Surfactant Protein-D Regulates Soluble CD14 through Matrix Metalloproteinase-12
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免疫学杂志 2005年第8期
Abstract
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 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.
Introduction
Surfactant protein D (SP-D)3 is a member of the collectin family of innate defense polypeptides that include surfactant protein A, mannose-binding lectin, and conglutinin (1, 2, 3). Collectins form multimeric structures consisting of a collagenous N-terminal domain and a globular C-terminal carbohydrate binding domain (4) that bind carbohydrate surfaces of many microorganisms mediating phagocytosis and killing by phagocytic cells (5).
SP-D gene-inactivated mice (SP-D–/–) develop progressive emphysema that is characterized by chronic inflammation, accumulation of surfactant phospholipids, and infiltration with lipid-laden alveolar macrophages (6). Phagocytosis of bacteria, viruses, and apoptotic cells is impaired in alveolar macrophages from SP-D–/– mice (7, 8, 9, 10). SP-D–/– mice also mount an exacerbated inflammatory response when challenged with bacteria (7). SP-D modulates lung inflammation by interaction with cell surface receptors on the macrophage including signal-inhibitory regulatory protein (11), CD91 (10, 11), calreticulin (10, 11), and CD14 (12).
CD14 is a 55-kDa pattern recognition receptor that is present on the surface of monocytes, macrophages, and neutrophils. CD14 is a GPI-linked receptor that lacks a cytoplasmic signaling domain and, therefore, requires interaction with other receptors to elicit its biological responses. CD14 binds LPS and interacts with toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) enhancing MAPK signaling and production of cytokines and chemokines (13). CD14 mediates phagocytosis of bacteria (14), clearance of apoptotic cells (15, 16, 17), and transport of lipids (18, 19). The CD14 receptor exists as both a membrane GPI-anchored protein and soluble protein. Soluble CD14 (sCD14) induces biological responses in epithelial cells (20) and endothelial cells (21) by interaction with TLRs present on the cell surface. sCD14 serves to down-modulate monocyte and macrophage activation (22, 23, 24).
sCD14 is produced by proteolytic cleavage (23, 24, 25, 26), lipolytic cleavage of the GPI linker (22, 27), or secreted without the GPI moiety (28, 29). Increased production of matrix metalloprotease (MMP) -2, -9, and -12 was detected in alveolar macrophages of SP-D–/– mice (6). Although it has not been determined whether MMP-2, -9, or -12 proteolytically cleave CD14 to form sCD14, several studies suggest that metalloproteases cleave receptors from cell surfaces. Treatment with collagenase, a MMP, reduced cell associated CD14 (30). MMP-12 cleaved the GPI-linked urokinase-type plasminogen activator receptor from the cell surface (31). In addition, the general metalloprotease inhibitor 1,10-phenantroline inhibits the formation of sCD16 (32, 33).
Although there is clear evidence that SP-D binds to phagocytic receptors and phagocytosis is impaired in SP-D–/– mice, mechanisms by which SP-D regulates innate host defense activities of alveolar macrophages remain unclear. The present study was undertaken to identify mechanisms whereby SP-D regulates cell surface CD14 and its function.
Materials and Methods
Animals
SP-D–/– mice were generated by targeted gene inactivation as previously described (34). SP-D was conditionally replaced in the respiratory epithelium of SP-D–/– mice by crossing SP-D–/– mice with CCSP-rtTA+ and (tetO)7-rSPD+ mice to generate triple-transgenic mice (CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/–) as previously described (35). Triple-transgenic mice were fed doxycycline containing food to induce the expression of the rSP-D protein. MMP9–/–/SP-D–/– and MMP12–/–/SP-D–/– mice were generated by crossing MMP9–/– (36) (kindly provided by Dr. R. Senior, Washington University School of Medicine, St. Louis, MO) and MMP-12–/– (36) mice with SP-D–/– mice. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/–, MMP9–/–/SP-D–/–, MMP12–/–/SP-D–/–, and SP-D–/– mice survive and breed normally in the vivarium under barrier containment facilities at the Cincinnati Children’s Hospital Medical Center. Experimental procedures were reviewed and approved by the Children’s Hospital Institutional Animal Care and Use Committee. Male and female mice 56–70 days old were used for this study.
Preparation of bacteria
Group B streptococcus (GBS) and Hemophilus influenzae from clinical isolates were cultured as previously described (7). A stock culture of Klebsiella pneumoniae strain K2 was a generous gift of Dr. I. Ofek (Tel Aviv University, Tel Aviv, Israel). Bacteria were suspended in medium containing 20% glycerol, and frozen in aliquots at –70°C. Bacteria from the same passage were used to minimize variations in virulence related to culture conditions. Before each experiment, an aliquot was thawed and plated on blood agar plates (Baxter Healthcare), inoculated into trypticase soy broth (Difco Laboratories), and grown for 14–16 h at 37°C with continuous shaking. The broth was centrifuged, and the bacteria were washed in PBS (pH 7.2) and resuspended in 4 ml of buffer. To facilitate studies, a growth curve was generated so the bacterial concentration could be determined spectrophotometrically and confirmed by quantitative culture of the intratracheal inoculum.
Intratracheal inoculation
Administration of bacteria into the respiratory tract was performed by intratracheal inoculation of GBS (106 CFU), H. influenzae (108 CFU), or K. pneumoniae (108 CFU) as previously described (37). Sham-treated mice were intratracheally injected with nonpyrogenic PBS.
