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(American Journal of Pathology. 2007;170:1152-1164.)
© 2007 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.060649

Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

Elizabeth M. Pierce*, Kristin Carpenter*, Claudia Jakubzick*, Steven L. Kunkel*, Kevin R. Flaherty{dagger}, Fernando J. Martinez{dagger} and Cory M. Hogaboam*

From the Department of Pathology,* Division of Pulmonary Medicine, and the Department of Internal Medicine,{dagger} University of Michigan Medical School, Ann Arbor, Michigan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 x 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7.

A persistently high mortality rate plagues idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP), the severest form of idiopathic interstitial pneumonia (IIP).1 This is due to the failure of conventional immunomodulatory therapies, such as corticosteroids, azathioprine, and cyclophosphamide, to halt effectively the aggressive profibrotic and tissue repair processes in this disease.2 Newly diagnosed IPF/UIP patients face respiratory failure and a plethora of complications including cardiovascular disease, lung cancer, and pulmonary embolism; most succumb to this disease within 3 to 5 years of diagnosis.3 With its growing prevalence worldwide,4 the need for novel therapies for IPF/UIP has become a major research focus, but a poor understanding of the etiopathogenesis of this disease has slowed progress in this regard.5 The aberrant parenchymal remodeling in IIP is characterized by the expansion of fibroblasts and myofibroblasts, and previous studies have shown that IPF/UIP fibroblasts have unique proliferation and synthetic properties relative to fibroblasts from other IIPs or normal lung tissues.6-9 These cells may arise from alterations in their microenvironment (due to dysregulated epithelial cell/mesenchymal cell interactions10 ) or be recruited out of the bone marrow (see reviews.11,12 ). Regardless of the source of these cells, controlling their activity in the lung is paramount, and ongoing genomic and proteomic analyses of intact lung biopsies13,14 and biopsy-derived fibroblasts15 have revealed putative targets to achieve this regulation.

Mouse models of pulmonary fibrosis have provided experimental paradigms with which to address abnormal tissue remodeling and scarring in the respiratory system.16 A number of approaches have been used to induce pulmonary fibrosis, and these include transgenic and gene transfer, radiation, inorganic irritants such as silica, and drugs promoting oxidant-induced inflammatory injury such as bleomycin.17 Of these models, the bleomycin model remains the most widely used because of its reproducibility and pathological similarity to human pulmonary fibrosis.17 Accordingly, the bleomycin model has been used to assess a number of targets of interest in IPF/UIP.13,14 Unfortunately, no animal model exists that fully recapitulates the clinico-pathological features of IPF/UIP, and debate still exists pertaining to the relative importance of ongoing inflammatory injury (the primary mode for inducing experimental fibrosis) to end-stage UIP.18,19

Given this dilemma, the present study addressed an alternative strategy for inducing experimental pulmonary fibrosis. Mice that are genetically immunodeficient due to severe combined immunodeficiency (scid) gene mutation or recombinase-activating gene 1 (rag-1) knockout have been extensively used as in vivo hosts of adoptively transferred normal or diseased human cells. Herein, we report that the adoptive intravenous (i.v.) transfer of either IPF/UIP or nonspecific interstitial pneumonia (NSIP; another less severe form of IIP20 ) but not normal fibroblasts into C.B-17 mice with the scid-beige (C.B-17SCID/bg) mutation initiated and maintained pulmonary fibrosis. Histological and biochemical evidence of fibrosis was first evident in both IIP fibroblast groups of C.B-17SCID/bg mice at day 35 and was prominent at day 63 after fibroblast injection. Cytokines and chemokines both seem to have major roles in the pathogenesis of IIP,21 and enzyme-linked immunosorbent assay (ELISA) analysis of whole-lung samples from C.B-17SCID/bg mice that received either IPF/UIP or NSIP fibroblasts revealed significant elevations in murine interleukin (IL)-13, CC ligand (CCL) 6, and CCL21 at day 63 compared with whole-lung levels measured at the earlier time point or in lung tissue from mice that did not receive fibroblasts. IL-1322,23 and CCL624 are mediators of pulmonary fibrosis, but the role of CCL21 in pulmonary remodeling events was unknown. Impetus to examine the role of CCL21 and CC chemokine receptor 7 (CCR7) in the pulmonary remodeling events precipitated by human IIP fibroblasts stemmed from our recent finding that CCR7 expression was increased in IIP biopsies,25 and the migratory, synthetic, and proliferative properties of IIP fibroblasts are significantly enhanced by CCL21 (E.M.P. and C.M.H., unpublished data). In separate immunoneutralization studies, the targeting of either human CCL21 or CCR7 (the receptor for CCL2126 ) from days 35 to 63 after IIP fibroblast injection into C.B-17SCID/bg mice significantly reduced all parameters of pulmonary fibrosis compared with groups of C.B-17SCID/bg mice receiving IIP fibroblasts and IgG. Together, these data highlight the creation of a new murine model of pulmonary fibrosis initiated and maintained by the i.v. introduction of IIP fibroblasts into C.B-17SCID/bg mice and demonstrate a novel role for CCL21 and CCR7 in the maintenance of fibrosis in this model.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female, ICR-scid (ICRSCID), C.B-17-scid (C.B-17SCID), and C.B-17-scid-beige (C.B-17SCID/bg) mice (6 to 8 weeks old) were purchased from Taconic Farms (Germantown, NY), and all SCID mice were housed in a gnotobiotic barrier facility at the University of Michigan Medial School. The first two groups of mice have the scid mutation leading to a lack of both T and B lymphocytes due to a V(D)J recombination defect, whereas C.B-17SCID/bg mice have two mutations: the first is the scid mutation, and the second is a beige mutation leading to a major defect in cytotoxic T-cell and macrophage function and a selective impairment in NK cell function. All mice had access to autoclaved water and pelleted mouse diet ad libitum. All procedures described below were performed in a sterile, laminar environment and were approved by an animal care and use committee at the University of Michigan Medical School.

