The rabbit anti-Mfge8 monoclonal antibody (4F6) (9
) was used for immunohistochemistry, and anti-Mfge8 antibody from R&D was used for Western blotting. Lactadherin antibody was purchased from R&D; Crk antibody was purchased from BD Biosciences; MMP8 and myeloperoxidase heavy chain antibodies were purchased from Santa Cruz Biotechnology Inc.; anti-Gr1 antibody was purchased from eBioscience; and α–smooth muscle antibody was purchased from Sigma-Aldrich. FITC–collagen type I was purchased from Invitrogen and rat tail type I collagen from Sigma-Aldrich. rMfge8 was purchased from R&D. GRGDSP and GRGESP were purchased from Anaspec.
Mice functionally deficient in Mfge8 were created using a gene trap vector and have been previously characterized (9
). Initial studies were conducted in a mixed-strain background (C57BL/6 × 129/Ola Mfge8+/–
littermates used as controls), and subsequent studies were conducted in mice backcrossed 6 or 10 generations into the C57BL/6 background (Mfge8+/+
used as controls) or 10 generations into the 129/SvEv background. Mice in the 129/SvEv background were used only in studies in Supplemental Figure 4. All experimental protocols were approved by the UCSF IACUC for animal studies.
Tissue sample accrual and processing.
Written informed consent was obtained from all subjects, and the study was approved by the UCSF Committee on Human Research. IPF lung tissues were obtained at the time of diagnostic lung biopsy. IPF patients underwent a history, physical examination, high-resolution computed tomography, pulmonary function testing, and diagnostic lung biopsy. In all cases, the pathologic diagnosis was usual interstitial pneumonia (UIP), and the consensus clinical diagnosis was IPF. Normal human lung tissue was obtained from lungs not used by the Northern California Transplant Donor Network. After harvest, lung tissue was directly snap-frozen in liquid nitrogen. Samples were stored at –80°C until use for experiments. For immunoblotting, frozen lung tissue was pulverized in a stainless steel tissue pulverizer (Fisher Scientific), pre-cooled in liquid nitrogen, then immediately lysed in RIPA buffer prior to separation by SDS-PAGE.
Tissues were fixed with 4% paraformaldehyde or zinc-based formalin (Z-FIX, ANATECH) and embedded in paraffin, and 5-μm sections were treated with 0.2% trypsin for 15 minutes for antigen retrieval. After blocking for 1 hour with 1% BSA/5% goat serum, sections were incubated at room temperature for 1–2 hours with primary antibody, followed by a biotinylated secondary antibody (Vector) for 45 minutes, followed by ABC reagent (Vector) for 30 minutes and liquid diaminobenzidine (Sigma-Aldrich). Sections were counterstained with hematoxylin or methyl green. α–Smooth muscle actin–positive cells taken from 25 randomly selected high-power fields (×200) were quantified. For frozen sections, lungs were inflated with 1.5 ml of OCT medium after BAL was performed, and 5-μm sections were air dried, washed in PBS, and fixed in acetone. After 1 hour of blocking with 5% rat serum, anti-Gr1 antibody was added for 60 minutes and washed off; Alexa Fluor 594 anti-rat (Invitrogen) was added for 30 minutes and washed off; and sections were coverslipped with anti-fade solution containing DAPI (Vector). The number of Gr1-positive cells per 10 randomly selected ×200 fields was quantified by an investigator blinded to the genotype of tissue sections.
Bleomycin model of pulmonary fibrosis.
Eight- to- 10-week-old sex-matched mice were anesthetized, and bleomycin (1.1 U/kg Blenoxane) was instilled directly into the trachea after cut-down. Twenty-eight (C57BL/6 or mixed-strain) or 56 (129/SvEv strain) days after treatment, lungs were removed, homogenized, precipitated with trichloroacetic acid, and baked overnight at 110°C in HCl. Samples were reconstituted with 2 ml of water, and hydroxyproline content was measured using a colorimetric chloramine T assay. Some lungs were inflated with 4% PFA to a pressure of 25 cm H2O, processed, and embedded in paraffin, and then 5-μm sections were stained with picrosirius red for evaluation of fibrosis.
Evaluation of collagen production with deuterated water.
