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-Inducible Protein-10 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via Inhibition of Angiogenesis1
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, 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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-inducible protein-10 (IP-10). To test this
hypothesis, we measured IP-10 by specific ELISA in whole lung
homogenates in either bleomycin-treated or control mice and correlated
these levels with lung hydroxyproline. We found that lung tissue from
mice treated with bleomycin, compared with that from saline-treated
controls, demonstrated a decrease in the presence of IP-10 that was
correlated to a greater angiogenic response and total lung
hydroxyproline content. Systemic administration of IP-10 significantly
reduced BPF without any alteration in lung lymphocyte or NK cell
populations. This was also paralleled by a reduction in angiogenesis.
Furthermore, IP-10 had no direct effect on isolated pulmonary
fibroblasts. These results demonstrate that the angiostatic CXC
chemokine, IP-10, inhibits fibroplasia and deposition of extracellular
matrix by regulating angiogenesis. |
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 interstitial fibrosis (IPF) and demonstrated neovascularization leading to anastomoses between the systemic and pulmonary microvasculatures and evidence of extensive vascular remodeling in areas of fibrosis. Further evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of bleomycin-induced pulmonary fibrosis (BPF) (4).
Our laboratory has 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). Although 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). In contrast, IP-10, a non-ELR CXC chemokine inhibits angiogenesis and endothelial cell chemotaxis (5, 9, 10).
We have previously shown that the CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in IPF (11). To demonstrate proof of this principle, we extended these studies to a murine model of BPF, and we have recently shown that neutralization of MIP-2, a murine functional homologue of IL-8, attenuates BPF via inhibition of angiogenesis (12).
In the current study we hypothesized that net angiogenesis during the pathogenesis of fibroplasia and the deposition of extracellular matrix during BPF is dependent in part on a relative deficiency of the angiostatic CXC chemokine, IP-10. We found that lung tissue from mice treated with bleomycin, compared with that from saline-treated controls, demonstrated a significant decrease in the presence of the CXC chemokine, IP-10, that was inversely correlated to total lung collagen and a greater angiogenic response compared with control lung tissue. Furthermore, pulmonary fibrosis was significantly reduced when bleomycin-exposed animals were treated with recombinant IP-10 related to a reduction in pulmonary fibrosis. These results demonstrate that the angiostatic CXC chemokine, IP-10, is an important factor that regulates angiogenesis in BPF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal anti-murine IP-10 Abs and murine IP-10 were
purchased from R & D (R & D Systems, Minneapolis, MN). The specificity
of the Ab was assessed by either Western blot analysis or ELISA against
a panel of other recombinant cytokines. Abs were specific in our
sandwich ELISA without cross-reactivity to a panel of cytokines,
including IL-1R antagonist protein (IRAP), IL-1, IL-2, IL-4, IL-6,
TNF-
, IFN-, MIG, and other members of the CXC and CC chemokine
families (10, 13). The anti-protease buffer for tissue
homogenization consisted of 1x PBS with one Complete tablet
(Boehringer Mannheim, Indianapolis, IN) per 50 ml. Rabbit
anti-factor VIII-related Ag Abs were purchased from (Biomeda,
Foster City, CA)
Animal model of pulmonary fibrosis
Female C57B6 and CBA/J mice (6–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in specific pathogen-free conditions and 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, 14). Control animals received only sterile saline as previously described (12, 14). Briefly, 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, 8, 12, and 20 days postinstillation, animals were euthanized, and both lungs were removed for homogenization as described below. In separate experiments bleomycin-treated mice were given daily i.m. injections of recombinant IP-10 (1 µg in 20 µl of 0.25% HSA) or HSA (20 µl 0.25% HSA) until day 12. Mice were sacrificed on day 12 for hydroxyproline assay.
Lung tissue preparation
Bleomycin- or saline (control)-treated lungs were homogenized and sonicated in anti-protease buffer using a method previously described (10, 12, 15). Specimens were centrifuged at 900 x g for 15 min, filtered through 1.2-µm Sterile Acrodiscs (Gelman Sciences, Ann Arbor, MI), and frozen at -70°C until thawed for assay by specific IP-10 ELISA. A portion of the specimen was lyophilized (Speed-Vac, Savant, Farmingdale, NY), and 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.