Human Fibroblast Culture

A mixed cell population was obtained from mechanically dissociated IPF/UIP and NSIP surgical lung biopsies, and pure human fibroblast cultures were derived as previously described in detail.9 Normal lung fibroblasts were purified in the same manner from cell suspensions of normal margins associated with resected lung tumor tissue. In the present study, a total of 10 IPF/UIP, six NSIP, and four normal fibroblast lines were used after the fourth passage in the initial, model characterization, and therapeutic intervention studies described herein. An institutional review board at the University of Michigan Medical School approved this study.

Intravenous Introduction of Human Pulmonary Fibroblasts into SCID Mice

Single-cell preparations of IPF/UIP, NSIP, and normal fibroblasts were obtained after trypsinization of 150-cm2 tissue culture flasks and labeled with PKH26 dye according to the manufacturer’s directions (Sigma Co., St. Louis, MO). Each labeled fibroblast line was diluted to 1 x 106 cells/ml of phosphate-buffered saline (PBS), and 1 ml of this suspension was injected via a tail vain into groups of five SCID mice. Other groups of five SCID mice were injected intravenously with PKH26 and PBS labeling solution alone (ie, control group). Mice were euthanized by anesthesia overdose at days 7, 21, 35, 49, and 63 after the i.v. human pulmonary fibroblast transfer. Whole-lung tissue was dissected at these times for molecular, histological, biochemical, and/or proteomic analysis (see below).

Assessing the Role of CCL21 and CCR7 in C.B-17SCID/bg Mice after the Adoptive Transfer of Human Pulmonary Fibroblasts

To address the role of CCL21 and CCR7, its receptor, in the pulmonary remodeling response after the i.v. adoptive transfer of human fibroblasts, groups of C.B-17SCID/bg mice received IPF/UIP (n = 35 mice), NSIP (n = 20 mice), normal fibroblasts (n = 15 mice), or vehicle (ie, PBS) alone (n = 15 mice). Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 µg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63. At day 63, all mice were euthanized by anesthesia overdose, and whole-lung tissue was dissected for molecular, histological, biochemical, and proteomic analysis (see below).

Molecular Analysis

Total RNA was isolated and cDNA generated from whole-lung samples as previously described in detail.27 Changes in gene profiles for human and murine chemokine and chemokine receptors were analyzed in pooled samples (n = 5) using nonradioactive GEArray gene array membranes according to the manufacturer’s instructions (SuperArray, Inc., Bethesda, MD), and signal intensities were determined as previously described in detail.28 Individual whole-lung cDNA samples (n ≥ 5) were analyzed for human CCR7, collagen I, cathepsin E, matrix metalloproteinase (MMP)-2, MMP-9, MMP-19, fibronectin, tissue inhibitors of metalloproteinases (TIMP)-1, and glyceraldehyde-3-phosphate dehydrogenase expression by real-time quantitative RT-PCR procedure using an 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) as previously described.27

Histological Analysis

After anesthesia-induced euthanasia, the right lobes from each mouse were dissected, fully inflated with 10% formalin solution, and placed in fresh formalin for 24 hours. Standard histological techniques were used to paraffin-embed each lobe, and 5-µm sections were stained with hematoxylin and eosin and Mason trichrome for histological analysis. Additional unstained whole-lung tissue sections were analyzed via fluorescent microscopy.

Biochemical Analysis

Whole-left lung samples were homogenized in 1x PBS and pelleted by centrifugation. The cell-free supernatants were removed for ELISA analysis, and the pellets were vacuum-dried and resuspended in 0.5 mol/L glacial acetic acid. The tissue was then processed for hydroxyproline concentration as previously described.29

Proteomic Analysis

Murine IL-13, CCL6, CCL21, interferon-{gamma}, IL-12, IL-4, CCL2, CCL7, CCL17, CCL3, CXC chemokine ligand (CXCL)13, tumor necrosis factor-{alpha}, CXCL10, CXCL9, and CXCL2 proteins were analyzed in 50 µl of cell-free supernatants from homogenized whole-lung samples using a standardized sandwich ELISA technique (R&D Systems) as previously described in detail.27

Statistical Analysis

All results are expressed as mean ± SEM. One-way analysis of variance analysis and Tukey-Kramer or Dunnett’s multiple comparisons tests were used to detect statistical differences between UIP, NSIP, normal, and control SCID mouse groups. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The Adoptive Intravenous Transfer of Either IPF/UIP or NSIP, but Not Normal, Pulmonary Fibroblasts Promoted Lung Histopathology and Remodeling in C.B-17SCID/bg Mice

