THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 41, Issue of October 12, pp. 37846–37852, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Induction of Macrophage Biosynthesis by Surfactant Protein D*

Received for publication, March 21, 2001, and in revised form, July 23, 2001 Published, JBC Papers in Press, July 31, 2001, DOI 10.1074/jbc.M102524200

Barbara Crippes Trask‡, Mark J. Malone‡, Esther H. Lum‡, Howard G. Welgus§, Erika C. Crouch¶, and Steven D. Shapiro‡ʈ From the ‡Departments of Pediatrics, Cell Biology and Physiology, and Medicine and the ¶Department of Pathology and Immunology, Washington University School of Medicine at St. Louis Children’s and Barnes-Jewish Hospitals, St. Louis, Missouri 63110 and §Pfizer Global Development, Ann Arbor, Michigan 48105

Recent studies strongly suggest that surfactant pro- addition to membership in this family of immune mediators, tein D (SP-D) plays important roles in pulmonary host there is growing evidence suggesting that SP-D can act as a defense and the regulation of immune and inflammatory mediator of host defense within the alveolar spaces and distal reactions in the lung. Although SP-D can bind to alveo- airways of the lung. Downloaded from lar macrophages and can elicit their chemotaxis, rela- SP-D is secreted as a homotrimer, each trimer consisting of tively little is known about the direct cellular conse- four structural domains: the amino-terminal, cysteine-rich, quences of SP-D on the function of these cells. Because cross-linking region; the triple-helical collagenous domain; the matrix metalloproteinases (MMPs) are synthesized in coiled-coil “neck” (or linking) region; and the globular, carboxyl- increased amounts in response to various proinflamma- terminal carbohydrate recognition domain (CRD) (1). While tory stimuli, we investigated the capacity of SP-D to

trimerization is sufficient for some of the functions of the pro- www.jbc.org modulate the production of MMPs by freshly isolated tein, SP-D in the alveolar space exists predominantly as a human alveolar macrophages. Unexpectedly we found dodecamer, consisting of four identical homotrimeric subunits. that recombinant rat SP-D dodecamers selectively in- This higher order multimerization is thought to mediate bridg- duce the biosynthesis of -1 (MMP-1), strome- lysin (MMP-3), and macrophage elastase (MMP-12) with- ing interactions, enabling simultaneous interaction with mul- at Washington University on February 27, 2009 out significantly increasing the production of tumor tiple binding partners. necrosis factor ␣ and interleukin-1␤. SP-D did not alter Trimerization is required for high affinity SP-D binding to the production of these MMPs by fibroblasts. Phosphati- various carbohydrate ligands, and the association with glyco- dylinositol, a surfactant-associated ligand that interacts conjugates on bacterial, fungal, and viral surfaces has been with the carboxyl-terminal neck and carbohydrate rec- attributed to the trimeric CRD region of SP-D (1, 4). These ognition domains of SP-D, inhibited the SP-D-dependent carbohydrate-mediated interactions have been suggested to as- increase in MMP biosynthesis. A trimeric, recombinant sist in the clearance of pathogens and of certain organic par- protein consisting of only the neck and carbohydrate ticulate antigens. In addition to carbohydrates, the SP-D car- recognition domain did not augment metalloproteinase boxyl-terminal domain is also capable of binding to the production, suggesting that the stimulatory effect on surfactant lipid phosphatidylinositol in part via the inositol MMP production depends on an appropriate spatial moiety of the lipid (5–8). presentation of trimeric lectin domains. Although SP-D SP-D has also been shown to interact with alveolar macro- dodecamers can selectively augment metalloproteinase phages (9, 10). Macrophage association with this surfactant activity in vitro, this effect may be competitively inhib- protein has been demonstrated to affect cellular function by ited by tissue inhibitors of metalloproteinases or surfac- several parameters. SP-D elicits directional polymerization of tant-associated ligands in vivo. F-actin to regulate macrophage chemotaxis, may induce the production of superoxide radicals (11, 12), and enhances the internalization and killing of some microorganisms in vitro (13, Surfactant protein D (SP-D)1 is a member of the collectin 14). In addition, SP-D-null mice show decreased alveolar subfamily of C-type lectins that also includes surfactant pro- macrophage phagocytosis of certain bacteria in vivo (15). tein A and the serum protein mannose-binding protein (1–3). In Macrophages are known to mediate host defense by their pro- duction of inflammatory cytokines, nitric oxide, and toxic oxygen * This work was supported by National Institutes of Health Grants metabolites. These cells also have the capacity to secrete several P01-HL29594 (to E. C. C. and S. D. S.), HL-44015 (to E. C. C.), and members of the matrix metalloproteinase family including colla- T32-HL07873 (to B. C. T.) and by a Glaxo-Wellcome fellowship (to genase-1 (MMP-1, Ref. 16), stromelysin (MMP-3, Ref. 17), gela- M. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby tinase B (MMP-9, Ref. 18), and macrophage elastase (MMP-12, marked “advertisement” in accordance with 18 U.S.C. Section 1734 Ref. 19). While the activity of these proteinases likely contributes solely to indicate this fact. to both the host defense function of macrophages and to normal ʈ To whom correspondence should be addressed: Dept. of Pediatrics, tissue remodeling and repair, ectopic or excessive MMP proteol- Washington University School of Medicine, Campus Box 8208, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-286-2778; Fax: 314- ysis has been associated with several pathological conditions 286-2895; E-mail: [email protected]. including arthritis, cancer growth and metastasis, and chronic 1 The abbreviations used are: SP-D, surfactant protein D; MMP, obstructive pulmonary disease (20–22). matrix metalloproteinase; IL, interleukin; TNF, tumor necrosis factor; To date, few modulators that induce macrophage MMP bio- PI, phosphatidylinositol; CRD, carbohydrate recognition domain; LPS, synthesis have been identified, particularly those of host deri- lipopolysaccharide; DMEM, Dulbecco’s modified Eagle’s medium; F12, Ham’s F12; FBS, fetal bovine serum; PMA, phorbol 12-myristate vation. Both lipopolysaccharide (LPS) and zymosan are known 13-acetate. to induce MMP production by alveolar macrophages (23, 24).