Bronchoalveolar lavage (BAL)
Mice were exsanguinated after a lethal i.p. injection of sodium pentobarbital and the lungs were lavaged three times with 1 ml of PBS. BAL cells were recovered by centrifugation at 800 x g and then resuspended in FACS buffer (PBS, pH 7.4, containing 0.1% NaN3, and 1% BSA) for flow cytometry or cell lysis buffer (10 mM Tris-HCl, pH 7.5, 15 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, 0.4 mM PMSF) for Western immunoblot analysis. The BAL supernatant was stored at –80°C.
Peritoneal lavage
Mice were sacrificed by administration of a lethal dose of sodium pentobarbital. The peritoneal cavity was lavaged three times with 5 ml of PBS. Cells in the peritoneal lavage fluid were recovered by centrifugation at 800 x g and resuspended in FACS buffer.
Isolation of blood and alveolar neutrophils
SP-D+/+ and SP-D–/– mice were intratracheally infected with K. pneumonia to recruit neutrophils to the lung. Alveolar and blood neutrophils were isolated by discontinuous Percoll gradient separation with the density of the lower fraction at 0.9881 g/ml and the density of the upper fraction at 0.7497 g/ml. After administration of a lethal dose of sodium pentobarbital, 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 collected as described above. Whole blood and BAL was placed on the gradient and centrifuged at 800 x g for 40 min at 20°C. Neutrophils forming a discrete band at the interface between the two gradient layers after centrifugation were recovered, washed twice in PBS, and resuspended in FACS buffer. Approximately 70% of BAL cells were neutrophils 6 h after infection with 108 CFU of K. pneumoniae.
Production of sCD14 by MMP-12 in vitro
The murine macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection and maintained in Dulbecco’s MEM containing 10% FBS, 10 mM HEPES, 50 U/ml penicillin, and 50 μg/ml streptomycin. RAW cells (1 x 106) in six-well plates were treated with 150 ng of MMP-12 (Biomol) in PBS. PBS supernatants were recovered, treated with protease inhibitor mixture (Sigma-Aldrich), and centrifuged at 10,000 x g for 10 min. After centrifugation, the supernatant was recovered and each sample was concentrated using a Centricon YM-10 (Millipore) per the manufacturer’s specification.
Flow cytometry analysis
Isolated macrophages and neutrophils were resuspended in 200 μl of FACS buffer and incubated with purified mouse IgG for 15 min on ice. Cells were incubated with FITC-conjugated anti-mouse CD14 (BD Pharmingen) for 1 h on ice and washed three times with FACS buffer. Cell-associated fluorescence was measured on a FACScan flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). For each sample, 10,000 events were acquired, and the results are expressed as mean fluorescence intensity.
LPS endocytosis
Alveolar macrophages from SP-D+/+ and SP-D–/– mice obtained by BAL were placed in culture at a concentration of 5 x 105 cells per well in serum-free RPMI 1640 medium (Invitrogen Life Technologies). Cultured alveolar macrophages were incubated with 100 ng of BODIPY-conjugated LPS from Salmonella minnesota (Molecular Probes) for 2 h. A separate culture of alveolar macrophages from SP-D+/+ mice were incubated with 1 μg of the inhibitory anti-mouse CD14 Ab, biG53, clone SPAK3, (Cellsciences) for 30 min before LPS treatment to demonstrate CD14 specificity. Macrophages were washed five times with PBS and removed by scraping. Cell-associated fluorescence was measured on a FACScan flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). For each sample, 10,000 events were acquired and the results are expressed as mean fluorescence intensity.
ELISA for TNF-
Alveolar macrophages from SP-D+/+ and SP-D–/– mice obtained by BAL were placed in culture at a concentration of 5 x 105 cells per well in serum-free RPMI 1640 medium (Invitrogen Life Technologies). Macrophages were incubated with 100 ng of LPS (List Biological Laboratories) for 18 h. TNF- levels were measured in triplicate with 50 μl of macrophage-conditioned medium using murine sandwich ELISA kits (R&D Systems) according to the manufacturer’s directions. All plates were read on a microplate spectrophotometer (Bio-Tek Instruments) and analyzed with the use of a computer-assisted analysis program (KC Junior; Bio-Tek Instruments). Only assays having standard curves with a calculated regression line value >0.95 were accepted for analysis.
Western immunoblot analysis of CD14
BAL macrophages were lysed in cell lysis buffer for the determination of total cellular CD14. Cell lysates were centrifuged at 10,000 x g for 10 min and supernatant was collected. To assess levels of sCD14, BAL fluid was concentrated using a Centricon YM-10 (Millipore) per the manufacturer’s specification. Protein concentration of the macrophage lysates and BAL fluid was determined using a BCA protein assay kit (Pierce). Equal protein amounts of all samples were resolved on 8–16% SDS-Tris-glycine-polyacrylamide gels (NOVEX). Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TTBS) and incubated overnight at 4°C with rat anti-mouse CD14 (rmC5-3) antiserum (BD Pharmingen), diluted 1/2,000; 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 International), diluted 1/2,000 in TTBS containing 1% BSA. Blots were washed with TTBS and incubated with peroxidase-conjugated goat anti-rat IgG or goat anti-rabbit IgG Ab (Amersham Biosciences) diluted 1/10,000 in TTBS containing 1% BSA. After washing, blots were developed with a chemiluminescence detection system (Amersham Biosciences).
