Turnover in Fibroblasts and Regulate MMP14 Levels and Collagen -Ig-H3

Macrophages Produce TGF-β-Induced (β -ig-h3) following Ingestion of Apoptotic Cells and Regulate MMP14 Levels and Collagen Turnover in Fibroblasts This information is current as of September 26, 2021. Natalia Nacu, Irina G. Luzina, Kendrick Highsmith, Virginia Lockatell, Kerill Pochetuhen, Zachary A. Cooper, Michael P. Gillmeister, Nevins W. Todd and Sergei P. Atamas J Immunol 2008; 180:5036-5044; ; doi: 10.4049/jimmunol.180.7.5036 Downloaded from http://www.jimmunol.org/content/180/7/5036

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Macrophages Produce TGF-␤-Induced (␤-ig-h3) following Ingestion of Apoptotic Cells and Regulate MMP14 Levels and Collagen Turnover in Fibroblasts1

Natalia Nacu,* Irina G. Luzina,*† Kendrick Highsmith,† Virginia Lockatell,* Kerill Pochetuhen,* Zachary A. Cooper,* Michael P. Gillmeister,† Nevins W. Todd,*† and Sergei P. Atamas2*†

Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of Downloaded from collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-␤ following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-␤-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and http://www.jimmunol.org/ up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen. The Journal of Immunology, 2008, 180: 5036–5044.

acrophages are important functional contributors to in such animals), the wounds heal with signiﬁcantly less inﬂam-

normal wound healing/repair process; they participate mation, lower levels of TGF-␤, and less fibrosis in the absence of by guest on September 26, 2021 M in the clearance of apoptotic debris that accumulates macrophages (6). Macrophages appear to be intimately involved in as a result of primary injury and subsequent inflammatory re- the regulation of tissue fibrosis in the lung (7–9), kidney (10, 11), sponse. Clearance of apoptotic debris is a major nonphlogistic and liver (12); the apoptotic mechanisms are often involved in the function of macrophages; ingestion of apoptotic debris causes dra- mechanism of tissue fibrosis in the lung (13), kidney (14), and liver matic phenotypic changes in these cells. Of particular importance, (15). the production of TGF-␤ by macrophages is accelerated following Little is known about pro- and anti-fibrotic regulation by mac- phagocytosis of apoptotic debris, suggesting potential anti- rophages in relation to phagocytotic clearance of apoptotic debris. inflammatory (1–4) and profibrotic (see 5 for a review) effects of It is unclear whether TGF-␤ production following the uptake of this phenomenon. Based on these observations, it is reasonable to apoptotic debris by macrophages is sufficient to drive tissue fibro- hypothesize that tissue fibrosis may be a consequence of disturbed sis, or more complex mechanisms, such as so-called alternative clearance mechanisms of apoptotic debris by macrophages. Fibro- macrophage activation (8, 16) or yet unknown novel mechanisms sis, often viewed as an exaggerated repair process, is a major de- are necessary. Of note, the levels of TGF-␤ production by mac- bilitating factor and a cause of death in patients with various dis- rophages following phagocytosis of apoptotic debris are relatively eases (5). Although macrophages may not be absolutely necessary low (within 100 pg/ml, see Ref. 4), and thus may be insufficient for for wound healing in macrophageless (PU.1 null) mice (apoptotic direct activation of collagen production in fibroblasts (17, 18). debris in the wounds is cleared by “stand-in” fibroblast phagocytes In this study, we sought to investigate the effects of phagocytosis of apoptotic or necrotic cells by macrophages on the rate of collagen production by primary fibroblasts in cell culture, and to *University of Maryland School of Medicine and †Baltimore Veterans Affairs Med- begin addressing the molecular mechanisms of such effects. We ical Center, Baltimore, MD 21201 report that phagocytosis of apoptotic but not necrotic debris by Received for publication August 8, 2007. Accepted for publication January 29, 2008. monocyte-derived but not alveolar macrophages stimulates colla- The costs of publication of this article were defrayed in part by the payment of page gen production in cocultures with primary fibroblasts. This regu- charges. This article must therefore be hereby marked advertisement in accordance ␤ 3 with 18 U.S.C. Section 1734 solely to indicate this fact. lation is mediated by TGF- induced (TGFBI) protein also called 1 This work was supported by a National Institutes of Health Grant 1 R01 HL074067 (to S.P.A.), a Veterans Administration Merit Review grant (to S.P.A.), and Maryland Chapter of Arthritis Foundation Research Awards (to I.G.L. and S.P.A.). 3 Abbreviations used in this paper: TGFBI, TGF-␤ induced; BIGH3, ␤-ig-h3; MDM, 2 Address correspondence and reprint requests to Dr. Sergei P. Atamas, University of monocyte-derived macrophage; AM, alveolar macrophage; TDM, THP-1-derived Maryland School of Medicine, 10 South Pine Street, MSTF 8-34, Baltimore, MD macrophage; siRNA, small interfering RNA; CAT, chloramphenicol acetyltrans- 21201. E-mail address: [email protected] ferase; rh, recombinant human. www.jimmunol.org The Journal of Immunology 5037 keratoepithelin, or ␤-ig-h3 (BIGH3). We report that the production Macrophage-fibroblast cocultures and conditioned medium of TGFBI by macrophages leads to up-regulation of collagen pro- experiments tein but not mRNA in primary fibroblasts. This effect of TGFBI is Fibroblasts were seeded in 6 well-tissue culture plates (BD Biosciences) at mediated by a decrease in the levels of MMP14 mRNA and protein a subconfluent density of 150,000 cells/well and grown for 24 h in the same in a p53-dependent and PU.1-dependent fashion. Thus, we de- conditions as described above, except that low-serum RPMI 1640 medium scribe a novel mechanism by which macrophages that ingest ap- supplemented with 50 ␮M ascorbic acid, and 50 ␮M BAPN (␤-aminopro- optotic cells may regulate normal wound healing and, if exagger- pionitrile) was used. Then, macrophages were added to each well at a concentration of 250,000 cells/well for additional 24 h, followed by adding ated, possibly fibrosis. This mechanism may be a novel target for 1.5 million apoptotic Jurkat cells for 2 h. After 2 h, the adherent cells were future therapies aimed at facilitating repair or preventing and treat- washed to remove noningested apoptotic cells and fresh medium was ing fibrosis. added for additional 24 h before analyzing these cultures for collagen or cytokine production. In separate experiments, fibroblasts were stimulated with the conditioned media collected from macrophage cultures following Materials and Methods phagocytosis assays, without or with neutralizing anti-TGF-␤ Ab 1D11 or isotype control Ig (both from R&D Systems). Fibroblast proliferation was Macrophage and fibroblast cell culture tested as described in Ref. 24. In brief, after 5 to 7 days of coculture, Macrophages were derived from human peripheral blood monocytes macrophages were removed and fibroblast proliferation tested using Cell- (monocyte-derived macrophages; MDM), bronchoalveolar lavage fluids Titer Aqueous 96 Non-Radioactive Cell Proliferation Assay (Promega) per (alveolar macrophages; AM), or a human monocytic cell line THP-1 (THP- the manufacturer’s recommendations. 1-derived macrophages; TDM). To produce MDM, PBMC were isolated Transwell assay from freshly drawn peripheral blood by density gradient centrifugation using Ficoll-Paque (Amersham Biosciences) and resuspended in RPMI To determine whether cell-to-cell contacts are necessary to mediate the Downloaded from 1640 medium supplemented with 20% human serum, 10 mM HEPES (pH effects of macrophages on fibroblasts, or whether soluble factors are suf- 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential ficient for the interactions between these two cell types, Transwell assays amino acid mix, 5 ϫ 10Ϫ5 M 2-ME, and 5 ␮g/ml gentamicin sulfate. The were performed. In these assays, macrophages were separated from fibro- cells were cultured overnight in 6-well plates (BD Biosciences), in a 5% blast monolayers by a membrane with 3.0-␮m pore size in the 6 well- CO2 humidified air atmosphere at 37°C. The nonadherent cells were re- Transwell plates (Corning Costar). Primary lung fibroblasts were seeded in moved and the adherent cells were cultured for an additional 5 days, and the lower chamber whereas macrophages that engulfed apoptotic cells were were termed monocyte-derived macrophages. Alveolar macrophages were placed in the upper chamber, using the same cell culture medium as de- http://www.jimmunol.org/ obtained from bronchoalveolar lavage fluids derived from two adult scribed above. These cocultures were incubated for 24 h before analyzing healthy individuals or from eight patients with interstitial lung disease as- the levels of collagen or cytokines. sociated with systemic sclerosis (19–21). The protocols for drawing blood and for bronchoalveolar lavage procedures were approved by the Univer- Western blotting sity of Maryland Institutional Review Board. Human monocytic line Preparation of cell lysates, immunoprecipitation of BIGH3 protein, nor- THP-1 was obtained from the American Type Culture Collection (ATCC; malization of protein concentration in the samples with BioRad assays, Manassas, VA) and maintained in the same medium, except that 10% FBS electrophoretic separation, and Western blotting were performed as previ- was used instead of human serum. The THP-1-derived macrophages were ously described (22). Goat Abs for BIGH3 were purchased from R&D obtained by stimulating these cells with 200 nM/ml PMA purchased from Systems. Goat Abs for MMP14 were purchased from Santa Cruz Biotech- Cell Signaling Technology.