IP-10 ELISA
Antigenic murine IP-10 was quantitated using a modification of
an ELISA as previously described (10, 13). The sensitivity
of our ELISA is 
50 pg/ml. Briefly, flat-bottom 96-well microtiter
plates (Nunc, Copenhagen, Denmark) were coated with 50 µl/well of the
polyclonal anti-murine IP-10 Ab (1 ng/µl in 0.6 M NaCl, 0.26 M
H3B04, 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 half-log
dilutions of recombinant IP-10 from 100 ng to 1 pg/ml (50
µl/well).
Hydroxyproline assay
Total lung collagen was determined by analysis of hydroxyproline as previously described (12, 14). Briefly, lungs were harvested on days 2, 8, 12, and 20 postbleomycin administration and homogenized in 2 ml of PBS, pH 7.4, with a Tissue Tearor (PRO-Scientific, Monroe, CT). 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 of 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, Rochester, NY) 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, St. Louis, MO) concentrations from 0–10 µg/ml were used to construct a standard curve.
Histology
Lungs were perfused in situ through the right ventricle with saline and then inflated under a constant pressure of 30 cm H2O with 1 ml of 4% paraformaldehyde. Lungs were ligated at the trachea, removed en bloc, and immersed in 4% paraformaldehyde for 24 h at which time they were changed to 70% alcohol before paraffin embedding, followed by sectioning and hematoxylin and eosin staining. Briefly, sections were dewaxed, rehydrated, and placed in hematoxylin solution (Sigma) for 5–10 min. Sections were washed in running water and differentiated in 1% acid-alcohol (using microscopic control) to ensure that only the nuclei were stained, followed by washing in water. Sections were then rinsed in ammonia water for 1 min, followed by brief rinsing in distilled water. Slides were then counterstained in eosin (Sigma) for 2–5 min, washed well in water, dehydrated, and mounted. Ten fields of random hematoxylin- and eosin-stained lung sections (n = 4) from three lungs of mice treated with either IP-10 or HSA were examined for the presence of fibrosis using a modification of the method described by Ashcroft (16). Ten fields of random sections (n = 4) from three lungs per condition were examined using an Olympus BH-2 microscope (New Hyde Park, NY) coupled to a Sony 3CCD camera (Tokyo, Japan) and a Macintosh IIfx computer. The total area of fibrosis was quantitated using NIH Image 1.55 software as previously described (10). For the purpose of quantitation, fibrosis was defined as areas that had Ashcroft grade 7 or 8 (16). These grades represent severe distortion of structure and large fibrous areas, including honeycomb lung, or total fibrous obliteration of the field. Results were expressed as square pixels at x400 magnification.
Fibroblast proliferation
Murine lung fibroblasts were cultured as previously described
(11, 12). Briefly, murine 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 murine
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 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 IP-10 and/or PDGF, and fibroblasts were cultured for
an additional 24, 48, and 72 h.
[3H]Thymidine was added 12 h before
harvesting using a cell harvester (Brandel, Gaithersburg, MD).
[3H]Thymidine incorporation was assessed using
a Beckman LS 1801 scintillation counter (Schaumburg, IL) and was
expressed as counts per minute.
Corneal micropocket (CMP) 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, 10, 11, 12, 13, 15). 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 in-growth of capillary sprouts and hairpin loops toward the implant were 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.
FACS analysis of CD31, factor VIII-related Ag, and leukocyte populations
Lung single-cell suspension preparations were made using a method previously described (12, 17). Briefly, lungs were harvested on day 12 from bleomycin-treated animals who had been treated with either IP-10 or HSA. Lungs were minced with scissors to a fine slurry in 15 ml of digestion buffer (RPMI, 5% FCS, 1 mg/ml collagenase (Boehringer Mannheim), 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) to allow live gating on either CD45-positive cells for analysis of leukocytes or CD45-negative cells for analysis of the nonleukocyte cell populations. CD31 Abs directly conjugated to PE (PharMingen, San Diego, CA) and primary rabbit anti-factor VIII-related Ag Abs (Sigma) followed by FITC-labeled goat anti-rabbit Abs (PharMingen) were added for further analysis of the nonleukocyte cellular populations. We have demonstrated that our digestion buffer does not alter the expression of either CD31 or factor VIII-related Ag from endothelial cells in culture. For analysis of lung lymphocyte or NK cell populations the following Abs directly conjugated to FITC or PE (PharMingen) were used: CD4, CD8, CD19, and NK1.1. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson).