Eight- to-10 week-old sex-matched mice were injected with a single bolus i.p. dose of 100% deuterated water (Isotec, Sigma-Aldrich) with 0.9% NaCl, an isosmotic solution delivered at 35 μl/g body mass, at the same time as they received bleomycin. Subsequently, normal drinking water was replaced with 8% deuterated water to maintain the animal’s total body water at 5% enrichment of deuterated water for a period of 14 days. Mice were then euthanized, and their lungs removed and processed by mass spectrometry analysis.
Preparation of sample for mass spectrometric analysis.
The derivatization of hydroxyproline for GC/MS analysis has been described previously (19
). Briefly, tissue was homogenized with a bead mill in normal abundance (non-deuterated) water, and the homogenate was subjected to 2 rounds of acetone precipitation at –20°C in order to obtain the total tissue protein for hydroxyproline assessment. The proteins were hydrolyzed by incubation in 6N HCl, dried under vacuum, and then suspended in a solution of 50% acetonitrile, 50 mM K2
, and pentafluorobenzyl bromide before incubation. Derivatives were extracted into ethyl acetate, and the top layer was removed and dried by vacuum centrifugation. In order to acetylate the hydroxyl moiety of hydroxyproline, we incubated samples with a solution of acetonitrile, N
-butyldimethyl-silyl]trifluoroacetamide, and methylimidazole. This material was extracted in petroleum ether and dried with Na2
GC/MS analysis of derivatized hydroxyproline.
Analysis of the derivatized hydroxyproline was performed on a standard quadrupole GC/MS instrument (Agilent 5973/6980) in negative chemical ionization mode (NCI-GC/MS), with helium as carrier and methane as reagent gas. The column used was a DB17 ZB-50 column (J&W Scientific, Agilent). Selected ion monitoring was performed on ions with mass-to-charge ratios (m/z) of 424 and 425, which will include all of the carbon-hydrogen bonds from hydroxyproline. The mole fraction of the M1 mass isotopomer was calculated as the ratio of peak areas: M1/(M0 + M1). Incorporation of 2
H into hydroxyproline was calculated as the excess mole fraction of M1 (EM1) above the M1 mole fraction in unlabeled standards at the same abundance. Fractional turnover (f
), the fraction of newly synthesized hydroxyproline, was calculated as EM1/EM1*, where EM1* represents the EM1 of a newly synthesized molecule. EM1* was calculated from measured body water 2
O enrichments as previously described (19
Analysis of body 2H2O enrichments in body water.
O enrichment in plasma samples was measured as previously described (51
) using a Series 3000 Cycloidal mass spectrometer (Monitor Instruments).
Evaluation of phagocytic index in vivo.
Mice were treated with bleomycin, and at indicated time points alveolar macrophages were obtained by BAL. Cytospin preparations were stained with Diff-Quick (Fisher Scientific) and the number of ingestions per macrophage quantified. The phagocytic index represents the number of ingestions per macrophages counted. A minimum of 300 macrophages were counted, and the investigator was blinded to the genotype of each sample.
Mice were euthanized, the trachea cannulated, and serial 0.9-ml lavage was performed with ice cold PBS with 1 mM EDTA for a total of 4.5 ml for evaluation of inflammation after bleomycin treatment. Cells were treated with rbc lysis buffer, after which a cell count was obtained by hemocytometer. Cytospin slides were prepared and stained with Diff-Quick reagent and the percentage of inflammatory cell types determined using a light microscope (Supplemental Figure 1, A and B).
Five-micrometer sections taken from mice treated with bleomycin were stained with TUNEL assay (ApopTag, Chemicon), and the number of apoptotic cells taken from 15 randomly selected high-power fields (×200) was quantified. For determination of the number of free apoptotic cells recovered by BAL, cytospin preparations were stained with TUNEL assay and the number and proportion of apoptotic cells quantified.
Expression arrays/real-time PCR.
Seven and 14 days after treatment with bleomycin (1.1 U/kg), mice were sacrificed, and lungs were removed and RNA extracted using a QIAGEN RNeasy Midi kit following the manufacturer’s instructions. After confirming acceptable RNA quality with Agilent nanotechnology, expression arrays were done using an Agilent Mouse One-Color 4×44 K array platform by the UCSF Functional Genomics Core Facility. Complementary DNA was generated from total RNA using a first-strand cDNA synthesis kit (Invitrogen). Real-time PCR was performed using SYBR Green PCR Master Mix (Invitrogen) and results analyzed on an AB Prism 7700 analyzer (Applied Biosystems). Real-time PCR values were normalized to β-actin RNA and expressed as fold increase in mRNA above saline-treated controls.
