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Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, and
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathology of IPF demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, fibroproliferation, and deposition of extracellular matrix, leading to progressive fibrosis and loss of lung function. The contribution of neovascularization to the progression of fibrosis in IPF has been largely ignored. The existence of neovascularization in IPF was originally identified by Turner-Warwick (3), who examined the lungs of patients with widespread IPF and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculature. Further evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of bleomycin-induced pulmonary fibrosis. Peao et al. perfused the vascular tree of rat lungs with methacrylate resin at a time of maximal pulmonary fibrosis after bleomycin treatment (4). Using scanning electron microscopy, these investigators demonstrated major vascular modifications that included neovascularization of an elaborate network of microvasculature located in the peribronchial regions of the lungs and distortion of the architecture of the alveolar capillaries. The location of neovascularization was closely associated with regions of pulmonary fibrosis, similar to the findings for human lungs.
We have shown that members of the CXC chemokine family exert disparate
effects in mediating angiogenesis as a function of the presence or the
absence of three amino acid residues (Glu-Leu-Arg; the ELR motif) that
immediately precedes the first cysteine amino acid of the primary
structure of these cytokines (5, 6). While IL-8, an ELR CXC chemokine,
was initially discovered on the basis of chemotaxis and activation of
neutrophils, our laboratory and others have found that IL-8 in vitro
has endothelial cell chemotactic activity and in vivo induces
neovascularization in the cornea of rats and rabbits, without
inflammation (6, 7, 8). Macrophage inflammatory protein-2 (MIP-2) is a
murine functional homologue of IL-8 and a structural homologue of human
GRO-ß/
, both ELR CXC chemokines. Furthermore, similar to IL-8, it
is chemotactic for endothelial cells and induces neovascularization in
the cornea of rats without associated inflammation (our
unpublished observations).
We have recently shown that the CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in IPF (9). To demonstrate proof of this principle, we extended these studies to a murine model of bleomycin-induced pulmonary fibrosis. We hypothesized that net angiogenesis during the pathogenesis of fibroplasia and deposition of extracellular matrix during bleomycin-induced pulmonary fibrosis is dependent in part upon an overexpression of the angiogenic CXC chemokine, MIP-2.
We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant increase in the presence of the CXC chemokine, MIP-2, that was correlated to a greater angiogenic response. In addition, this angiogenic activity was significantly attenuated in the presence of neutralizing Abs to MIP-2. Furthermore, pulmonary fibrosis was significantly reduced when bleomycin-exposed animals were passively immunized with anti-MIP-2 Abs. The reduction of pulmonary fibrosis was also associated with a reduction in angiogenesis. These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis in bleomycin-induced pulmonary fibrosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal anti-murine MIP-2-specific antiserum was produced
by the immunization of a rabbit with murine MIP-2 (R&D Systems,
Minneapolis, MN) in multiple intradermal sites with CFA (10, 11). The
specificity of the Ab was assessed by either Western blot analysis or
ELISA against a panel of other recombinant cytokines. This Ab was found
to neutralize 30 ng of MIP-2 at a dilution of 1/1000. Neutralizing
capacity was assessed using two strategies: 1) blocking i.p.
MIP-2-induced neutrophil chemotaxis in mice, and 2) inhibiting
MIP-2-mediated angiogenesis using the rat corneal micropocket assay of
neovascularization. Abs were specific in our sandwich ELISA without
cross-reactivity to a panel of cytokines, including IL-1R antagonist
protein, IL-1, IL-2, IL-4, IL-6, TNF-
, IFN-, and other members of
the CXC and CC chemokine families (10, 11). The anti-protease
buffer for tissue homogenization consisted of 1x PBS with 2 mM PMSF
and 1 µg/ml each of antipain, aprotinin, leupeptin, and pepstatin A.
Rabbit anti-factor VIII-related Ag Abs were purchased from Biomeda
(Foster City, CA).
Animal model of pulmonary fibrosis
Female CBA/J mice (6–8 wk old; The Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Mice were maintained in specific pathogen-free conditions and were provided with food and water ad libitum. To induce pulmonary fibrosis, mice were treated with intratracheal bleomycin (Blenoxane, a gift from Bristol Myers, Evansville, IN; 0.15 U/kg) on day 0 as previously described (12). Control animals received only sterile saline as previously described (12). Briefly, CBA/J mice were anesthetized with 250 µl of 12.5 µg/ml ketamine injected i.p. followed by intratracheal instillation of 0.025 U of bleomycin in 25 µl of sterile isotonic saline. At 2, 4, 8, 12, 16, and 20 days postinstillation, animals were euthanized, and both lungs were removed for homogenization as described below. In separate experiments bleomycin-treated mice were passively immunized by i.p. injection of 0.5 ml of anti-MIP-2 serum or preimmune serum on days 8, 10, 12, and 14, before maximal fibrosis.
Lung tissue preparation
Bleomycin- or saline (control)-treated lungs were homogenized and sonicated in anti-protease buffer using a method previously described (11, 13). Specimens were centrifuged at 900 x g for 15 min, filtered through 1.2-µm pore size Sterile Acrodiscs (Gelman Sciences, Ann Arbor, MI), and frozen at -70°C until thawed for assay by specific MIP-2 ELISA. A portion of the specimen was concentrated approximately 12-fold by lyophilization (Speed-Vac, Savant, Farmingdale, NY) and was used in the corneal micropocket model of neovascularization for analysis of angiogenic activity. Additionally, some lungs were fixed in 4% paraformaldehyde and embedded in paraffin for histologic and immunohistochemical analyses.