37846 This paper is available on line at http://www.jbc.org SP-D Induction of Macrophage Metalloproteinases 37847

Diethyl maleate (25) and granulocyte-macrophage colony-stim- binant protein was mixed with chloroform-solubilized lipid (Avanti ulating factor (26) have also been shown to enhance production Polar Lipids, Inc., Alabaster, AL) in a microcentrifuge tube and allowed to incubate at room temperature for 15 min prior to addition to cells. of macrophage elastase by mouse peritoneal macrophages. Human peripheral blood monocytes were obtained from healthy vol- Given the co-localization of SP-D with human alveolar unteers. Cells were isolated by Ficoll-Hypaque density gradient centrif- macrophages in vivo (27), the ability of macrophages and SP-D ugation followed by countercurrent centrifugal elutriation of the mono- to interact in vitro (6, 9), and evidence that SP-D can modulate nuclear layer in a Beckman J2–21 centrifuge as described previously the response of alveolar macrophage to certain pulmonary (33). Isolated monocytes were plated in six-well plates at a density of ϫ 6 pathogens (13–15), we examined the effect of this surfactant 2 10 cells/ml/well. Cells were allowed to adhere for 1–3 h after which medium was changed to DMEM/F12 containing 5% FBS. Cells were protein on various parameters of alveolar macrophage activa- then left untreated or were treated with LPS (2.5 ␮g/ml) or SP-D (5 tion, including metalloproteinase biosynthesis. By contrast ␮ g/ml) and were maintained at 37 °C, 5% CO2. In vitro differentiation of with the more generalized activation response elicited by bac- macrophages from peripheral blood monocytes was accomplished by terial LPS, SP-D dodecamers caused a selective increase in the culturing isolated cells for 7 days in DMEM supplemented with gluta- production of specific MMPs without concomitant stimulation mine, antibiotics, and 10% autologous human serum with only one of proinflammatory cytokine secretion. These effects were de- medium change at day 1 postplating. Human fibroblasts (American Type Culture Collection no. CRL 1905) pendent on the state of multimerization of the SP-D CRDs and were plated in 75-cm2 tissue culture flasks and were maintained in were inhibited with phosphatidylinositol. DMEM containing 10% FBS in a humidified incubator at 37 °C, 5% ϳ CO2. When cells reached confluence ( 3 days postplating), the media MATERIALS AND METHODS were changed to DMEM containing 5% FBS, and cells were then left ␮ ␮ Chemicals and Reagents—All chemicals were purchased from Sigma untreated or were treated with LPS (2.5 g/ml), SP-D (10 g/ml), or Downloaded from Chemical Co. unless otherwise indicated. All radioisotopes were pur- PMA (20 ng/ml). chased from Amersham Pharmacia Biotech. Immunologic Assay of Secreted Proteins—Human alveolar macro- Isolation of Surfactant Protein D—Recombinant rat SP-D was iso- phages were obtained and plated in six-well plates as described above. 1–3 lated as described previously (28). Briefly, CHO-K1 cells were trans- h postplating, media were changed, and cells were left untreated or were ␮ fected with a full-length rat SP-D cDNA in the pEE14 mammalian treated with SP-D (10 g/ml). Media were harvested at the indicated expression vector. Cells were selected for incorporation of the plasmid, times, nonadherent cells were removed by centrifugation, and media samples were stored at Ϫ80 °C until assayed. An immunosorbent assay cloned by serial dilution, and screened for production of recombinant www.jbc.org was used to measure collagenase-1 protein in the macrophage-conditioned protein. Conditioned medium from a clonal population was harvested medium after 72 h and to determine the time course of collagenase-1 and dialyzed against Tris-buffered saline containing 10 mM EDTA. The induction (34). For measurement of macrophage cytokine production, cells dialysate was recalcified and applied to a maltosyl-agarose column were left untreated or were incubated in the presence of either LPS (2.5 equilibrated with Tris-buffered saline containing 2 mM calcium chlo- ␮ ␮

