Surfactant Protein-D Regulates Soluble CD14 through Matrix -12 Albert P. Senft, Thomas R. Korfhagen, Jeffrey A. Whitsett, Steven D. Shapiro and Ann Marie LeVine This information is current as of September 25, 2021. J Immunol 2005; 174:4953-4959; ; doi: 10.4049/jimmunol.174.8.4953 http://www.jimmunol.org/content/174/8/4953 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Surfactant Protein-D Regulates Soluble CD14 through -121

Albert P. Senft,* Thomas R. Korfhagen,* Jeffrey A. Whitsett,* Steven D. Shapiro,‡ and Ann Marie LeVine2*†

Surfactant protein D (SP-D) and CD14 are important innate immune defense molecules that mediate clearance of pathogens and apoptotic cells from the lung. To test whether CD14 expression and function were influenced by SP-D, the surface expression of CD14 was assessed on alveolar macrophages from SP-D؊/؊ mice. CD14 was reduced on alveolar macrophages from SP-D؊/؊ mice and was associated with reduced uptake of LPS and decreased production of TNF-␣ after LPS stimulation. CD14 is proteolytically cleaved from the cell surface to form a soluble peptide. Soluble CD14 (sCD14) was increased in the bronchoalveolar lavage fluid ,from SP-D؊/؊ mice. Because matrix metalloproteinase (MMP)-9 and -12 activities were increased in the lungs of SP-D؊/؊ mice ؊ ؊ ؊ ؊ the role of these metalloproteases in the production of sCD14 was assessed. sCD14 was decreased in both MMP9 / /SP-D / and Downloaded from MMP12؊/؊/SP-D؊/؊ mice demonstrating MMP-9 and MMP-12 contribute to proteolytic shedding of CD14. The increased sCD14 seen in SP-D؊/؊ mice was dependent upon the activation of MMP-12 via an MMP-9-dependent mechanism. Supporting this observation, MMP-12 caused the release of sCD14 from RAW 264.7 cells in vitro. In conclusion, SP-D influences innate host defense, in part, by regulating sCD14 in a process mediated by MMP-9 and MMP-12. The Journal of Immunology, 2005, 174: 4953–4959. http://www.jimmunol.org/ urfactant protein D (SP-D)3 is a member of the collectin a GPI-linked receptor that lacks a cytoplasmic signaling domain family of innate defense polypeptides that include surfac- and, therefore, requires interaction with other receptors to elicit its S tant protein A, mannose-binding lectin, and conglutinin biological responses. CD14 binds LPS and interacts with toll-like (1–3). Collectins form multimeric structures consisting of a col- receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) lagenous N-terminal domain and a globular C-terminal carbohy- enhancing MAPK signaling and production of cytokines and che- drate binding domain (4) that bind carbohydrate surfaces of many mokines (13). CD14 mediates phagocytosis of bacteria (14), clear- microorganisms mediating phagocytosis and killing by phagocytic ance of apoptotic cells (15–17), and transport of lipids (18, 19). cells (5). The CD14 receptor exists as both a membrane GPI-anchored pro- SP-D -inactivated mice (SP-DϪ/Ϫ) develop progressive em- tein and soluble protein. Soluble CD14 (sCD14) induces biological by guest on September 25, 2021 physema that is characterized by chronic inflammation, accumulation responses in epithelial cells (20) and endothelial cells (21) by in- of surfactant phospholipids, and infiltration with lipid-laden alveolar teraction with TLRs present on the cell surface. sCD14 serves to macrophages (6). Phagocytosis of bacteria, viruses, and apoptotic down-modulate monocyte and macrophage activation (22–24). cells is impaired in alveolar macrophages from SP-DϪ/Ϫ mice (7–10). sCD14 is produced by proteolytic cleavage (23–26), lipolytic SP-DϪ/Ϫ mice also mount an exacerbated inflammatory response cleavage of the GPI linker (22, 27), or secreted without the GPI when challenged with bacteria (7). SP-D modulates lung inflamma- moiety (28, 29). Increased production of matrix metalloprotease tion by interaction with cell surface receptors on the macrophage in- (MMP) -2, -9, and -12 was detected in alveolar macrophages of cluding signal-inhibitory regulatory protein ␣ (11), CD91 (10, 11), SP-DϪ/Ϫ mice (6). Although it has not been determined whether calreticulin (10, 11), and CD14 (12). MMP-2, -9, or -12 proteolytically cleave CD14 to form sCD14, CD14 is a 55-kDa pattern recognition receptor that is present on several studies suggest that metalloproteases cleave receptors from the surface of monocytes, macrophages, and . CD14 is cell surfaces. Treatment with , a MMP, reduced cell associated CD14 (30). MMP-12 cleaved the GPI-linked urokinase- type plasminogen activator receptor from the cell surface (31). In † Divisions of *Neonatology and Pulmonary Biology and Critical Care Medicine, addition, the general metalloprotease inhibitor 1,10-phenantroline Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and ‡Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Boston, inhibits the formation of sCD16 (32, 33). MA 02115 Although there is clear evidence that SP-D binds to phagocytic Ϫ Ϫ Received for publication November 11, 2004. Accepted for publication February receptors and phagocytosis is impaired in SP-D / mice, mecha- 1, 2005. nisms by which SP-D regulates innate host defense activities of The costs of publication of this article were defrayed in part by the payment of page alveolar macrophages remain unclear. The present study was un- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. dertaken to identify mechanisms whereby SP-D regulates cell sur- 1 This work was supported by National Institutes of Health Grants RO1HL71522 (to face CD14 and its function. A.M.L.), HL58759 (to T.R.K.), HL63329 (to J.A.W.), and T32HL07752, and by an American Lung Association Research Award (to A.M.L.). Materials and Methods 2 Address correspondence and reprint requests to Dr. Ann Marie LeVine, Divisions of Neo- Animals natology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: [email protected] SP-DϪ/Ϫ mice were generated by targeted gene inactivation as previously 3 Abbreviations used in this paper: SP-D, surfactant protein D; TLR4, toll-like re- described (34). SP-D was conditionally replaced in the respiratory epithe- Ϫ/Ϫ Ϫ/Ϫ ϩ ceptor 4; MD-2, myeloid differentiation protein 2; MMP, matrix metalloprotease, lium of SP-D mice by crossing SP-D mice with CCSP-rtTA and ϩ ϩ GBS, group B streptococcus; BAL, bronchoalveolar lavage; sCD14, soluble CD14. (tetO)7-rSPD mice to generate triple-transgenic mice (CCSP-rtTA /