RNA isolation and real-time PCR analysis
Alveolar macrophages recovered by BAL were immediately lysed in 4 M guanidinium isothiocyanate, 0.5% laurylsarcosine, and 0.1 M 2-ME in 25 mM sodium citrate buffer and total cellular RNA was isolated by ultracentrifugation through a 5.7 M CsCl cushion at 150,000 x g for 18 h at 20°C. cDNA templates were made by reverse transcription (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen Life Technologies). PCR mixes consisted of template, 0.5 μM of each primer (1.0 μM of each primer), 2.5 mM MgCl2, and 1x DNA Master SYBR Green I (Roche Molecular Biochemicals) that contained Taq polymerase, dNTPs, SYBR Green dye, and buffer. Reaction conditions differed slightly, depending on the primers used, and generally were 95°C for 120–150 s followed by 35–40 cycles of amplification (95°C for 6–10 s, 59–62°C for 10–15 s, and 72°C for 15–25 s). Amplification product size and forward and reverse primer sequences were as follows: L32 (257 bp) 5'-GTGAAGCCCAAGATCGTC-3', 5'-AGCAATCTCAGCACAGTAAG-3', CD14 (118 bp) 5'-AACATCTTGAACCTCCGCAACG-3', 5'-TGAGTGAGTGTGCTTGGGCAATAC-3'. Measurement of amplified product was made for 6 s every cycle at a temperature above that of the melting temperature of possible nonspecific products and 1–2°C below the melting temperature of the specific product. Melt curve analyses were performed after every run to ensure that a single amplified product was produced. Relative quantitation was obtained by measuring the cycle at which the greatest accumulation of product occurred (cycle threshold) and plotting that against the cycle thresholds of a dilution series of positive control samples. Only experiments in which the regression analysis of the dilution series gave an r2 value 0.985 were used to determine quantitation.
Statistical methods
Results were compared using ANOVA and Student’s t test. Findings were considered statistically significant at probability levels <0.05. Results are presented as the mean ± SEM.
Results
Decreased surface expression of CD14 on alveolar macrophages from SP-D–/– mice
Surface expression of CD14 was assessed by flow cytometry on alveolar macrophage isolated from BAL 2 h after saline treatment or intratracheal infection with GBS or H. influenzae. BAL fluid from SP-D+/+ and SP-D–/– mice contained >90% macrophages after infection (data not shown). Significantly less CD14 was detected on alveolar macrophages from uninfected, GBS-, or H. influenzae-infected SP-D–/– mice compared with SP-D+/+ mice (Fig. 1A). Alveolar macrophage CD14 was reduced 2 h after infection with H. influenzae, and peaked at 24 h postinfection in both SP-D–/– and SP-D+/+ mice. CD14 was significantly decreased on alveolar macrophages from SP-D–/– mice at all time points examined (Fig. 1B). No difference in CD14 was observed on peritoneal macrophages from SP-D–/– and SP-D+/+ mice (mean fluorescence intensity = 11.7 ± 1.1 and 14.6 ± 1.1 respectively; n = 8, mean ± SEM).
FIGURE 1. CD14 is reduced on alveolar macrophages from SP-D–/– 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 uninfected mice). A, CD14 was significantly decreased on alveolar macrophages from uninfected, GBS-infected, and H. influenzae-infected SP-D–/– () compared with SP-D+/+ mice (). B, CD14 was significantly decreased on alveolar macrophages from SP-D–/– (?) compared with the SP-D+/+ mice () at all time points examined after infection with H. influenzae. Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice with similar treatment conditions.
Impaired CD14-mediated endocytosis of LPS by macrophages from SP-D–/– mice
Endocytosis of LPS was assessed by flow cytometry to determine whether decreased levels of surface CD14 were associated with impaired macrophage endocytic function. Endocytosis of LPS was reduced by 66% in alveolar macrophages isolated from SP-D–/– mice (Fig. 2A). LPS uptake was inhibited by an Ab against the CD14 receptor, indicating that endocytosis of the LPS was a CD14-dependent process (Fig. 2B). These data indicate that reduced surface expression of CD14 on alveolar macrophage from SP-D–/– mice contributes to impaired CD14-mediated endocytosis.
FIGURE 2. Impaired uptake of LPS by SP-D–/– alveolar macrophages. Flow cytometry analysis was performed on cultured alveolar macrophages treated with BODIPY-LPS to determine LPS uptake. A, Alveolar macrophages from SP-D–/– mice () endocytosed significantly less LPS than those from SP-D+/+ mice (). B, Pretreatment with the CD14 blocking Ab significantly inhibited LPS uptake in the SP-D+/+ mice indicating CD14 specificity of LPS endocytosis. Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ alveolar macrophages.
Impaired TNF- production by macrophages from SP-D–/– mice
LPS-induced TNF- production by alveolar macrophages from SP-D–/– mice was reduced (Fig. 3), indicating that reduced surface expression of CD14 on alveolar macrophage from SP-D–/– mice was associated with impaired CD14-TLR4 signaling.
FIGURE 3. Alveolar macrophages from SP-D–/– mice produce less 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 TNF- was measured in culture medium by ELISA. LPS-challenged alveolar macrophage from SP-D–/– mice () produced significantly less TNF- than those from SP-D+/+ mice (). No differences in TNF- levels were detected in unchallenged alveolar macrophage from SP-D–/– mice () and SP-D+/+ mice (). Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ alveolar macrophages.
Whole cell CD14 levels are reduced in alveolar macrophages from SP-D–/– mice
Whole cell CD14 levels were assessed by Western immunoblot analysis in uninfected and H. influenzae-infected mice to determine whether reduced surface expression of CD14 was caused by reduced cellular content of CD14. Significantly less CD14 was detected in alveolar macrophage lysates from both uninfected and H. influenzae-infected SP-D–/– mice (Fig. 4).
