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Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo, Japan; and
Department of Chest Medicine, School of Medicine, Chiba University, Chiba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and RANTES. Immunohistochemical study showed that CCR1-positive cells
accumulated in the interstitial inflammatory site. Furthermore, the
treatment of anti-CCR1 Ab significantly reduced the accumulation of
inflammatory cells and collagen deposition, resulting in dramatic
improvement of survival. These results suggest that CCR1-positive cells
play significant roles in the pathogenesis of pulmonary fibrosis
subsequent to bleomycin-induced lung injury, and that CCR1 could be a
novel molecular target for intervention therapy against pulmonary
fibrosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a key role in this model (2, 3). Neutralization of TNF- with anti-TNF- Ab or
soluble TNF- receptors diminished the development of lung fibrosis
in mice (4, 5). TGF-ß mainly participates in fibrotic
responses subsequent to inflammation (6, 7), i.e.,
neutralization of TGF-ß with anti-TGF-ß Ab reduced lung
fibrosis in a murine model (6).
Chemokines, including macrophage-inflammatory protein-1
(MIP-1),3 monocyte
chemoattractant protein-1 (MCP-1)/JE, RANTES, MIP-2, and IFN-inducible
protein-10 (IP-10) might also contribute to the inflammatory, fibrotic,
and angiogenic responses in a bleomycin model (8, 9, 10).
Furthermore, analyses of bronchoalveolar lavage (BAL) and open-lung
biopsies from patients with idiopathic pulmonary fibrosis have
demonstrated elevated levels of MIP-1, RANTES, and MCP-1 compared
with that of healthy volunteers (11, 12, 13). A CC chemokine,
MIP-1, promotes leukocyte accumulation and activation, leading to
fibrosis. Treatment of bleomycin-challenged mice with anti-MIP-1
Ab reduced accumulation of pulmonary mononuclear phagocytes and
fibrosis (8). RANTES, another CC chemokine, is a potent
eosinophil and lymphocyte attractant and may mediate T lymphocyte
influx in fibrosing alveolitis (14).
Although it is clear that macrophages, lymphocytes, neutrophils, eosinophils, and their chemoattractants participate in the pathogenesis of lung fibrosis, it is still controversial which subtype of leukocyte plays an essential role (15, 16). Since chemokines induce their biological effects by interacting with specific receptors on the target cell surface, the chemokine receptors have been expected to be the targets to prevent leukocyte migration or activation causing fibrosis.
CCR1 is constitutively expressed on monocytes, neutrophils,
lymphocytes, and eosinophils (17, 18) and interacts with
MIP-1, RANTES, leukotactin-1, MCP-3, and hemofiltrate-derived CC
chemokine-1. Recent studies showed that immature dendritic cells also
express CCR1 and regulate interaction of dendritic cells with T cells
in the process of Ag presentation (19, 20).
In mouse, MIP-1 has been implicated in multiple pathologies
including pulmonary fibrosis, influenza A alveolitis, and experimental
allergic encephalomyelitis; however, specific roles of CCR1 are not
defined yet. In the present study, we investigated the role of
CCR1-expressing cells in bleomycin-induced lung injury, including
expression kinetics along with the production of its ligands and
immunohistochemical localization. Furthermore, we examined the effects
of neutralizing anti-CCR1 Ab on bleomycin-induced lung fibrosis
in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan). The mice were maintained in a pathogen-free mouse facility at the Department of Molecular Preventive Medicine, University of Tokyo. Female New Zealand White rabbits were purchased from CLEA Japan (Tokyo, Japan). All experiments complied with approved animal care protocols of the University of Tokyo. Bleomycin sulfate was kindly gifted from Nipponkayaku (Tokyo, Japan).
Bleomycin administration
Bleomycin sulfate was administered to C57BL/6J female mice aged 8–10 wk. Briefly, C57BL/6J mice were anesthetized with 200 µl of 5 mg/ml pentobarbital injected i.p., followed by intratracheal instillation of 3 mg/kg bleomycin sulfate in 50 µl sterile saline.