nology. Western blotting assays for collagen were performed using rabbit by guest on September 26, 2021 Four primary pulmonary fibroblast cultures (PF1-PF4) derived from dif- affinity purified anti-collagen type I Ab (Rockland). ferent adult healthy donors were purchased from Cambrex and each tested separately in independent experiments. Fibroblast cultures were maintained Nucleofection of primary fibroblast cultures in T75 culture flasks as previously described (22–24). In all experiments fibroblast cell lines were tested in passages three to seven. Nucleofection with collagen promoter-chloramphenicol acetyltransferase (CAT) reporter constructs (25), MMP14- or p53-encoding constructs (un- der control of CMV promoter, OriGene Technologies), small interfering Apoptotic and necrotic cellular debris RNA (siRNA) directed against collagen ␣2(I) or MMP14 or PU.1, or non- targeting control siRNA (all from Santa Cruz Biotechnology) was per- Jurkat cells (human T cell line) were purchased from ATCC and main- formed using Basic Nucleofector kit reagents from Amaxa, following the tained according to the supplier’s recommendations. Apoptosis of Jurkat manufacturer’s recommendations. Transfected fibroblasts were cultured for cells was induced by incubation with 0.5 ␮g/ml staurosporine (Sigma- 48 h before treatment with 300 ng/ml TGFBI (R&D Systems). The effi- Aldrich) at 37°C for 6–8 h or, alternatively, by exposure to UV irradiation ciency of target depletion was assessed by measuring the levels of corre- at 90 mJ/cm2 followed by culture for 3–4 h. The percentage of apoptotic sponding mRNA by quantitative (real-time) PCR. cells was quantified by flow cytometry analysis by using Annexin V and propidium iodide staining (Sigma-Aldrich) and was within 70–80%. Ne- Collagen production assays crotic debris was generated by three cycles of freezing-thawing involving freezing the cells in liquid nitrogen and then thawing them at 37°C. Production of collagen was measured in cell cultures using the metabolic la- beling of collagen with 14C-proline as described in (25, 26). In brief, fibroblast 14 monolayers were pulsed with L-[U- C]-proline (Amersham Biosciences) at 1 Phagocytosis assays ␮Ci/ml for the final 12 h of incubation. Purified bacterial collagenase type III Jurkat cells were labeled with the dye TAMRA (Molecular Probes). was purchased from Sigma-Aldrich. Fibroblasts were then ruptured by re- TAMRA-labeled cells were added to cultured macrophages at a ratio of 5:1 peated freeze-thawing, and part of each sample digested with collagenase type and incubated for2hat37°C. For confocal microscopy experiments, vital III. The samples were pelletted with 20% TCA containing 0.1% L-proline, and staining of macrophages was performed with calcein AM (Molecular then the pellets were resuspended and washed twice with 5% TCA and 95% ice-cold ethanol. The samples were assayed in a liquid scintillation counter to Probes) immediately before assays. At the end of the incubation period, the 14 monolayer was vigorously washed with ice-cold PBS to remove unbound determine the amount of collagenase-digestible and nondigestible C-labeled and bound but unengulfed apoptotic cells. The complete removal of non- protein. Alternatively, collagen protein levels in cell culture supernatants were measured in Western blotting assays as described above. ingested apoptotic debris after washing was confirmed by colocalization of ␣ TAMRA-stained apoptotic material and calcein AM-stained macrophages The activity of the collagen 2(I) promoter was measured in primary via confocal microscopy at ϫ400 magnification using Zeiss LSM 510 laser fibroblasts transfected with collagen promoter-chloramphenicol acetyl- scanning confocal microscope. The phagocytosis was assessed by fluores- transferase (CAT) reporter constructs as described (25). cent or confocal microscopy. The percentage of macrophages that ingested ELISA TAMRA-labeled apoptotic cells was determined as the percent phagocytosis (number of macrophages, per 100, that ingested at least one apoptotic ELISA kits for TGF-␤1, IL-4, and IL-13, were purchased from R&D Sys- particle) in three different wells. Also, the conditioned supernatant media tems and assays performed following the manufacturer’s recommenda- from these cell cultures were collected and used for stimulation of fibro- tions. Fibroblast culture supernatants and whole cell lysates were activated blast cultures as described below. by acidification before the assay to quantify the levels of IL-4, IL-13, and 5038 MACROPHAGES REGULATE COLLAGEN IN FIBROBLASTS THROUGH TGFBI AND MMP14 total (active and latent) TGF-␤1. All samples were assessed in duplicates. Low-serum cell culture medium containing 0.5% dialyzed FBS had no detectable TGF-␤1, IL-4, or IL-13 and was used as a negative control in these assays. Profiling of gene expression with DNA arrays Expression of 367 genes for cytokines and cytokine receptors in macrophages was profiled with cDNA macroarrays (SuperArray) at 0, 2, 6, 12, and 24 h of exposure to apoptotic debris. Developed membranes were scanned and hybridization intensities for each spot were measured using Image Quant software (Molecular Dynamics) and background subtracted. Numeric spot density data were exported into spreadsheet software for data analyses. Results were confirmed by real-time PCR assays for selected genes as indicated in the Results section. Analyses of DNA binding by transcription factors Nuclear extracts from TGFBI-activated and control fibroblast cultures were prepared using nuclear extraction kit from Active Motif and adjusted for total protein content using Bio-Rad assays. DNA binding by 345 different transcription factors was evaluated using protein/DNA TranSignal system (Panomics), following the manufacturer’s recommendations. To validate selected results of the protein-DNA array experiments, electromobility Downloaded from shift assay kit (Active Motiff), including p53-specific consensus sequence probe and corresponding mutant probe, was used as described (23). Real-time PCR quantification of mRNA levels Total RNA purification, reverse transcription, and real-time PCR were performed using LightCycler (Roche), as previously described (22). Quanti-