g/ml) or SP-D (5 g/ml). Media were harvested at 48 h post-treatment at Washington University on February 27, 2009 ride. The column was washed with the same buffer prior to elution of and stored at Ϫ80 °C until assayed. Human TNF␣ and IL-1␤ immunoas- the bound protein with Tris-buffered saline containing 10 mM EDTA. say kits were purchased from R&D Systems (Minneapolis, MN) and were SP-D dodecamers were then resolved from larger multimers and minor used according to the manufacturer’s specifications. lower molecular weight contaminants by gel filtration chromatography Immunoprecipitation Assays—Human alveolar macrophages were under nondenaturing conditions as described previously (28). The obtained, plated, and maintained overnight as described above. At 24 h eluted proteins were visualized by SDS-polyacrylamide gel electro- postplating, media were aspirated, and cells were starved in serum- phoresis and silver staining, and proteins in the desired fractions were free, methionine-free DMEM/F12. After 1 h, media were again aspi- concentrated by rechromatography on maltosyl-agarose. For most stud- rated, and newly synthesized proteins were metabolically labeled by ies, the bound protein was eluted with Hepes-buffered saline containing incubating the cells in methionine-free DMEM/F12 containing 5% dia- 10 mM EDTA and was stored at Ϫ80 °C. lyzed FBS to which 50 ␮Ci/ml [35S]methionine (Amersham Pharmacia Recombinant rat SP-D CRD containing an amino-terminal His tag 6 Biotech) had been added. Human peripheral blood monocytes were was expressed in Escherichia coli (29) and was purified by nickel affin- labeled 24 h postisolation and plating as for the macrophages above. In ity chromatography. Characterization of the recombinant protein indi- all cases, cells were maintained at 37 °C, 5% CO2 for 48 h before cated that it self-associated to form trimers as it was eluted from a gel radiolabeled media were harvested for immunoprecipitation of radiola- filtration column at the size expected for a trimeric protein. Carbohy- beled proteins. drate binding activity of the recombinant CRD was confirmed by its Radiolabeled conditioned media were diluted 1:1 with a buffer con- ability to bind maltosyl-agarose. Previous studies have shown that taining 50 mM Tris, pH 8, 20 mM EDTA, and 0.8% Triton X-100. these trimeric mutants retain their ability to elicit chemotaxis and to Samples were then “precleared” to diminish nonspecifically binding bind various microbial ligands in vitro (30, 31). proteins by rotating end-over-end for1hatroom temperature with 45 The level of endotoxin contamination for both recombinant proteins ␮l of a 50% slurry of Protein A-Sepharose (Zymed Laboratories Inc., San was quantified using a sensitive, end point chromogenic microplate Francisco, CA). Protein A-Sepharose was removed by centrifugation, assay (Chromogenix, Gothenberg, Sweden) with E. coli O111:B4 endo- and media were transferred to new tubes containing polyclonal anti- toxin as a standard. The endotoxin content of the purified recombinant body for collagenase-1 (35), 92-kDa (36), stromelysin 1 (37), Ͻ Ͻϳ ␮ proteins used for these experiments was 2 ng/ml or 10 pg/ g SP-D or macrophage elastase (38). 35 ␮l of a 50% slurry of protein A-Sepha- for our stock solutions. For individual assays, the stock was further rose was added to each tube, and immunoprecipitations were allowed to diluted at least 25-fold in endotoxin-free medium, giving final endotoxin proceed by rotation end-over-end overnight at 4 °C. Beads were pelleted concentrations of Ͻ80 pg/ml, several orders of magnitude less than the by centrifugation and washed four times with detergent buffer contain- amount associated with detectable macrophage activation in our ing 0.1% SDS, 0.25% deoxycholate, 2 mM EDTA, 0.5% (w/v) Triton system. X-100, 150 mM NaCl, and 50 mM Tris, pH 8. Immunoprecipitated Cells and Cell Culture—Human alveolar macrophages were obtained proteins were separated on 10–12% polyacrylamide gels. Following by bronchoalveolar lavage of healthy adult volunteer cigarette smokers electrophoresis, gels were fixed, treated with En3Hance (Amersham (minimum, 1 pack/day). For each harvest, 600 ml of sterile saline was Pharmacia Biotech), and dried before visualization of immunoprecipi- introduced and recovered from the right middle lobe and lingular sub- tated proteins by fluorography. segment as described previously (32). Cells were pelleted by centrifu- RNA Analysis—Human alveolar macrophages obtained by bron- gation and washed three times with serum-free Dulbecco’s modified choalveolar lavage as described above were plated in 75-cm2 tissue Eagle’s medium/Ham’s F12 (DMEM/F12, 1:1, v/v, BioWhittaker, Walk- culture flasks (Costar, Corp., Cambridge, MA) at a density of 3 ϫ 107 ersville, MD) before plating in Linbro six-well plates (Flow Laborato- cells/flask. Cells were allowed to adhere for 1–3 h after which media ries, Inc., McLean, VA) at a density of 2 ϫ 106 cells/ml/well. Cells were were replaced. Cells were then left untreated or were treated with LPS