Collagen uptake assay.
Freshly isolated alveolar macrophages were cultured for 60 minutes on glass inserts and RPMI with 0.1% BSA in either 10% mouse serum from the same genotype (Figure , B and C, Figure F, and Supplemental Figure 4, A and B) or under serum-free conditions (Figure D). FITC-conjugated type I collagen (50 μg/ml) was added for 30 minutes at 37°C. After 30 minutes, inserts were washed several times to remove unbound/uningested collagen, counterstained with DAPI, and mounted on slides. Slides were examined with fluorescence microscopy using a Leica DM 5000B camera with Spot 4.5 acquisition software; images were obtained at ×200 magnification, and a minimum of 500 cells were analyzed for evidence of collagen binding/ingestion. Investigators were blinded to the experimental conditions when quantifying collagen uptake. The collagen uptake index represented the number of ingestions per macrophages counted and was expressed as percentage of wild-type uptake. Absolute uptake numbers ranged from 1.5% to 4% for wild-type macrophages. Only cells that visually had collagen surrounded by a rim of cytoplasm were considered to have uptake. The software program Merge 2 (Venning Graphic Utilities) was used to merge images of FITC (representing collagen) and phase contrast (to see cytoplasm) to determine whether collagen was internalized. In preliminary studies, cells that were visually considered as having uptake were further analyzed with Z-plane stacked imaging using a Leica CTR 6000 camera and Image-Pro 5.1 (Media Cybernetics) acquisition software to determine whether collagen was ingested or bound. For collagen to be considered ingested, the maximum fluorescence signal of the FITC (representing collagen) and DAPI (representing macrophage nuclei) had to be present at the same level, and there had to be a clear rim of cytoplasm between the FITC signal and the outside of the cell. Using these criteria, 70% of cells (of 100 counted) that were considered to have uptake by visual analysis had uptake according to Z-plane analysis. Therefore, the in vitro collagen uptake index represents both ingested (70%) and bound (30%) collagen.
For all inhibitor studies and studies with recombinant protein, compounds were added to macrophages 30 minutes prior to initiation of the uptake assay.
For the in vivo uptake assay, 60 μg of collagen in 60 μl H2O was placed intratracheally; 30 minutes later, alveolar macrophages were recovered by BAL and cytospin preparations made with DAPI counterstain, and the proportion of macrophages with ingestions was quantified by fluorescence microscopy and expressed as percent control of wild-type macrophage uptake. The actual percentage of wild-type macrophage uptake in vivo was 39. Mouse lungs were further lavaged with 10 ml PBS to remove residual collagen and then inflated with 1.5 cc of OCT medium and prepared for frozen sectioning. Five-micrometer sections were counterstained with DAPI, and the number of collagen particles per total number of nuclei counted in the section was quantified from 5 randomly selected high-power fields (×200) and expressed as percent control of wild-type. The actual proportion of retained nuclei in wild-type sections was 15%.
Fibroblast collagen uptake assay.
For fibroblast uptake assays, lung fibroblasts were isolated by digesting whole lung with Blendzyme 3 (Roche). After 4 passages in vitro, fibroblasts were plated at a concentration of 50,000 cells per 24-well plate overnight in 10% FCS DMEM. Fibroblasts were then washed 3 times with PBS and incubated in media containing 10% mouse serum of the same genotype as the fibroblasts. FITC-conjugated type I collagen (50 μg/ml) was added for 90 minutes, after which the cells were washed vigorously with PBS. Fibroblasts were then incubated with 50 μg/ml trypsin and 50 μg/ml proteinase K at 37°C for 5 minutes, after which they were removed and centrifuged, and the supernatant containing the membrane-bound collagen cleaved by proteolytic treatment was separated. The remaining pellet was lysed with 0.1 M NaOH to release intracellular collagen, and fluorescence was measured in both the supernatant and pellet using a spectrofluorometer (Tecan).
Collagen degradation assays.