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4954 MMP MEDIATES SP-D-DEPENDENT EXPRESSION OF CD14

ϩ Ϫ/Ϫ (tetO)7-rSPD /SP-D ) as previously described (35). Triple-transgenic Flow cytometry analysis mice were fed doxycycline containing food to induce the expression of the Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ␮ rSP-D protein. MMP9 / /SP-D / and MMP12 / /SP-D / mice were Isolated macrophages and neutrophils were resuspended in 200 lof generated by crossing MMP9Ϫ/Ϫ (36) (kindly provided by Dr. R. Senior, FACS buffer and incubated with purified mouse IgG for 15 min on ice. Washington University School of Medicine, St. Louis, MO) and MMP- Cells were incubated with FITC-conjugated anti-mouse CD14 (BD Pharm- Ϫ/Ϫ Ϫ/Ϫ ϩ ϩ ingen) for1honiceandwashed three times with FACS buffer. Cell- 12 (36) mice with SP-D mice. CCSP-rtTA /(tetO)7-rSPD /SP- DϪ/Ϫ, MMP9Ϫ/Ϫ/SP-DϪ/Ϫ, MMP12Ϫ/Ϫ/SP-DϪ/Ϫ, and SP-DϪ/Ϫ mice sur- associated fluorescence was measured on a FACScan flow cytometer (BD vive and breed normally in the vivarium under barrier containment Biosciences) using CellQuest software (BD Biosciences). For each sample, facilities at the Cincinnati Children’s Hospital Medical Center. Experimen- 10,000 events were acquired, and the results are expressed as mean fluo- tal procedures were reviewed and approved by the Children’s Hospital rescence intensity. Institutional Animal Care and Use Committee. Male and female mice LPS endocytosis 56–70 days old were used for this study. Alveolar macrophages from SP-Dϩ/ϩ and SP-DϪ/Ϫ mice obtained by BAL Preparation of bacteria were placed in culture at a concentration of 5 ϫ 105 cells per well in Group B streptococcus (GBS) and Hemophilus influenzae from clinical serum-free RPMI 1640 medium (Invitrogen Life Technologies). Cultured isolates were cultured as previously described (7). A stock culture of Kleb- alveolar macrophages were incubated with 100 ng of BODIPY-conjugated LPS from Salmonella minnesota (Molecular Probes) for 2 h. A separate siella pneumoniae strain K2 was a generous gift of Dr. I. Ofek (Tel Aviv ϩ/ϩ University, Tel Aviv, Israel). Bacteria were suspended in medium contain- culture of alveolar macrophages from SP-D mice were incubated with ␮ ing 20% glycerol, and frozen in aliquots at Ϫ70°C. Bacteria from the same 1 g of the inhibitory anti-mouse CD14 Ab, biG53, clone SPAK3, (Cell- passage were used to minimize variations in virulence related to culture sciences) for 30 min before LPS treatment to demonstrate CD14 specific- conditions. Before each experiment, an aliquot was thawed and plated on ity. Macrophages were washed five times with PBS and removed by scrap- blood agar plates (Baxter Healthcare), inoculated into trypticase soy broth ing. Cell-associated fluorescence was measured on a FACScan flow (Difco Laboratories), and grown for 14–16 h at 37°C with continuous cytometer (BD Biosciences) using CellQuest software (BD Biosciences). Downloaded from shaking. The broth was centrifuged, and the bacteria were washed in PBS For each sample, 10,000 events were acquired and the results are expressed (pH 7.2) and resuspended in 4 ml of buffer. To facilitate studies, a growth as mean fluorescence intensity. curve was generated so the bacterial concentration could be determined ELISA for TNF-␣ spectrophotometrically and confirmed by quantitative culture of the intra- tracheal inoculum. Alveolar macrophages from SP-Dϩ/ϩ and SP-DϪ/Ϫ mice obtained by BAL were placed in culture at a concentration of 5 ϫ 105 cells per well in Intratracheal inoculation serum-free RPMI 1640 medium (Invitrogen Life Technologies). Macro- http://www.jimmunol.org/ Administration of bacteria into the respiratory tract was performed by in- phages were incubated with 100 ng of LPS (List Biological Laboratories) ␣ ␮ tratracheal inoculation of GBS (106 CFU), H. influenzae (108 CFU), or K. for 18 h. TNF- levels were measured in triplicate with 50 l of macroph- pneumoniae (108 CFU) as previously described (37). Sham-treated mice age-conditioned medium using murine sandwich ELISA kits (R&D Sys- were intratracheally injected with nonpyrogenic PBS. tems) according to the manufacturer’s directions. All plates were read on a microplate spectrophotometer (Bio-Tek Instruments) and analyzed with Bronchoalveolar lavage (BAL) the use of a computer-assisted analysis program (KC Junior; Bio-Tek In- struments). Only assays having standard curves with a calculated regres- Mice were exsanguinated after a lethal i.p. injection of sodium pentobar- sion line value Ͼ0.95 were accepted for analysis. bital and the lungs were lavaged three times with 1 ml of PBS. BAL cells were recovered by centrifugation at 800 ϫ g and then resuspended in Western immunoblot analysis of CD14