allowed to adhere for 1–3 h in a humidified incubator (37 °C, 5% CO2) (2.5 ␮g/ml) or SP-D (10 ␮g/ml). Approximately 26 h postplating (24 h after which the medium was changed to DMEM/F12 containing 5% fetal post-treatment), media were aspirated, and RNA was isolated using bovine serum (FBS). This treatment resulted in a population of cells TRIZOL total RNA isolation reagent (Life Technologies, Inc.) according that were Ͼ98% macrophages. Cells were then left untreated or were to the manufacturer’s specifications. treated with experimental reagents as indicated and were maintained Human fibroblasts were cultured and treated with LPS, SP-D, or

at 37 °C, 5% CO2. In the case of SP-D preincubation with lipids, recom- PMA as described above. Approximately 24 h post-treatment, media 37848 SP-D Induction of Macrophage Metalloproteinases

FIG.1.Effect of SP-D on collagenase-1 (MMP-1) biosynthesis. Immunoprecipitation of 35S metabolically labeled collagenase-1 from the medium of untreated (lane 1), LPS-treated (lane 2), or SP-D-treated alveolar macrophages (lanes 3–5) shows that the surfactant protein FIG.2.Effect of SP-D on collagenase-1 steady-state mRNA lev- induces collagenase-1 biosynthesis by these cells. Final SP-D concen- els. Panel a, macrophages were cultured in the presence of no agent trations are 0.1 (lane 3),1(lane 4), and 10 ␮g/ml (lane 5). (lane 1), LPS (lane 2), PMA (lane 3), and SP-D (lane 4). Total cellular RNA was isolated, and a Northern blot was performed. Panel b shows the ethidium bromide-stained gel prior to transfer, indicating that each were aspirated, and RNA was isolated using TRIZOL total RNA isola- lane contains similar amounts of total RNA. Downloaded from tion reagent as described above. Total RNA was separated by electrophoresis through a 1% agarose gel containing 1 M formaldehyde as described previously (39). Equal loading and RNA integrity were confirmed by staining with ethidium bromide. RNA was transferred to a Hybond-Nϩ membrane (Amersham Pharmacia Biotech) by capillary transfer and was cross-linked to the filter by drying under vacuum at 80 °C for 1 h. The filter was prehy- www.jbc.org bridized for1hat65°C in a buffer containing 250 mM Na2HPO4,7% SDS, 1 mM EDTA, and 0.5% bovine serum albumin. Hybridization was performed in the same buffer under the same conditions overnight. cDNA probes for collagenase-1 (40) and ␤-actin were random prime- labeled with [32P]dCTP using the Redivue labeling kit (Amersham at Washington University on February 27, 2009 Pharmacia Biotech) according to the manufacturer’s specifications. Fol- lowing hybridization, the filter was washed three times (10 min/wash) in a buffer containing 40 mM Na2HPO4, 1% SDS, and 1 mM EDTA at 60 °C. The filter was exposed overnight at Ϫ80 °C to Kodak XAR-5- Omat film.