MIP-2 ELISA
Antigenic MIP-2 was quantitated using an ELISA as previously described (10, 11). Briefly, flat-bottom 96-well microtiter plates (Nunc, Copenhagen, Denmark) were coated with 50 µl/well of the appropriate polyclonal Ab (1 ng/µl in 0.6 M NaCl, 0.26 M H3BO4, and 0.08 N NaOH, pH 9.6) for 24 h at 4°C and then washed with PBS and 0.05% Tween 20 (wash buffer). Nonspecific binding sites were blocked with 2% BSA. Plates were rinsed, and samples were added (50 µl/well), followed by incubation for 1 h at 37°C. Plates were then washed, and 50 µl/well of the appropriate biotinylated polyclonal Ab (3.5 ng/µl in wash buffer and 2% FCS) was added for 45 min at 37°C. Plates were washed three times, streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added, and the plates were incubated for 30 min at 37°C. Chromogen substrate (Dako, Carpinteria, CA) was then added, and the plates were incubated at room temperature to the desired extinction. Plates were read at 490 nm in an automated microplate reader (Bio-Tek Instruments, Winooski, VT). Standards were 1/2-log dilutions of recombinant MIP-2 from 100 ng to 1 pg/ml (50 µl/well).
Immunohistochemistry of MIP-2
Paraffin-embedded tissue from control and bleomycin-treated lung was processed for immunohistochemical localization of MIP-2, using a method previously described (10). Briefly, tissue sections were dewaxed with xylene and rehydrated through graded concentrations of ethanol. Tissue-nonspecific binding sites were blocked using normal goat serum (BioGenex, San Ramon, CA). Tissue sections were overlaid with a 1/500 dilution of either control (rabbit) or polyclonal rabbit anti-MIP-2 Abs. The tissue sections were washed with Tris-buffered saline, incubated for 60 min with secondary goat anti-rabbit biotinylated Abs (BioGenex), washed in Tris-buffered saline, and incubated with alkaline phosphatase conjugated to streptavidin (BioGenex). Fast Red (BioGenex) reagent was used for chromogenic localization of MIP-2. After optimal color development, tissue sections were immersed in sterile water, counterstained with Lerner’s hematoxylin, and coverslipped using an aqueous mounting solution.
Immunolocalization of factor VIII-related Ag
Paraffin-embedded tissue from control and bleomycin-treated lung was processed for immunohistochemical localization of factor VIII-related Ag as previously described (10). Briefly, tissue sections were dewaxed with xylene and rehydrated through graded concentrations of ethanol. Slides were blocked with normal goat serum (BioGenex) and overlaid with a 1/500 dilution of either control (rabbit) or polyclonal rabbit anti-factor VIII-related Ag Abs. Slides were then rinsed and overlaid with secondary biotinylated goat anti-rabbit IgG (1/35) and incubated for 60 min. After washing with Tris-buffered saline, slides were overlaid with a 1/35 dilution of alkaline phosphatase conjugated to streptavidin (BioGenex) and incubated for 60 min. Fast Red (BioGenex) reagent was used for chromogenic localization of factor VIII-related Ag. After optimal color development, sections were immersed in sterile water, counterstained with Lerner’s hematoxylin, and coverslipped using an aqueous mounting solution.
Western blot analysis of factor VIII-related Ag
Total protein extracts were made by homogenizing lungs in lysis buffer (20 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40, and 2.5 mM EDTA) supplemented with 2 ng/ml aprotinin and 35 ng/ml PMSF. Cell extracts were incubated on ice for 30 min, followed by centrifugation at 4°C for 30 min. Supernatants were then removed and assayed for total protein content using BCA protein assay reagents (Pierce, Rockford, IL) and comparison to known amounts of BSA. One microgram of total protein was loaded in each well of a 12% polyacrylamide gel, and extracts were subjected to SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride membrane (Pierce) by electrophoretic transfer overnight in Tris-glycine buffer (20 mM Tris and 150 mM glycine (pH 8.0) with methanol added to a final concentration of 20% (v/v)). Blots were blocked in 5% skim milk in TBST buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween-20) for 2 h at room temperature, followed by incubation in rabbit primary Ab serum against factor VIII-related Ag (Sigma, St. Louis, MO) diluted 1/200 in blocking solution for 2 h at room temperature. Blots were washed for three 10-min washes in TBST and were incubated for 1 h at room temperature in goat anti-rabbit horseradish peroxidase-conjugated secondary Ab (Bio-Rad, Hercules, CA) at a 1/20,000 dilution. Blots were again washed four times, 10 min each time, in TBST, and proteins were visualized following incubation of the blots in SuperSignal chemiluminescent substrate solution according to the manufacturer’s protocol (Pierce) and exposure to XAR-5 film (Eastman Kodak, Rochester, NY).
FACS analysis of factor VIII-related Ag
Single lung cell suspension preparations were made using a method previously described (14). Briefly, lungs were harvested on day 16 from bleomycin-treated animals who had been treated with either normal rabbit serum or anti-MIP-2 Abs. Lungs were minced with scissors to a fine slurry in 15 ml of digestion buffer (RPMI, 5% FCS, 1 mg/ml collagenase (Boehringer Mannheim, Indianapolis, IN), and 30 µg/ml DNase (Sigma)). Lung slurry was enzymatically digested for 45 min at 37°C. Any undigested fragments were further dispersed by drawing the solution up and down through the bore of a 10-ml syringe. The total lung cell suspension was pelleted, resuspended, and spun through a 20% Percoll gradient. Cell counts and viability were determined using trypan blue exclusion on a hemocytometer. Single cell suspensions were stained with anti-CD45 Tricolor (Caltag, South San Francisco, CA) and primary rabbit anti-factor VIII-related Ag Abs (Sigma) followed by FITC-labeled goat anti-rabbit Abs (PharMingen) to allow live gating on CD45-negative cells, thus selecting out the nonleukocyte population for further analysis of expression of factor VIII-related Ag. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson).
Hydroxyproline assay
Total lung collagen was determined by analysis of hydroxyproline as previously described (12). Briefly, lungs were harvested on days 2, 4, 8, 12, 16, and 20 postbleomycin administration and homogenized in 2 ml of PBS, pH 7.4, with a Tissue Tearor. One-half milliliter of each sample (both lungs) was then digested in 1 ml of 6 N HCl for 8 h at 120°C. Five microliters of citrate/acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid, pH 6.0) and 100 µl of chloramine-T solution (282 mg chloramine-T, 2 ml of n-propanol, 2 ml of H2O, and 16 ml of citrate/acetate buffer) were added to 5 µl of sample, and the samples were left at room temperature for 20 min. Next, 100 µl of Ehrlich’s solution (2.5 g of 4-(dimethylamino)benzaldehyde; Aldrich, Milwaukee, WI), 9.3 ml of n-propanol, and 3.9 ml of 70% perchloric acid (Eastman Kodak)) were added to each sample, and the samples were incubated for 15 min at 65°C. Samples were cooled for 10 min and read at 550 nm on a Beckman DU 640 spectrophotometer (Fullerton, CA). Hydroxyproline (Sigma) concentrations from 0 to 100 µg/ml were used to construct a standard curve.