FACS buffer (PBS, pH 7.4, containing 0.1% NaN3, and 1% BSA) for flow by guest on September 25, 2021 cytometry or cell lysis buffer (10 mM Tris-HCl, pH 7.5, 15 mM NaCl, BAL macrophages were lysed in cell lysis buffer for the determination of ϫ 0.5% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, pH 8.0, total cellular CD14. Cell lysates were centrifuged at 10,000 g for 10 min 0.2 mM sodium orthovanadate, 0.4 mM PMSF) for Western immunoblot and supernatant was collected. To assess levels of sCD14, BAL fluid was analysis. The BAL supernatant was stored at Ϫ80°C. concentrated using a Centricon YM-10 (Millipore) per the manufacturer’s specification. Protein concentration of the macrophage lysates and BAL Peritoneal lavage fluid was determined using a BCA protein assay kit (Pierce). Equal protein amounts of all samples were resolved on 8–16% SDS-Tris-glycine-poly- Mice were sacrificed by administration of a lethal dose of sodium pento- acrylamide gels (NOVEX). Proteins were transferred to a nitrocellulose barbital. The peritoneal cavity was lavaged three times with 5 ml of PBS. membrane. The membrane was blocked with 3% BSA in Tris-buffered Cells in the peritoneal lavage fluid were recovered by centrifugation at saline with 0.1% Tween 20 (TTBS) and incubated overnight at 4°C with rat ϫ 800 g and resuspended in FACS buffer. anti-mouse CD14 (rmC5-3) antiserum (BD Pharmingen), diluted 1/2,000; Isolation of blood and alveolar neutrophils rabbit anti-mouse SP-D antiserum, diluted 1/5,000; rabbit anti-human MMP-12 (Biomol), diluted 1/500, or rabbit anti-rat MMP-9 (Chemicon SP-Dϩ/ϩ and SP-DϪ/Ϫ mice were intratracheally infected with K. pneu- International), diluted 1/2,000 in TTBS containing 1% BSA. Blots were monia to recruit neutrophils to the lung. Alveolar and blood neutrophils washed with TTBS and incubated with peroxidase-conjugated goat anti-rat were isolated by discontinuous Percoll gradient separation with the density IgG or goat anti-rabbit IgG Ab (Amersham Biosciences) diluted 1/10,000 of the lower fraction at 0.9881 g/ml and the density of the upper fraction in TTBS containing 1% BSA. After washing, blots were developed with a at 0.7497 g/ml. After administration of a lethal dose of sodium pentobar- chemiluminescence detection system (Amersham Biosciences). bital, blood from the descending aorta was drawn into a 1-ml syringe fitted with a 27-gauge needle and filled with 100 U of heparin. BAL was col- RNA isolation and real-time PCR analysis lected as described above. Whole blood and BAL was placed on the gra- Alveolar macrophages recovered by BAL were immediately lysed in 4 M ϫ dient and centrifuged at 800 g for 40 min at 20°C. Neutrophils forming guanidinium isothiocyanate, 0.5% laurylsarcosine, and 0.1 M 2-ME in 25 a discrete band at the interface between the two gradient layers after cen- mM sodium citrate buffer and total cellular RNA was isolated by ultracen- trifugation were recovered, washed twice in PBS, and resuspended in trifugation through a 5.7 M CsCl cushion at 150,000 ϫ g for 18 h at 20°C. FACS buffer. Approximately 70% of BAL cells were neutrophils 6 h after 8 cDNA templates were made by reverse transcription (SuperScript First- infection with 10 CFU of K. pneumoniae. Strand Synthesis System for RT-PCR; Invitrogen Life Technologies). PCR Production of sCD14 by MMP-12 in vitro mixes consisted of template, 0.5 ␮M of each primer (1.0 ␮M of each ϫ primer), 2.5 mM MgCl2, and 1 DNA Master SYBR Green I (Roche The murine macrophage cell line, RAW 264.7, was obtained from the Molecular Biochemicals) that contained Taq polymerase, dNTPs, SYBR American Type Culture Collection and maintained in Dulbecco’s MEM Green dye, and buffer. Reaction conditions differed slightly, depending on containing 10% FBS, 10 mM HEPES, 50 U/ml penicillin, and 50 ␮g/ml the primers used, and generally were 95°C for 120–150 s followed by streptomycin. RAW cells (1 ϫ 106) in six-well plates were treated with 150 35–40 cycles of amplification (95°C for 6–10 s, 59–62°C for 10–15 s, and ng of MMP-12 (Biomol) in PBS. PBS supernatants were recovered, treated 72°C for 15–25 s). Amplification product size and forward and reverse with inhibitor mixture (Sigma-Aldrich), and centrifuged at primer sequences were as follows: L32 (257 bp) 5Ј-GTGAAGCCCAA 10,000 ϫ g for 10 min. After centrifugation, the supernatant was recovered GATCGTC-3Ј,5Ј-AGCAATCTCAGCACAGTAAG-3Ј, CD14 (118 bp) and each sample was concentrated using a Centricon YM-10 (Millipore) 5Ј-AACATCTTGAACCTCCGCAACG-3Ј,5Ј-TGAGTGAGTGTGCTT per the manufacturer’s specification. GGGCAATAC-3Ј. Measurement of amplified product was made for 6 s The Journal of Immunology 4955