fication of internal control 18S ribosomal RNA was performed as reported http://www.jimmunol.org/ previously (23). The PCR reaction mixture included the recommended components of the FastStart DNA Master Hybridization Probes Hot Start Reaction Mix (Roche). The fold difference in gene expression relative to 18S ribosomal RNA between treated and untreated cultures was calculated using the 2Ϫ⌬CT method (26). The primers and the hybridization probes for collagen ␣2(I) mRNA were designed and prepared by TIB Molbiol. The primers for collagen ␣2(I) mRNA were: forward, 5Ј-GAT GGT GAA GAT GGT CCC ACA GG-3Ј and reverse, 5Ј-GGT CGT CCG GGT TTT CCA FIGURE 1. Confocal microscopy of calcein AM-stained THP-1 mac- GGG T-3Ј. The hybridization probes were labeled with fluorescein at the rophages (green) after 2-h coculture with TAMRA-labeled apoptotic cells 3Ј-terminus (3FL) of one probe and with LightCycler Red at the 5Ј-termi- (red) followed by washing. Four serial sections of a macrophage are shown nus (5LC) of the other probe. The probes were 3FL 5Ј-TTC CAA GGA in A1–A4, top to bottom; and two sections of a different macrophage are by guest on September 26, 2021 CCT GCT GGT GAG CCT-3Ј and 5LC 5Ј-TGA ACC TGG TCA AAC shown in B1 and B2, top to bottom. Notice that ingested apoptotic material TGG TCC TGC AG-3Ј. TGFBI-specific primers and PU.1-specific primers remains inside macrophages whereas noningested debris is removed by were designed and tested for specificity by SuperArray, and their specific- washing. ity has been additionally confirmed in our preliminary experiments. Real- time PCR assays (RT2 Profiler, SuperArray) were used to measure expression of 84 genes related to extracellular matrix in fibroblasts, following the manufacturer’s recommendations. gen production in cocultures, as judged by 14C-proline incorporation and Western blotting assays (Fig. 2). These experiments were Results repeated on thirteen independent occasions, in duplicates or trip- Collagen production but not proliferation of fibroblasts is up- licates, using 14C-proline incorporation, and on eight independent regulated in cocultures with macrophages following ingestion occasions using Western blotting for collagen in primary fibroblast of apoptotic cells cultures from four different unrelated donors, with consistent re- TAMRA-labeled apoptotic cells were cocultured with either TDM, sults. These observations suggested that ingestion of apoptotic de- MDM, or AM for 2 h. The subsequent washing completely re- bris by macrophages may have a profibrotic effect. Additional ex- moved the noningested apoptotic material (Fig. 1). On average, periments included monocyte-derived and alveolar macrophages. 31 Ϯ 6% of macrophages have engulfed at least one apoptotic Coculturing primary fibroblasts with MDM did not significantly particle. There was no significant difference in the percent phago- influence collagen production in fibroblasts when compared with cytosis between the three types of macrophages ( p Ͼ 0.05, one- fibroblasts cultured alone ( p Ͼ 0.05, Fig. 3). Coculturing MDM way ANOVA). Because fibroblast proliferation and collagen turn- that have ingested apoptotic cells with fibroblasts caused a signif- over jointly define fibrosis, we tested whether ingestion of icant increase in collagen production (Fig. 3). These results were apoptotic debris affects these two processes in the macrophage- consistently observed in two independent experiments using pri- fibroblast cocultures. Phagocytosis of apoptotic debris by TDM, mary fibroblast cultures from four different unrelated donors. A MDM, or AM did not affect proliferation rates in the macrophages- different pattern of modulation of collagen production was ob- fibroblast cocultures ( p Ͼ 0.05, two-tailed Student’s t test com- served when primary fibroblasts were cocultured with AM from paring cocultures of primary pulmonary fibroblasts with macro- two healthy volunteers or eight patients with interstitial lung dis- phages that engulfed or did not engulf apoptotic cells). ease. Collagen production was increased when primary fibroblasts The initial experiments were performed with TDM. The results were cocultured with nonstimulated AM (Fig. 3). There was no suggested that these nonactivated macrophages or those exposed to further increase in collagen production in fibroblast cocultures necrotic Jurkat cells failed to affect collagen production in cocul- with AM that have ingested apoptotic cells (Fig. 3). No statistically tures with pulmonary fibroblasts (Fig. 2). However, ingestion of significant difference was observed between AM macrophages the apoptotic cells by TDM caused a significant increase in colla- from healthy donors and from patients with interstitial lung disease The Journal of Immunology 5039