Myeloperoxidase assay
Pulmonary neutrophil sequestration was quantitated using a myeloperoxidase (MPO) assay as previously described (15). Briefly, at the time of sacrifice lungs were perfused free of blood with 10 ml of 0.9% saline via the spontaneously beating right ventricle. The lungs were excised and placed in a 50 mM potassium phosphate buffer solution (pH 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma). The lung tissue was homogenized, sonicated, and centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was then assayed for MPO activity using a spectrophotometric reaction with o-dianisidine hydrochloride (Sigma) at 460 nm.
Fibroblast proliferation
Fibroblasts were cultured as previously described (9). Briefly,
fibroblasts were isolated from CBA/J mouse lungs. Pulmonary fibroblasts
were grown to 80% confluence and passaged. At the time of the fourth
passage, pulmonary fibroblast purity was >99% as determined by the
absence of nonspecific esterase, factor VIII-related Ag, or cytokeratin
immunostaining. The cells were >90% positive for vimentin, laminin,
and fibronectin and were >90% negative for -smooth muscle actin
and desmin. This technique allowed the establishment of pulmonary
fibroblast cell lines. On the day of use, pulmonary fibroblasts were
plated out in 96-well plates at a concentration of 5000 cells/well and
were allowed to adhere overnight. Fibroblasts were then washed free of
serum and cultured for 24 h under serum-free conditions. At
24 h serum was again added along with varying concentrations of
MIP-2 or platelet-derived growth factor, and fibroblasts were cultured
for a further 24, 48, and 72 h. [3H]thymidine was
added 12 h before harvesting using a cell harvester (Brandel,
Gaithersburg, MD). The percent incorporation of
[3H]thymidine was assessed using a Beckman LS 1801
scintillation counter (Beckman, Schaumburg, IL).
RT-PCR for collagen gene expression
Fibroblasts were isolated as described above. At passage 4, fibroblasts were plated into 60-mm dishes. When they reached 80% confluence they were stimulated in the presence of 1-30 ng/ml of MIP-2 or 20 ng/ml of TGF-ß. Total RNA was extracted from fibroblasts using Trizol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. RT-PCR was performed using an Access RT-PCR kit (Promega, Madison, WI). ß-Actin was used as a housekeeping gene. For ß-actin the sense primer used was 5'-GTGGGGCGCCCCAGGCACCA; the antisense primer was 5'-GCTCGGCCGTGGTGGTGAAGC. For type I collagen the sense primer used was 5'-TGGTGCCAAGGGTCTCACTGGC; the antisense primer was 5'-GGACCTTGTACACCACGTTCACC. For type III collagen the sense primer was 5'-GCAGTCCAACGTAGATGAATTGG; the antisense primer was 5'-GAAGGCCTGGTGGACCAGCTGG. PCR products were visualized by agarose gel electrophoresis.
Corneal micropocket assay of angiogenesis
Angiogenic activity of lung homogenates was assayed in vivo in the avascular cornea of hooded Long-Evans rat eyes, as previously described (5, 9, 10, 11, 13). Briefly, equal volumes of lyophilized lung tissue specimens normalized to total protein were combined with sterile Hydron (Interferon Sciences, New Brunswick, NJ) casting solution. Five-microliter aliquots were pipetted onto the flat surface of an inverted sterile polypropylene specimen container and polymerized overnight in a laminar flow hood under UV light. Before implantation, pellets were rehydrated with normal saline. Animals were anesthetized with ketamine (150 mg/kg) and atropine (250 µg/kg) i.p. Rat corneas were anesthetized with 0.5% proparacaine hydrochloride ophthalmic solution followed by implantation of the Hydron pellet into an intracorneal pocket (1–2 mm from the limbus). Six days after implantation, animals received 1000 U of heparin and ketamine (150 mg/kg) i.p., followed by a 10-ml perfusion of colloidal carbon via the left ventricle. Corneas were harvested and photographed. No inflammatory response was observed in any of the corneas treated with the above specimens. Positive neovascularization responses were recorded only if sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant was observed. Negative responses were recorded when either no growth was observed or only an occasional sprout or hairpin loop displaying no evidence of sustained growth was detected. All animals were handled in accordance with the University of Michigan unit for laboratory animal medicine.
Statistical analysis
Data were analyzed on a Macintosh IIci computer using the
StatView 4.5 statistical package (Abacus Concepts, Berkeley, CA).
Comparisons were made using the unpaired t test. Data were
considered statistically significant at p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We obtained lung tissue from bleomycin-treated mice
(n = 6) at each time point or from saline-treated
controls (n = 6) at each time point and measured MIP-2
by specific ELISA standardized per lung. Lung tissue from
bleomycin-treated animals demonstrated greater levels of MIP-2 compared
with that from saline-treated controls on days 16 and 20
(p < 0.05; Fig. 1
). Furthermore, these greater levels of
MIP-2 continued in parallel with the time of maximal pulmonary fibrosis
(days 16 and 20) as determined by total lung hydroxyproline (Fig. 2). These results suggest a temporal
relationship between elevated levels of MIP-2 and the development of
pulmonary fibrosis.
