In addition, other receptors such as CRTh2 have also been detected in human Th2 cells (5). However, the expression of these chemokine receptors is not restricted to Th2 cells. For example, CCR3 is primarily expressed in eosinophils and binds its ligand, CCL11/eotaxin (6). A recent study reported that CCR3-deficient mice are defective in pulmonary eosinophilia (7). CCR4 binds to two known ligands, CCL17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine, and neutralization of ligand activity in vivo using blocking Abs alleviates the development of allergic airway inflammation (8, 9). The chemokine receptor CCR8 binds to its ligand CCL1/I-309, which induces calcium flux and migration of receptor-expressing cells in vitro (10, 11). The mouse ligand for CCR8 has been identified to be TCA-3 (12). Expression of TCA-3 is elevated in inflamed lung tissues in a mouse model of asthma (13). A recent report has also demonstrated that CCR8 is selectively induced in Ag-activated eosinophils and can regulate migration and activation of these cells (14). Furthermore, CCR8- and CCR4-positive T cells are detected in human lung tissues from asthmatic patients (15). In addition to Th2 cells, CCR8 is also expressed in a variety of other cell types, such as monocytes/macrophages, IL-2-activated NK cells, endothelial cells, and IL-10-producing regulatory T cells (16, 17, 18, 19, 20). A recent study showed that CCR8-deficient mice have defective pulmonary eosinophilia and Th2 responses in a mouse model of asthma (21). Collectively, these studies suggest that recruitment of various inflammatory cells to affected sites could potentially be mediated by a chemokine receptor-dependent process.
The in vivo roles of CCR8 and its ligand TCA-3 in affecting the pathogenesis of allergic airway inflammation have not been established. In this study, CCR8-deficient mice were generated to explore its importance in airway inflammation. In addition, we examined the importance of TCA-3 in allergic airway inflammation by neutralizing its activity in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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OVA and alum were purchased from Pierce (Rockford, IL). Diff-Quik stain set was purchased from Dade Behring (Newark, DE). The following were obtained from R&D Systems (Minneapolis, MN): rTCA-3, recombinant I-309, anti-TCA-3 goat polyclonal Ab, control goat IgG, and ELISA kits for IL-4, IL-5, and IL-13. Hexadecyltrimethyl ammonium bromide, o-phenylenediamine, and hydrogen peroxide were purchased from Sigma-Aldrich (St. Louis, MO).
Generation of CCR8-deficient mice
CCR8-deficient mice were generated at Deltagen (Menlo Park, CA)
by replacing the 46 bp upstream and the first 178 bp of the CCR8 coding
sequence with a IRES-lacZ Neo cassette (Fig. 1
B). The targeting vector was electroporated into embryonic
stem cells, and drug-resistant clones were selected. Chimeric mice were
generated after injection of targeted embryonic stem cell clones into
the blastocysts of the 129/OlaHsd mouse substrain.
F1 mice were generated by breeding chimeric mice
to C57BL/6 females. Heterozygous and homozygous mutant animals were
obtained by intercrossing males and females from the
F1 generation. The genotype of the
mice was identified by PCR analysis of tail genomic DNA
usingthe following primers: GS(E)-GGAGGTGTTTAAGGGCTTCAATCTG;
GS(T,E)-CAGGTTCAGGAGGTAGATATCTGTG; Neo(T)
GGGGATCGATCCGTCCTGTAAGTCT. The expected sizes for the endogenous
and targeting alleles were 305 and 606 bp, respectively. Eight- to
twelve-week-old CCR8-/- and wild-type
(WT)2 littermates from
F2 generation were used for further
characterization. To confirm the absence of CCR8 message in homozygous
mutant mice, RT-PCR analysis was performed on RNA prepared from
thymus.