Initial studies were undertaken to assess the impact of adoptively transferred normal and IIP fibroblasts on the lung architecture in various strains of SCID mice including ICRSCID, C.B-17SCID, and C.B-17SCID/bg (n = 5 per time point). These initial studies were undertaken using three IPF/UIP and two normal human fibroblast lines. All human fibroblast lines were labeled with PKH26 before injection into the SCID mouse groups thereby allowing for the detection of labeled cells in histological sections. At days 7 (Figure 1, B and C) and 21 (not shown) after injection, collections of human fibroblasts were detected in pulmonary blood vessels in each SCID group. However, the intensity of this marker diminishes after 21 days (information from the provided Sigma data sheet), and hence, we failed to detect PKH26 fluorescence in histological sections from each at day 35 after fibroblast injection (not shown). Other organs (ie, liver, spleen, and kidney) presumably contained human pulmonary fibroblasts, but we observed no evidence of gross macroscopic alterations to these organs. Because IIP is a lung-specific disease,30 which does not exhibit fibrogenesis in any other organ, a detailed histological analysis of other organs was not undertaken in the present study.


Figure 1
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  		Figure 1. Representative whole-lung tissues sections from C.B-17SCID/bg mice that received PBS + PKH26 (A) or IPF/UIP fibroblasts + PBS + PKH26 (B and C). The presence of fluorescently labeled human fibroblasts in the lung was observed in C.B-17SCID/bg mice that received human IPF/UIP fibroblasts 7 days previously (B and C). The clumped human fibroblasts were typically detected in the walls of pulmonary blood vessels (B and C). Original magnifications: x100 (A), x200 (B), and x400 (C).

 
Examination of whole-lung sections in groups of SCID mice at later time points revealed that marked lung remodeling was only present in C.B-17SCID/bg mice. Specifically, no fibroproliferation was observed at day 49 after the introduction of IPF/UIP fibroblasts into ICRSCID mice (n = 5 mice; not shown). Likewise, the i.v. adoptive transfer of IPF/UIP fibroblasts into C.B-17SCID mice failed to elicit histologically evident pulmonary remodeling at days 7 (n = 5 mice), 21 (n = 10 mice), 35 (n = 5 mice), or 49 (n = 5 mice) after the i.v. injection of IPF/UIP fibroblasts.

Given the presence of marked pulmonary histopathology in the C.B-17SCID/bg group, all subsequent studies described below involved the adoptive transfer of normal and IIP fibroblasts into C.B-17SCID/bg mice. In the model characterization study, four IPF/UIP, four NSIP, and one normal fibroblast lines were adoptively transferred into separate groups of C.B17-SCID/bg mice, and pulmonary histopathological, genomic, and proteomic alterations were analyzed at days 35 and 63 after fibroblast transfer.

Little or no pulmonary histopathology was observed in C.B-17SCID/bg mice that received normal pulmonary fibroblasts (Figure 2A) . However, C.B-17SCID/bg mice exhibited significant pulmonary histopathology, which was evident at day 35 after the i.v. injection of either NSIP (Figure 2B) or IPF/UIP (Figure 2C) fibroblast lines. The pulmonary histopathology in C.B-17SCID/bg mice following NSIP or IPF/UIP pulmonary fibroblasts was characterized by disruption of the alveolar space, apparent fibroproliferation, and the presence of eosinophilic granulocytes (Figure 2, B and C) . Mason trichrome staining of histological tissue sections from the lungs of C.B-17SCID/bg mice at day 35 after the adoptive transfer of normal (Figure 2D) , NSIP (Figure 2E) , or IPF/UIP (Figure 2F) human pulmonary fibroblasts revealed the presence of extracellular matrix (stained light blue) in remodeled areas in C.B-17SCID/bg groups that received the IIP but not normal fibroblasts.


Figure 2
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  		Figure 2. Representative hematoxylin and eosin-stained histological sections from C.B-17SCID/bg mice that received normal (A), NSIP (B), or IPF/UIP (C) fibroblasts. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (D), NSIP (E), or IPF/UIP (F) fibroblasts are shown. Lung samples were removed at day 35 after the adoptive i.v. transfer of human pulmonary fibroblasts into C.B-17SCID/bg mice. Original magnification, x200 (A, C–F) or x400 (B).

 
Later analysis of histological sections from C.B-17SCID/bg groups revealed major differences in the extent and appearance of the pulmonary remodeling precipitated by the introduction of IPF/UIP or NSIP fibroblasts. The lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts 63 days previously exhibited a heterogeneous appearance with areas of relatively normal-appearing lung tissue (Figure 3A) adjacent to areas of severe interstitial disruption and remodeling (Figure 3B) . In addition, foci of human fibroblasts were apparent in the lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts (Figure 3B , inset), but these foci were detected in blood vessels and not in interstitial areas as in clinical UIP.1 The histological pattern in whole-lung tissue sections from C.B-17SCID/bg mice that received NSIP fibroblasts 63 days previously was characterized by interstitial thickening and overtly fibrotic areas, and the remodeling in these mice appeared to involve most of the lung (Figure 3, C and D) . Together, these data showed that the introduction of human IIP fibroblasts into C.B-17SCID/bg mice caused fibrotic lesions in these mice.