FIGURE 4. Cellular CD14 levels are reduced in alveolar macrophage from SP-D–/– mice. Immunoblot was used to compare CD14 levels from SP-D–/– and SP-D+/+ alveolar macrophages that were uninfected or infected with H. influenzae. CD14 levels were reduced in alveolar macrophages from uninfected and H. influenzae-infected SP-D–/– compared with SP-D+/+ mice. Each lane represents alveolar macrophage cell lysates from one animal.
SP-D does not influence CD14 mRNA in alveolar macrophages
Because cellular CD14 was reduced in alveolar macrophages from the SP-D–/– mice, CD14 mRNA was measured by real-time PCR analysis. Alveolar macrophage CD14 mRNA levels were similar in SP-D–/– and SP-D+/+ mice (CD14/L32 = 1.0 ± 0.2 and 1.0 ± 0.2, respectively; n = 8, mean ± SEM).
sCD14 is increased in the BAL fluid from SP-D–/– mice
To determine whether reduced macrophage CD14 levels were caused by receptor shedding, sCD14 levels were assessed by Western immunoblot analysis on BAL fluid. Significantly more sCD14 was detected in BAL fluid from SP-D–/– mice (Fig. 5).
FIGURE 5. Increased sCD14 in BAL from SP-D–/– mice. Immunoblot was used to measure sCD14 in BAL fluid. sCD14 was increased in BAL fluid from SP-D–/– compared with SP-D+/+ mice. Each lane of the immunoblot was loaded with 20 μg of concentrated BAL fluid protein from an individual animal.
MMPs regulate sCD14 levels in SP-D–/– mice
MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– mice were used to determine the involvement of MMP-9 and MMP-12 in the production of sCD14. sCD14 was assessed by immunoblot analysis of BAL fluid from SP-D+/+, SP-D–/–, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice. sCD14 was significantly reduced in BAL fluid from MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– compared with SP-D–/– mice (Fig. 6A). However, loss of MMP-9 or MMP-12 in the SP-D–/– genetic background did not fully restore BAL sCD14 levels to those observed in SP-D+/+ mice (Fig. 6A). Immunoblot analysis for active forms of MMP-9 and MMP-12 was performed on BAL samples from SP-D–/–, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice to confirm that both MMP-9 and MMP-12 are involved in the formation of sCD14 in the SP-D–/– mice. MMP-9 was not detected in the BAL from the MMP-12–/–/SP-D–/– mice and MMP-12 was significantly reduced in the MMP-9–/–/SP-D–/– mice suggesting that MMP-12 was necessary for increased sCD14 observed in SP-D–/– mice and that MMP-9 played a role in the increased activity of MMP-12.
FIGURE 6. MMP-12 mediates SP-D-dependent changes in CD14. A, BAL sCD14 was determined by immunoblot analysis on samples from SP-D–/–, SP-D+/+, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice. sCD14 levels were reduced in BAL from MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– compared with SP-D–/– mice but remained increased when compared with SP-D+/+ mice. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal. B, The immunoblot shown is representative of three independent experiments that are summarized in the bar graph. Immunoreactive band densitometry data are expressed as mean ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice, #, p < 0.05 compared with SP-D–/– mice. C, Immunoblot analysis for active MMP-9 (92 kDa) and MMP-12 (22 kDa) was performed on BAL from SP-D–/–, MMP9–/–/SP-D–/–, and MMP12–/–/SP-D–/– mice. Active forms of MMP-9 and MMP-12 were detected in BAL from SP-D mice. MMP-9 was not detected in BAL from MMP-12–/–/SP-D–/– mice and MMP-12 was significantly reduced in the MMP-9–/–/SP-D–/– mice. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal.
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 was analyzed by Western immunoblot analysis. MMP-12 treatment of RAW 264.7 cells increased sCD14 in the cell culture medium (Fig. 7). The electrophoretic mobility of in vivo and in vitro generated sCD14 was compared by Western immunoblot analysis and was similar for the two samples (data not shown). This further supports the observation that MMP-12 is important for the formation of sCD14 in the SP-D–/– mouse lung.
FIGURE 7. MMP12 increases sCD14 in vitro. sCD14 in cell culture medium from sham- and MMP-12-treated RAW264.7 cells was compared by Western immunoblot analysis. MMP12 increased sCD14 in cell culture medium from RAW 264.7 cells. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated culture medium from one cell culture well. The immunoblot is representative of three independent experiments.
Kinetics of reduced surface and elevated sCD14 after loss of SP-D in vivo
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, conditionally expressing SP-D in the bronchiolar and respiratory epithelium under control of doxycycline (35), were used to determine the time course of decreased alveolar macrophage surface CD14 and increased BAL fluid sCD14 after the loss of SP-D. As previously reported (35), BAL SP-D concentration decreased rapidly after removal from doxycycline. Alveolar macrophage CD14 decreased (Fig. 8A) and sCD14 increased (Fig. 8B) within 3 days, findings that persisted thereafter. Immunoreactive bands for active MMP-12 were increased 36 h after the removal of doxycycline (Fig. 8B).
FIGURE 8. Conditional regulation of alveolar macrophage CD14 and sCD14 by SP-D. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D, were used to determine the kinetics of decreased membrane CD14 and increased BAL fluid sCD14 after the loss of SP-D. Alveolar macrophage CD14 was determined by flow cytometry analysis and BAL sCD14, SP-D and MMP-12 by immunoblot analysis. Alveolar macrophage CD14 decreased (A) while MMP-12 (22 kDa) and sCD14 increased (B) in CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice in association with the loss of SP-D after removal from doxycycline. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid from an individual animal. The immunoblot shown is representative of three independent experiments. Flow cytometry data are expressed as mean ± SEM, n = 6; *, p < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with doxycycline.