Preparation of a recombinant GST protein fused with the NH2-terminal portion of murine CCR1
cDNA encoding the NH2-terminal extracellular portion of murine CCR1 was obtained by PCR using the full-length cDNA as a template and a set of oligonucleotides (5'-GCGGATCCATGGAGATTTCAGATTTCACAG-3' and 5'-GCGCGGCCGCCTACCCAAAGGCTCTTACAGC-3') as primers (17). The resulting fragment was digested with BamHI and NotI and subcloned into a GST fusion protein expression vector, pGEM4T-3, which was predigested with BamHI and NotI. Expression and purification of the GST fusion protein was performed as described previously (17). Similarly, we prepared the NH2-terminal portion of murine CCR2 using the full-length cDNA as a template and a set of oligonucleotides (5'-GCGGATCCATGGAAGACAATAATATGTTAC-3' and 5'-GCGCGGCCGCCTATCCAATTTGCTTCACACT-3') as primers (17).
Preparation of polyclonal Ab to a GST protein fused with the NH2-terminal portion of murine CCR1
Rabbit anti-CCR1 Ab was prepared by multiple-site
immunization of New Zealand White rabbits with GST fusion protein in
CFA (Iatron Laboratories, Tokyo, Japan). One week after the final
immunization, rabbits were bled and sera were obtained and fractionated
into IgG using a column packed with protein G-agarose (Pharmacia
Biotech, Uppsala, Sweden). The anti-CCR1 Ab stained only CCR1
transfectants, but not CCR2, CCR7, or CXCR4 transfectants, establishing
the binding specificity. Furthermore, 50% inhibition of
MIP-1-induced in vitro splenocytes chemotaxis was obtained at 10
µg/ml anti-CCR1 Ab. Similarly, we prepared blocking anti-CCR2
Ab. The anti-CCR2 Ab stained only CCR2 transfectants, but not CCR1,
CCR7, or CXCR4 transfectants. Sixty-six percent inhibition of
MCP-1/JE-induced in vitro chemotaxis was obtained at 100 µg/ml
anti-CCR2 Ab.
Passive immunization with anti-CCR1 Ab
To evaluate the effects of anti-CCR1 Ab or anti-CCR2 Ab on bleomycin-induced lung injury, we treated mice with Ab three times. Mice were injected i.v. with 500 µg anti-CCR1 Ab, anti-CCR2 Ab, or normal rabbit IgG in 200 µl PBS 1 h before intratracheal administration of bleomycin, and additional anti-CCR1 Ab, anti-CCR2 Ab, or normal rabbit IgG was injected i.p. at days 3 and 6.
Histopathology
Tissues were fixed in 10% buffered Formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined by light microscopy.
Assessment of BAL cells and lung-infiltrating leukocytes
BAL cells and lung-infiltrating leukocytes were prepared as described elsewhere (8), with some modification. In brief, after anesthesia, 1 ml PBS was instilled and withdrawn five times from the lung via an intratracheal cannula. The BAL fluids were collected and after RBC lysis total leukocyte counts were determined. Cell differentials were determined after cytospin centrifuge. Specimens were stained with Diff-Quik products (Baxter, Miami, FL).
To get the lung-infiltrating leukocytes, lungs were perfused with saline, dissected from the chest cavity, and then minced with scissors. Each sample was incubated for 30 min at 37°C on a rocker in 15 ml digesting buffer (10% FCS in RPMI 1640 with 1% collagenase; Wako Pure Chemical, Osaka, Japan). Next, the sample was pressed through nylon mesh and suspended in 10% FCS-RPMI 1640 after being rinsed. The cell suspension was treated with Histopaque-1119 (Sigma, St. Louis, MO) and centrifuged at 2000 rpm for 20 min to remove lung parenchymal cells and RBC. The pellet was resuspended in 2.5% FCS-PBS after being rinsed. After cell counts were performed, flow cytometric immunofluorescence analyses were conducted.