Figure 3
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  		Figure 3. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received IPF/UIP (A and B) or NSIP (C and D) fibroblasts 35 or 63 days previously. Normal-appearing areas (A), intensely fibrotic areas (B), and foci of human fibroblasts (observed at day 35 after fibroblast injection; inset to B) were apparent in whole-lung histological sections from C.B-17SCID/bg mice that received IPF/UIP fibroblasts. In contrast, whole-lung histological sections from C.B-17SCID/bg mice that received NSIP showed a more generalized involvement of the lung tissue examined with varying degrees of fibrosis and remodeling (C and D). Lung samples were removed at days 35 or 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, x200.

 
Hydroxyproline Levels Were Significantly Altered in a Temporally Dependent Manner after the Introduction of IIP Fibroblasts into C.B-17SCID/bg Mice

Hydroxyproline is a commonly used marker of de novo collagen synthesis in experimental models involving pulmonary remodeling.31 In the present study, hydroxyproline levels were measured in whole-lung samples from C.B-17SCID/bg mice that had received no fibroblasts or normal, NSIP, or IPF/UIP human pulmonary fibroblasts either 35 or 63 days previously. As shown in Figure 4 , hydroxyproline levels were unchanged at these time points after the introduction of normal fibroblasts. The levels of hydroxyproline in these C.B-17SCID/bg groups were 4.7 ± 0.3 and 5.6 ± 0.5 µg/mg protein at days 35 and 63, respectively, and these hydroxyproline levels were similar to those detected in C.B-17SCID/bg mouse groups that did not receive human fibroblasts (4.5 ± 1.3 µg/mg protein). However, the lungs of mice contained greater amounts of hydroxyproline at day 35 after intravenously transferred NSIP (11.4 ± 3.7 µg/mg protein) or IPF/UIP (8.2 ± 2 µg/mg protein) fibroblasts compared with the normal fibroblast group. In addition, hydroxyproline levels were further increased 3- and 2.5-fold in the NSIP (31 ± 11 µg/mg protein) and IPF/UIP (22 ± 4 µg/mg protein) fibroblast groups, respectively, at day 63 after i.v. adoptive transfer (Figure 4) . At the day-63 time point, the increase in hydroxyproline levels in the C.B-17SCID/bg groups that received either NSIP or IPF/UIP human fibroblasts reached statistical significance compared with the C.B-17SCID/bg group that received normal fibroblasts. Thus, the intravenous injection of human IIP fibroblasts enhanced hydroxyproline levels at day 35 and, most demonstrably, at day 63 after adoptive transfer.


Figure 4
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  		Figure 4. Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ± SEM. *P ≤ 0.05 compared with hydroxyproline levels measured at day 63 after adoptive transfer of normal fibroblasts into C.B-17SCID/bg mice.

 
Whole-Lung Cytokine Analysis Showed That Murine IL-13, CCL6, and CCL21 Were Significantly Elevated in the Lungs of C.B-17SCID/bg Mice That Received IIP Fibroblasts

Whole-lung ELISA analysis of several cytokines, CC ligand, and CXC ligand chemokines at days 35 and 63 after the i.v. adoptive transfer of human normal or IIP fibroblasts revealed a number of statistically significant changes in murine IL-13, CCL6, and CCL21 (Figure 5) . Although the whole-lung levels of IL-13 in the C.B-17SCID/bg group that received normal fibroblasts were below the level of ELISA detection, whole-lung IL-13 levels were significantly greater in the C.B-17SCID/bg group that received either NSIP or IPF/UIP fibroblasts compared with the C.B-17SCID/bg group that received normal fibroblasts 35 and/or 63 days previously (Figure 5 , top). In addition, significantly more IL-13 was detected in the lungs of C.B-17SCID/bg mice that received IPF/UIP fibroblasts at day 63 versus 35 after adoptive transfer of fibroblasts. At days 63 after fibroblast injection, whole-lung CCL6 levels were significantly greater in the C.B-17SCID/bg groups that received either IPF/UIP or NSIP fibroblasts compared with the C.B-17SCID/bg group that received normal fibroblasts (Figure 5 , middle). Significantly greater CCL6 was detected in the lungs of C.B-17SCID/bg mice that received IPF/UIP or NSIP fibroblasts at day 63 versus 35 after adoptive transfer of fibroblasts (Figure 5 , middle). The only other murine CC ligand that was altered by the introduction of human fibroblasts into C.B-17SCID/bg mice was CCL21. This chemokine was significantly greater in the NSIP and IPF/UIP fibroblast C.B-17SCID/bg groups at day 63 compared with the normal fibroblast C.B-17SCID/bg groups at day 63. In addition, whole-lung murine CCL21 levels were significantly elevated in IPF/UIP fibroblast groups at day 63 after fibroblast transfer compared with the NSIP fibroblast group at the same time (Figure 5 , bottom). Finally, significantly higher levels of CCL21 were present in whole-lung samples from the IIP fibroblasts C.B-17SCID/bg group at day 63 after adoptive transfer compared with the IIP fibroblasts C.B-17SCID/bg groups at day 35 after adoptive transfer. Thus, taken together, these data suggested that the presence of IIP fibroblasts, but not normal fibroblasts, in C.B-17SCID/bg mice significantly altered the whole-lung levels of murine cytokines and chemokines with established (ie, IL-1322,23 and CCL624 ) and putative (ie, CCL21) profibrotic roles in the lung.