FIGURE 2. Regulation of collagen levels in macrophage-fibroblast co- Downloaded from cultures. Primary fibroblasts (Fib) were cultured with or without THP-1- derived macrophages (TDM). The macrophages were or were not exposed to apoptotic (Apopt) or necrotic (Necr) Jurkat cells. A, Fold change in total (f) or collagenase-sensitive (Ⅺ) levels of metabolic incorporation of 14C- proline (mean cpm Ϯ SD of quadruplicate cultures). Significant increases in 14C-proline incorporation (p Ͻ 0.05) are indicated with asterisks. B, Representative Western blotting results for collagen type I in these http://www.jimmunol.org/ cocultures. FIGURE 4. Effect of macrophage-derived soluble factors on 14C-pro- in these relatively small subsets of volunteers (data not shown; p Ͼ line incorporation by primary fibroblasts. The bars represent fold change in f Ⅺ 14 0.05). These observations suggested that macrophages derived total ( ) or collagenase-sensitive ( ) metabolic incorporation of C-pro- Ϯ from monocytes (TDM or MDM) respond to apoptotic cells by line (mean cpm SD of quadruplicate cultures). A, macrophages that have ingested apoptotic debris were separated from fibroblast monolayers with increasing their profibrotic potential, whereas alveolar macro- Transwell membranes. B, the medium conditioned by macrophages that phages appear to be preactivated by the pulmonary milieu in their have ingested apoptotic cells [(TDMϩApopt) sup] was transferred into ability to induce accumulation of collagen, and fail to further up- fibroblast cultures, without or with added anti-TGF-␤ Ab or isotype control by guest on September 26, 2021 regulate collagen levels following exposure to apoptotic cells. Ig. Single and double asterisks indicate significant differences (p Ͻ 0.05) from fibroblasts cultured alone, whereas double asterisks indicate the sig- The profibrotic effect of macrophages on fibroblasts is mediated nificant difference between cocultures treated with isotype control and anti- by soluble factors TGF-␤ Ab. Direct contact between macrophages and fibroblasts is not We then sought to determine whether direct cell-to-cell contacts necessary to up-regulate the collagen levels; the effect is mediated, at least between macrophages and fibroblasts are necessary to stimulate in part, by soluble factors produced by macrophages.

collagen production, or secretion of soluble profibrotic factors by macrophages may be sufficient to mediate such an effect. To address this question, two types of experiments were performed. Macrophages were either cocultured with fibroblasts with or without separation with a semipermeable membrane (Transwell assays) (Fig. 4A), or, separately, the conditioned supernatant from the macrophage cultures were transferred into fibroblast cultures, and the effect on collagen production was evaluated (Fig. 4B). Macro- phages that have ingested apoptotic debris stimulated collagen production in fibroblast monolayers even when separated by a Trans- well membrane (Fig. 4A). Also, the conditioned medium from macrophages that have ingested apoptotic debris stimulated collagen production in fibroblast monolayers (Fig. 4B). These observations suggested that soluble factors produced by macrophages fol- FIGURE 3. Regulation of collagen levels in cocultures of MDM or AM lowing ingestion of apoptotic debris drive the increase in collagen f and fibroblasts. The bars represent fold change in total ( ) or collagenase- levels. To further investigate this mechanism, we considered a Ⅺ 14 Ϯ sensitive ( ) metabolic incorporation of C-proline (mean cpm SD of well-known up-regulation in production of TGF-␤, a potent pro- quadruplicate cultures). The collagen production was up-regulated when fibrotic cytokine, by macrophages following ingestion of apoptotic fibroblasts were cocultured with MDM or AM alone and the levels of ␤ collagen increased further significantly when MDM have ingested apopto- debris (4). A potent neutralizing anti-TGF Ab 1D11 clone added tic cells (Apopt). The levels of collagen did not change in cocultures of to the conditioned medium from macrophages that have ingested fibroblasts with AM that have ingested apoptotic cells. Significant in- apoptotic debris, had a statistically significant, yet relatively mod- creases in 14C-proline incorporation (p Ͻ 0.05) are indicated with asterisks. est attenuating effect on the up-regulation of collagen levels (Fig. 4B). ELISA assays revealed that indeed the levels of total TGF-␤1 5040 MACROPHAGES REGULATE COLLAGEN IN FIBROBLASTS THROUGH TGFBI AND MMP14

repeated on two different occasions with consistent results ( p Ͻ 0.05, one-way ANOVA). In the THP-1-derived macrophages, en- gulfment of apoptotic Jurkat T cells stimulated an increase in TGFBI protein levels, as judged by the density of the bands in Western blotting analyses (Fig. 6C). No differences in steady-state levels of TGFBI mRNA and TGFBI protein were observed between activated and control cultures of alveolar macrophages (AM) (Fig. 6, A and D). The Western blotting experiments were repeated on two different occasions in each of these macrophage FIGURE 5. Production of total TGF-␤1 in cultures of MDM after 2 or types. 24 h of exposure to apoptotic cells (Apopt) measured by ELISA, mean Others have previously reported that so-called alternatively ac- Ϯ ␤ pg/ml values SD of triplicate cultures. Levels of active TGF- were tivated macrophages express elevated levels of TGFBI (27). An- below detection level in these assays (data not shown). other group also reported that such macrophages stimulate collagen production in cocultures with fibroblasts (28). Therefore, a were increased in macrophage cultures at 2 and 24 h of exposure possibility was considered that phagocytosis of apoptotic debris to apoptotic debris (Fig. 5). Of important notice, the levels of may convert macrophages toward an alternative phenotype that is TGF-␤1 production are consistent with the previous observation characterized by the cell surface expression of CD163 and CD206 (4), and may not be sufficient to directly drive collagen production (29). We conducted flow cytometric analyses of TDM and MDM in fibroblasts (17, 18, 25), particularly because no active TGF-␤ macrophages before and after phagocytosis of apoptotic debris and Downloaded from was detected in the cocultures by ELISA assays. Also, TGF-␤ is observed no increase in the cell surface expression of CD163 and known to increase collagen production transcriptionally, but our CD 206 at 24 h postincubation, as judged by lack of change in the real-time PCR experiments for collagen ␣2(I) mRNA revealed no mean fluorescence intensity (data not shown). Levels of IL-4 and increase in collagen mRNA in the macrophage-fibroblast cocul- IL-13, cytokines known to promote alternative activation of mac- tures (data not shown). Therefore, a possibility was considered that rophages, were below detection thresholds of 0.25 pg/ml (IL-4) factors other than TGF-␤ might contribute to up-regulation of col- and 62.5 pg/ml (IL-13) in all macrophage culture supernatants; http://www.jimmunol.org/ lagen levels by the macrophages following ingestion of apoptotic these concentrations are significantly lower than those necessary debris. for alternative activation of macrophages (28) or for up-regulation of collagen production in fibroblasts (5, 24). Therefore, ingestion TGFBI is generated by macrophages following exposure to of apoptotic cells causes a selective increase in the expression apoptotic cells levels of TGFBI without alternative activation of macrophages. Transcriptomic profiling of macrophages using cDNA arrays revealed that out of nearly 400 cytokine, chemokine, and their re- TGFBI up-regulates collagen levels in primary fibroblasts without activation of collagen production

ceptor genes represented on the array, only one, TGFBI, also by guest on September 26, 2021 known as keratoepithelin, or BIGH3, was consistently increased The finding of increased TGFBI production by macrophages fol- shortly (2 h) following ingestion of apoptotic debris (data not lowing ingestion of the apoptotic debris suggested that this cyto- shown). Reverse transcriptase-real-time PCR assays confirmed kine may directly regulate collagen accumulation in fibroblast cul- that the steady-state levels of TGFBI mRNA increased signifi- tures. To test this possibility, fibroblast cultures were incubated for cantly when TDM and MDM engulfed apoptotic cells compared various times with various doses of recombinant human (rh) with nonstimulated macrophages (Fig. 6A). MDM were tested on TGFBI, and the levels of collagen accumulation were measured. two independent occasions, and TDM and AM were tested on four The experiments showed that TGFBI directly regulated collagen independent occasions, in duplicate cultures, with consistent re- levels in cultured primary fibroblasts in a dose- (Fig. 7, A and B) sults. Additional experiments defined the dynamics of increase in and time- (Fig. 7, C and D) dependent fashion. These experiments levels of TGFBI mRNA in macrophage cultures following inges- were repeated on at least two occasions, in duplicates, for each of tion of apoptotic debris (Fig. 6B). The latter experiments were the four primary fibroblast cultures, with consistent results.