Ϫ Ϫ ϩ ϩ every cycle at a temperature above that of the melting temperature of pos- SP-D / and SP-D / mice. CD14 was significantly decreased on sible nonspecific products and 1–2°C below the melting temperature of the alveolar macrophages from SP-DϪ/Ϫ mice at all time points ex- specific product. Melt curve analyses were performed after every run to amined (Fig. 1B). No difference in CD14 was observed on peri- ensure that a single amplified product was produced. Relative quantitation Ϫ/Ϫ ϩ/ϩ was obtained by measuring the cycle at which the greatest accumulation of toneal macrophages from SP-D and SP-D mice (mean flu- product occurred (cycle threshold) and plotting that against the cycle orescence intensity ϭ 11.7 Ϯ 1.1 and 14.6 Ϯ 1.1 respectively; n ϭ thresholds of a dilution series of positive control samples. Only experi- 8, mean Ϯ SEM). ments in which the regression analysis of the dilution series gave an r2 value Ն 0.985 were used to determine quantitation. Impaired CD14-mediated endocytosis of LPS by macrophages Ϫ/Ϫ Statistical methods from SP-D mice Results were compared using ANOVA and Student’s t test. Findings were Endocytosis of LPS was assessed by flow cytometry to determine considered statistically significant at probability levels Ͻ0.05. Results are whether decreased levels of surface CD14 were associated with presented as the mean Ϯ SEM. impaired macrophage endocytic function. Endocytosis of LPS was reduced by 66% in alveolar macrophages isolated from SP-DϪ/Ϫ Results mice (Fig. 2A). LPS uptake was inhibited by an Ab against the Decreased surface expression of CD14 on alveolar CD14 receptor, indicating that endocytosis of the LPS was a Ϫ Ϫ macrophages from SP-D / mice CD14-dependent process (Fig. 2B). These data indicate that re- Surface expression of CD14 was assessed by flow cytometry on duced surface expression of CD14 on alveolar macrophage from Ϫ/Ϫ alveolar macrophage isolated from BAL 2 h after saline treatment SP-D mice contributes to impaired CD14-mediated or intratracheal infection with GBS or H. influenzae. BAL fluid endocytosis. Downloaded from ϩ/ϩ Ϫ/Ϫ from SP-D and SP-D mice contained Ͼ90% macrophages Ϫ Ϫ Impaired TNF-␣ production by macrophages from SP-D / after infection (data not shown). Significantly less CD14 was de- mice tected on alveolar macrophages from uninfected, GBS-, or H. in- fluenzae-infected SP-DϪ/Ϫ mice compared with SP-Dϩ/ϩ mice LPS-induced TNF-␣ production by alveolar macrophages from Ϫ/Ϫ (Fig. 1A). Alveolar macrophage CD14 was reduced 2 h after in- SP-D mice was reduced (Fig. 3), indicating that reduced sur- Ϫ Ϫ

/ http://www.jimmunol.org/ fection with H. influenzae, and peaked at 24 h postinfection in both face expression of CD14 on alveolar macrophage from SP-D mice was associated with impaired CD14-TLR4 signaling.

Whole cell CD14 levels are reduced in alveolar macrophages from SP-DϪ/Ϫ mice Whole cell CD14 levels were assessed by Western immunoblot analysis in uninfected and H. influenzae-infected mice to deter- mine whether reduced surface expression of CD14 was caused by by guest on September 25, 2021

FIGURE 1. CD14 is reduced on alveolar macrophages from SP-DϪ/Ϫ mice. Flow cytometry analysis was used to detect cell surface CD14 on alveolar macrophages recovered 2 h after intratracheal infection with GBS or H. influenzae, and on alveolar macrophages recovered 2, 4, 6, and 24 h after intratracheal infection with H. influenzae (0 h time point represents FIGURE 2. Impaired uptake of LPS by SP-DϪ/Ϫ alveolar macrophages. uninfected mice). A, CD14 was significantly decreased on alveolar mac- Flow cytometry analysis was performed on cultured alveolar macrophages rophages from uninfected, GBS-infected, and H. influenzae-infected SP- treated with BODIPY-LPS to determine LPS uptake. A, Alveolar macro- DϪ/Ϫ (Ⅺ) compared with SP-Dϩ/ϩ mice (o). B, CD14 was significantly phages from SP-DϪ/Ϫ mice (Ⅺ) endocytosed significantly less LPS than decreased on alveolar macrophages from SP-DϪ/Ϫ (F) compared with the those from SP-Dϩ/ϩ mice (o). B, Pretreatment with the CD14 blocking Ab SP-Dϩ/ϩ mice (f) at all time points examined after infection with H. significantly inhibited LPS uptake in the SP-Dϩ/ϩ mice indicating CD14 p Ͻ 0.05 ,ء ;p Ͻ 0.05 compared with specificity of LPS endocytosis. Data are means Ϯ SEM, n ϭ 6 ,ء ;influenzae. Data are means Ϯ SEM, n ϭ 6 SP-Dϩ/ϩ mice with similar treatment conditions. compared with SP-Dϩ/ϩ alveolar macrophages. 4956 MMP MEDIATES SP-D-DEPENDENT EXPRESSION OF CD14