RESULTS

Exposure of Alveolar Macrophages to SP-D Induces the Bio- FIG.3.Time course of collagenase-1 secretion in response to synthesis and Secretion of Collagenase-1 (MMP-1)—Although SP-D. Macrophages were cultured in the presence of SP-D, conditioned activated macrophages have the capacity to produce several media was collected at various times, and collagenase secretion was quantified by -linked immunosorbent assay. Note, collagenase-1 matrix metalloproteinases, unstimulated cells secrete nearly secretion peaks at 48 h postexposure. undetectable levels of several MMPs, including collagenase-1 (Ref. 18; Fig. 1, lane 1). MMP synthesis by macrophages is induced upon their activation as evidenced by collagenase-1 and macrophage elastase (MMP-12). Under in vitro culture secretion after treatment with lipopolysaccharide (Fig. 1, lane conditions, MMP-9 is secreted constitutively, while MMP-3 and 2). Incubation with purified recombinant SP-D also induces -12 are synthesized following treatment with an activating biosynthesis of collagenase-1 by alveolar macrophages in a agent such as LPS. Immunoprecipitation assays were per- dose-dependent manner (Fig. 1, lanes 3–5). Maximal stimula- formed to assess whether the biosynthesis of these proteinases tion was in the physiologically relevant range of 1–10 ␮g/ml was also modulated in response to SP-D. Fig. 4 indicates that SP-D (11). Even 1 ␮g/ml SP-D resulted in collagenase-1 pro- production of both MMP-3 and -12 were induced by SP-D treat- duction roughly equivalent to that seen with maximal LPS ment (Fig. 4, panels C and B, respectively), while the biosyn- stimulation. It should be noted that, based upon electrophoretic thesis of MMP-9 was unchanged (Fig. 4, panel A). mobility, treatment with either reagent yielded the catalyti- Exposure of Alveolar Macrophages to SP-D Does Not Result cally active form of the metalloproteinase. Northern analysis of in Generalized Cellular Activation—Macrophage activation, as total cellular RNA demonstrated induction of collagenase-1 classically defined by tumoricidal and phagocytic capacity, is steady-state mRNA levels by SP-D (Fig. 2). fully induced by treatment with both LPS and interferon ␥. A time course of collagenase-1 secretion was determined LPS alone induces macrophage production of reactive oxygen using an enzyme-linked immunosorbent assay with SP-D- species and nitric oxide (41) as well as secretion of various treated, macrophage-conditioned medium. Fig. 3 shows that no inflammatory mediators including TNF␣ and IL-1␤ (42). SP-D collagenase-1 was detected during the first4hoftreatment. treatment of macrophages has also been reported to induce the Secretion increased linearly after 24 h post-treatment, yielding production of reactive oxygen (12) but does not significantly a final production that was calculated to be 0.95 ␮g of colla- increase the production of nitric oxide in the absence of con- genase-1/106 cells/24 h. taminating endotoxin (43). To further investigate the ability of Biosynthesis of Stromelysin (MMP-3) and Macrophage Elas- SP-D to induce a more generalized cellular activation, secretion tase (MMP-12) Is Also Induced by SP-D—In addition to colla- of both TNF␣ and IL-1␤ were measured using enzyme-linked genase-1 secretion, alveolar macrophages can synthesize and immunosorbent assays following exposure of alveolar macro- secrete several additional metalloproteinases, including gela- phages to either recombinant surfactant protein or optimal tinase B (92-kDa gelatinase or MMP-9), stromelysin (MMP-3), concentrations of LPS. Results for each of these assays are SP-D Induction of Macrophage Metalloproteinases 37849

lated by the trimeric neck ϩ CRD or whether it is dependent upon the dodecameric structure of SP-D, cells were treated with a recombinant trimeric SP-D protein consisting of the neck and carbohydrate recognition domains. The recombinant trimeric protein, although active as a lectin, failed to induce collagenase-1 production as demonstrated by our inability to immunoprecipitate significant amounts of this MMP from the medium of CRD-treated cells (Fig. 8).