Statistical analysis
Data were analyzed on a Power Macintosh 7500 computer (Apple
Computer, Cupertino, CA) using the StatView 4.5 statistical package
(Abacus Concepts, Berkeley, CA). Comparisons were made using the
unpaired t test. Data were considered statistically
significant if p 
0.05. All values represent the
mean ± SEM.
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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) or from saline-treated controls
(n = 6) at each time point and measured IP-10 by
specific ELISA standardized per lung on days 2, 8, 12, and 20. We chose
these time points because fibrosis does not occur before day 7 in this
model (18, 19, 20, 21, 22, 23). Lung tissue from bleomycin-treated animals
demonstrated lower levels of IP-10 compared with saline-treated
controls on days 12 and 20 (p < 0.05; Fig. 1
). Furthermore, the levels of IP-10 were
inversely correlated with pulmonary fibrosis as determined by total
lung hydroxyproline on days 2, 8, 12, and 20 (Fig. 1). These results
suggest a temporal relationship between decreased levels of IP-10 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 12 days
using the rat CMP assay of neovascularization (Fig. 2). We found that lung tissue from
bleomycin-treated mice (Fig. 2, IB) induced a greater
angiogenic response than lung tissue from saline-treated controls (Fig. 2, IA; n = 6 for each manipulation). Taken
together with our previous findings of vascular remodeling as evidenced
by the immunolocalization of factor VIII-related Ag in the bleomycin
model (12), these results are further evidence of a
significant role for neovascularization 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, which was directly and inversely
correlated to the development of fibrosis and IP-10 levels,
respectively, we next assessed whether administration of exogenous
IP-10, by repeated i.m. injection, during BPF would attenuate the
fibrotic response. Administration of IP-10 on days 1–12 led to reduced
total lung hydroxyproline on day 12 compared with that in controls
(Fig. 3A). Furthermore, these
findings correlated with histopathologic findings of reduced fibrosis.
For the purpose of quantitation, fibrosis was defined as areas that had
Ashcroft grade 7 or 8 (16). These grades represent severe
distortion of structure and large fibrous areas, including honeycomb
lung, or total fibrous obliteration of the field (Figs. 3B
and 4).
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Having shown that administration of IP-10 led to a reduction in
bleomycin-induced fibrosis we next assessed the effect of
administration of IP-10 on in vivo angiogenesis. Lungs from mice
treated with IP-10 demonstrated decreased angiogenic activity in the
CMP assay (Fig. 2
, IIB; zero of six corneas (0%) positive)
compared with lungs from HSA-treated control mice (Fig. 2, IIA; five of six corneas (83%) positive). Furthermore,
IP-10 led to a reduction in the total number of endothelial cells in
the lung as assessed by dual expression of CD31 and factor VIII-related
Ag using FACS analysis (Fig. 3C).
Systemic administration of IP-10 does not alter lung lymphocyte populations
IP-10 is a potent T cell and NK cell chemoattractant, and BPF has
been associated with T cell infiltration. To exclude the possibility
that the beneficial effect of systemic (i.m.) administration of IP-10
was due to the creation of a gradient preventing lymphocytes and NK
cells from entering the lung, we assessed lymphocyte populations in the
lungs of mice treated with either IP-10 or HSA. We found no difference
in the presence of CD45-, CD4-, CD8-, CD19-, or NK1.1-positive
leukocytes between the two groups (Table I).
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Having shown that IP-10 reduces BPF, we were next interested to
determine whether IP-10 had any direct action on pulmonary fibroblasts.
Pulmonary fibroblasts were isolated from mouse lungs and stimulated
with various concentrations of IP-10 in combination with PDGF, whereas
PDGF alone was used as a positive control. Proliferation was measured
using the incorporation of [3H]thymidine. As
shown in Fig. 5, IP-10 had no effect on
fibroblast proliferation, nor did it inhibit PDGF-induced
proliferation. The same concentrations of IP-10 alone had no effect on
proliferation compared with that in controls (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although angiogenesis has been shown to play a role in the evolution of tissue repair and fibroplasia associated with acute lung injury and sarcoidosis (24, 25), the contribution of neovascularization to the pathogenesis of fibrosis in IPF has until recently been largely ignored. The existence of morphological neovascularization in IPF was originally identified by Turner-Warwick (3), who performed postmortem studies on the lungs of patients with widespread IPF and demonstrated neovascularization/vascular remodeling that was often associated with anastomoses between the systemic and pulmonary microvasculatures.