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To induce airway inflammation, WT and CCR8-/- mice were immunized with OVA using two different protocols. In protocol A, mice were immunized with i.p. injection of 100 µg of OVA in alum (1:1 in alum in 0.1 ml final volume) on days 0 and 14, as previously described (22). On days 14, 24, 25, and 26, mice were challenged intranasally with 100 µg of OVA in 0.1 ml of normal saline. In protocol B, mice were immunized on days 0 and 5 with 8 µg of OVA in alum (1:1 in alum in 0.1 ml), as described earlier (23). On day 12, mice received 100 µg of OVA in 0.1 ml of normal saline. For Ab neutralization studies, 6- to 8-wk-old BALB/c mice (D&K, Seattle, WA) were immunized according to protocol A. Fifty micrograms of anti-TCA-3 Ab or control goat Ab was administered 1 h before Ag challenge on days 14, 24, 25, and 26. All animal procedures for this part of the study were approved by the University of Washington Animal Care Committee.
Analysis of bronchoalveolar lavage (BAL) fluid
Two days after the last OVA challenge in protocols A and B, BAL fluid was collected according to a previously described protocol (22). Briefly, the left lung was ligated with a suture thread, and the right lung was lavaged three times with 0.4 ml of normal saline each. The cells were pelleted by centrifugation at 1500 x g for 5 min, and the supernatants from all three flushes were pooled and later used to quantify different Th2 cytokines. The total number of cells in the pellet was counted, and cytospin slides were stained using Diff-Quik stain set for estimating the differential counts of various cell types. A total of 200 cells were counted in each slide to calculate the percentages of eosinophils in the BAL from different groups of mice. Cytokine levels in the BAL fluid were quantified using 50 µl of BAL fluid (assayed in duplicate) using IL-4, IL-5, and IL-13 ELISA kits, according to the manufacturer’s instructions. The lower level of sensitivity for all three cytokines was 10 pg/ml. Plasma IgE levels were measured using a commercial kit (BD PharMingen, San Diego, CA). Results were expressed as mean +/- SD values for each group.
Lung histology
Left lung from WT or CCR8-/- mice was fixed in 10% neutral buffered Formalin. Tissues were cut into 5-µm sections and stained using H&E. The number of eosinophils were counted at x400 magnification.
Quantitative RT-PCR analysis
Total RNA from lung was prepared using the TRIzol method (Invitrogen, San Diego, CA). One-step quantitative RT-PCR was performed using real-time fluorogenic 5'-nuclease PCR using an ABI Prism 7700 Sequence BioDetector (PE Biosystems, Foster City, CA), according to the manufacturer’s instructions (TaqMan; PerkinElmer, Foster City, CA). Primers and probes for murine CCR8 and GAPDH were purchased from ABI/Applied Biosystems (Foster City, CA). Cycling conditions were 30 min at 48°C for the reverse-transcription step, and 12 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Control experiments established >95% efficiency over serial 10-fold dilutions for each of the primer pairs and probes. Reactions were set up concurrently for CCR8 and GAPDH genes, and the expression of the CCR8 gene was normalized to GAPDH abundance. The specific signals for each of the transcripts were at least 45-fold (5.5 cycles) over nonspecific background from RNA without prior reverse transcription.
Migration assay
The neutralizing ability of anti-TCA-3 Ab was tested by its ability to block TCA-3-dependent migration in a BI-CD4 T cell lymphoma cell line (kind gift from C. Miceli, University of California School of Medicine, Los Angeles, CA). Migration assays were performed using 96-well migration chambers (5 µM pore size; Neuroprobe, Gaithersburg, MD). TCA-3 (10 ng/ml) was preincubated in the presence of 10 or 100 µg/ml of anti-TCA-3 Ab for 30 min at 37°C. A total of 32 µl of ligand (preincubated in the presence or absence of anti-TCA-3 Ab) was added to the lower chamber, and the filter was assembled on the top of this chamber. Fifty microliters of cells (5 x 106/ml) were placed on top of the filters and incubated at 37°C for 2 h. After removing the filter, 5 µl of Alamar blue was added to each well and incubated for an additional 30 min at 37°C. The relative fluorescence was measured at 530- to 580-nm excitation wavelength and 590-nm emission wavelength. The number of cells that migrated to TCA-3 stimulation was 10–15% of the total input of cells.