Flow cytometry
Immunofluorescence analyses of peripheral blood leukocytes and lung-infiltrating leukocytes were performed with the use of an Epics Elite cell sorter (Coulter Electronics, Hialeah, FL) as described previously (21, 22). Peripheral blood leukocytes were prepared from normal mice with RBC lysis buffer. After incubation with Fc Block (anti-CD16/32; PharMingen, San Diego, CA) for 10 min, cells were stained with PE-conjugated mAb against CD3, CD4, CD8, CD11b, CD11c, and Gr-1 (PharMingen), and also stained with 20 µg/ml of rabbit anti-CCR1 polyclonal Ab followed by staining with FITC-conjugated goat anti-rabbit IgG (Leinco Technologies, St. Louis, MO). Before analyses propidium iodide (Sigma) staining was performed to remove the dead cells.
Chemokine and chemokine receptor gene expression analysis in bleomycin-challenged lung
Total RNA was isolated from lung specimens using RNAzol B
(Tel-Test, Friendswood, TX) according to manufacturer’s instructions.
It was then reversely transcribed into cDNA and amplified. The PCR
products of MIP-1, RANTES, CCR1, CCR2, and CCR5 were examined by 2%
agarose gel electrophoresis. After ethidium bromide staining, bands
were visible only at the expected size for each mRNA product. The sense
primer for CCR1 was 5'-GTGTTCATCATTGGAGTGGTGG-3' and the antisense
primer was 5'-GGTTGAACAGGTAGATGCTGGTC-3'. The sense primer for CCR2 was
5'-TGTTACCTCAGTTCATCCACGG-3' and the antisense primer was
5'-CAGAATGGTAATGTGAGCAGGAAG-3'. The sense primer for MCP-1/JE was
5'-ATGCAGGTCCCTGTCATGC-3' and the antisense primer was
5'-GTTCACTGTCACACTGGTCA-3'. The other primers were described previously
(21, 22). The expression of CCR1 was determined by
real-time quantitative PCR using the ABI 7700 sequence detector system
(PE Applied Biosystems, Foster City, CA) (21, 22). The
CCR1 fluorescence-labeled probe was 5'-TGGTGCTCATGCAGCAT
AGGAGGCTT-3'.
Immunohistochemistry
The preparation of lung specimens was performed as described previously (21, 22). Briefly, lung specimens were fixed in periodate-lysine-paraformaldehyde, washed with PBS containing sucrose, embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and cut into 7-µm-thick sections with a cryostat. After inhibition of endogenous peroxidase activity, the sections were incubated with the first Ab. The Abs used were rabbit anti-CCR1 Ab, rat anti-F4/80 (BMA Biomedicals, Geneva, Switzerland), rat anti-CD4, rat anti-CD8, rat anti-Gr-1 (PharMingen), rat anti-nonlymphoid dendritic cell (NLDC)-145, and rat anti-MHC class II (BMA Biomedicals). As a negative control either a rabbit IgG or a rat IgG was used, respectively. They were treated sequentially with either HRP-conjugated goat anti-rabbit IgG (Cedarlane Laboratories, Hornby, Ontario, Canada) or a HRP-conjugated goat anti-rat IgG (BioSource International, Camarillo, CA). After staining with 3,3'-diaminobenzidine (Wako Pure Chemical) or 3-amino-9-ethylcarbazole substrate kit (Vector Laboratories, Burlingame, CA), samples were counterstained with Mayer’s hematoxylin.
Collagen assay
Total lung collagen content was determined by assaying total soluble collagen using the Sircol Collagen Assay kit (Biocolor, Northern Ireland) according to the manufacturer’s instructions. Briefly, lungs were harvested at day14 after bleomycin administration and homogenized in 10 ml 0.5 M acetic acid containing about 1 mg pepsin/10 mg tissue residue. Each sample was incubated for 24 h at 4°C with stirring. After centrifugation, 200 µl of each supernatant was assayed. One milliliter of Sircol dye reagent that binds to collagen was added to each sample and then mixed for 30 min. After centrifugation, the pellet was suspended in 1 ml of the alkali reagent included in the kit and read at 540 nm by a spectrophotometer. Collagen standard solutions were utilized to construct a standard curve. Collagens contain about 14% hydroxyproline by weight, and collagen contents obtained with this method correlate well with the hydroxyproline content according to the manufacturer’s data.