Reversibility of sCD14 by SP-D in vivo
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were used to determine whether the elevated BAL sCD14 after loss of SP-D was reversible with replacement of SP-D. sCD14 was increased in the BAL fluid 3 days after conditional loss of SP-D. Surprisingly, sCD14 remained increased when expression of SP-D was restored for 5 days (Fig. 9). Thus the loss of SP-D increased MMP-12 activity in the lung. However, restoration of SP-D for 5 days was not sufficient to reduce the increase in MMP-12, perhaps indicating a generalized inflammation in the lung during this time.
FIGURE 9. Conditional replacement of SP-D does not reduce BAL sCD14. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were used to used to determine whether increased BAL fluid sCD14 associated with lack of SP-D was reversed by restoration of SP-D. BAL sCD14 and MMP-12 were assessed by Western immunoblot analysis. The active forms of MMP-12 and sCD14 were increased in the BAL 3 days after the conditional removal of SP-D (–Dox 3d) compared with wild-type (+Dox). Restoration of SP-D for 5 days (–Dox 3d, +Dox 5d) did not reduce the levels of BAL sCD14 nor did it reduce the active form of MMP-12 (22 kDa) in the BAL (when compared with –Dox 3d). Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal.
Decreased cell surface expression of CD14 on alveolar neutrophils from SP-D–/– mice
Cell surface expression of CD14 was assessed by flow cytometry on neutrophils isolated from blood and BAL fluid from mice intratracheally infected with K. pneumoniae for 6 h. Significantly less CD14 was detected on alveolar neutrophils from SP-D–/– mice compared with SP-D+/+ mice (Fig. 10A). In contrast, CD14 was similar on blood neutrophils from SP-D–/– and SP-D+/+ mice (Fig. 10B). These results indicate that SP-D also has a tissue-specific effect on neutrophil CD14 expression.
FIGURE 10. CD14 is reduced on alveolar neutrophils from SP-D–/– mice. Intratracheal infection with K. pneumonia was used to cause neutrophil influx into the lung. Flow cytometry analysis was used to measure CD14 on alveolar and blood neutrophils. A, Blood neutrophil CD14 was similar for SP-D–/– () and SP-D+/+ mice (). B, CD14 was significantly decreased on alveolar neutrophils from SP-D–/– () compared with SP-D+/+ mice (). Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice with similar treatment conditions.
Discussion
CD14 in association with TLR4 and MD-2 comprise a tripartite receptor that mediates cellular responses to LPS and other pathogen recognition motifs (13). Studies were undertaken to examine the possible contributions of SP-D to the regulation of CD14 expression by alveolar macrophages. Loss of SP-D from the lung resulted in a rapid loss of CD14 from the alveolar macrophage cell surface and an accumulation of sCD14 in BAL. Accumulation of sCD14 was dependent, in part, on MMP-12 and appears not to be readily reversible with the reintroduction of SP-D into the lung. Reduced CD14 on the surface of the alveolar macrophage resulted in a functional impairment of LPS uptake and LPS-induced cytokine production. The present study identifies a novel mechanism by which SP-D influences CD14 levels on alveolar macrophages that in turn, regulates responses to LPS.
In the current study, sCD14 was significantly increased in BAL fluid from the SP-D–/– mice. Cleavage of CD14 from the cell surface is an important physiologic event that serves to down-modulate monocyte-macrophage activation (22, 23, 24). Previous studies demonstrated that collagenase (30), neutrophil elastase (23), and cathepsin G (26) cleave CD14 from the cell surface. Metalloproteases, including MMP-12, function as sheddases for a variety of cell surface receptors (30, 31, 32, 33). Because previous studies indicated that MMP-2, -9, and -12 were increased in the SP-D–/– mice (6), the present study sought to determine whether these metalloproteases were involved in the elevated sCD14 observed in BAL fluid from SP-D–/– mice. Loss of MMP-9 or MMP-12 in the SP-D–/– genetic background did not correct the foamy macrophage infiltrate or emphysema characteristic of the SP-D–/– mice (38). However, in the present study, targeted ablation of MMP-9 or MMP-12 significantly reduced BAL sCD14 levels in SP-D–/– mice (38). MMP-9 was not present in the MMP-12–/–/SP-D–/– mice. This observation is in agreement with findings by Lanone et al. (39) in which MMP-12 was required for optimal accumulation of MMP-9 after IL-13 stimulation. Surprisingly, MMP-12 was greatly reduced in the MMP-9–/–/SP-D–/– mice, suggesting that MMP-9 influences the accumulation of active MMP-12 in the SP-D–/– mouse lung. In addition, increased sCD14 in the cell culture medium from RAW 264.7 cells treated with active MMP-12 supports the concept that MMP-12 cleaves CD14 from the alveolar macrophage cell surface. MMP-12-dependent cleavage of CD14 accounts for formation of 60% of the sCD14 in BAL fluid from SP-D–/– mice. Mechanisms explaining sCD14 formation that was not MMP-12 dependent in the MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– mice have not been elucidated. However, cleavage of CD14 by MMP-2, a collagenase, is likely since previous studies have demonstrated that MMP-2 is increased in the SP-D–/– mice (6, 40) and collagenase reduced the level of CD14 on the macrophage cell surface (30).