Figure 5
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  		Figure 5. Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice, and all soluble proteins were measured by specific ELISA. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with appropriate the C.B-17SCID/bg group that received normal fibroblasts. {tau}{tau}P ≤ 0.01, {tau}{tau}{tau}P ≤ 0.001 compared with whole-lung cytokine and chemokine levels at the day 35 time point in the C.B-17SCID/bg groups with either IPF/UIP or NSIP fibroblasts The whole-lung cytokine and chemokine levels in control C.B-17SCID/bg group that did not receive fibroblasts were as follows: IL-13, 0.18 ± 0.014 ng/mg protein; CCL6, 0.37 ± 0.05 ng/mg protein; and CCL21, 2.0 ± 0.1 ng/mg protein.

 
Human CCR7 and CCL21 Gene Transcripts Were Present in the Lungs of C.B-17SCID/bg Mice That Received Human Pulmonary Fibroblasts 35 Days Previously

The changes in whole-lung levels of murine CCL21 in C.B-17SCID/bg mice that received either NSIP or IPF/UIP fibroblasts were intriguing in light of previous studies demonstrating the important remodeling role of this CC ligand in the kidney32 and liver.33 This prompted further analysis of this CC ligand and its receptor, CCR7, during the pulmonary remodeling responses elicited by human IIP fibroblasts. Human transcripts for CCR7 and CCL21 were detected by SuperArray gene analysis, and among the three C.B-17SCID/bg groups, the greatest transcript expression for CCR7 and CCL21 was present in lung samples from the IPF/UIP fibroblast group (Table 1) . Furthermore, the presence of CCR7 was confirmed by TaqMan analysis, and again the IPF/UIP group exhibited the highest CCR7 transcript expression among the C.B-17SCID/bg groups (Table 1) . SuperArray and TaqMan analysis of murine CCR7 and CCL21 also confirmed the presence of these transcript products in all three C.B-17SCID/bg groups that received human fibroblasts, and the highest levels of both transcripts were present in the C.B-17SCID/bg groups that received IPF/UIP fibroblasts (not shown). Thus, the i.v. adoptive transfer of normal and IIP fibroblasts resulted in the presence of human gene transcripts for CCR7 and CCL21.


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  		Table 1. SuperArray Analysis of Human CCR7 and CCL21 and TaqMan Analysis of Human CCR7 in Whole-Lung Samples from C.B-17SCID/bg Mice at Day 35 after i.v. Human Fibroblast Injection

 
Quantitative TaqMan PCR Analysis of Murine Extracellular Matrix-Associated Genes after the Adoptive Transfer of Human Fibroblasts into C.B-17SCID/bg Mice

Lung alterations in murine extracellular matrix-associated genes were analyzed using quantitative PCR analysis. As shown in Figure 6 , top, the expression of collagen 1, cathepsin E, MMP-19, and TIMP-1 was present in C.B-17SCID/bg mice that received normal, NSIP, and IPF/UIP human fibroblasts 63 days previously. Most importantly, when transcript levels for these genes in fibroblast-challenged mice were compared with transcript levels in control C.B-17SCID/bg mice, the increases in collagen 1 and cathepsin transcript expression in C.B-17SCID/bg mice that received either type of IIP fibroblast and MMP-19 and TIMP-1 transcript expression in mice that received IPF/UIP fibroblasts reached statistical significance when compared with the transcript increases for these genes in C.B-17SCID/bg mice that received normal human fibroblasts (Figure 6 , top). Other extracellular matrix genes were analyzed by TaqMan, and the presence of MMP-2, MMP-9, and fibronectin was confirmed. No significant differences in levels of these transcripts were observed among the groups of C.B-17SCID/bg mice, but the greatest increase in MMP-2 and fibronectin was observed in the IPF/UIP C.B-17SCID/bg group, whereas the smallest increase in MMP-9 was observed in this same C.B-17SCID/bg group (Figure 6 , bottom). Thus, these data showed that transcript levels for murine extracellular matrix-associated genes were altered by the presence of human fibroblasts in C.B-17SCID/bg mice.


Figure 6
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  		Figure 6. Quantitative TaqMan PCR analysis of extracellular matrix-associated genes, collagen 1, cathepsin E, MMP-19, TIMP-1, MMP-2, MMP-9, and fibronectin in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ± SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS and PKH26 alone. *P ≤ 0.05, **P ≤ 0.01 compared with the C.B-17SCID/bg group that received normal fibroblasts.