Eosinophil peroxidase (EPO) assay
Whole lungs were homogenized in a buffer A containing 5% HBSS diluted in 1x PBS (pH 7.2). Samples were centrifuged at 2000 x g for 10 min, and the supernatant was discarded. The pellet was resuspended in 1 ml of RBC lysis buffer and centrifuged at 3000 x g for 5 min at 4°C. Next, the pellet was resuspended in buffer A containing 0.5% (w/v) hexadecyltrimethyl ammonium bromide and snap frozen in LN2. The samples were then thawed in a 37°C water bath. After a total of three freeze-thaw cycles, samples were centrifuged at 3000 x g for 10 min, and 50 µl of supernatant was incubated with 50 µl of substrate (1.5 mM o-phenylenediamine and 6.6 mM hydrogen peroxide in 0.05 M Tris-HCl, pH 7.5) for 10 min at room temperature. Reactions were stopped by addition of 4 M sulfuric acid, and the absorbance was read at 490 nm to determine EPO activity.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We used an established mouse model of allergic asthma to
examine the expression of CCR8 during the course of induction of an
airway inflammatory response using real-time quantitative RT-PCR
analysis. BALB/c mice were sensitized with OVA on days 0 and 14, and
were subsequently rechallenged with the same Ag on days 24, 25, and 26
(protocol A). Very low levels of CCR8 expression were detected in
nonchallenged lungs (Fig. 1
A).
In contrast, three consecutive rechallenges of OVA resulted in a
consistent 2-fold increase in CCR8 expression in the inflamed lungs
compared with lungs obtained from unchallenged mice (Fig. 1A).
Generation of CCR8-deficient mice
To understand the in vivo function of CCR8 in allergic
airway inflammation, CCR8-deficient mice were generated using
homologous recombination (Fig. 1, B and C). The
lack of expression of CCR8 in homozygous deficient mice was confirmed
by RT-PCR analysis of thymus RNA (Fig. 1D). Phenotypic
analysis revealed that CCR8-/- mice were viable
and indistinguishable from heterozygous or WT littermate controls. The
total cell numbers and percentages of different subpopulations in the
spleen, thymus, and lymph nodes were comparable between WT and
CCR8-/- mice (data not shown).
Characterization of allergic inflammation in CCR8-/- mice
WT and CCR8 homozygous deficient mice were sensitized and
challenged with OVA, as described above (protocol A). Forty hours after
the last OVA challenge, BAL fluid was collected from
CCR8-/- and WT control mice for further
analysis. In contrast to saline-treated mice, Ag-challenged mice had a
significant infiltration of inflammatory cells in BAL fluid, composed
primarily of eosinophils (70–90% of total cells). The total number of
infiltrating cells and the percentage of eosinophils in the BAL fluid
were comparable in CCR8-/- mice and WT mice
(Fig. 2, A and B).
There was no difference in the number of lymphocytes or macrophages
present in the BAL fluid between both groups of mice (data not shown).
Additionally, the amount of IL-5 protein detected in the BAL fluid of
allergen-challenged animals was comparable between
CCR8-/- and WT mice (data not shown).
Histological analysis of affected lungs revealed significant
perivascular and peribronchiolar infiltration of inflammatory cells,
primarily of mononuclear cells and eosinophils, which was
indistinguishable between CCR8-/- and WT
littermates (data not shown). In addition, there was no difference in
the elevated IgE levels in response to OVA challenge between both
groups of mice (Fig. 2C).
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The dosage and timing of OVA immunization can affect the magnitude
and nature of allergic airway inflammation in mouse models of asthma
(24). For example, a critical role for mast cells for the
development of airway hyperresponsiveness was abolished when mice were
challenged with multiple doses of OVA (24). Therefore, we
examined whether the requirement for CCR8 to affect the outcome of
airway inflammation was masked by the dosage and timing of Ag
challenge. In protocol B, mice were immunized with lower doses of Ag,
as described previously (23).