For the in vitro assay, isolated alveolar macrophages were cultured in black 96-well black plates in triplicate. Cells were plated at a concentration of 50,000 cells per well in PBS with 1 μg/ml of FITC-conjugated type I collagen (Invitrogen) in a total volume of 100 μl. The FITC-conjugated collagen from Invitrogen is designed for enzymatic assays and is supersaturated with FITC. When the collagen is cleaved, the fluorescent signal increases. After 30 minutes, fluorescence was quantified using a spectrofluorometer (Tecan). The fluorescent signal from control wells containing only collagen was subtracted from that from wells containing macrophages and collagen.
For the assay using in vivo samples, 10 μg of total lung homogenates from saline- and bleomycin-treated mice were incubated at 37°C with FITC-conjugated type I collagen (10 μg/ml) in duplicate. After 60 minutes, fluorescence was quantified using a spectrofluorometer (Tecan). The fluorescent signal from control wells containing only collagen was subtracted from that from wells containing tissue lysates and collagen.
RNA taken from the involuting mouse mammary gland was extracted using TRIzol following the manufacturer’s instructions (Invitrogen). Full-length Mfge8 was cloned into the pMIB/V5-His vector (Invitrogen) using the sequences 5′-GGCATGCTAAGCTTGTCTGGTGACTTCTGTGACTCCAGCCTGTGC-3′ (Mfge8 forward primer) and 5′-GGCGGCACTAGTTCTGCCTTCGATACAGCCCAGCAGCTCCAG-3′ (Mfge8 reverse primer). The huFc domain was cloned into the vector using the sequences 5′-GGCGGCACTAGTGCACCTGAACTCCTGGGGGGACCGTC-3′ (Fc forward primer) and 5′-TATCTGCAGAATTCTCATTTACCCGGAGACAGGGAGAGGCTC-3′ (Fc reverse primer). For the Dd1 and Ndd construct, the primers 5′-GGCATGCTAAGCTTGTCTGGTGACTTCTGTGACTCCAGCCTGTGC-3′ (Mfge8 forward primer), 5′-GGCGGCACTAGTTCTGCCTTCGATGCCCAGGAGCTCGAAGCG-3′ (Dd1 reverse primer), and 5′-GGCGGCACTAGTTCTGCCTTCGATGGAGGCTAGGTTGTTGGA-3′ (Ndd reverse primer) were used to obtain cDNA that was then inserted into the pMIB/V5-His vector containing huFc after enzymatic digestion and removal of the full-length construct. High Five cells were transfected with each vector using Cellfectin reagent (Invitrogen) and recombinant protein isolated by binding on a protein G column and eluting with a pH gradient.
Surface plasmon resonance.
Proteins were immobilized on different flow cells of a Biacore CM5 chip and analyzed on a Biacore T100. The dextran surface on the chip was first activated by injection of a 1:1 mixture of N-hydroxysuccinimide and N-ethyl-NP-[(3-dimethylamino)-propyl]-carbodiimide hydrochloride (GE Healthcare). Proteins were then diluted to 20 μg/ml in 10 mM sodium acetate at pH 4.5 and injected to equal mass density of 3,000 response units (RU) on the surface. The remaining active sites on the surface were then blocked with 1 M ethanolamine-HCl, and washed with HEPES-buffered saline with 0.05% P-20. Type I collagen was diluted into running buffer (PBS with 0.05% Tween-20) and injected at different concentrations at a flow rate of 30 μl/min in duplicate with intermittent blank injections of running buffer alone. The surfaces were regenerated after each injection to remove residual bound collagen by injection of 2 M NaCl and washing with running buffer. The response for each flow cell was represented in reference to a flow cell with EGFR1-Fc immobilized and subtracted by the average response of the blank injections over each surface.
Evaluation of vascular permeability.
Five days after treatment with intratracheal bleomycin (5 U/kg), mice were administered 0.5 μCi of [125
I]albumin by i.p. injection. Four hours later, they were sacrificed and their lungs removed and homogenized. A blood sample was used to calculate the hematocrit. Vascular permeability was expressed as extravascular plasma equivalents (EVPE), the ratio of radioactive counts in the lung (after subtraction of counts attributable to the blood content within the lung) to counts in the plasma (52
Paired data columns were evaluated using Student’s t test with Microsoft Office Excel 2007. One-way ANOVA was used for comparison of multiple data columns using SigmaStat 3.11 (SYSTAT), and when differences were statistically significant, this was followed with a Bonferroni t test for subsequent pairwise analysis. Data were tested for normality and variance, and a P value less than 0.05 was considered significant.