Statistical analysis
Results are expressed as means ± SD. Comparisons between the two groups were analyzed using paired Student’s t test or ANOVA. A generalized Wilcoxon test of Kaplan-Meier curves was used to evaluate the significance of survival rates. p < 0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine which subpopulation of leukocytes expresses CCR1,
two-color immunofluorescence analysis was performed (Fig. 1
). A majority of CD11b-or a part of
Gr-1-positive cells was strongly stained with anti-CCR1Ab, whereas
CD3-, CD4-, or CD8-positive cells were weakly stained with
anti-CCR1 Ab. There were a small number of CCR1 and CD11c double
positive cells. These results demonstrated that murine peripheral blood
monocytes, granulocytes, and possibly a part of dendritic precursor
cells in peripheral blood express CCR1 on their surface, whereas
lymphocytes expressed CCR1 at low levels. These data are consistent
with the previous reports that MIP-1 exhibits chemotactic activity
against a wide range of leukocytes, including immature dendritic cells
(19, 20, 23). Furthermore, there were some differences of
CCR1 distribution between murine leukocytes and human leukocytes. In
murine peripheral blood, CCR1 is expressed on wide range of leukocytes
including neutrophils (Fig. 1) (24). In contrast, human peripheral
blood neutrophils lack expression of CCR1 (17).
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To determine chemokine or chemokine receptor expression, RT-PCR
and quantitative PCR of whole-lung homogenate were performed (Fig. 2). Peripheral blood contamination was
minimized by lung perfusion with saline. Low levels of CCR1, CCR2, and
CCR5 mRNA were detected in the untreated lung. After bleomycin
treatment, the expression levels were subsequently enhanced and peaked
at day 7. Their ligands, MIP-1, RANTES, and MCP-1/JE also peaked at
day 7. Real-time quantitative PCR of CCR1 confirmed the above
expression patterns (Fig. 2B). These results are consistent
with the observation that leukocytes accumulate mainly in the first
week (1).
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The cells infiltrating into the lung after bleomycin treatment
were identified using immunohistochemical staining of frozen sections.
At day 7 after bleomycin treatment, CCR1 protein was detected on large
mononuclear cells present in the subepithelial and interstitial
inflammatory area and alveolar macrophages (Fig. 3, a–c). In
immunohistochemical staining, few number of alveolar macrophages was
detected in untreated mice, which were CCR1 positive in
immunofluorescence analysis (data not shown). F4/80-positive large
cells, which were considered to be macrophages, were detected in the
interstitial inflammatory area (Fig. 3d). The invading
lymphocytes were mainly CD4-positive cells (Fig. 3e), with a
few CD8-positive cells (Fig. 3f). Gr-1-positive small cells,
which were considered to be granulocytes, were scattered in the
interstitial inflammatory area (Fig. 3g). Dendritic cells
were detected as NLDC-145-positive large cells in the upper layer of
the epithelium and interstitial inflammatory site (Fig. 3h).
MHC class II-positive cells were also detected in a similar area (Fig. 3i). Immunohistochemical staining of untreated lung barely
detected NLDC-145- or MHC class II-positive cells (data not shown).
These findings suggest that dendritic cells besides macrophages,
lymphocytes, and granulocytes may be also involved in these lines of
pathogenesis. Moreover, many of CCR1-positive cells were considered to
be macrophages or dendritic cells in both their morphology and
distribution, confirming the results of immunofluorescence analysis of
peripheral blood leukocytes (Fig. 1
).
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To determine the involvement of CCR1-positive cells causing lung
injury, neutralizing anti-CCR1 Ab was administered before and after
bleomycin challenge. When the mice were treated with control Ab, they
were getting thin and from day 7 they began to die with high lethality
(45% at day 14) (Fig. 4A).
Anti-CCR1 Ab treatment provided a significant protection against
lethality (6.25% at day 14). In contrast, neutralization of CCR2,
receptor for MCP-1/JE, showed no effect on survival (Fig. 4B).