FIG.4. Effect of SP-D on biosynthesis of other macrophage DISCUSSION MMPs. Macrophages were cultured in the presence of increasing con- We found that SP-D selectively and potently stimulates the centrations of SP-D, LPS, or no agent, and the 35S metabolically labeled conditioned medium was immunoprecipitated for the presence of synthesis and secretion of three metalloproteinases produced MMPs. Note that SP-D is able to induce the biosynthesis of MMP-12 by human alveolar macrophages. In contrast to LPS-mediated (panel b) and MMP-3 (panel c) but has no effect on macrophage MMP-9 macrophage activation, there was no concomitant increase in biosynthesis (panel a). In each of the three panels, lane 1 shows un- the production of TNF␣ or IL-1␤ with SP-D treatment. The treated cells, lane 2 shows cells incubated in the presence of LPS, and inhibitory activity of a known SP-D ligand, PI, strongly sug- lanes 3–5 show macrophages treated with increasing concentrations of SP-D. Final SP-D concentrations are 0.1 (lane 3),1(lane 4), and 10 gests that the effects of SP-D involve an interaction between ␮g/ml (lane 5). the carboxyl-terminal domain of SP-D and the macrophage

either on the cell surface or following its internalization (47). Downloaded from shown in Fig. 5. As expected, LPS induced a significant in- The inability of recombinant trimeric CRD to stimulate MMP crease in the production of both TNF␣ and IL-1␤. By contrast, biosynthesis suggests that dodecameric multimerization is re- neither cytokine was significantly up-regulated in response to quired for this effect. SP-D. Given that LPS can stimulate MMP production and that SP-D Treatment Has No Effect on Fibroblast MMP Produc- SP-D binds to LPS (48), we were initially concerned that the tion—While MMPs are secreted by a number of different cell effects of SP-D were mediated by contaminating endotoxin www.jbc.org types, the mediators that regulate production are often cell- either directly or through SP-D-dependent presentation of specific. To investigate the cell type specificity of the SP-D LPS to the cell. In this regard, it has been demonstrated that response, interstitial fibroblasts, peripheral blood monocytes the apparent effects of soluble SP-D on macrophage NO pro- and macrophages derived from in vitro differentiation of mono- duction can be attributed to endotoxin (43); it is also possible cytes were treated with the surfactant protein, and immuno- that LPS is responsible for the reported effects of human at Washington University on February 27, 2009 precipitation assays for MMPs were performed. The results alveolar proteinosis-derived SP-D on oxygen radical produc- shown in Fig. 6 demonstrate that in contrast to both alveolar tion by macrophages (12). To address this possibility, we used and in vitro differentiated macrophages of nonsmokers, periph- a sensitive assay to quantify endotoxin levels in highly puri- eral blood monocytes do not secrete MMP-1 and minimal fied SP-D dodecamer preparations. The final LPS concentra- amounts of MMP-12 after stimulation with SP-D (but not LPS) tions in our assays were orders of magnitude below the (not shown). Fibroblasts do not produce MMP-1 in response to threshold of LPS required for detectable macrophage MMP SP-D. Fibroblasts do, however, synthesize this proteinase in production. That MMP biosynthesis is an SP-D-mediated response to treatment with PMA (Fig. 6, panel a, lane 2). rather than an endotoxin-mediated effect is further sup- Phosphatidylinositol Inhibits SP-D Induction of Collagen- ported by the failure of purified SP-D to induce macrophage ase-1 Biosynthesis—To further characterize the mechanism of IL-1␤ and TNF␣ production at the same concentration that MMP induction, we examined the effects of a known competing induced metalloproteinase production. The effects of higher, ligand, phosphatidylinositol (PI), on macrophage MMP biosyn- likely supraphysiologic, concentrations of SP-D on cytokine thesis. The interactions of SP-D with PI have been well char- production were not investigated. acterized and are mediated by the carboxyl-terminal domain of The mechanism by which SP-D associates with the macro- SP-D and, at least in part, by the inositol moiety of the lipid phage to elicit MMP biosynthesis is unclear. The surfactant (5–8). To assess the possible role of the carboxyl-terminal lectin protein could be mediating its effects via an interaction with a domain of SP-D in this induction we incubated SP-D with PI specific macrophage cell surface receptor (10). At present, GP- prior to the addition of the carboxyl-terminal domain of SP-D to 340 is the only putative cell surface receptor described for the cells. An immunoprecipitation assay for collagenase-1 fol- SP-D. This protein has been immunologically localized to the lowing treatment with PI-incubated SP-D is shown in Fig. 7. surface of human alveolar macrophages and interacts with the These experiments demonstrate that phosphatidylinositol can carbohydrate recognition domain, although by a lectin-inde- inhibit the SP-D-mediated induction of collagenase-1 synthesis pendent mechanism (49, 50). In addition, lung GP-340 appears and strongly implicates the carboxyl-terminal domain of SP-D to lack a transmembrane domain, and recent studies suggest as the effector. Of note, we observed some interindividual var- that it is identical to salivary agglutinin (51). Thus, the role of iability in that 10 ␮g/ml PI entirely inhibited macrophage GP-340 as a cellular receptor is uncertain. In any case, the MMP production in some volunteers (not shown), while the ability of an SP-D ligand, PI, to inhibit MMP biosynthesis rules effect was only partial (as in Fig. 7) in others. against GP-340-dependent activation. Furthermore, in prelim- Recombinant Trimeric SP-D CRD Does Not Induce Macro- inary studies, we found that an antibody that blocks the inter- phage Collagenase-1 Biosynthesis—Previous studies have action of SP-D with GP-340 was unable to inhibit the macro- shown that some CRD-dependent activities of SP-D, such as phage response (data not shown). Taken together, the available macrophage chemotaxis (30), inhibition of IL-2-dependent T- data argue against a role for GP-340 in the induction of MMP lymphocyte proliferation (44), and neutralization of respiratory biosynthesis. syncytial virus in vivo (45), can be mediated by the trimeric Although we believe GP-340 can be discounted as the medi- neck ϩ CRD domains. Other activities, such as the enhanced ator of the MMP biosynthetic effect of SP-D, it is possible that internalization of viral particles, require multimerization of SP-D binds via its CRD to another transmembrane receptor in trimeric subunits into dodecamers (46). To determine whether a lectin-dependent manner. In this regard, SP-D has recently the effect of SP-D on macrophage MMP biosynthesis is modu- been shown to bind to N-linked sugars associated with soluble 37850 SP-D Induction of Macrophage Metalloproteinases