reduced in the MMP-9Ϫ/Ϫ/SP-DϪ/Ϫ mice suggesting that MMP-12 was necessary for increased sCD14 observed in SP-DϪ/Ϫ mice and that MMP-9 played a role in the increased activity of MMP-12.

MMP12 cleaves CD14 in vitro Because MMP-12 appeared to be important for the formation of sCD14 in vivo, RAW 264.7 cells were treated with recombinant MMP-12. sCD14 in the culture medium after MMP-12 treatment FIGURE 3. Alveolar macrophages from SP-DϪ/Ϫ mice produce less was analyzed by Western immunoblot analysis. MMP-12 treat- TNF-␣ after LPS challenge. Cultured BAL alveolar macrophages were plated at the same density and treated with 100 ng/ml LPS for 18 h and ment of RAW 264.7 cells increased sCD14 in the cell culture TNF-␣ was measured in culture medium by ELISA. LPS-challenged al- medium (Fig. 7). The electrophoretic mobility of in vivo and in veolar macrophage from SP-DϪ/Ϫ mice (Ⅺ) produced significantly less vitro generated sCD14 was compared by Western immunoblot TNF-␣ than those from SP-Dϩ/ϩ mice (s). No differences in TNF-␣ levels analysis and was similar for the two samples (data not shown). were detected in unchallenged alveolar macrophage from SP-DϪ/Ϫ mice This further supports the observation that MMP-12 is important for ϩ ϩ .p Ͻ 0.05 the formation of sCD14 in the SP-DϪ/Ϫ mouse lung ,ء ;f) and SP-D / mice (o). Data are means Ϯ SEM, n ϭ 6) compared with SP-Dϩ/ϩ alveolar macrophages. Kinetics of reduced surface and elevated sCD14 after loss of SP-D in vivo Downloaded from reduced cellular content of CD14. Significantly less CD14 was CCSP-rtTAϩ/(tetO) -rSPDϩ/SP-DϪ/Ϫ mice, conditionally ex- detected in alveolar macrophage lysates from both uninfected and 7 Ϫ Ϫ pressing SP-D in the bronchiolar and respiratory under H. influenzae-infected SP-D / mice (Fig. 4). control of doxycycline (35), were used to determine the time SP-D does not influence CD14 mRNA in alveolar macrophages course of decreased alveolar macrophage surface CD14 and in- creased BAL fluid sCD14 after the loss of SP-D. As previously Because cellular CD14 was reduced in alveolar macrophages from http://www.jimmunol.org/ Ϫ Ϫ reported (35), BAL SP-D concentration decreased rapidly after the SP-D / mice, CD14 mRNA was measured by real-time PCR removal from doxycycline. Alveolar macrophage CD14 decreased analysis. Alveolar macrophage CD14 mRNA levels were similar Ϫ Ϫ ϩ ϩ (Fig. 8A) and sCD14 increased (Fig. 8B) within 3 days, findings in SP-D / and SP-D / mice (CD14/L32 ϭ 1.0 Ϯ 0.2 and 1.0 Ϯ that persisted thereafter. Immunoreactive bands for active 0.2, respectively; n ϭ 8, mean Ϯ SEM). MMP-12 were increased 36 h after the removal of doxycycline sCD14 is increased in the BAL fluid from SP-DϪ/Ϫ mice (Fig. 8B).

To determine whether reduced macrophage CD14 levels were Reversibility of sCD14 by SP-D in vivo caused by receptor shedding, sCD14 levels were assessed by West- ϩ ϩ Ϫ Ϫ