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To substantiate that this CXC chemokine may be modulating lung
tissue-derived angiogenic activity, we next assessed the in vivo
angiogenic activity of six random pooled samples of either
bleomycin-treated lung tissue or saline control lung tissue at 16 days
in the presence or the absence of preimmune (control) or neutralizing
MIP-2 Abs, using the rat corneal micropocket model of
neovascularization (Fig. 3). These Abs
did not contain significant quantities of LPS contamination as assessed
by Limulus assay, and all samples were normalized to total
protein. We found that lung tissue from bleomycin-treated mice (Fig. 3B) induced a greater angiogenic response than lung tissue
from saline-treated controls (Fig. 3A; n = 6
for each manipulation). Neutralizing Abs to MIP-2 significantly
attenuated the angiogenic activity of bleomycin-treated lung tissue
(Fig. 3C) compared with control Abs (Fig. 3B).
These findings suggested that MIP-2 is a significant angiogenic factor
in bleomycin-induced pulmonary fibrosis.
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Since MIP-2 was significantly elevated in bleomycin-treated lung
tissue and was temporally related to the development of pulmonary
fibrosis, we next assessed whether MIP-2 was associated with areas of
pulmonary fibrosis. Using immunohistochemistry, we found that MIP-2
expression was localized to areas of pulmonary fibrosis (Fig. 4). Interestingly, the areas associated
with MIP-2 localization were essentially devoid of infiltrating
neutrophils and demonstrated significant vascular remodeling as
evidenced by the immunolocalization of factor VIII-related Ag (Fig. 5). Taken together these results are
strong evidence for a significant role for MIP-2 in the development of
the pulmonary fibrotic response to bleomycin.
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Based on our findings of increased angiogenic activity from
bleomycin-treated lung tissue that was significantly attributable to
MIP-2, which itself correlated to the development of fibrosis, we next
assessed whether neutralization of MIP-2 in the in vivo model of
bleomycin-induced pulmonary fibrosis would attenuate the fibrotic
response. Passive immunization with neutralizing MIP-2 Abs on days 8,
10, 12, and 14 led to reduced total lung hydroxyproline compared with
control values (Fig. 6). Control
serum-treated mice had similar hydroxyproline levels as mice treated
with bleomycin alone (data not shown). We chose these time points as
they were the times of maximum MIP-2 expression in our model. In
addition, these time points proceed the time of maximum pulmonary
fibrosis.
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Having shown that the neutralization of MIP-2 led to a reduction
in bleomycin-induced fibrosis we next assessed the effect of passive
immunization and neutralization of MIP-2 on in vivo angiogenesis. We
demonstrated that in vivo neutralization of MIP-2 led to a reduction in
lung-derived angiogenic activity as assessed by the corneal micropocket
assay (Fig. 7). Furthermore,
neutralization of MIP-2 led to a reduction in the total number of
endothelial cells in the lung as assessed by 1) expression of factor
VIII-related Ag using FACS analysis (Table I) and 2) Western blot analysis of
expression of factor VIII-related Ag compared with that in control
treated mice (Fig. 8).
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Having shown that MIP-2 has an important role in the development
of the fibrotic response to bleomycin our next step was to substantiate
that neutralization of MIP-2 was attenuating fibrosis through an
antiangiogenic mechanism by assessing its effect on total lung
neutrophil infiltration. As shown in Fig. 9, neutralization of MIP-2 did not lead
to a reduction in neutrophil presence in the lungs of treated animals
as assessed by MPO activity. These results support the contention that
MIP-2 has an important role in the development and maintenance of
pulmonary fibrosis that is independent of neutrophil chemotactic or
activating properties.
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Having shown that MIP-2 reduces fibrosis through a
neutrophil-independent mechanism we were next interested to determine
whether MIP-2 had any direct action on pulmonary fibroblasts. Pulmonary
fibroblasts were isolated from CBA/J mouse lungs and stimulated with
various concentrations of MIP-2, whereas platelet-derived growth factor
was used as a positive control. Proliferation was measured using
incorporation of tritiated thymidine. As shown in Fig. 10, MIP-2 had no effect on fibroblast
proliferation. Furthermore, when isolated pulmonary fibroblasts were
stimulated with either MIP-2 or TGF-ß as a positive control, there
was no increase in either collagen I or collagen III gene expression
(Fig. 11). Taken together these
findings are further evidence for an alternative biological role for
MIP-2 in the pathogenesis of bleomycin-induced pulmonary fibrosis other
than through an effect on fibroblast proliferation, collagen gene
expression, or neutrophil recruitment or activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While angiogenesis has been shown to play a role in the evolution of tissue repair and fibroplasia associated with acute lung injury and sarcoidosis (16, 17), the contribution of neovascularization to the pathogenesis of fibrosis in IPF has been largely ignored. The existence of morphological neovascularization in IPF was originally identified by Turner-Warwick (3), who examined the lungs of patients with widespread IPF and demonstrated neovascularization/vascular remodeling that was often associated with anastomoses between the systemic and pulmonary microvasculature.
Further evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of bleomycin-induced pulmonary fibrosis (4). Peao and associates perfused the vascular tree of rat lungs with methacrylate resin at a time of maximal pulmonary fibrosis (4). Using scanning electron microscopy, these investigators demonstrated major vascular modifications that included neovascularization of an elaborate network of microvasculature located in the peribronchial regions of the lungs and distortion of the architecture of the alveolar capillaries. The location of neovascularization was closely associated with regions of pulmonary fibrosis, similar to the findings for human lungs (3), and this neovascularization appeared to lead to the formation of systemic-pulmonary anastomoses (4).
We have recently shown that the CXC chemokines, IL-8 and IP-10, are important factors that regulate angiogenic activity in IPF and that an imbalance exists in their expression that favors net angiogenesis in this disease (9). We found that levels of IL-8 were greater from tissue specimens of IPF patients than in those from controls. In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects compared with IPF patients. When IL-8 or IP-10 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced or enhanced, respectively. These findings support the idea that IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF.