FIGURE 6. TGFBI protein and mRNA production by macrophage cultures. A, Steady-state levels of TGFBI mRNA increased significantly in TDM and MDM cultures following ingestion of apoptotic debris for 2 h, but not in AM cultures. Significant changes (p Ͻ 0.05) are indicated with asterisks. B, Fold changes in the steady-state levels of TGFBI mRNA (measured by reverse transcriptase – real time PCR) in THP-1 derived macrophage cultures following exposure to apoptotic debris for indicated times. Significant changes (p Ͻ 0.05) are indicated with asterisks. C and D, Western blotting for TGFBI protein in THP-1 derived (C) and alveolar (D) macrophage cultures. The Journal of Immunology 5041 Downloaded from http://www.jimmunol.org/

FIGURE 7. Time and dose-dependent effect of rhTGFBI on collagen protein levels in primary lung fibroblast cultures. Collagen levels were measured by metabolic incorporation of 14C-proline (A and C), or using Western blotting technique (B, D, and E). Significant increases in 14C-proline incorporation (p Ͻ 0.05) are indicated with asterisks. In A and B, fibroblasts were activated for 48 h with increasing concentrations of rhTGFBI as indicated. C, Fibroblast cultures were incubated for indicated times without or with 100 ng/ml rhTGFBI. D, Fibroblasts were cultured for 72 h, with 100 ng/ml rhTGFBI added to the cultures for the final 24, 48, or 72 h as indicated. E, Fibroblasts were transfected with collagen ␣2(I) siRNA or control siRNA as indicated, and the effect of rhTGFBI on collagen protein levels was assessed. Notice the decline in basal collagen production but persistent up-regulation in collagen level

in response to TGFBI stimulation (repeated on two independent occasions with consistent results). by guest on September 26, 2021

The experiments were performed to determine whether the in- 300 ng/ml TGFBI. The expression levels of 84 genes related to crease in collagen levels stimulated by TGFBI was due to tran- connective tissue biology were analyzed using reverse transcrip- scriptional up-regulation of the collagen gene expression. To test tase-real time PCR approach (SuperArray). Expression of MMP14 whether collagen gene expression was transcriptionally up-regu- was consistently decreased following stimulation with TGFBI lated by TGFBI, collagen ␣2(I) mRNA steady-state levels were (Fig. 8A). Western blotting analyses confirmed that MMP14 pro- measured by quantitative (real-time) PCR and showed no differ- tein levels decreased significantly in fibroblast cultures activated ences in steady-state levels of collagen ␣2(I) mRNA between TG- with TGFBI for 24 h or 48 h, compared with nonactivated fibro- FBI-treated and control cultures (data not shown). The response of blasts (Fig. 8B). These experiments were repeated on three inde- the collagen ␣2(I) promoter-CAT reporter constructs was evalu- pendent occasions with primary fibroblast cultures from different ated and no differences were observed (data not shown). Finally, donors, with consistent results. MMP14 is a critical factor for col- activation of DNA binding by transcription factors known to regu- lagen turnover by fibroblasts (30). This decrease in MMP14 levels late the activity of collagen gene promoter (Smad3/4, Sp1, AP1, Ets) (Fig. 8) is consistent with the increase in collagen levels (Fig. 7), was tested using transcription factor array approach (TranSignal Pro- as collagen turnover is likely to be down-regulated due to lower tein/DNA system, Panomics), and again, no differences were detected levels of MMP14. To further confirm the inverse link between the (data not shown). These results suggested that the observed up-regu- levels of MMP14 and collagen, primary fibroblasts were trans- lation of collagen protein levels by TGFBI (see Fig. 7) was not due to fected with either an MMP14-encoding plasmid construct or the transcriptional regulation at the level of the collagen gene promoter. corresponding “blank” plasmid (Fig. 9, B and C). As expected, the To further confirm this conclusion, siRNA-mediated inhibition of col- levels of MMP14 following the transfection were increased (Fig. lagen ␣2(I) was performed (Fig. 7E). Despite the decrease in the total 9B) and the levels of collagen reciprocally decreased (Fig. 9C). collagen level, the responsiveness to stimulation with TGFBI was not Reciprocally, siRNA-mediated inhibition of MMP14 expression in affected, further supporting the notion that collagen level regulation by fibroblasts led to an increase in collagen levels compared with TGFBI does not occur at the level of production. control siRNA-transfected fibroblasts (Fig. 9C).

Stimulation with TGFBI inhibits expression of MMP14 Regulation of MMP14 and collagen by TGFBI is dependent on A possibility was considered that TGFBI up-regulates collagen PU1 and p53 levels in ﬁbroblast cultures by attenuating collagen turnover. To Parallel semiquantitative screening of DNA binding by transcription address such a possibility, the experiments were performed in factors (TranSignal Protein/DNA Array, Panomics) revealed a signif- which primary ﬁbroblasts were incubated for 6 h with or without icant up-regulation of DNA binding by PU.1 and a down-regulation 5042 MACROPHAGES REGULATE COLLAGEN IN FIBROBLASTS THROUGH TGFBI AND MMP14

transfection technique. Real-time PCR analyses revealed that levels of PU.1 mRNA were decreased 3-fold in PU.1 siRNA-transfected fibroblasts compared with control siRNA-transfected fibroblasts (data not shown). Simultaneously, expression levels of MMP14 increased (Fig. 9B) and levels of collagen decreased in PU.1 siRNA-transfected fibroblasts (Fig. 9C). Transient transfection of fibroblast cultures with p53-encoding plasmid construct was performed to determine whether overexpression of p53 leads to increase in MMP14 and a decrease in collagen protein levels. As expected, MMP14 protein levels increased (Fig. 9B) and collagen levels decreased (Fig. 9C) in fibroblasts transfected with p53-encoding plasmid. These observations suggest that PU.1 and p53 are involved in the regulation of MMP14 and collagen levels in fibroblasts.