/ by guest on September 25, 2021 ern immunoblot analysis on BAL fluid. Significantly more sCD14 CCSP-rtTA /(tetO)7-rSPD /SP-D mice were used to deter- was detected in BAL fluid from SP-DϪ/Ϫ mice (Fig. 5). mine whether the elevated BAL sCD14 after loss of SP-D was reversible with replacement of SP-D. sCD14 was increased in the MMPs regulate sCD14 levels in SP-DϪ/Ϫ mice BAL fluid 3 days after conditional loss of SP-D. Surprisingly, Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ sCD14 remained increased when expression of SP-D was restored MMP-9 / /SP-D / and MMP-12 / /SP-D / mice were used for 5 days (Fig. 9). Thus the loss of SP-D increased MMP-12 to determine the involvement of MMP-9 and MMP-12 in the pro- activity in the lung. However, restoration of SP-D for 5 days was duction of sCD14. sCD14 was assessed by immunoblot analysis of ϩ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ not sufficient to reduce the increase in MMP-12, perhaps indicating BAL fluid from SP-D / , SP-D / , MMP-9 / /SP-D / , and Ϫ Ϫ Ϫ Ϫ a generalized inflammation in the lung during this time. MMP-12 / /SP-D / mice. sCD14 was significantly reduced in BAL fluid from MMP-9Ϫ/Ϫ/SP-DϪ/Ϫ and MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ Ϫ/Ϫ Decreased cell surface expression of CD14 on alveolar compared with SP-D mice (Fig. 6A). However, loss of MMP-9 Ϫ Ϫ neutrophils from SP-D / mice or MMP-12 in the SP-DϪ/Ϫ genetic background did not fully re- store BAL sCD14 levels to those observed in SP-Dϩ/ϩ mice (Fig. Cell surface expression of CD14 was assessed by flow cytometry 6A). Immunoblot analysis for active forms of MMP-9 and on neutrophils isolated from blood and BAL fluid from mice in- MMP-12 was performed on BAL samples from SP-DϪ/Ϫ, MMP- tratracheally infected with K. pneumoniae for 6 h. Significantly Ϫ Ϫ 9Ϫ/Ϫ/SP-DϪ/Ϫ, and MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ mice to confirm that less CD14 was detected on alveolar neutrophils from SP-D / ϩ ϩ both MMP-9 and MMP-12 are involved in the formation of sCD14 mice compared with SP-D / mice (Fig. 10A). In contrast, CD14 Ϫ Ϫ ϩ ϩ in the SP-DϪ/Ϫ mice. MMP-9 was not detected in the BAL from was similar on blood neutrophils from SP-D / and SP-D / the MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ mice and MMP-12 was significantly mice (Fig. 10B). These results indicate that SP-D also has a tissue- specific effect on CD14 expression.

FIGURE 4. Cellular CD14 levels are reduced in alveolar macrophage from SP-DϪ/Ϫ mice. Immunoblot was used to compare CD14 levels from SP-DϪ/Ϫ and SP-Dϩ/ϩ alveolar macrophages that were uninfected or in- FIGURE 5. Increased sCD14 in BAL from SP-DϪ/Ϫ mice. Immunoblot fected with H. influenzae. CD14 levels were reduced in alveolar macro- was used to measure sCD14 in BAL fluid. sCD14 was increased in BAL phages from uninfected and H. influenzae-infected SP-DϪ/Ϫ compared fluid from SP-DϪ/Ϫ compared with SP-Dϩ/ϩ mice. Each lane of the im- with SP-Dϩ/ϩ mice. Each lane represents alveolar macrophage cell lysates munoblot was loaded with 20 ␮g of concentrated BAL fluid protein from from one animal. an individual animal. The Journal of Immunology 4957 Downloaded from