 
Immunoneutralization of Human CCR7 or Human CCL21 Abrogated Pulmonary Remodeling in C.B-17SCID/bg Mice That Received Human IIP Fibroblasts

In the next series of experiments, the roles of human CCR7 and CCL21 were assessed in C.B-17SCID/bg mice that received human normal (n = 1 line) or IIP fibroblasts (n = 3 IPF/UIP and n = 2 NSIP lines). Although attempts to measure human CCL21 in whole-lung samples were unsuccessful, presumably because of the presence of this CC ligand at levels below the level of ELISA detection (not shown), previous studies have shown that both mouse and human CCL21 can promote cellular calcium flux via human CCR7.34 In addition, the immunoneutralization of mouse CCL21 using a polyclonal antibody has been shown to abrogate the migration of human dendritic cells and the priming of human T cells in a humanized model of house dust mite-induced allergic airway disease.35 Given the reported cross-reactivity of mouse and human CCL21, a therapeutic protocol was implemented in which either human CCL21 or human CCR7 was targeted by monoclonal antibody administration from days 35 to 63 after the adoptive transfer of fibroblasts. A representative histological survey of whole-lung tissues at day 63 in the three C.B-17SCID/bg groups with the three treatment modalities tested is shown in Figure 7 . In the normal fibroblast C.B-17SCID/bg group, no evidence of interstitial pulmonary remodeling was evident in any of the treatment groups (IgG treatment, Figure 7A ; anti-CCL21 antibody treatment, Figure 7B ; and anti-CCR7 antibody treatment, Figure 7C ). However, the vascular accumulation of normal fibroblasts was histologically apparent in these C.B-17SCID/bg groups, and an example is shown in Figure 7B . None of the treatments altered this feature in C.B-17SCID/bg mice that received normal fibroblasts. Interstitial remodeling was apparent in the NSIP fibroblast C.B-17SCID/bg group that received IgG (Figure 7D) , but this remodeling response was abrogated by either anti-CCL21 antibody (Figure 7E) or anti-CCR7 antibody (Figure 7F) administration from days 35 to 63 after adoptive transfer. Likewise, the interstitial remodeling apparent in the IgG C.B-17SCID/bg group that received IPF/UIP fibroblasts (Figure 7G) was absent in groups of C.B-17SCID/bg mice that received anti-CCL21 antibody (Figure 7H) or anti-CCR7 antibody (Figure 7I) administration from days 35 to 63 after adoptive transfer IPF/UIP fibroblasts. Thus, therapeutic targeting of either CCL21 or CCR7 abrogated histological evidence of interstitial remodeling in C.B-17SCID/bg mice that received IIP fibroblasts.


Figure 7
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  		Figure 7. Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (A–C), NSIP (D–F), or IPF/UIP (G–I) fibroblasts. No interstitial remodeling was apparent in C.B-17SCID/bg mice that received normal fibroblasts, but vascular anomalies were observed in this group (B), and the IgG (A), anti-CCL21 monoclonal antibody (B), and anti-CCR7 monoclonal antibody (C) therapies did not alter the lung histological appearance in this group. Pulmonary remodeling was apparent in C.B-17SCID/bg mice that received NSIP fibroblasts, and this pattern was not altered by IgG (D), whereas the anti-CCL21 antibody (E) or anti-CCR7 antibody (F) therapies markedly reduced the interstitial remodeling in whole-lung samples. Interstitial pulmonary fibrosis was apparent in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and this pattern was not altered by IgG (G), whereas the anti-CCL21 antibody (H) or anti-CCR7 antibody (I) therapies markedly reduced the interstitial remodeling in whole-lung samples. Monoclonal antibody therapies began at day 35 and continued to day 63, and lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, x400.

 
Quantitative TaqMan PCR Analysis of Murine Extracellular Matrix-Associated Genes after the Therapeutic Targeting of Either Human CCL21 or CCR7 in C.B-17SCID/bg Mice That Received Human Normal and IIP Fibroblasts

Quantitative TaqMan analysis of the antibody treatment groups also confirmed that the anti-CCL21 and anti-CCR7 treatments also significantly altered the transcript levels of MMP-2 and MMP-19. Transcript levels for these MMPs in antibody-treated, control C.B-17SCID/bg mice were compared with the levels of these transcripts in antibody-treated, fibroblast-challenged C.B-17SCID/bg mice. Anti-CCL21 antibody treatment significantly reduced the fold increase in MMP-2 transcript levels in C.B-17SCID/bg groups that received normal fibroblasts compared with the fold increase in the transcript levels in C.B-17SCID/bg groups that received IgG (Figure 8 , top). The anti-CCR7 antibody treatment significantly reduced the fold increase in MMP-2 transcript levels compared with the appropriate IgG group. Quantitative TaqMan analysis of MMP-19 revealed that anti-CCR7 significantly increased the fold change in the C.B-17SCID/bg group that received normal fibroblasts, whereas both antibody treatments significantly increased the fold change in this transcript compared with the appropriate IgG group (Figure 8 , bottom). Thus, the antibody treatments used in C.B-17SCID/bg mice challenged with human fibroblasts markedly altered transcript expression for murine extracellular matrix-associated genes.


Figure 8
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  		Figure 8. Quantitative TaqMan PCR analysis of extracellular matrix-associated genes MMP-2 (top) and MMP-19 (bottom) in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ± SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS, PKH26, and one of IgG, anti-CCL21 antibody, and anti-CCR7 antibody. *P ≤ 0.05, ***P ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment.