Further evidence of neovascularization during the pathogenesis of pulmonary fibrosis has been seen in a rat model of BPF (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 lung (3), and this neovascularization appeared to lead to the formation of systemic-pulmonary anastomoses (4).
We have 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, which favors net angiogenesis in this disease (11). We found that levels of IL-8 were greater from tissue specimens of IPF patients compared with those in control tissue (11). In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects compared with those from IPF patients (11). When IL-8 or IP-10 were depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced or enhanced, respectively (11). 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, we have recently extended our studies to an animal model of fibrotic lung disease (12). 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). Although these pathologic changes occur in a more rapid fashion than human IPF, the rodent pulmonary inflammatory response to intratracheal bleomycin challenge constitutes a representative model of human IPF. 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 MIP-2 that was correlated to a greater angiogenic response and total lung hydroxyproline content (12). When MIP-2 was depleted in vivo by passive immunization, fibrosis was significantly reduced without a change in the presence of pulmonary neutrophils, fibroblast proliferation, or increases in collagen mRNA levels in isolated fibroblast cultures (12). This was also paralleled by a reduction in angiogenesis (12). These results demonstrate that the angiogenic CXC chemokine, MIP-2, is an important factor that regulates angiogenesis/fibrosis in pulmonary fibrosis (12).
In the current study we have demonstrated that during the pathogenesis of BPF there is a relative deficiency of the CXC chemokine, IP-10, which favors augmented net angiogenic activity. Lung tissue from bleomycin-treated animals had decreased levels of IP-10 compared with controls and demonstrated in vivo angiogenic activity that could be significantly attenuated in the presence of IP-10. These findings support our previous observations of increased angiogenic activity in IPF lung tissue, which was associated with a deficiency of IP-10 in the presence of elevated levels of IL-8, and our recent demonstration that neutralization of the angiogenic CXC chemokine, MIP-2, attenuates the fibrotic response to bleomycin via inhibition of angiogenesis (11, 12).
Systemic administration of recombinant IP-10 attenuated the fibrotic response to bleomycin. The magnitude of reduction in hydroxyproline was similar to that previously shown in cytokine neutralization studies (12, 14, 26, 27, 28). These findings correlated with histopathologic findings. Furthermore, systemic administration of IP-10 in our in vivo model led to a significant reduction in lung-derived angiogenic activity. Further indirect support for the role of IP-10 as an angiostatic factor during the pathogenesis of BPF is the lack of a direct effect of IP-10 on fibroblast proliferation. This supports the contention that IP-10 has no direct effect on pulmonary fibroblasts, suggesting that the beneficial effects of systemic IP-10 are mediated through the regulation of angiogenesis. Moreover, IP-10 transgenic mice have impaired wound healing, with abnormal blood vessel formation (29). This demonstrates an important role for IP-10 in modulating the repair process through its angiostatic properties. Interestingly, angiogenesis has been implicated in the pathogenesis of other chronic inflammatory disorders, such as psoriasis and rheumatoid arthritis, and the inhibition of angiogenesis improves Ag-induced arthritis in rabbits (30, 31, 32, 33).
These findings are similar to our previous studies of IP-10 in the
regulation of angiogenesis in association with tumorigenesis of human
non-small cell lung carcinoma and its potential to inhibit tumor growth
and metastases via its angiostatic activity (10).
Interestingly, IFN-, a major inducer of IP-10 from a number of cells
(34, 35, 36, 37, 38), is a known inhibitor of wound repair, in part
due to its angiostatic properties (39), and has been shown
to attenuate fibrosis in BPF (40). Moreover, the
anti-tumor effect of IL-12 appears to be mediated via a cytokine
cascade involving IFN-, and hence IP-10 and MIG through their
inhibitory effects on tumor vasculature (41, 42). This
supports the idea that the distal mediator of the effects of IFN- is
in part related to IP-10.