CCR8-/- mice and WT littermates were sensitized
on days 0 and 5 with 8 µg of OVA, and later challenged with a single
intranasal dose of OVA on day 12. On day 14, mice were sacrificed, and
the total number of infiltrating cells in the BAL fluid was enumerated.
As shown in Fig. 3, A and
B, there was no significant difference in the total number
of cells or percentage of eosinophils present in the BAL fluid in
OVA-challenged CCR8-/- mice compared with WT
mice. EPO activity was also measured in lung homogenates after allergen
challenge as an additional parameter of pulmonary eosinophilia. Lung
EPO levels were elevated to similar levels between WT and
CCR8-/- mice (Fig. 3C). EPO activity
was not detected in saline-treated animals (data not shown).
Histological examination of lung sections also did not reveal any
differences in the degree of lung inflammation and tissue eosinophilia
between the two groups of mice (Fig. 4, A–E). Furthermore, the levels of BAL Th2 cytokines (IL-4,
IL-5, and IL-13) were also similar between
CCR8-/- and WT mice (Fig. 5, A–C).
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TCA-3 has been identified as the ligand for CCR8 based on its
ability to bind CCR8 and induce migration of CCR8-expressing cells
(12). Increased expression of TCA-3 has also been observed
in the affected lungs, after adoptive transfer of Ag-specific Th2 cells
to induce airway inflammation (13). To address the role of
TCA-3 in allergic asthma, we examined the effect of neutralizing
anti-TCA-3 Ab in the mouse asthma model. Initially, we tested the
ability of anti-TCA-3 Ab to block CCR8-dependent migration under in
vitro stimulation conditions. As shown in Fig. 6A, the presence of excess
amounts of anti-TCA-3 Ab completely inhibited TCA-3-dependent
migration.
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, B and C,
there was no detectable difference in the total number of infiltrating
cells or percentage of eosinophils in the BAL fluid of anti-TCA-3
or control Ab-treated mice. Moreover, there was no reduction in the
levels of IL-5 and IL-13 in the BAL fluid between the two groups of
mice (Fig. 6, D and E). Administration of TCA-3
Ab also failed to inhibit methacholine-induced airway hyperreactivity
in OVA-challenged mice as determined by whole body in vivo
plethysmography (25) (data not shown). In summary, the present study was conducted to explore the in vivo biological role of CCR8 and its ligand TCA-3, in allergic airway inflammation. Using two different protocols of Ag challenge, we were unable to demonstrate an essential role for CCR8 in the mouse model of allergen-induced airway inflammation. Although CCR8 is selectively expressed by Th2 cells and Ag-activated eosinophils, we were not able to detect any impairment in pulmonary Th2 cytokine responses and eosinophilia in CCR8-/- mice. Studies using the neutralizing anti-TCA-3 Ab also provided additional support for the lack of an important role for CCR8 in allergic airway inflammation.
When this study was in progress, Chensue et al. (21) reported that CCR8-/- mice had impaired pulmonary eosinophilia and Th2 responses in a mouse asthma model. Although we have used an OVA immunization protocol (protocol B) and mouse strain background (C57BL/6 x 129/OlaHsd F2) similar to their report, no defect was observed in the development of allergic airway inflammation in CCR8-/- mice. At present, we are unable to resolve this discrepancy. Although CCR8-positive T cells have been localized in the lungs from asthma patients, it is not clear whether this population represents the pool of Th2 cells (15). It is possible that other receptors involved in Th2 cell migration such as CCR3, CCR4, or CRTh2 could compensate for the absence of CCR8 in promoting recruitment of Th2 cells to sites of allergic inflammation. Future studies are necessary to simultaneously disrupt the function of these receptors to elucidate the mechanisms that drive Th2-mediated inflammation in asthma and allergy.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; EPO, eosinophil peroxidase.
Received for publication August 22, 2002. Accepted for publication November 4, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-chemokine I-309: cloning and molecular characterization of murine CCR8 as the receptor for TCA-3. J. Immunol. 160:1975. (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8. J. Leukocyte Biol. 71:1019.
4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J. Clin. Invest. 100:3083.[Medline]
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