FIG.5. Effect of LPS and SP-D on macrophage cytokine expression. Macrophages were cultured in the pres- ence of no agent, LPS, or SP-D, and the conditioned medium was subjected to en- zyme-linked immunosorbent assay using antibodies directed against TNF␣ (left)or IL-1␤ (right). Note that alveolar macro- phages do not secrete significant amounts of TNF␣ (left) or IL-1␤ (right) in response to SP-D. LPS treatment resulted in macrophage secretion of both inflamma- tory cytokines. Downloaded from www.jbc.org FIG.6. Effect of SP-D and other agents on biosynthesis of fibroblast and monocyte collagenase-1. Immunoprecipitation as- says of 35S metabolically labeled collagenase-1 from the medium of fibroblasts (panel a) and peripheral blood monocytes (panel b) show that

the MMP biosynthetic effect of SP-D is specific to macrophages. Panel a at Washington University on February 27, 2009 shows untreated fibroblasts (lane 1) and cells treated with PMA (lane FIG.8.Effect of CRD on biosynthesis of collagenase-1. Macro- 2), LPS (lane 3), or 10 ␮g/ml SP-D (lane 4). Panel b shows untreated phages were cultured in the presence of varying concentrations of the monocytes (lane 1) and cells treated with LPS (lane 2)or10␮g/ml SP-D recombinant trimeric SP-D CRD, and immunoprecipitation of 35S met- (lane 3). abolically labeled medium for collagenase-1 was performed. This do- main was unable to independently induce MMP-1 biosynthesis. Lane 1 shows untreated cells, lane 2 shows macrophages treated with LPS, lane 3 shows cells treated with 10 ␮g/ml dodecameric SP-D, lane 4 shows treatment with 1 ␮g/ml trimeric CRD, and lane 5 shows cells treated with 5 ␮g/ml CRD.