IP-10 is a potent T cell and NK cell chemoattactant (38), and BPF is associated with T cell infiltration, in which CD4 or CD8 lymphocytes modulate production of profibrotic mediators from mononuclear phagocytes (43, 44). Furthermore, the fibrotic response to bleomycin is significantly reduced in athymic mice (43). Depletion of CD4 or CD8 lymphocytes attenuates BPF (44). Interestingly, combined depletion of CD4 and CD8 lymphocytes has a greater than additive benefit over depletion of either subset alone (44). Systemic administration of IP-10 did not lead to any alteration in lung lymphocyte populations or NK cells. These studies suggest an alternative biological role for IP-10 in BPF other than lymphocyte recruitment, specifically in the regulation of angiogenesis. We cannot exclude that other chemokines or chemoattractants may be recruiting T cells and NK cells in this model. Moreover, recent work calls into question the role of the adaptive immune response in BPF and supports the idea that IP-10 is working in a T cell-independent manner (45)
In conclusion, we have shown that in the context of BPF, a proangiogenic environment exists. This proangiogenic environment may be important in supporting fibroplasia and deposition of extracellular matrix during the pathogenesis of BPF. This persistent proangiogenic environment contrasts with the normal process of tissue repair, in which angiogenesis is usually rapidly expressed, transient, and tightly regulated (46, 47, 48). In addition, administration of IP-10 attenuates the fibrotic response to bleomycin through a mechanism that is independent of fibroblast proliferation or alteration of lung lymphocyte or NK cell populations. Our findings support the idea that IP-10 inhibits 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 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; BPF, bleomycin-induced pulmonary fibrosis; CMP, corneal micropocket; HSA, human serum albumin; MIP-2, macrophage inflammatory protein-2; IP-10, IFN--inducible protein; ELR, Glu-Leu-Arg; GRO, growth-related oncogene; ULAM, unit for laboratory animal medicine; PDGF, platelet-derived growth factor.
Received for publication June 2, 1999. Accepted for publication September 7, 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-mouse model of lung fibrosis: downregulation of TGF-ß and procollagen I and III gene expression. Exp. Lung Res. 21:791.[Medline]
vß3 antagonist. J. Clin. Invest. 103:47.[Medline]
interferon-induced protein (IP-10) in delayed immune responses in human skin. J. Exp. Med. 166:1098.-inducible protein (-IP-10) gene expression in cultured normal human keratinocytes. Arch. Dermatol. Res. 286:471.[Medline]
-inducible protein 10 in lymphoid organs and inducible expression in T cells and thymocytes. J. Exp. Med. 179:1373./ß injection. Int. J. Exp. Pathol. 74:79.[Medline]
interferon on the cellular responses to bleomycin in mice. Exp. Lung Res. 14:687.[Medline]
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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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 K. M. Antoniou, A. Tzouvelekis, M. G. Alexandrakis, K. Sfiridaki, I. Tsiligianni, G. Rachiotis, N. Tzanakis, D. Bouros, J. Milic-Emili, and N. M. Siafakas Different angiogenic activity in pulmonary sarcoidosis and idiopathic pulmonary fibrosis. Chest, October 1, 2006; 130(4): 982 - 988. [Abstract] [Full Text] [PDF]
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 S. Boodoo, E. W. Spannhake, J. D. Powell, and M. R. Horton Differential regulation of hyaluronan-induced IL-8 and IP-10 in airway epithelial cells Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L479 - L486. [Abstract] [Full Text] [PDF]
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J. Pan, M. D. Burdick, J. A. Belperio, Y. Y. Xue, C. Gerard, S. Sharma, S. M. Dubinett, and R. M. Strieter CXCR3/CXCR3 Ligand Biological Axis Impairs RENCA Tumor Growth by a Mechanism of Immunoangiostasis J. Immunol., February 1, 2006; 176(3): 1456 - 1464. [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. 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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 K. Chen, Y. Wei, A. Alter, G. C. Sharp, and H. Braley-Mullen Chemokine expression during development of fibrosis versus resolution in a murine model of granulomatous experimental autoimmune thyroiditis J. Leukoc. Biol., September 1, 2005; 78(3): 716 - 724. [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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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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A. M. Tager, R. L. Kradin, P. LaCamera, S. D. Bercury, G. S. V. Campanella, C. P. Leary, V. Polosukhin, L.-H. Zhao, H. Sakamoto, T. S. Blackwell, et al. Inhibition of Pulmonary Fibrosis by the Chemokine IP-10/CXCL10 Am. J. Respir. Cell Mol. Biol., October 1, 2004; 31(4): 395 - 404. [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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J. H. Dauber, K. F. Gibson, and N. Kaminski Interferon-{gamma} 1b in Idiopathic Pulmonary Fibrosis: What We Know and What Must We Learn Am. J. Respir. Crit. Care Med., July 15, 2004; 170(2): 107 - 108. [Full Text] [PDF]