To effectively assess the relevance of these mechanisms during the pathogenesis of pulmonary fibrosis in vivo, it is necessary to use an animal model of fibrotic lung disease. Bleomycin sulfate has been used in rodents to initiate fibrotic lung lesions, which have many of the histologic components of IPF (18, 19). Bleomycin administration results in a route-, dose-, and strain-dependent pulmonary inflammatory response characterized by increases in leukocyte accumulation, fibroblast proliferation, and collagen content (18, 19, 20, 21, 22, 23). In addition, the lungs of these animals typically exhibit necrosis of type I pneumocytes within the first 24 h postchallenge, acute alveolitis 2–3 days postchallenge, and intense interstitial inflammation 4–12 days postchallenge (12, 19, 24, 25). Moreover, fibroblast proliferation and extracellular matrix synthesis are initiated 4–14 days postbleomycin challenge, with collagen content elevated approximately twofold at 3 wk postchallenge (12, 19, 20, 21, 22, 23, 24, 25, 26). Although these pathologic changes occur in a more rapid fashion than those in human IPF, the rodent pulmonary inflammatory response to intratracheal bleomycin challenge constitutes a representative model of human IPF.
Our studies have demonstrated that during the pathogenesis of bleomycin-induced pulmonary fibrosis there is increased expression of the CXC chemokine, MIP-2, which favors augmented net angiogenic activity. Lung tissue from bleomycin-treated animals had elevated levels of MIP-2 compared with control values and demonstrated in vivo angiogenic activity that could be significantly attenuated in the presence of neutralizing MIP-2 Abs. Immunolocalization of MIP-2 showed it to be associated with areas of fibrosis, and areas of MIP-2 expression were essentially devoid of neutrophil infiltration. The diffuse nature of the staining is consistent with the fact that CXC chemokines are heparin binding proteins that bind glycosoaminoglycans and is evidence for a significant presence of MIP-2 in the extracellular matrix (27). These findings support our previous observations of increased angiogenic activity in IPF lung tissue, which was significantly attributable to IL-8 (9). Similarly, areas of IL-8 expression were essentially devoid of neutrophil infiltration (9). Moreover, while neutralization of MIP-2 attenuated the fibrotic response, this did not appear to be mediated by its effect on neutrophil recruitment as there was no difference in MPO activity. This supports an alternative biologic role for MIP-2 in the pathogenesis of bleomycin-induced pulmonary fibrosis other than neutrophil recruitment. Further support for this idea is seen in the study by Thrall et al. (28). They showed that neutrophil depletion augmented the fibrotic response to bleomycin, suggesting a beneficial role for neutrophils in bleomycin-induced pulmonary fibrosis. Our results demonstrate that neutralization of MIP-2 leads to a reduction in the expression of factor VIII-related Ag in the lung, which correlates with the reduction in fibrosis. This supports the idea that MIP-2 is an important angiogenic factor during the pathogenesis of bleomycin-induced pulmonary fibrosis. In further support of the role of MIP-2 as an angiogenic factor is the role of the human functional homologue, IL-8, as an angiogenic factor in psoriasis and in the development of neovascularization of the pannus of rheumatoid arthritis (29, 30, 31).
Passive immunization with neutralizing MIP-2 Abs attenuated the
fibrotic response to bleomycin. The magnitude of reduction in
hydroxyproline was of a similar magnitude, as has previously been shown
with other cytokine neutralization studies (12, 32, 33, 34). The lack of
complete reduction of fibrosis suggests that there are potentially
other angiogenic factors present in bleomycin-induced fibrosis. Our
laboratory has previously shown that CXC chemokines other than MIP-2,
such as GRO- (human structural homologue of KC) display
angiogenic activity (5). Furthermore, there are a variety of non-CXC
chemokine molecules that can induce angiogenesis, and we cannot exclude
their potential contribution to neovascularization in bleomycin-induced
pulmonary fibrosis. Moreover, blocking angiogenesis may not be a
complete way of blocking fibrosis, as a variety of mediators do have a
direct effect on fibroblast proliferation and collagen gene expression
(35, 36, 37, 38). Further indirect support for the role of MIP-2 as an
angiogenic factor during the pathogenesis of bleomycin-induced
pulmonary fibrosis is the lack of effect of MIP-2 on fibroblast
proliferation or on fibroblast collagen gene expression. This supports
the contention that MIP-2 has no direct effect on pulmonary
fibroblasts, suggesting that the beneficial effects of neutralization
of MIP-2 are mediated through the regulation of angiogenesis.
In conclusion, we have shown that in the context of bleomycin-induced pulmonary fibrosis, a proangiogenic environment exists. This proangiogenic environment may be important in supporting fibroplasia and deposition of extracellular matrix during the pathogenesis of bleomycin-induced pulmonary fibrosis. This persistent proangiogenic environment contrasts with the normal process of tissue repair in which angiogenesis is usually rapidly expressed, transient, and tightly regulated (39, 40, 41). In addition, passive immunization with neutralizing MIP-2 Abs attenuates the fibrotic response to bleomycin through a mechanism that is independent of fibroblast proliferation, fibroblast collagen gene expression, or neutrophil recruitment or activation. Our findings support the idea that MIP-2 supports fibroplasia and deposition of extracellular matrix by regulating angiogenesis. These results suggest that targeting the regulation of angiogenesis may represent a viable therapeutic option for the treatment of pulmonary fibrosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert M. Strieter, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, 6200 MSRB III, Box 0642, 1150 W. Medical Center Dr., University of Michigan Medical Center, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; ELR, Glu-Leu-Arg; MIP-2, macrophage inflammatory protein-2; IP-10, IFN--inducible protein; GRO, growth-related oncogene; MPO, myeloperoxidase.