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). Control Ab-treated mice showed
severely damaged lung, including inflammatory cell accumulation at the
alveolar septa and disruption of the alveolar architecture associated
with collagen deposition (Fig. 5, A-2). In contrast,
after anti-CCR1 Ab treatment, those inflammatory and fibrotic
changes were significantly reduced (Fig. 5, A-3). To
further directly evaluate the collagen deposition, the total lung
collagen content was determined (Fig. 5B). After bleomycin
administration, the total lung collagen content was increased almost
7-fold at day 14 compared with that in untreated mice. Anti-CCR1Ab
treatment reduced this collagen deposition by half. In contrast,
anti-CCR2 Ab showed no significant effect, confirming the outcome
of survival (Fig. 4B). These results suggest that
CCR1-positive cells, rather than CCR2 positive cells, could play an
essential role in bleomycin-induced fibrotic responses.
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We next evaluated the effects of anti-CCR1 Ab on leukocyte
infiltration in bleomycin-induced inflammatory processes. We estimated
the inflammatory cell populations of both BAL cells and
lung-infiltrating leukocytes at 6 days after bleomycin challenge (Fig. 6). The total number of BAL cells in
anti-CCR1 Ab-treated mice was significantly decreased (by 39%)
compared with that of control Ab-treated mice. Differential counts of
lavage cells from anti-CCR1 Ab-treated mice showed a significant
reduction in macrophages (by 38%) but not in lymphocyte and
granulocyte populations compared with those of control Ab-treated mice
(Fig. 6A).
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B). The total
number of infiltrating leukocytes in anti-CCR1 Ab-treated mice was
significantly decreased (by 42%) compared with that of control
Ab-treated mice. Differential counts of infiltrating leukocytes were
performed with immunofluorescence analyses. The number of
CD4,CD11c-positive cells was significantly decreased, whereas the
number of CD11b,CD8,Gr-1-positive cells tended to be decreased but did
not reach significant differences. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß6, could be a major target (6, 7). Third, to control the unremitting inflammation characterized
by leukocyte infiltration may be one of the hopeful therapeutic
approaches. To control inflammation, cytokines or chemokines could be
potent molecular targets. Although neutralization of TNF-, MIP-2, or
MIP-1 improved fibrotic changes (5, 8, 10), the role of
their receptors in regulating inflammation and fibrosis so far remains
unclear. The specific chemokine receptor expression and regulation on
each type of leukocyte may potentially control the selective leukocyte
recruitment and activation at the inflammatory site (26, 27). Although
MIP-1 and RANTES have been implicated in the pathogenesis of
pulmonary fibrosis, our data suggest that their common receptor CCR1
will be novel therapeutic target in this disease.
After bleomycin challenge the expression of CCR1 mRNA as well as its
ligands MIP-1 and RANTES mRNA increased and peaked at day 7 (Fig. 2). Immunohistochemical study (Fig. 3) and immunofluorescence analysis
(Fig. 1) showed that CCR1-positive cells may be mainly macrophages and
dendritic cells accumulated in the subepithelial and interstitial areas
(Fig. 3, a–c). Although involvement of macrophages and
lymphocytes was reported, the contribution of dendritic cells has not
been explored. We recognized NLDC-145- or MHC class II-positive cells
in the subepithelial and interstitial areas after bleomycin challenge
(Fig. 3, h and i). Immature dendritic cells are
attracted toward the inflammatory site by MIP-1, RANTES, and
MIP-3 through their receptors CCR1 and CCR6. Dendritic cells are the
sources of activated T cell chemoattractants, such as EBI-1 ligand
chemokine, thymus and activation-regulated chemokine, and
macrophage-derived chemokine (19). After anti-CCR1 Ab
treatment, the number of CD4- as well as CD11c-positive cells was
reduced (Fig. 6B). These results suggest that dendritic
cells expressing CCR1 might play a role in maintaining chronic
inflammation including pulmonary fibrosis.