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R. M. Strieter, K. M. Starko, R. I. Enelow, I. Noth, V. G. Valentine, and the other members of the Idiopathic Pulmonary Fibr Effects of Interferon-{gamma} 1b on Biomarker Expression in Patients with Idiopathic Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., July 15, 2004; 170(2): 133 - 140. [Abstract] [Full Text] [PDF]
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 N R Simler, P E Brenchley, A W Horrocks, S M Greaves, P S Hasleton, and J J Egan Angiogenic cytokines in patients with idiopathic interstitial pneumonia Thorax, July 1, 2004; 59(7): 581 - 585. [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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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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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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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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S. G. Kallapur, A. H. Jobe, M. Ikegami, and C. J. Bachurski Increased IP-10 and MIG Expression after Intra-amniotic Endotoxin in Preterm Lamb Lung Am. J. Respir. Crit. Care Med., March 1, 2003; 167(5): 779 - 786. [Abstract] [Full Text] [PDF]
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E. A. Renzoni, D. A. Walsh, M. Salmon, A. U. Wells, P. Sestini, A. G. Nicholson, S. Veeraraghavan, A. E. Bishop, H. M. Romanska, P. Pantelidis, et al. Interstitial Vascularity in Fibrosing Alveolitis Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 438 - 443. [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 (2009): 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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Z. Zhu, B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, and J. A. Elias IL-13-Induced Chemokine Responses in the Lung: Role of CCR2 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling J. Immunol., March 15, 2002; 168(6): 2953 - 2962. [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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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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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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B. Ebrahimi, B. M. Dutia, D. G. Brownstein, and A. A. Nash Murine Gammaherpesvirus-68 Infection Causes Multi-Organ Fibrosis and Alters Leukocyte Trafficking in Interferon-{{gamma}} Receptor Knockout Mice Am. J. Pathol., June 1, 2001; 158(6): 2117 - 2125. [Abstract] [Full Text] [PDF]
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 F. Recchia, S. De Filippis, M. Rosselli, G. Saggio, A. Cesta, L. Fumagalli, and S. Rea Phase 1B Study of Subcutaneously Administered Interleukin 2 in Combination with 13-cis Retinoic Acid as Maintenance Therapy in Advanced Cancer Clin. Cancer Res., May 1, 2001; 7(5): 1251 - 1257. [Abstract] [Full Text]
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 C. Choi, X. Xu, J.-W. Oh, S. J. Lee, G. Y. Gillespie, H. Park, H. Jo, and E. N. Benveniste Fas-induced Expression of Chemokines in Human Glioma Cells: Involvement of Extracellular Signal-regulated Kinase 1/2 and p38 Mitogen-activated Protein Kinase Cancer Res., April 1, 2001; 61(7): 3084 - 3091. [Abstract] [Full Text]
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M. T. Liu, D. Armstrong, T. A. Hamilton, and T. E. Lane Expression of Mig (Monokine Induced by Interferon-{{gamma}}) Is Important in T Lymphocyte Recruitment and Host Defense Following Viral Infection of the Central Nervous System J. Immunol., February 1, 2001; 166(3): 1790 - 1795. [Abstract] [Full Text] [PDF]
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M. Itakura, A. Tokuda, H. Kimura, S. Nagai, H. Yoneyama, N. Onai, S. Ishikawa, T. Kuriyama, and K. Matsushima Blockade of Secondary Lymphoid Tissue Chemokine Exacerbates Propionibacterium acnes-Induced Acute Lung Inflammation J. Immunol., February 1, 2001; 166(3): 2071 - 2079. [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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M. T. Liu, B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, and T. E. Lane Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease J. Immunol., September 1, 2000; 165(5): 2327 - 2330. [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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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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