Received for publication September 10, 1998. Accepted for publication February 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981. in bleomycin-induced lung injury. J. Immunol. 153:4704.[Abstract]
in bleomycin-mouse model of lung fibrosis: downregulation of TGF-ß and procollagen I and III gene expression. Exp. Lung Res. 21:791.[Medline]
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 M. P. Keane The role of chemokines and cytokines in lung fibrosis Eur. Respir. Rev., December 1, 2008; 17(109): 151 - 156. [Abstract] [Full Text] [PDF]
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 E. C. Keeley, B. Mehrad, and R. M. Strieter Chemokines as Mediators of Neovascularization Arterioscler. Thromb. Vasc. Biol., November 1, 2008; 28(11): 1928 - 1936. [Abstract] [Full Text] [PDF]
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D. Pilling, D. Roife, M. Wang, S. D. Ronkainen, J. R. Crawford, E. L. Travis, and R. H. Gomer Reduction of Bleomycin-Induced Pulmonary Fibrosis by Serum Amyloid P J. Immunol., September 15, 2007; 179(6): 4035 - 4044. [Abstract] [Full Text] [PDF]
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 J. Sanchez, A. Moldobaeva, J. McClintock, J. Jenkins, and E. Wagner The role of CXCR2 in systemic neovascularization of the mouse lung J Appl Physiol, August 1, 2007; 103(2): 594 - 599. [Abstract] [Full Text] [PDF]
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 I. Dell'Aica, R. Niero, F. Piazza, A. Cabrelle, L. Sartor, C. Colalto, E. Brunetta, G. Lorusso, R. Benelli, A. Albini, et al. Hyperforin Blocks Neutrophil Activation of Matrix Metalloproteinase-9, Motility and Recruitment, and Restrains Inflammation-Triggered Angiogenesis and Lung Fibrosis J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 492 - 500. [Abstract] [Full Text] [PDF]
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M. Watanabe, W. Matsuyama, Y. Shirahama, H. Mitsuyama, K.-i. Oonakahara, S. Noma, I. Higashimoto, M. Osame, and K. Arimura Dual Effect of AMD3100, a CXCR4 Antagonist, on Bleomycin-Induced Lung Inflammation J. Immunol., May 1, 2007; 178(9): 5888 - 5898. [Abstract] [Full Text] [PDF]
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 Y. Ishida, A. Kimura, T. Kondo, T. Hayashi, M. Ueno, N. Takakura, K. Matsushima, and N. Mukaida Essential Roles of the CC Chemokine Ligand 3-CC Chemokine Receptor 5 Axis in Bleomycin-Induced Pulmonary Fibrosis through Regulation of Macrophage and Fibrocyte Infiltration Am. J. Pathol., March 1, 2007; 170(3): 843 - 854. [Abstract] [Full Text] [PDF]
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J. M. Thurman, A. M. Lenderink, P. A. Royer, K. E. Coleman, J. Zhou, J. D. Lambris, R. A. Nemenoff, R. J. Quigg, and V. M. Holers C3a Is Required for the Production of CXC Chemokines by Tubular Epithelial Cells after Renal Ishemia/Reperfusion J. Immunol., February 1, 2007; 178(3): 1819 - 1828. [Abstract] [Full Text] [PDF]
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S. J. Park, M. T. Wiekowski, S. A. Lira, and B. Mehrad Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease J. Immunol., February 15, 2006; 176(4): 2538 - 2545. [Abstract] [Full Text] [PDF]
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 O. Kollmar, C. Scheuer, M. D. Menger, and M. K. Schilling Macrophage Inflammatory Protein-2 Promotes Angiogenesis, Cell Migration, and Tumor Growth in Hepatic Metastasis Ann. Surg. Oncol., February 1, 2006; 13(2): 263 - 275. [Abstract] [Full Text] [PDF]
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I. Dell'Aica, L. Sartor, P. Galletti, D. Giacomini, A. Quintavalla, F. Calabrese, C. Giacometti, E. Brunetta, F. Piazza, C. Agostini, et al. Inhibition of Leukocyte Elastase, Polymorphonuclear Chemoinvasion, and Inflammation-Triggered Pulmonary Fibrosis by a 4-Alkyliden-beta-lactam with a Galloyl Moiety J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 539 - 546. [Abstract] [Full Text] [PDF]
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W. Matsuyama, M. Watanabe, Y. Shirahama, R. Hirano, H. Mitsuyama, I. Higashimoto, M. Osame, and K. Arimura Suppression of Discoidin Domain Receptor 1 by RNA Interference Attenuates Lung Inflammation J. Immunol., February 1, 2006; 176(3): 1928 - 1936. [Abstract] [Full Text] [PDF]
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 C. Agostini and C. Gurrieri Chemokine/Cytokine cocktail in idiopathic pulmonary fibrosis. Proceedings of the ATS, January 1, 2006; 3(4): 357 - 363. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. P. Keane, M. D. Burdick, B. N. Gomperts, Y. Y. Xue, K. Hong, J. Mestas, D. Zisman, A. Ardehali, R. Saggar, et al. CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury J. Immunol., November 15, 2005; 175(10): 6931 - 6939. [Abstract] [Full Text] [PDF]
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 H. Uemura, H. Ishiguro, Y. Nagashima, T. Sasaki, N. Nakaigawa, H. Hasumi, S. Kato, and Y. Kubota Antiproliferative activity of angiotensin II receptor blocker through cross-talk between stromal and epithelial prostate cancer cells Mol. Cancer Ther., November 1, 2005; 4(11): 1699 - 1709. [Abstract] [Full Text] [PDF]
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J. Mestas, M. D. Burdick, K. Reckamp, A. Pantuck, R. A. Figlin, and R. M. Strieter The Role of CXCR2/CXCR2 Ligand Biological Axis in Renal Cell Carcinoma J. Immunol., October 15, 2005; 175(8): 5351 - 5357. [Abstract] [Full Text] [PDF]
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N. Hamada, K. Kuwano, M. Yamada, N. Hagimoto, K. Hiasa, K. Egashira, N. Nakashima, T. Maeyama, M. Yoshimi, and Y. Nakanishi Anti-Vascular Endothelial Growth Factor Gene Therapy Attenuates Lung Injury and Fibrosis in Mice J. Immunol., July 15, 2005; 175(2): 1224 - 1231. [Abstract] [Full Text] [PDF]