Anti-CCR1 Ab treatment significantly improved the survival in
association with the reduction of collagen deposition (Figs. 4 and 5).
Anti-CCR1 Ab treatment reduced the total cell count of BAL fluid as
well as the infiltrating leukocyte count in tissue (Fig. 6). Smith et
al. (8) reported that in the bleomycin model
neutralization of MIP-1 reduced the macrophage accumulation without
modulating other leukocytes such as lymphocytes or granulocytes. Our
data showed that anti-CCR1 Ab treatment reduced not only the number
of macrophages in BAL fluid but also other leukocyte subpopulations
(Fig. 6). The participation of other ligands of CCR1 such as RANTES and
leukotactin-1 could explain the difference in the effects of
anti-MIP-1 Ab and anti-CCR1 Ab in this model. In
CCR1-deficient mice, the size of Schistosoma mansoni-induced
granuloma was reduced, but there were no differences in the cellular
composition of the granuloma (28). These data suggest a
possible effect of CCR1 on leukocyte migration for many leukocyte
subtypes in vivo.
Compared with CCR1Ab treatment, neutralization of CCR2 failed to
modulate fibrotic responses (Figs. 4B and
5B), although its ligand MCP-1/JE may participate
in this pathogenesis (Fig. 2A). Immunofluorescence analysis
of CCR2 showed a similar distribution pattern to CCR1, i.e., the
majority of CD11b-positive cells were strongly stained and CD3-positive
cells were weakly stained with anti-CCR2 Ab (data not shown).
Failure of anti-CCR2 Ab treatment in this model in spite of the
similar distribution pattern might be considered as follows. First, the
timing of the Ab treatment might not be appropriate. However, in our
preliminary experiments, administration of anti-CCR2 Ab at every 2
days from days 0 to 12 failed to improve the survival rate and collagen
deposition (data not shown). This result may be able to exclude the
possibility of the inappropriate treatment. Second, other chemokine
receptors on the CCR2-expressing cells might compensate; therefore,
anti-CCR2 Ab could not block the migration of the CCR2- expressing
cells in vivo. However, CCR2-deficient mice failed to recruit
macrophages in an experimental peritoneal inflammation
(29), suggesting that CCR2 has a nonredundant role. Third,
the MIP-1–CCR1 pathway plays more of an essential role than the
MCP-1–CCR2 pathway does in this model. CCR1 and CCR2 could be
expressed on the same cells. Although both receptors participate in
leukocyte chemotaxis and activation, they might act in different way.
In bleomycin-induced lung injury, anti-MIP-1- Ab reduced the
total number of CD11b-positive cells and showed significant reduction
of fibrosis (1). Although anti-MCP-1 Ab also reduced
total lung inflammatory cell count, there has been no reports
demonstrating an antifibrotic effect (1). Chemokine
receptor-deficient mice would provide additional information. In
CCR1-deficient mice, the size of S. mansoni-induced
granuloma was reduced (28). In contrast, in CCR2-deficient
mice, the granuloma size could not be reduced, although the size and
macrophage number were reduced only in the early phase
(30).
Despite chemokine redundancy, the neutralization of a single chemokine
receptor could affect a remarkable decrease in inflammatory responses
and fibrotic responses. There are two considerable explanations. First,
the neutralization of CCR1 results in an overall decrease of
CCR1-expressing leukocyte subpopulations, including monocytes,
lymphocytes, neutrophils, eosinophils, and immature dendritic cells.
Second, neutralization of CCR1 would initially lead to a decrease of
the infiltration and activation in the CCR1-expressing key cells, such
as alveolar macrophages (Fig. 3c), which are the potentially
important source of cytokines and chemokines, including TNF-,
MIP-1, RANTES, and MCP-1/JE (1, 4, 8, 9, 11, 12).
Anti-CCR1 Ab reduced the alveolar macrophages in number by 38% (Fig. 6A), which may cause the reduction of these cytokines and
chemokines resulting in a remarkable decrease in the inflammatory
responses, through blocking the autocrine process. Gerard et al.