Discussion This study addressed the issue of macrophages’ involvement in the regulation of extracellular matrix following ingestion of apoptotic

cells. Macrophages derived from a monocytic cell line THP-1 (see Downloaded from Fig. 2), as well as macrophages derived from primary monocytes FIGURE 8. MMP14 mRNA (A) and protein (B) levels in primary fi- (see Fig. 3), stimulated up-regulation of collagen protein levels in broblasts. A, mRNA was purified from control or TGFBI-stimulated fibro- cocultures with fibroblasts, following ingestion of apoptotic cells. blasts and reverse transcribed into cDNA; real-time PCR amplification was In contrast, primary alveolar macrophages stimulated up-regula- performed using primers for a housekeeping gene GAPDH and for MMP14 tion of collagen levels even without exposure to apoptotic cells, gene. The amplification curves for GAPDH corresponding to control and and this effect was not further increased following ingestion of http://www.jimmunol.org/ TGFBI-stimulated fibroblasts closely overlapped, whereas amplification of apoptotic debris (see Fig. 3). The apparent “preactivation” of al- MMP14 template in cDNA from TGFBI-stimulated fibroblasts occurred veolar macrophages may be due to the specialized TGF-␤-rich two cycles later, suggesting 22 ϭ 4 lower levels of MMP14 mRNA. B, Primary fibroblasts were cultured for indicated times without or with 300 pulmonary environment (31) that predisposes alveolar macro- ng/ml rhTGFBI. Lysates were normalized for total protein, and Western phages to a more profibrotic basal phenotype. The effect of inges- blotting assays conducted using anti-MMP14 Ab. tion of apoptotic cells by macrophages on collagen production by fibroblasts in cocultures was preserved if the macrophages and fibroblasts were separated by a Transwell membrane (see Fig. 4A), of DNA binding by p53 in response to stimulation of fibroblasts suggesting that soluble factors produced by macrophages mediated

with 300 ng/ml rhTGFBI (data not shown). These observations the effect on collagen levels. If macrophages were exposed to ap- by guest on September 26, 2021 suggested that PU.1 might be a repressor, whereas p53 might be an optotic cells separately, and the conditioned medium was then activator of MMP14 expression. To confirm the validity of these transferred into the cultures of the primary fibroblast monolayers, observations in protein/DNA arrays, electromobility shift assays the effect was still preserved (see Fig. 4B), further supporting the were performed to assess DNA binding by p53 (Fig. 9A). To test notion that soluble factor(s) were produced by macrophages fol- whether PU.1 is involved in the regulation of MMP14 and collagen lowing ingestion of apoptotic cells, and that those soluble factors levels, PU.1 expression in fibroblasts was inhibited using siRNA regulated accumulation of collagen.

FIGURE 9. DNA binding by p53 (A) and effects of MMP14 or p53 overexpression, or MMP14 or PU.1 siRNA inhibition on the expression levels of MMP14 and collagen (B and C). A, Electromobility shift assays were used, on two independent occasions, to validate the results of the protein-DNA array experiments. The p53-specific 32P-labeled probe was incubated with nuclear lysates from nonstimulated control or TGFBI-activated fibroblasts, as indicated, or in the presence of 10-fold excess of the mutant (scrambled) or unlabeled (cold) specific probe, as indicated. The cold but not the scrambled probe inhibited p53 binding (arrows), suggesting that the binding is specific. B and C, Fibroblast cultures were transfected with MMP14-encoding or p53-encoding plasmid, or with MMP14 or PU.1 siRNA as indicated. Western blotting assays were performed using Abs against MMP14 or collagen. At least three independent experiments were conducted for each panel shown, with consistent results. The Journal of Immunology 5043

An obvious candidate for such a soluble factor would be TGF-␤, a factor whose expression in macrophages does increase following ingestion of apoptotic debris (4), and that is a known potent profibrotic factor (5). Indeed, ELISA assays revealed that total TGF-␤ was elevated in the macrophage culture supernatants following ingestion of apoptotic debris (see Fig. 5). However, such an increase in TGF-␤ is unlikely to explain the observed increase in collagen for the following reasons. First, a concentration of at least 500 pg/ml active TGF-␤ is required for up-regulation of collagen production (17, 18, 25), whereas in these assays, total TGF-␤ did not exceed 325 pg/ml, and active TGF-␤ was not detectable. Sec- ond, TGF-␤ is known to up-regulate collagen production transcriptionally, leading to elevated steady-state levels of collagen mRNA, but no such increase in collagen ␣2(I) mRNA was observed by real-time PCR in fibroblast-macrophage cocultures following ingestion of apoptotic cells. Finally, the increase in collagen levels was somewhat attenuated but still significant in the presence of neutralizing anti-TGF-␤ Ab (see Fig. 4B). Together, these observations suggest that an additional soluble factor may promote up- Downloaded from regulation of collagen protein levels. To address this possibility, profiling of gene expression for cytokines and related factors has been performed in fibroblasts from cocultures with macrophages. In three independent experiments, expression of mRNA for TGFBI, also termed BIGH3, or keratoepithelin, was increased. Real-time PCR experiments confirmed http://www.jimmunol.org/ this observation in THP-1-derived and primary monocyte-derived, but not in alveolar macrophages (see Fig. 6A), in a time-dependent fashion (see Fig. 6B). Western blotting analyses for TGFBI confirmed that expression of this protein increased following ingestion of apoptotic but not necrotic cells (see Fig. 6C). This effect on TGFBI expression was observed in THP-1-derived macrophages (see Fig. 6C) but not in alveolar macrophages (see Fig. 6D). Thus, FIGURE 10. Proposed outline of the sequential regulation of collagen ex- expression of TGFBI in macrophages and the effect on collagen pression in fibroblasts by macrophages following exposure to apoptotic cells. by guest on September 26, 2021 accumulation in cocultures with fibroblasts are both up-regulated following ingestion of apoptotic cells in THP-1-derived and primary monocyte-derived, but not in alveolar macrophages (com- that TGFBI regulates collagen levels through a mechanism that is pare Fig. 6 with Figs. 2 and 3). The observed nonresponsiveness of different from up-regulation of collagen production. alveolar macrophages may be due to their “preactivation” in the A possibility was considered that TGFBI may regulate collagen ␤ TGF- -rich pulmonary environment (31). These observations sug- turnover, e.g., by inhibiting the levels of matrix metalloproteinases gest that TGFBI may be an important factor mediating the profi- in fibroblasts. To address such a possibility, real-time PCR exper- brotic effect of macrophages that ingested apoptotic cells. While pro- iments were conducted in fibroblasts that were or were not acti- ducing TGFBI (27), and acting profibrotically (28) similarly to vated with rhTGFBI, to compare steady-state levels of mRNA for alternatively activated macrophages, the macrophages that ingested genes related to extracellular matrix. Real-time PCR-based system apoptotic cells did not express other markers of alternative activation, (RT2 Profiler, SuperArray) was used to simultaneously compare such as CD163 or CD206 (29). There was no increase in the levels of expression of 84 extracellular matrix relevant genes. The differ- cytokines IL-4 or IL-13 that are known to facilitate alternative mac- ences between TGFBI-activated and control cells were observed for rophage activation or up-regulation of collagen in fibroblasts. MMP14 (also termed MT1-MMP), whose mRNA levels decreased To investigate the possible profibrotic involvement of TGFBI, 4-fold following activation with TGFBI (see Fig. 8A). Western blot- rhTGFBI was added to cultured primary fibroblasts and collagen ting assays revealed a similar decrease in MMP14 protein following levels assessed (see Fig. 7). Increases in collagen protein levels stimulation of fibroblasts with TGFBI (see Fig. 8B). were observed in response to stimulation with rhTGFBI in a dose- MMP14 is able to directly degrade various extracellular matrix and time-dependent fashion (see Fig. 7, A–D). However, TGFBI components, including type I, II, and III collagens, gelatin, fi- did not stimulate an increase in collagen mRNA according to real- bronectin, vitronectin, tenascin, entactin, and laminin-1, as well as time PCR data (data not shown). Also, a collagen promoter-CAT to directly activate pro-MMP-2 (32, 33). The loss of MMP14 leads reporter construct that has been previously described to respond to to significant disturbances of connective tissue metabolism. Fibro- TGF-␤ activation (25) was transfected into fibroblast and was re- blasts from MMP14-deficient mice completely lose the ability to sponsive to TGF-␤ stimulation but nonresponsive to TGFBI stim- degrade collagen fibrils, resulting in severe and progressive fibro- ulation (data not shown). Profiling of DNA binding by transcrip- sis in many tissues of MMP14-deficient mice (34). In our study, tion factor arrays showed no increase in the activity of Smad3/4, overexpression of MMP14 in cultured fibroblasts caused down- Sp1, AP1, and Ets (factors known to regulate activity of the col- regulation of collagen levels, whereas siRNA-mediated inhibition lagen gene promoter). Finally, siRNA-mediated inhibition of col- of MMP14 expression led to increased collagen levels (Fig. 9C), lagen production (see Fig. 7E) did not affect the TGFBI-stimulated confirming previous observations of MMP14 involvement in col- increase in collagen levels. Together, these observations suggest lagen turnover by fibroblasts (30). 5044 MACROPHAGES REGULATE COLLAGEN IN FIBROBLASTS THROUGH TGFBI AND MMP14