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A. Moldobaeva and E. M. Wagner Difference in proangiogenic potential of systemic and pulmonary endothelium: role of CXCR2 Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1117 - L1123. [Abstract] [Full Text] [PDF]
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S. Arora, Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Role of IFN-{gamma} in Regulating T2 Immunity and the Development of Alternatively Activated Macrophages during Allergic Bronchopulmonary Mycosis J. Immunol., May 15, 2005; 174(10): 6346 - 6356. [Abstract] [Full Text] [PDF]
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D. C.J. Howell, R. H. Johns, J. A. Lasky, B. Shan, C. J. Scotton, G. J. Laurent, and R. C. Chambers Absence of Proteinase-Activated Receptor-1 Signaling Affords Protection from Bleomycin-Induced Lung Inflammation and Fibrosis Am. J. Pathol., May 1, 2005; 166(5): 1353 - 1365. [Abstract] [Full Text] [PDF]
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M. D. Burdick, L. A. Murray, M. P. Keane, Y. Y. Xue, D. A. Zisman, J. A. Belperio, and R. M. Strieter CXCL11 Attenuates Bleomycin-induced Pulmonary Fibrosis via Inhibition of Vascular Remodeling Am. J. Respir. Crit. Care Med., February 1, 2005; 171(3): 261 - 268. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. Dy, L. Murray, M. D. Burdick, Y. Y. Xue, R. M. Strieter, and M. P. Keane The Role of the Th2 CC Chemokine Ligand CCL17 in Pulmonary Fibrosis J. Immunol., October 1, 2004; 173(7): 4692 - 4698. [Abstract] [Full Text] [PDF]
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M. P. Keane Angiogenesis and Pulmonary Fibrosis: Feast or Famine? Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 207 - 209. [Full Text] [PDF]
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G. P. Cosgrove, K. K. Brown, W. P. Schiemann, A. E. Serls, J. E. Parr, M. W. Geraci, M. I. Schwarz, C. D. Cool, and G. S. Worthen Pigment Epithelium-derived Factor in Idiopathic Pulmonary Fibrosis: A Role in Aberrant Angiogenesis Am. J. Respir. Crit. Care Med., August 1, 2004; 170(3): 242 - 251. [Abstract] [Full Text] [PDF]
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E. A. Renzoni Neovascularization in Idiopathic Pulmonary Fibrosis: Too Much or too Little? Am. J. Respir. Crit. Care Med., June 1, 2004; 169(11): 1179 - 1180. [Full Text] [PDF]
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R. D. Sue, J. A. Belperio, M. D. Burdick, L. A. Murray, Y. Y. Xue, M. C. Dy, J. J. Kwon, M. P. Keane, and R. M. Strieter CXCR2 Is Critical to Hyperoxia-Induced Lung Injury J. Immunol., March 15, 2004; 172(6): 3860 - 3868. [Abstract] [Full Text] [PDF]
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M. P. Keane, J. A. Belperio, Y. Y. Xue, M. D. Burdick, and R. M. Strieter Depletion of CXCR2 Inhibits Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer J. Immunol., March 1, 2004; 172(5): 2853 - 2860. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. P. Keane, M. D. Burdick, J. P. Lynch III, D. A. Zisman, Y. Y. Xue, K. Li, A. Ardehali, D. J. Ross, and R. M. Strieter Role of CXCL9/CXCR3 Chemokine Biology during Pathogenesis of Acute Lung Allograft Rejection J. Immunol., November 1, 2003; 171(9): 4844 - 4852. [Abstract] [Full Text] [PDF]
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N. Kaminski, J. A. Belperio, P. B. Bitterman, L. Chen, S. W. Chensue, A. M.K. Choi, S. Dacic, J. H. Dauber, R. M. du Bois, J. J. Enghild, et al. Idiopathic Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): S1 - 105. [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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S. Srisuma, S. S. Biswal, W. A. Mitzner, S. J. Gallagher, K. H. Mai, and E. M. Wagner Identification of Genes Promoting Angiogenesis in Mouse Lung by Transcriptional Profiling Am. J. Respir. Cell Mol. Biol., August 1, 2003; 29(2): 172 - 179. [Abstract] [Full Text] [PDF]
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N. Mukaida Pathophysiological roles of interleukin-8/CXCL8 in pulmonary diseases Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L566 - L577. [Abstract] [Full Text] [PDF]
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M. P. Keane, S. C. Donnelly, J. A. Belperio, R. B. Goodman, M. Dy, M. D. Burdick, M. C. Fishbein, and R. M. Strieter Imbalance in the Expression of CXC Chemokines Correlates with Bronchoalveolar Lavage Fluid Angiogenic Activity and Procollagen Levels in Acute Respiratory Distress Syndrome J. Immunol., December 1, 2002; 169(11): 6515 - 6521. [Abstract] [Full Text] [PDF]
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 R. M. Strieter, J. A. Belperio, and M. P. Keane CXC Chemokines in Angiogenesis Related to Pulmonary Fibrosis Chest, December 1, 2002; 122(6_suppl): 298S - 301S. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. Dy, M. D. Burdick, Y. Y. Xue, K. Li, J. A. Elias, and M. P. Keane Interaction of IL-13 and C10 in the Pathogenesis of Bleomycin-Induced Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., October 1, 2002; 27(4): 419 - 427. [Abstract] [Full Text] [PDF]
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 T. Hehlgans, B. Stoelcker, P. Stopfer, P. Muller, G. Cernaianu, M. Guba, M. Steinbauer, S. A. Nedospasov, K. Pfeffer, and D. N. Mannel Lymphotoxin-{beta} Receptor Immune Interaction Promotes Tumor Growth by Inducing Angiogenesis Cancer Res., July 15, 2002; 62(14): 4034 - 4040. [Abstract] [Full Text] [PDF]
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J. A. Belperio, M. P. Keane, M. D. Burdick, J. P. Lynch III, Y. Y. Xue, K. Li, D. J. Ross, and R. M. Strieter Critical Role for CXCR3 Chemokine Biology in the Pathogenesis of Bronchiolitis Obliterans Syndrome J. Immunol., July 15, 2002; 169(2): 1037 - 1049. [Abstract] [Full Text] [PDF]
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Y. Tanino, H. Makita, K. Miyamoto, T. Betsuyaku, Y. Ohtsuka, J. Nishihira, and M. Nishimura Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice Am J Physiol Lung Cell Mol Physiol, July 1, 2002; 283(1): L156 - L162. [Abstract] [Full Text] [PDF]