 
Immunoneutralization of Human CCR7 or Human CCL21 Significantly Reduced Whole-Lung Hydroxyproline Levels in C.B-17SCID/bg Mice That Received Human IIP Fibroblasts

Hydroxyproline levels in whole-lung samples from IgG-, anti-CCL21-, or anti-CCR7-treated control C.B-17SCID/bg mice (ie, mice that did not receive human fibroblasts) and groups of treated mice that received normal, NSIP, or IPF/UIP fibroblasts are shown in Figure 9 . Hydroxyproline levels were increased in whole-lung samples from C.B-17SCID/bg mice that received normal fibroblasts and IgG, but neither antibody treatment altered these levels (Figure 9) . In the NSIP fibroblast C.B-17SCID/bg groups, hydroxyproline levels were significantly increased above those levels measured in the control C.B-17SCID/bg group, and the anti-CCL21 antibody and anti-CCR7 antibody therapies significantly reduced hydroxyproline levels by 52 ± 6.7 and 51 ± 5.6%, respectively, compared with the IgG treatment group (Figure 9) . In the IPF/UIP fibroblast C.B-17SCID/bg group that received IgG, hydroxyproline levels were again significantly increased above those levels measured in the control C.B-17SCID/bg group. In addition, hydroxyproline levels in IPF/UIP fibroblast challenged mice were reduced by 66 ± 7.2 and 59 ± 7.1% in the anti-CCL21 and anti-CCR7 antibody treatment groups, respectively, compared with the IgG-treated C.B-17SCID/bg IPF/UIP fibroblast group (Figure 9) . Thus, these data confirmed that the targeting of either CCL21 or CCR7 markedly and significantly reduced pulmonary remodeling precipitated by the adoptive transfer of NSIP or IPF/UIP fibroblasts in C.B-17SCID/bg mice.


Figure 9
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  		Figure 9. Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received no fibroblasts (ie, control) or received normal, NSIP, or IPF/UIP fibroblasts. All groups of mice received either IgG or monoclonal antibody therapy. IgG, anti-CCL21, and anti-CCR7 monoclonal antibody therapies began in separate groups of C.B-17SCID/bg mice at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ± SEM. *P ≤ 0.05 compared with the control C.B-17SCID/bg group, which did not receive any human fibroblasts; {tau}{tau}{tau}P ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment; {tau}P ≤ 0.05 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgC treatment.

 
Whole-Lung Cytokine Analysis Showed That Murine CCL21 Levels Were Significantly Reduced in the Lungs of C.B-17SCID/bg Mice That Received IPF/UIP Fibroblasts and Anti-CCR7 Antibody Therapy

Because whole-lung levels of murine IL-13, CCL6, and CCL21 were altered and/or increased by the presence of NSIP and IPF/UIP fibroblasts in C.B-17SCID/bg mice, the effects of the IgG and monoclonal antibody treatments on these mediators at day 63 were assessed. As shown in Figure 10 , the anti-CCR7 and anti-CCL21 antibody therapies did not affect whole-lung levels of IL-13 or CCL6 in any of the C.B-17SCID/bg fibroblast groups. However, the anti-CCR7 antibody therapy significantly reduced whole-lung levels of murine CCL21 in the C.B-17SCID/bg group that received IPF/UIP fibroblasts compared with the C.B-17SCID/bg group that received IPF/UIP fibroblasts + IgG (Figure 10) . Thus, the therapeutic targeting of CCR7 reduced murine CCL21 levels but did not affect the levels of the other two mouse mediators found to be elevated in the IIP fibroblast models.


Figure 10
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  		Figure 10. Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts and IgG or monoclonal antibody therapies. Anti-CCL21 and anti-CCR7 monoclonal antibody therapies began at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Specific ELISA was used to measure all soluble proteins. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 compared with indicated protein levels measured in whole-lung samples from C.B-17SCID/bg mice that received human fibroblasts.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present study addressed the following two questions: Do adoptively transferred human pulmonary fibroblasts remodel the lung architecture in SCID mice, and what role do CCL21 and CCR7 exert in the remodeling response precipitated by the adoptive transfer of human fibroblasts? The response to the first question was affirmative because the i.v. adoptive transfer of either 1 x 106 IPF/UIP or NSIP fibroblasts into mice C.B-17SCID/bg mice lacking both adaptive and innate immune features caused fibrosis, which was confirmed using histological, molecular, and biochemical analyses. This fibrosis was abrogated by the therapeutic administration of monoclonal antibodies directed against either human CCL21 or CCR7, thereby demonstrating a major role for this ligand and its receptor in pulmonary fibrosis similar to their roles in renal32 and liver33 fibrosis. Although murine equivalents of known and putative profibrotic mediators were increased in C.B-17SCID/bg mice that had received either IPF/UIP or NSIP fibroblasts, the role of these mediators in the fibrotic process is questionable because their levels were unchanged in many groups of C.B-17SCID/bg mice, which showed a significant decrease in lung remodeling. Thus, the present study provides evidence that the adoptive transfer of IIP fibroblasts promotes fibrosis in C.B-17SCID/bg mice and identifies a novel therapeutic target in IIP.