Because the levels of MMP14 decreased at both mRNA and 11. Chow, F. Y., D. J. Nikolic-Paterson, R. C. Atkins, and G. H. Tesch. 2004. Mac- protein levels, the possibility of transcriptional regulation of rophages in streptozotocin-induced diabetic nephropathy: potential role in renal fibrosis. Nephrol. Dial. Transplant. 19: 2987–2996. MMP14 by TGFBI was considered. To assess possible transcrip- 12. Duffield, J. S., S. J. Forbes, C. M. Constandinou, S. Clay, M. Partolina, tion factors that might be involved in down-regulation of MMP14, S. Vuthoori, S. Wu, R. Lang, and J. P. Iredale. 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. profiling of DNA binding activity by various transcription factors J. Clin. Invest. 115: 56–65. was assessed in TGFBI-stimulated in comparison with control fi- 13. Wang, L., J. F. Scabilloni, J. M. Antonini, Y. Rojanasakul, V. Castranova, and broblasts. Experiments with transcription factor arrays revealed R. R. Mercer. 2006. Induction of secondary apoptosis, inflammation, and lung fibrosis after intratracheal instillation of apoptotic cells in rats. Am. J. Physiol. that out of 345 transcription factors analyzed, two factors have 290: L695–L702. significantly changed their DNA binding activity in fibroblasts in 14. Docherty, N. G., O. E. O’Sullivan, D. A. Healy, J. M. Fitzpatrick, and response to activation with TGFBI. Activity of PU.1 was increased R. W. Watson. 2006. Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction. and activity of p53 was decreased following fibroblast activation Am. J. Physiol. 290: F4–F13. with TGFBI. Inhibition of PU.1 expression with siRNA in cultured 15. Canbay, A., H. Higuchi, S. F. Bronk, M. Taniai, T. J. Sebo, and G. J. Gores. 2002. fibroblasts led to an increase in MMP14 expression levels and a Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology 123: 1323–1330. decrease in collagen levels (see Fig. 9, B and C). This observation is 16. Mora, A. L., E. Torres-Gonzalez, C. Corredor, J. Ritzenthaler, J. Xu, J. Roman, consistent with the notion that PU.1 is a repressor of MMP14 expres- K. Brigham, and A. Stecenko. 2006. Activation of alveolar macrophages via the alternative pathway in herpesvirus-induced lung fibrosis. Am. J. Respir. Cell Mol. sion, as stimulation with TGFBI activates DNA binding by PU.1 and Biol. 35: 466–473. simultaneously causes a decrease in MMP14 expression (see 17. Kahari, V. M., Y. Q. Chen, M. W. Su, F. Ramirez, and J. Uitto. 1990. Tumor Fig. 8). Overexpression of p53, a possible activator of MMP14 necrosis factor-␣ and interferon-␥ suppress the activation of human type I collagen gene expression by transforming growth factor-␤1: evidence for two dis- expression, led to a similar increase in MMP14 and decrease in tinct mechanisms of inhibition at the transcriptional and posttranscriptional lev- Downloaded from collagen expression (see Fig. 9, B and C). The latter observation els. J. Clin. Invest. 86: 1489–1495. is consistent with the previous report (35) that suggests an in- 18. Fine, A., and R. H. Goldstein. 1987. The effect of transforming growth factor-␤ on cell proliferation and collagen formation by lung fibroblasts. J. Biol. Chem. hibitory effect of p53 on TGF-␤-stimulated collagen expression. 262: 3897–3902. Based on the observations summarized above, a sequence of 19. Atamas, S. P., V. V. Yurovsky, R. Wise, F. M. Wigley, C. J. Goter Robinson, P. Henry, W. J. Alms, and B. White. 1999. Production of type 2 cytokines by events connecting ingestion of apoptotic cells by macrophages ϩ CD8 lung cells is associated with greater decline in pulmonary function in with collagen accumulation in fibroblasts appears to be as pre- patients with systemic sclerosis. Arthritis Rheum. 42: 1168–1178. http://www.jimmunol.org/ sented in Fig. 10. Physiologically, the studied mechanisms are 20. Luzina, I. G., S. P. Atamas, R. Wise, F. M. Wigley, H. Q. Xiao, and B. White. 2002. Gene expression in bronchoalveolar lavage cells from scleroderma patients. likely to be relevant to inflammation resolution and healing, but, if Am. J. Respir. Cell Mol. Biol. 26: 549–557. exaggerated, may possibly contribute to tissue fibrosis. Pharmaco- 21. Luzina, I. G., S. P. Atamas, R. Wise, F. M. Wigley, J. Choi, H. Q. Xiao, and logical targeting of this pathway may be useful in developing fu- B. White. 2003. Occurrence of an activated, profibrotic pattern of gene expression in lung CD8ϩ T cells from scleroderma patients. Arthritis Rheum. 48: 2262–2274. ture therapies for delayed wound healing or for tissue fibrosis. 22. Atamas, S. P., I. G. Luzina, J. Choi, N. Tsymbalyuk, N. H. Carbonetti, I. S. Singh, M. Trojanowska, S. A. Jimenez, and B. White. 2003. Pulmonary and activation- Acknowledgments regulated chemokine stimulates collagen production in lung fibroblasts. Am. J. Respir. Cell Mol. Biol. We thank Drs. Barry S. Handwerger, Jeffrey D. Hasday, and Simeon E. 29: 743–749. 23. Luzina, I. G., N. Tsymbalyuk, J. Choi, J. D. Hasday, and S. P. Atamas. 2006. Goldblum for critical discussions of this work and Jung Choi for CCL18-stimulated upregulation of collagen production in lung fibroblasts re- by guest on September 26, 2021 technical help. quires Sp1 signaling and basal Smad3 activity. J. Cell. Physiol. 