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D in the bronchiolar and respiratory epithelium (35), were used to understand the sequence of events after the loss of SP-D that lead to reduced alveolar macrophage surface CD14, elevated BAL sCD14, and appearance of the active form of MMP-12 in the BAL. In support of the concept that MMP-12 mediates production of a large portion of sCD14 found in the BAL of the SP-D–/– mice, increased levels of BAL MMP-12 and sCD14 as well as reduced surface expression of alveolar macrophage CD14 was observed three days after loss of SP-D. Thus changes in alveolar macrophages seen in SP-D–/– mice occur rapidly after the loss of SP-D. A possible explanation for the rapid changes is based upon recent findings that SP-D binds to CD14 via its carbohydrate recognition domain, inhibiting CD14-LPS interactions (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 restoration of SP-D expression. Surprisingly, sCD14 did not decrease five days after SP-D expression was restored. Interestingly, MMP-12 also remained elevated five days after SP-D expression was restored. This supports the concept that MMP-12 is integral is the proteolytic cleavage of CD14 in SP-D–/– mice. Although it is not clear why MMP-12 remained elevated, the observation is similar to that by Yoshida et al. (40) in which addition of mouse SP-D in vitro did not reduce MMP production by alveolar macrophages from SP-D–/– mice. Together the findings support the concept that activation of MMPs after the loss of SP-D occurs by initiation of a complex signaling pathway resulting in the formation of secondary mediators and that SP-D is not directly able to inhibit the secondary mediators activating the MMPs.
Decreased surface CD14 on macrophages from SP-D–/– mice was associated with decreased LPS-stimulated TNF- production. Macrophages from CD14–/– mice are also deficient in LPS-induced IL-6 and TNF- production (41) supporting the concept that reduced CD14 resulted in impaired LPS-induced cytokine production in the current study. The lack of TNF- production from SP-D–/– macrophage appears contradictory to previous in vivo studies that demonstrated elevated TNF-, IL-6, and IL-1 in H. influenzae-infected SP-D–/– mice (7). Interestingly, respiratory epithelial cells express TLRs (42) and when treated with LPS and sCD14 elicited IL-6 and IL-8 production (20). This suggests that the respiratory epithelium may be an important source of cytokines in SP-D–/– mice after bacterial challenge.
Phagocytic uptake of LPS was also impaired in alveolar macrophages from SP-D–/– mice. The reduced LPS uptake suggests that SP-D–/– mice are impaired in their ability to clear LPS from the lung; however, it is unlikely that phagocytic defect observed previously in SP-D–/– mice (7) is due solely to reduced CD14 because CD14-deficient macrophages were not impaired in their ability to phagocytose Escherichia coli (41).
The increased MMP-12, sCD14, and reduced LPS-induced 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 addition, human alveolar macrophages in culture produced significantly less TNF- and IL-6 after cigarette smoke exposure (46). This suggests MMP-12 may mediate reduction of alveolar macrophage 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 clearance and inflammatory processes that are important for host defense 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 macrophage responses to LPS.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Robert M. Senior for generously providing the MMP-9–/– mice. We thank Jaymi Semona, Victor LaFay, and Lindsey Malone for their assistance with animal husbandry. We thank Katy Davis and Theresa Richardson for their assistance in generating MMP/SP-D double knockouts.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants RO1HL71522 (to 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.).
2 Address correspondence and reprint requests to Dr. Ann Marie LeVine, Divisions of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: annmarie.levine{at}cchmc.org
3 Abbreviations used in this paper: SP-D, surfactant protein D; TLR4, toll-like receptor 4; MD-2, myeloid differentiation protein 2; MMP, matrix metalloprotease, GBS, group B streptococcus; BAL, bronchoalveolar lavage; sCD14, soluble CD14.
Received for publication November 11, 2004. Accepted for publication February 1, 2005.
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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 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.
Introduction
Surfactant protein D (SP-D)3 is a member of the collectin family of innate defense polypeptides that include surfactant protein A, mannose-binding lectin, and conglutinin (1, 2, 3). Collectins form multimeric structures consisting of a collagenous N-terminal domain and a globular C-terminal carbohydrate binding domain (4) that bind carbohydrate surfaces of many microorganisms mediating phagocytosis and killing by phagocytic cells (5).
SP-D gene-inactivated mice (SP-D–/–) develop progressive emphysema that is characterized by chronic inflammation, accumulation of surfactant phospholipids, and infiltration with lipid-laden alveolar macrophages (6). Phagocytosis of bacteria, viruses, and apoptotic cells is impaired in alveolar macrophages from SP-D–/– mice (7, 8, 9, 10). SP-D–/– mice also mount an exacerbated inflammatory response when challenged with bacteria (7). SP-D modulates lung inflammation by interaction with cell surface receptors on the macrophage including signal-inhibitory regulatory protein (11), CD91 (10, 11), calreticulin (10, 11), and CD14 (12).