either cell surface glycoconjugates or membrane lipids mediat- ing the observed effect. It is unclear how a lipid-mediated interaction would transduce a biosynthetic signal, but the proc- ess could involve PI-dependent alterations in signal transduc- tion mediated by other membrane-associated molecules. FIG.7.Effect of PI on SP-D-induced biosynthesis of collagen- Regardless of the cell surface molecule responsible for medi- 35 ase-1. Immunoprecipitation assays of S metabolically labeled colla- ating the MMP induction of SP-D, failure of the trimeric neck genase-1 from the medium of alveolar macrophages treated with 10 ϩ ␮g/ml SP-D (panel a, lanes 1–5)or2.5␮g/ml LPS (panel b, lanes 6–10) CRD recombinant protein to stimulate the MMP production show that co-incubation with PI inhibits the MMP-inducing effect of suggests that functional interactions leading to increased MMP SP-D in a dose-dependent manner but has no effect upon LPS-mediated production, unlike those associated with chemotaxis, involve stimulation of MMP biosynthesis. Alveolar macrophages treated with multivalent or bridging interactions between two or more bind- SP-D (lane 1) or LPS (lane 6) alone show that both reagents induce MMP-1 biosynthesis. The effects of mixing SP-D with decreasing con- ing sites on the phagocyte. Although the truncated protein was centrations of PI prior to addition to the cells are shown in lanes 2–5, produced in bacterial cells, a lack of post-translational modifi- while LPS mixed with decreasing concentrations of PI is shown in lanes cation is not likely responsible for the absence of a response as ␮ ␮ 7–10. Lanes 2 and 7,10 g/ml PI; lanes 3 and 8,5 g/ml PI; lanes 4 and there are no known modifications within this region of either 9,1␮g/ml PI; and lanes 5 and 10, 0.5 ␮g/ml PI. rat or human SP-D. Furthermore, bacterially expressed human SP-D neck ϩ CRD showed the expected structure when crys- CD14 (52). While macrophage CD14 is a possible SP-D cell tallized (60). Moreover, the CRD used in these studies bound to surface receptor, the pattern of cellular activation observed maltose, indicating carbohydrate binding functionality, and with SP-D is distinct from that for CD14-dependent macro- eluted as a trimer on gel filtration, showing that it retained the phage activation by LPS. ability to self-associate (29). While subtle differences due to Rather than a direct association with a transmembrane pro- structure cannot be completely discounted, failure of CRD-de- tein receptor, several studies have implicated cell surface gly- pendent MMP induction together with the lipid inhibition data coconjugates in the binding of SP-D to leukocytes (9, 53, 54). In lead us to speculate that CRD-dependent bridging interactions addition, Wright and co-workers (47) have reported prelimi- between membrane-associated glycolipids or other glycoconju- nary evidence suggesting that SP-D can bind to endogenous PI gates modulates specific aspects of macrophage function. associated with the outer leaflet of the alveolar macrophage The observed effects do appear to be specific for macrophages plasma membrane. The results presented here, including the as SP-D failed to induce MMP biosynthesis in either fibroblasts inhibitory effects of PI on MMP production, are consistent with or peripheral blood monocytes (Fig. 6). SP-D stimulation of SP-D Induction of Macrophage Metalloproteinases 37851

MMP production by in vitro differentiated macrophages (not edly abnormal with abundant lipid vacuoles, and the functional shown) suggests that SP-D-generated MMP production may be activities of these abnormal cells have not yet been fully char- limited by the state of mononuclear cell differentiation rather acterized. Thus, it is possible that SP-D deficiency indirectly than by cellular location or smoking status. augments MMP expression via aberrant surfactant turnover The functional role(s) of metalloproteinases in normal lung and the elaboration of proinflammatory cytokines. In this re- function have not been fully elucidated. It is clear that these gard, cross-breeding of SP-D-null mice with mice deficient in have a role in maintaining normal tissue architecture; granulocyte-macrophage colony-stimulating factor yields a host defense is another probable function. Secretion of serine double knock-out phenotype with offspring that lack many of proteinases by neutrophils has been shown to have a protective the features characteristic of the SP-D-null animals (59). effect in prevention against bacterial infection (55), and the In conclusion, we speculate that under normal, unchallenged synthesis of metalloproteinases by macrophages could have a circumstances in vivo, interaction of SP-D with airspace PI similar protective effect. Preliminary results do, in fact, dem- prevents the surfactant protein from activating macrophage onstrate that MMP-12 is involved in macrophage-mediated metalloproteinase biosynthesis. However, in injury or in path- killing of Gram-positive bacteria (56). Similarly, matrilysin has ological conditions where SP-D concentrations become elevated been shown to activate defensins in the gastrointestinal tract or when surfactant lipid concentrations are decreased relative (57), and production of matrix metalloproteinases by macro- to normal, an imbalance between the surfactant components phages in the airspace may induce activation of similar pulmo- could result in a local induction of these proteinases. While nary host defense molecules. such an induction, if excessive, might result in tissue damage,

While our data indicate that SP-D can modulate macrophage it might also be beneficial to the extent that MMPs participate Downloaded from metalloproteinase production in vitro, it is unknown whether in antimicrobial host defense or pulmonary repair. the surfactant protein stimulates the release of active enzyme Acknowledgments—We thank Dr. Uffe Holmskov for providing the in vivo. The mobility of the immunoreactive proteins on SDS- anti-GP-340 antibody and Dr. Andrew Limper for supplying the recom- polyacrylamide gel electrophoresis strongly suggests cleavage binant rat neck ϩ CRD used in this study. of the amino-terminal precursor domains, consistent with the generation of active protein. The most likely explanation for REFERENCES www.jbc.org our detection of activated MMPs is that macrophage production 1. Crouch, E. C. (2000) Respir. Res. 1, 93–108 2. Wright, J. R. (1997) Physiol. Rev. 77, 931–962 of urokinase results in the generation of plasmin, a known 3. Lawson, P. 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