206: 221–228. 24. Atamas, S. P., I. G. Luzina, H. Dai, S. G. Wilt, and B. White. 2002. Synergy Disclosures between CD40 ligation and IL-4 on fibroblast proliferation involves IL-4 receptor signaling. J. Immunol. 168: 1139–1145. The authors have no financial conflict of interest. 25. Luzina, I. G., K. Highsmith, K. Pochetuhen, N. Nacu, J. N. Rao, and S. P. Atamas. 2006. PKC␣ mediates CCL18-stimulated collagen production in References pulmonary fibroblasts. Am. J. Respir. Cell Mol. Biol. 35: 298–305. 1. Huynh, M. L., V. A. Fadok, and P. M. Henson. 2002. Phosphatidylserine-depen- 26. Luzina, I. G., J. C. Papadimitriou, R. Anderson, K. Pochetuhen, and S. P. Atamas. dent ingestion of apoptotic cells promotes TGF-␤1 secretion and the resolution of 2006. Induction of prolonged infiltration of T lymphocytes and transient T lym- inflammation. J. Clin. Invest. 109: 41–50. phocyte-dependent collagen deposition in mouse lungs following adenoviral gene 2. Xiao, Y. Q., K. Malcolm, G. S. Worthen, S. Gardai, W. P. Schiemann, transfer of CCL18. Arthritis Rheum. 54: 2643–2655. V. A. Fadok, D. L. Bratton, and P. M. Henson. 2002. Cross-talk between ERK 27. Gratchev, A., P. Guillot, N. Hakiy, O. Politz, C. E. Orfanos, K. Schledzewski, and and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by S. Goerdt. 2001. Alternatively activated macrophages differentially express fi- ␤ transforming growth factor-␤. J. Biol. Chem. 277: 14884–14893. bronectin and its splice variants and the extracellular matrix protein IG-H3. 3. McDonald, P. P., V. A. Fadok, D. Bratton, and P. M. Henson. 1999. Transcrip- Scand. J. Immunol. 53: 386–392. tional and translational regulation of inflammatory mediator production by en- 28. Song, E., N. Quyang, M. Horbelt, B. Antus, M. Wang, and M. S. Exton. 2000. dogenous TGF-␤ in macrophages that have ingested apoptotic cells. J. Immunol. Influence of alternatively and classically activated macrophages on fibrogenic 163: 6164–6172. activities of human fibroblasts. Cell. Immunol. 204: 19–28. 4. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and 29. Porcheray, V., S. Viaud, A. C. Rimaniol, C. Leone, B. Samah, N. Dereuddre- P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro Bosquet, D. Dormont, and G. Gras. 2005. Macrophage activation switching: an asset inhibit proinflammatory cytokine production through autocrine/paracrine mech- for the resolution of inflammation. Clin. Exp. Immunol. 142: 481–489. anisms involving TGF-␤, PGE2, and PAF. J. Clin. Invest. 101: 890–898. 30. Lee, H., C. M. Overall, C. A. McCulloch, and J. Sodek. 2006. A critical role for 5. Atamas, S. P., and B. White. 2003. Cytokine regulation of pulmonary fibrosis in the membrane-type 1 matrix metalloproteinase in collagen phagocytosis. Mol. scleroderma. Cytokine Growth Factor Rev. 14: 537–550. Biol. Cell 17: 4812–4826. 6. Martin, P., D. D’Souza, J. Martin, R. Grose, L. Cooper, R. Maki, and 31. Takabayshi, K., M. Corr, T. Hayashi, V. Redecke, L. Beck, D. Guiney, S. R. McKercher. 2003. Wound healing in the PU. 1 null mice: tissue repair is not D. Sheppard, and E. Raz. 2006. Induction of a homeostatic circuit in lung tissue dependent on inflammatory cells. Curr. Biol. 13: 1122–1128. by microbial compounds. Immunity 24: 475–487. 7. Reynolds, H. Y. 2005. Lung inflammation and fibrosis: an alveolar macrophage- 32. Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and centered perspective from the 1970s to 1980s. Am. J. Respir. Crit. Care Med. M. Seiki. 1994. A matrix metalloproteinase expressed on the surface of invasive 171: 98–102. tumour cells. Nature 370: 61–65. 8. Prasse, A., D. V. Pechkovsky, G. B. Toews, W. Jungraithmayr, F. Kollert, 33. Okada, A., C. Tomasetto, Y. Lutz, J. P. Bellocq, M. C. Rio, and P. Basset. 1997. T. Goldmann, E. Vollmer, J. Muller-Quernheim, and G. Zissel. 2006. A vicious Expression of matrix metalloproteinases during rat skin wound healing: evidence circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via that membrane type-1 matrix metalloproteinase is a stromal activator of pro- CCL18. Am. J. Respir. Crit. Care Med. 173: 781–792. gellatinase A. J. Cell Biol. 137: 67–77. 9. Zhang-Hoover, J., A. Sutton, N. van Rooijen, and J. Stein-Streilein. 2000. A 34. Holmbeck, K., P. Bianco, J. Caterina, S. Yamada, M. Kromer, S. A. Kuznetsov, critical role for alveolar macrophages in elicitation of pulmonary immune fibro- M. Mankani, P. G. Robey, A. R. Poole, I. Pidoux, et al. 1999. MT1-MMP- sis. Immunology 101: 501–511. deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue dis- 10. Yamate, J., K. Sato, M. Ide, M. Nakanishi, M. Kuwamura, S. Sakuma, and ease due to inadequate collagen turnover. Cell 99: 81–92. S. Nakatsuji. 2002. Participation of different macrophage populations and myofibro- 35. Ghosh, A. K., S. Bhattacharyya, and J. Varga. 2004. The tumor supressor p53 blastic cells in chronically developed renal interstitial fibrosis after cisplatin-induced abrogates Smad-dependent collagen gene induction in mesenchymal cells. renal injury in rats. Vet. Pathol. 39: 322–333. J. Biol. Chem. 279: 47455–47463.