FIGURE 6. MMP-12 mediates SP-D-dependent changes in CD14. A, BAL sCD14 was determined by immunoblot analysis on samples from SP-DϪ/Ϫ, SP-Dϩ/ϩ, MMP-9Ϫ/Ϫ/SP-DϪ/Ϫ, and MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ FIGURE 8. Conditional regulation of alveolar macrophage CD14 and Ϫ/Ϫ Ϫ/Ϫ ϩ ϩ Ϫ/Ϫ mice. sCD14 levels were reduced in BAL from MMP-9 /SP-D and sCD14 by SP-D. CCSP-rtTA /(tetO)7-rSPD /SP-D mice, which con- MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ compared with SP-DϪ/Ϫ mice but remained in- ditionally express SP-D, were used to determine the kinetics of decreased http://www.jimmunol.org/ creased when compared with SP-Dϩ/ϩ mice. Each lane of the immunoblot membrane CD14 and increased BAL fluid sCD14 after the loss of SP-D. was loaded with 20 ␮g of protein from concentrated BAL fluid of an Alveolar macrophage CD14 was determined by flow cytometry analysis individual animal. B, The immunoblot shown is representative of three and BAL sCD14, SP-D and MMP-12 by immunoblot analysis. Alveolar independent experiments that are summarized in the bar graph. Immuno- macrophage CD14 decreased (A) while MMP-12 (22 kDa) and sCD14 ϩ ϩ Ϫ/Ϫ increased (B) in CCSP-rtTA /(tetO)7-rSPD /SP-D mice in association ,ء ;reactive band densitometry data are expressed as mean Ϯ SEM, n ϭ 6 p Ͻ 0.05 compared with SP-Dϩ/ϩ mice, #, p Ͻ 0.05 compared with SP- with the loss of SP-D after removal from doxycycline. Each lane of the DϪ/Ϫ mice. C, Immunoblot analysis for active MMP-9 (92 kDa) and immunoblot was loaded with 20 ␮g of protein from concentrated BAL MMP-12 (22 kDa) was performed on BAL from SP-DϪ/Ϫ, MMP9Ϫ/Ϫ/SP- fluid from an individual animal. The immunoblot shown is representative DϪ/Ϫ, and MMP12Ϫ/Ϫ/SP-DϪ/Ϫ mice. Active forms of MMP-9 and of three independent experiments. Flow cytometry data are expressed as p Ͻ 0.05 compared with conditional CCSP- by guest on September 25, 2021 ,ء ;MMP-12 were detected in BAL from SP-D mice. MMP-9 was not detected mean Ϯ SEM, n ϭ 6 Ϫ/Ϫ Ϫ/Ϫ ϩ ϩ Ϫ/Ϫ in BAL from MMP-12 /SP-D mice and MMP-12 was significantly rtTA /(tetO)7-rSPD /SP-D mice treated with doxycycline. reduced in the MMP-9Ϫ/Ϫ/SP-DϪ/Ϫ mice. Each lane of the immunoblot was loaded with 20 ␮g of protein from concentrated BAL fluid of an Reduced CD14 on the surface of the alveolar macrophage resulted individual animal. in a functional impairment of LPS uptake and LPS-induced cyto- kine production. The present study identifies a novel mechanism Discussion by which SP-D influences CD14 levels on alveolar macrophages CD14 in association with TLR4 and MD-2 comprise a tripartite that in turn, regulates responses to LPS. In the current study, sCD14 was significantly increased in BAL receptor that mediates cellular responses to LPS and other patho- Ϫ/Ϫ gen recognition motifs (13). Studies were undertaken to examine fluid from the SP-D mice. Cleavage of CD14 from the cell the possible contributions of SP-D to the regulation of CD14 ex- surface is an important physiologic event that serves to down- pression by alveolar macrophages. Loss of SP-D from the lung modulate monocyte-macrophage activation (22–24). Previous resulted in a rapid loss of CD14 from the alveolar macrophage cell studies demonstrated that collagenase (30), neutrophil elastase surface and an accumulation of sCD14 in BAL. Accumulation of (23), and cathepsin G (26) cleave CD14 from the cell surface. sCD14 was dependent, in part, on MMP-12 and appears not to be Metalloproteases, including MMP-12, function as sheddases for a readily reversible with the reintroduction of SP-D into the lung. variety of cell surface receptors (30–33). Because previous studies indicated that MMP-2, -9, and -12 were increased in the SP-DϪ/Ϫ mice (6), the present study sought to determine whether these met- alloproteases were involved in the elevated sCD14 observed in BAL fluid from SP-DϪ/Ϫ mice. Loss of MMP-9 or MMP-12 in the SP-DϪ/Ϫ genetic background did not correct the foamy macro- phage infiltrate or emphysema characteristic of the SP-DϪ/Ϫ mice (38). However, in the present study, targeted ablation of MMP-9 or MMP-12 significantly reduced BAL sCD14 levels in SP-DϪ/Ϫ mice (38). MMP-9 was not present in the MMP-12Ϫ/Ϫ/SP-DϪ/Ϫ FIGURE 7. MMP12 increases sCD14 in vitro. sCD14 in cell culture mice. This observation is in agreement with findings by Lanone et medium from sham- and MMP-12-treated RAW264.7 cells was compared by Western immunoblot analysis. MMP12 increased sCD14 in cell culture al. (39) in which MMP-12 was required for optimal accumulation medium from RAW 264.7 cells. Each lane of the immunoblot was loaded of MMP-9 after IL-13 stimulation. Surprisingly, MMP-12 was Ϫ/Ϫ Ϫ/Ϫ with 20 ␮g of protein from concentrated culture medium from one cell greatly reduced in the MMP-9 /SP-D mice, suggesting that culture well. The immunoblot is representative of three independent MMP-9 influences the accumulation of active MMP-12 in the SP- Ϫ Ϫ experiments. D / mouse lung. In addition, increased sCD14 in the cell culture 4958 MMP MEDIATES SP-D-DEPENDENT EXPRESSION OF CD14