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M. P. KEANE, J. A. BELPERIO, M. D. BURDICK, J. P. LYNCH III, M. C. FISHBEIN, and R. M. STRIETER ENA-78 Is an Important Angiogenic Factor in Idiopathic Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., December 15, 2001; 164(12): 2239 - 2242. [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. Miura, K. Morita, H. Kobayashi, T. A. Hamilton, M. D. Burdick, R. M. Strieter, and R. L. Fairchild Monokine Induced by IFN-{gamma} Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection J. Immunol., September 15, 2001; 167(6): 3494 - 3504. [Abstract] [Full Text] [PDF]
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M. Q. Zhao, M. K. Amir, W. R. Rice, and R. I. Enelow Type II Pneumocyte-CD8+ T-Cell Interactions . Relationship between Target Cell Cytotoxicity and Activation Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 362 - 369. [Abstract] [Full Text] [PDF]
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T. Okazaki, A. Nakao, H. Nakano, F. Takahashi, K. Takahashi, O. Shimozato, K. Takeda, H. Yagita, and K. Okumura Impairment of Bleomycin-Induced Lung Fibrosis in CD28-Deficient Mice J. Immunol., August 15, 2001; 167(4): 1977 - 1981. [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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T. Kielian, B. Barry, and W. F. Hickey CXC Chemokine Receptor-2 Ligands Are Required for Neutrophil-Mediated Host Defense in Experimental Brain Abscesses1 J. Immunol., April 1, 2001; 166(7): 4634 - 4643. [Abstract] [Full Text] [PDF]
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 M. Selman, T. E. King Jr., and A. Pardo Idiopathic Pulmonary Fibrosis: Prevailing and Evolving Hypotheses about Its Pathogenesis and Implications for Therapy Ann Intern Med, January 16, 2001; 134(2): 136 - 151. [Abstract] [Full Text] [PDF]
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A. J. FISHER, S. C. DONNELLY, N. HIRANI, C. HASLETT, R. M. STRIETER, J. H. DARK, and P. A. CORRIS Elevated Levels of Interleukin-8 in Donor Lungs Is Associated with Early Graft Failure after Lung Transplantation Am. J. Respir. Crit. Care Med., January 1, 2001; 163(1): 259 - 265. [Abstract] [Full Text]
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K. Blease, B. Mehrad, T. J. Standiford, N. W. Lukacs, J. Gosling, L. Boring, I. F. Charo, S. L. Kunkel, and C. M. Hogaboam Enhanced Pulmonary Allergic Responses to Aspergillus in CCR2-/- Mice J. Immunol., September 1, 2000; 165(5): 2603 - 2611. [Abstract] [Full Text] [PDF]
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K. C. Fang Mesenchymal Regulation of Alveolar Repair in Pulmonary Fibrosis Am. J. Respir. Cell Mol. Biol., August 1, 2000; 23(2): 142 - 145. [Full Text]
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C. R. Brown and S. L. Reiner Bone-Marrow Chimeras Reveal Hemopoietic and Nonhemopoietic Control of Resistance to Experimental Lyme Arthritis J. Immunol., August 1, 2000; 165(3): 1446 - 1452. [Abstract] [Full Text] [PDF]
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K. Blease, B. Mehrad, T. J. Standiford, N. W. Lukacs, S. L. Kunkel, S. W. Chensue, B. Lu, C. J. Gerard, and C. M. Hogaboam Airway Remodeling Is Absent in CCR1-/- Mice During Chronic Fungal Allergic Airway Disease J. Immunol., August 1, 2000; 165(3): 1564 - 1572. [Abstract] [Full Text] [PDF]
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 J. A. Belperio, M. P. Keane, D. A. Arenberg, C. L. Addison, J. E. Ehlert, M. D. Burdick, and R. M. Strieter CXC chemokines in angiogenesis J. Leukoc. Biol., July 1, 2000; 68(1): 1 - 8. [Abstract] [Full Text]
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J. A. Belperio, M. D. Burdick, M. P. Keane, Y. Y. Xue, J. P. Lynch III, B. L. Daugherty, S. L. Kunkel, and R. M. Strieter The Role of the CC Chemokine, RANTES, in Acute Lung Allograft Rejection J. Immunol., July 1, 2000; 165(1): 461 - 472. [Abstract] [Full Text] [PDF]
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A. Tokuda, M. Itakura, N. Onai, H. Kimura, T. Kuriyama, and K. Matsushima Pivotal Role of CCR1-Positive Leukocytes in Bleomycin- Induced Lung Fibrosis in Mice J. Immunol., March 1, 2000; 164(5): 2745 - 2751. [Abstract] [Full Text] [PDF]
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C. M. Hogaboam, K. Blease, B. Mehrad, M. L. Steinhauser, T. J. Standiford, S. L. Kunkel, and N. W. Lukacs Chronic Airway Hyperreactivity, Goblet Cell Hyperplasia, and Peribronchial Fibrosis during Allergic Airway Disease Induced by Aspergillus fumigatus Am. J. Pathol., February 1, 2000; 156(2): 723 - 732. [Abstract] [Full Text] [PDF]
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K. A. Kernacki, R. P. Barrett, J. A. Hobden, and L. D. Hazlett Macrophage Inflammatory Protein-2 Is a Mediator of Polymorphonuclear Neutrophil Influx in Ocular Bacterial Infection J. Immunol., January 15, 2000; 164(2): 1037 - 1045. [Abstract] [Full Text] [PDF]
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M. P. Keane, J. A. Belperio, D. A. Arenberg, M. D. Burdick, Z. J. Xu, Y. Y. Xue, and R. M. Strieter IFN-{gamma}-Inducible Protein-10 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via Inhibition of Angiogenesis J. Immunol., November 15, 1999; 163(10): 5686 - 5692. [Abstract] [Full Text] [PDF]
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 S. Faouzi, B. E. Burckhardt, J. C. Hanson, C. B. Campe, L. W. Schrum, R. A. Rippe, and J. J. Maher Anti-Fas Induces Hepatic Chemokines and Promotes Inflammation by an NF-kappa B-independent, Caspase-3-dependent Pathway J. Biol. Chem., December 21, 2001; 276(52): 49077 - 49082. [Abstract] [Full Text] [PDF]
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, not statistically significant.