CD14 is a 55-kDa pattern recognition receptor that is present on the surface of monocytes, macrophages, and neutrophils. CD14 is a GPI-linked receptor that lacks a cytoplasmic signaling domain and, therefore, requires interaction with other receptors to elicit its biological responses. CD14 binds LPS and interacts with toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) enhancing MAPK signaling and production of cytokines and chemokines (13). CD14 mediates phagocytosis of bacteria (14), clearance of apoptotic cells (15, 16, 17), and transport of lipids (18, 19). The CD14 receptor exists as both a membrane GPI-anchored protein and soluble protein. Soluble CD14 (sCD14) induces biological responses in epithelial cells (20) and endothelial cells (21) by interaction with TLRs present on the cell surface. sCD14 serves to down-modulate monocyte and macrophage activation (22, 23, 24).
sCD14 is produced by proteolytic cleavage (23, 24, 25, 26), lipolytic cleavage of the GPI linker (22, 27), or secreted without the GPI moiety (28, 29). Increased production of matrix metalloprotease (MMP) -2, -9, and -12 was detected in alveolar macrophages of SP-D–/– mice (6). Although it has not been determined whether MMP-2, -9, or -12 proteolytically cleave CD14 to form sCD14, several studies suggest that metalloproteases cleave receptors from cell surfaces. Treatment with collagenase, a MMP, reduced cell associated CD14 (30). MMP-12 cleaved the GPI-linked urokinase-type plasminogen activator receptor from the cell surface (31). In addition, the general metalloprotease inhibitor 1,10-phenantroline inhibits the formation of sCD16 (32, 33).
Although there is clear evidence that SP-D binds to phagocytic receptors and phagocytosis is impaired in SP-D–/– mice, mechanisms by which SP-D regulates innate host defense activities of alveolar macrophages remain unclear. The present study was undertaken to identify mechanisms whereby SP-D regulates cell surface CD14 and its function.
Materials and Methods
Animals
SP-D–/– mice were generated by targeted gene inactivation as previously described (34). SP-D was conditionally replaced in the respiratory epithelium of SP-D–/– mice by crossing SP-D–/– mice with CCSP-rtTA+ and (tetO)7-rSPD+ mice to generate triple-transgenic mice (CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/–) as previously described (35). Triple-transgenic mice were fed doxycycline containing food to induce the expression of the rSP-D protein. MMP9–/–/SP-D–/– and MMP12–/–/SP-D–/– mice were generated by crossing MMP9–/– (36) (kindly provided by Dr. R. Senior, Washington University School of Medicine, St. Louis, MO) and MMP-12–/– (36) mice with SP-D–/– mice. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/–, MMP9–/–/SP-D–/–, MMP12–/–/SP-D–/–, and SP-D–/– mice survive and breed normally in the vivarium under barrier containment facilities at the Cincinnati Children’s Hospital Medical Center. Experimental procedures were reviewed and approved by the Children’s Hospital Institutional Animal Care and Use Committee. Male and female mice 56–70 days old were used for this study.
Preparation of bacteria
Group B streptococcus (GBS) and Hemophilus influenzae from clinical isolates were cultured as previously described (7). A stock culture of Klebsiella pneumoniae strain K2 was a generous gift of Dr. I. Ofek (Tel Aviv University, Tel Aviv, Israel). Bacteria were suspended in medium containing 20% glycerol, and frozen in aliquots at –70°C. Bacteria from the same passage were used to minimize variations in virulence related to culture conditions. Before each experiment, an aliquot was thawed and plated on blood agar plates (Baxter Healthcare), inoculated into trypticase soy broth (Difco Laboratories), and grown for 14–16 h at 37°C with continuous shaking. The broth was centrifuged, and the bacteria were washed in PBS (pH 7.2) and resuspended in 4 ml of buffer. To facilitate studies, a growth curve was generated so the bacterial concentration could be determined spectrophotometrically and confirmed by quantitative culture of the intratracheal inoculum.
Intratracheal inoculation
Administration of bacteria into the respiratory tract was performed by intratracheal inoculation of GBS (106 CFU), H. influenzae (108 CFU), or K. pneumoniae (108 CFU) as previously described (37). Sham-treated mice were intratracheally injected with nonpyrogenic PBS.
Bronchoalveolar lavage (BAL)
Mice were exsanguinated after a lethal i.p. injection of sodium pentobarbital and the lungs were lavaged three times with 1 ml of PBS. BAL cells were recovered by centrifugation at 800 x g and then resuspended in FACS buffer (PBS, pH 7.4, containing 0.1% NaN3, and 1% BSA) for flow cytometry or cell lysis buffer (10 mM Tris-HCl, pH 7.5, 15 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM sodium orthovanadate, 0.4 mM PMSF) for Western immunoblot analysis. The BAL supernatant was stored at –80°C.
Peritoneal lavage
Mice were sacrificed by administration of a lethal dose of sodium pentobarbital. The peritoneal cavity was lavaged three times with 5 ml of PBS. Cells in the peritoneal lavage fluid were recovered by centrifugation at 800 x g and resuspended in FACS buffer.
Isolation of blood and alveolar neutrophils
SP-D+/+ and SP-D–/– mice were intratracheally infected with K. pneumonia to recruit neutrophils to the lung. Alveolar and blood neutrophils were isolated by discontinuous Percoll gradient separation with the density of the lower fraction at 0.9881 g/ml and the density of the upper fraction at 0.7497 g/ml. After administration of a lethal dose of sodium pentobarbital, 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 collected as described above. Whole blood and BAL was placed on the gradient and centrifuged at 800 x g for 40 min at 20°C. Neutrophils forming a discrete band at the interface between the two gradient layers after centrifugation were recovered, washed twice in PBS, and resuspended in FACS buffer. Approximately 70% of BAL cells were neutrophils 6 h after infection with 108 CFU of K. pneumoniae.
Production of sCD14 by MMP-12 in vitro
The murine macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection and maintained in Dulbecco’s MEM containing 10% FBS, 10 mM HEPES, 50 U/ml penicillin, and 50 μg/ml streptomycin. RAW cells (1 x 106) in six-well plates were treated with 150 ng of MMP-12 (Biomol) in PBS. PBS supernatants were recovered, treated with protease inhibitor mixture (Sigma-Aldrich), and centrifuged at 10,000 x g for 10 min. After centrifugation, the supernatant was recovered and each sample was concentrated using a Centricon YM-10 (Millipore) per the manufacturer’s specification.