its carbohydrate recognition domain, inhibiting CD14-LPS inter- actions (12). Therefore, loss of SP-D quickly leads to the LPS- dependent activation of alveolar macrophage because LPS is in the environment and the lung is continually exposed. The current study also aimed to determine whether the elevated sCD14 observed in the absence of SP-D could be reversed with the FIGURE 9. Conditional replacement of SP-D does not reduce BAL ϩ ϩ Ϫ/Ϫ restoration of SP-D expression. Surprisingly, sCD14 did not de- sCD14. CCSP-rtTA /(tetO)7-rSPD /SP-D mice were used to used to determine whether increased BAL fluid sCD14 associated with lack of crease five days after SP-D expression was restored. Interestingly, SP-D was reversed by restoration of SP-D. BAL sCD14 and MMP-12 were MMP-12 also remained elevated five days after SP-D expression assessed by Western immunoblot analysis. The active forms of MMP-12 was restored. This supports the concept that MMP-12 is integral is Ϫ Ϫ and sCD14 were increased in the BAL 3 days after the conditional removal the proteolytic cleavage of CD14 in SP-D / mice. Although it is of SP-D (ϪDox 3d) compared with wild-type (ϩDox). Restoration of not clear why MMP-12 remained elevated, the observation is sim- SP-D for 5 days (ϪDox 3d, ϩDox 5d) did not reduce the levels of BAL ilar to that by Yoshida et al. (40) in which addition of mouse SP-D sCD14 nor did it reduce the active form of MMP-12 (22 kDa) in the BAL in vitro did not reduce MMP production by alveolar macrophages Ϫ (when compared with Dox 3d). Each lane of the immunoblot was loaded from SP-DϪ/Ϫ mice. Together the findings support the concept with 20 ␮g of protein from concentrated BAL fluid of an individual animal. that activation of MMPs after the loss of SP-D occurs by initiation of a complex signaling pathway resulting in the formation of sec- medium from RAW 264.7 cells treated with active MMP-12 sup- ondary mediators and that SP-D is not directly able to inhibit the secondary mediators activating the MMPs. Downloaded from ports the concept that MMP-12 cleaves CD14 from the alveolar Ϫ/Ϫ macrophage cell surface. MMP-12-dependent cleavage of CD14 Decreased surface CD14 on macrophages from SP-D mice ϳ was associated with decreased LPS-stimulated TNF-␣ production. accounts for formation of 60% of the sCD14 in BAL fluid from Ϫ/Ϫ SP-DϪ/Ϫ mice. Mechanisms explaining sCD14 formation that was Macrophages from CD14 mice are also deficient in LPS-in- ␣ not MMP-12 dependent in the MMP-9Ϫ/Ϫ/SP-DϪ/Ϫ and MMP- duced IL-6 and TNF- production (41) supporting the concept that 12Ϫ/Ϫ/SP-DϪ/Ϫ mice have not been elucidated. However, cleav- reduced CD14 resulted in impaired LPS-induced cytokine produc- tion in the current study. The lack of TNF-␣ production from SP- http://www.jimmunol.org/ age of CD14 by MMP-2, a collagenase, is likely since previous Ϫ/Ϫ studies have demonstrated that MMP-2 is increased in the SP- D macrophage appears contradictory to previous in vivo stud- Ϫ/Ϫ ies that demonstrated elevated TNF-␣, IL-6, and IL-1␤ in H. D mice (6, 40) and collagenase reduced the level of CD14 on Ϫ/Ϫ the macrophage cell surface (30). influenzae-infected SP-D mice (7). Interestingly, respiratory ϩ ϩ Ϫ/Ϫ epithelial cells express TLRs (42) and when treated with LPS and CCSP-rtTA /(tetO)7-rSPD /SP-D mice, which condition- ally express SP-D in the bronchiolar and respiratory epithelium sCD14 elicited IL-6 and IL-8 production (20). This suggests that the respiratory epithelium may be an important source of cytokines (35), were used to understand the sequence of events after the loss Ϫ/Ϫ of SP-D that lead to reduced alveolar macrophage surface CD14, in SP-D mice after bacterial challenge. Phagocytic uptake of LPS was also impaired in alveolar mac-

elevated BAL sCD14, and appearance of the active form of by guest on September 25, 2021 rophages from SP-DϪ/Ϫ mice. The reduced LPS uptake suggests MMP-12 in the BAL. In support of the concept that MMP-12 Ϫ/Ϫ mediates production of a large portion of sCD14 found in the BAL that SP-D mice are impaired in their ability to clear LPS from of the SP-DϪ/Ϫ mice, increased levels of BAL MMP-12 and the lung; however, it is unlikely that phagocytic defect observed Ϫ/Ϫ sCD14 as well as reduced surface expression of alveolar macro- previously in SP-D mice (7) is due solely to reduced CD14 phage CD14 was observed three days after loss of SP-D. Thus because CD14-deficient macrophages were not impaired in their changes in alveolar macrophages seen in SP-DϪ/Ϫ mice occur rap- ability to phagocytose Escherichia coli (41). idly after the loss of SP-D. A possible explanation for the rapid The increased MMP-12, sCD14, and reduced LPS-induced ␣ changes is based upon recent findings that SP-D binds to CD14 via TNF- production are similar to the effects reported in human studies examining the effects of cigarette smoking. Cigarette smokers exhibit reduced SP-D levels (43), elevated MMP levels (44), and reduced alveolar macrophage CD14 levels (45). In ad- dition, human alveolar macrophages in culture produced signifi- cantly less TNF-␣ and IL-6 after cigarette smoke exposure (46). This suggests MMP-12 may mediate reduction of alveolar macro- phage CD14 and reduced macrophage cytokine production in smokers and other pulmonary diseases characterized by elevated MMPs. In summary, SP-D is an important regulator of microbial clear- ance and inflammatory processes that are important for host de- fense and pulmonary homeostasis. The current study demonstrates that SP-D regulates MMP production that in turn alters the surface expression of the phagocytic receptor CD14 and associated mac- rophage responses to LPS. FIGURE 10. CD14 is reduced on alveolar neutrophils from SP-DϪ/Ϫ mice. Intratracheal infection with K. pneumonia was used to cause neutro- phil influx into the lung. Flow cytometry analysis was used to measure Acknowledgments CD14 on alveolar and blood neutrophils. A, Blood neutrophil CD14 was We thank Dr. Robert M. Senior for generously providing the MMP-9Ϫ/Ϫ similar for SP-DϪ/Ϫ (Ⅺ) and SP-Dϩ/ϩ mice (o). B, CD14 was significantly mice. We thank Jaymi Semona, Victor LaFay, and Lindsey Malone for decreased on alveolar neutrophils from SP-DϪ/Ϫ (Ⅺ) compared with SP- their assistance with animal husbandry. We thank Katy Davis and p Ͻ 0.05 compared with Theresa Richardson for their assistance in generating MMP/SP-D double ,ء ;Dϩ/ϩ mice (o). Data are means Ϯ SEM, n ϭ 6 SP-Dϩ/ϩ mice with similar treatment conditions. knockouts. The Journal of Immunology 4959

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