The modeling of clinical pulmonary fibrosis remains a major challenge in the laboratory.17 Although bleomycin sulfate is the agent of choice in the induction of experimental fibrosis, bleomycin-induced pulmonary fibrosis has been criticized as a less than ideal model of IPF/UIP.17 Recognizing that newer models of pulmonary fibrosis should incorporate as many of the features of clinical IIP as possible, the present study capitalized on the observation that the fibroblast is primarily responsible for the profound and often lethal remodeling in these diseases.4 The adoptive transfer of either IPF/UIP or NSIP fibroblasts initiated interstitial remodeling and histologically evident fibrosis was observed at day 35, but not earlier, after the i.v. adoptive transfer of these lines. The delay in appearance of fibrosis is not readily explainable, but these findings are consistent with previous studies in which the adoptive transfer of human endothelial cells into C.B-17SCID/bg mice required approximately 30 to 40 days before distinct blood vessels were apparent.36 Thus, the present study highlights that a clinically relevant model of pulmonary fibrosis can be initiated in immunodeficient mice with the adoptive transfer of human pulmonary fibroblasts.

The obvious benefit of the IPF/UIP and NSIP fibroblast C.B-17SCID/bg models described herein is their utility in the testing of novel therapeutics for these diseases. The present study addressed the roles of human CCL21 and CCR7 because we have observed that both are prominently expressed in IIP biopsies25 and cultured IIP fibroblasts (E.M.P. and C.M.H., unpublished observations). Our present hypothesis is that the therapeutic effect of anti-human CCL21 and anti-human CCR7 antibody treatments relates to their negations of the pro-proliferative effect of CCL21 on IPF/UIP and NSIP fibroblasts (E.M.P. and C.M.H., unpublished observations). Although it is unlikely that the monoclonal antibodies used herein affected the migration of i.v.-injected fibroblasts because the antibody treatments were delayed until a time point when fibrosis was histologically apparent in C.B-17SCID/bg mice, it is conceivable that this therapeutic approach, if used in other models of pulmonary fibrosis, may have an effect on the recruitment of fibrocytes into the lung. CCR7 is prominently expressed on fibrocytes,37,38 and CCL21 promotes their recruitment into various tissue sites.37 Studies are presently underway to address the role of CCR7-positive fibrocytes in bleomycin-induced pulmonary fibrosis using CCR7 wild-type and gene-deficient mice.

The precise contribution of the adoptively transferred human IIP fibroblasts and the mouse-associated fibrotic components (ie, cells and mediators) to the overall pulmonary remodeling response observed in C.B-17SCID/bg mice remains to be determined. In the present study, it would seem that there was an interaction between the transferred human fibroblasts and mouse components as evidenced by the dynamic changes in murine extracellular matrix-associated transcripts and murine-soluble profibrotic proteins in the lungs of C.B-17SCID/bg mice that most notably received either of the two IIP fibroblast types. Although the relative importance of the soluble murine proteins detected in the C.B-17SCID/bg fibrotic response is questionable at this point, the alterations in cathepsin E, MMPs, extracellular matrix components (ie, collagen and fibronectin), and the inhibitors of MMPs in the IIP fibroblast C.B-17SCID/bg groups relative to the normal fibroblast C.B-17SCID/bg group may indicate that the mouse-associated fibrotic components were actively involved in the remodeling response. Quantitative TaqMan PCR confirmed that collagen 1, cathepsin E, MMP-19, and TIMP-1 transcript levels were significantly increased in the IIP fibroblast C.B-17SCID/bg groups relative to changes in transcript levels for these genes in C.B-17SCID/bg mice that received normal fibroblasts. These transcript changes are relevant to clinical pulmonary fibrosis because cathepsins,39,40 MMPs and TIMPs (reviewed in Ref. 41 ), and extracellular matrix42,43 are increased in the more severe forms of IIP such as IPF/UIP and NSIP. MMP-19 was increased most notably in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and although this proteinase seems to have a major role in dermal wound healing responses (commentary by Mauch44 ), its role in pulmonary fibrosis is unknown. Almost without exception, the anti-CCL21 and anti-CCR7 antibody therapies reduced all of the abovementioned murine extracellular matrix-associated gene products analyzed using quantitative TaqMan PCR. The one exception was MMP-19, and further study of the relative importance of MMP-19 in the pulmonary fibrotic response evoked after the adoptive transfer of human IIP fibroblasts is warranted in light of this and our other findings.

In summary, the present study confirms that pulmonary fibrosis can be transferred to C.B-17SCID/bg mice after the i.v. adoptive transfer of either IPF/UIP or NSIP primary fibroblast lines. The utility of this model in the testing of novel therapeutic strategies was demonstrated, and our findings raise the possibility that the CCL21-CCR7 interaction may be an important target in clinical NSIP and IPF/UIP.


   Footnotes
 
Address reprint requests to Cory M. Hogaboam, Ph.D., Associate Professor, Immunology Program, Department of Pathology, University of Michigan Medical School, Room 4057, BSRB, 109 Zina Pitcher Pl., Ann Arbor, MI 48109-0602. E-mail: Hogaboam{at}med.umich.edu

Supported by a grant from the National Heart Lung and Blood Institute P50 HL56402 (to C.M.H., S.L.K., K.R.F., and F.J.M.).

Accepted for publication December 19, 2006.


   References
Top
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 Materials and Methods
 Results
 Discussion
 References
 

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B. B. Moore and C. M. Hogaboam
Murine models of pulmonary fibrosis
Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L152 - L160.
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