(31) reported that in pancreatitis-associated lung injury,
CCR1-deficient mice did not show any increase in TNF- levels in BAL
fluid. Neutralization of the chemokine receptor could modulate not only
direct leukocyte migration but also secondary response.
In this report, we detected time-dependent expression of CCR1 mRNA and
CCR1-positive cells accumulated in the inflammatory site, demonstrating
that chemokine receptor CCR1 contributes to the inflammatory responses
in bleomycin-induced lung injury. Furthermore, anti-CCR1 Ab
treatment in bleomycin-challenged mice significantly improved the
survival rate and decreased collagen deposition, corresponding with the
reduction of inflammatory cell accumulation. Anti-CCR1 Ab treatment
failed to achieve the complete inhibition of both inflammatory cell
infiltrations into lung and subsequent fibrosis, suggesting that other
chemokine receptors are also involved in the pathogenesis of the
disease. Nevertheless, our data suggest that chemokine receptors could
be a potent therapeutic target against pulmonary fibrosis. Additional
experiments are also expected to establish the therapeutic possibility
of targeting CCR1 in other inflammatory diseases involving MIP-1 and
RANTES such as rheumatoid arthritis and multiple sclerosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kouji Matsushima, Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage-inflammatory protein-1; MCP-1, monocyte chemoattractant protein-1; IP-10, IFN-inducible protein-10; BAL, bronchoalveolar lavage; NLDC, nonlymphoid dendritic cell.
Received for publication September 23, 1999. Accepted for publication December 23, 1999.
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A. Kitani, I. Fuss, K. Nakamura, F. Kumaki, T. Usui, and W. Strober Transforming Growth Factor (TGF)-{beta}1-producing Regulatory T Cells Induce Smad-mediated Interleukin 10 Secretion That Facilitates Coordinated Immunoregulatory Activity and Amelioration of TGF-{beta}1-mediated Fibrosis J. Exp. Med., October 20, 2003; 198(8): 1179 - 1188. [Abstract] [Full Text] [PDF]
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C. Jakubzick, E. S. Choi, B. H. Joshi, M. P. Keane, S. L. Kunkel, R. K. Puri, and C. M. Hogaboam Therapeutic Attenuation of Pulmonary Fibrosis Via Targeting of IL-4- and IL-13-Responsive Cells J. Immunol., September 1, 2003; 171(5): 2684 - 2693. [Abstract] [Full Text] [PDF]
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Y. Hamaguchi, Y. Nishizawa, M. Yasui, M. Hasegawa, Y. Kaburagi, K. Komura, T. Nagaoka, E. Saito, Y. Shimada, K. Takehara, et al. Intercellular Adhesion Molecule-1 and L-Selectin Regulate Bleomycin-Induced Lung Fibrosis Am. J. Pathol., November 1, 2002; 161(5): 1607 - 1618. [Abstract] [Full Text] [PDF]
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B. B. Moore, R. Paine III, P. J. Christensen, T. A. Moore, S. Sitterding, R. Ngan, C. A. Wilke, W. A. Kuziel, and G. B. Toews Protection from Pulmonary Fibrosis in the Absence of CCR2 Signaling J. Immunol., October 15, 2001; 167(8): 4368 - 4377. [Abstract] [Full Text] [PDF]
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 M. P. Keane, J. A. Belperio, M. D. Burdick, and R. M. Strieter IL-12 attenuates bleomycin-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L92 - L97. [Abstract] [Full Text] [PDF]
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M. N. Ajuebor, C. M. Hogaboam, S. L. Kunkel, A. E. I. Proudfoot, and J. L. Wallace The Chemokine RANTES Is a Crucial Mediator of the Progression from Acute to Chronic Colitis in the Rat J. Immunol., January 1, 2001; 166(1): 552 - 558. [Abstract] [Full Text] [PDF]
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 N. Onai, Y.-y. Zhang, H. Yoneyama, T. Kitamura, S. Ishikawa, and K. Matsushima Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow-hematopoietic progenitor cells expressing SDF-1-intrakine Blood, September 15, 2000; 96(6): 2074 - 2080. [Abstract] [Full Text] [PDF]
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