Flow cytometry analysis
Isolated macrophages and neutrophils were resuspended in 200 μl of FACS buffer and incubated with purified mouse IgG for 15 min on ice. Cells were incubated with FITC-conjugated anti-mouse CD14 (BD Pharmingen) for 1 h on ice and washed three times with FACS buffer. Cell-associated fluorescence was measured on a FACScan flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). For each sample, 10,000 events were acquired, and the results are expressed as mean fluorescence intensity.
LPS endocytosis
Alveolar macrophages from SP-D+/+ and SP-D–/– mice obtained by BAL were placed in culture at a concentration of 5 x 105 cells per well in serum-free RPMI 1640 medium (Invitrogen Life Technologies). Cultured alveolar macrophages were incubated with 100 ng of BODIPY-conjugated LPS from Salmonella minnesota (Molecular Probes) for 2 h. A separate culture of alveolar macrophages from SP-D+/+ mice were incubated with 1 μg of the inhibitory anti-mouse CD14 Ab, biG53, clone SPAK3, (Cellsciences) for 30 min before LPS treatment to demonstrate CD14 specificity. Macrophages were washed five times with PBS and removed by scraping. Cell-associated fluorescence was measured on a FACScan flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). For each sample, 10,000 events were acquired and the results are expressed as mean fluorescence intensity.
ELISA for TNF-
Alveolar macrophages from SP-D+/+ and SP-D–/– mice obtained by BAL were placed in culture at a concentration of 5 x 105 cells per well in serum-free RPMI 1640 medium (Invitrogen Life Technologies). Macrophages were incubated with 100 ng of LPS (List Biological Laboratories) for 18 h. TNF- levels were measured in triplicate with 50 μl of macrophage-conditioned medium using murine sandwich ELISA kits (R&D Systems) according to the manufacturer’s directions. All plates were read on a microplate spectrophotometer (Bio-Tek Instruments) and analyzed with the use of a computer-assisted analysis program (KC Junior; Bio-Tek Instruments). Only assays having standard curves with a calculated regression line value >0.95 were accepted for analysis.
Western immunoblot analysis of CD14
BAL macrophages were lysed in cell lysis buffer for the determination of total cellular CD14. Cell lysates were centrifuged at 10,000 x g for 10 min and supernatant was collected. To assess levels of sCD14, BAL fluid was concentrated using a Centricon YM-10 (Millipore) per the manufacturer’s specification. Protein concentration of the macrophage lysates and BAL fluid was determined using a BCA protein assay kit (Pierce). Equal protein amounts of all samples were resolved on 8–16% SDS-Tris-glycine-polyacrylamide gels (NOVEX). Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TTBS) and incubated overnight at 4°C with rat anti-mouse CD14 (rmC5-3) antiserum (BD Pharmingen), diluted 1/2,000; 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 International), diluted 1/2,000 in TTBS containing 1% BSA. Blots were washed with TTBS and incubated with peroxidase-conjugated goat anti-rat IgG or goat anti-rabbit IgG Ab (Amersham Biosciences) diluted 1/10,000 in TTBS containing 1% BSA. After washing, blots were developed with a chemiluminescence detection system (Amersham Biosciences).
RNA isolation and real-time PCR analysis
Alveolar macrophages recovered by BAL were immediately lysed in 4 M guanidinium isothiocyanate, 0.5% laurylsarcosine, and 0.1 M 2-ME in 25 mM sodium citrate buffer and total cellular RNA was isolated by ultracentrifugation through a 5.7 M CsCl cushion at 150,000 x g for 18 h at 20°C. cDNA templates were made by reverse transcription (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen Life Technologies). PCR mixes consisted of template, 0.5 μM of each primer (1.0 μM of each primer), 2.5 mM MgCl2, and 1x DNA Master SYBR Green I (Roche Molecular Biochemicals) that contained Taq polymerase, dNTPs, SYBR Green dye, and buffer. Reaction conditions differed slightly, depending on the primers used, and generally were 95°C for 120–150 s followed by 35–40 cycles of amplification (95°C for 6–10 s, 59–62°C for 10–15 s, and 72°C for 15–25 s). Amplification product size and forward and reverse primer sequences were as follows: L32 (257 bp) 5'-GTGAAGCCCAAGATCGTC-3', 5'-AGCAATCTCAGCACAGTAAG-3', CD14 (118 bp) 5'-AACATCTTGAACCTCCGCAACG-3', 5'-TGAGTGAGTGTGCTTGGGCAATAC-3'. Measurement of amplified product was made for 6 s every cycle at a temperature above that of the melting temperature of possible nonspecific products and 1–2°C below the melting temperature of the specific product. Melt curve analyses were performed after every run to ensure that a single amplified product was produced. Relative quantitation was obtained by measuring the cycle at which the greatest accumulation of product occurred (cycle threshold) and plotting that against the cycle thresholds of a dilution series of positive control samples. Only experiments in which the regression analysis of the dilution series gave an r2 value 0.985 were used to determine quantitation.
Statistical methods
Results were compared using ANOVA and Student’s t test. Findings were considered statistically significant at probability levels <0.05. Results are presented as the mean ± SEM.
Results
Decreased surface expression of CD14 on alveolar macrophages from SP-D–/– mice
Surface expression of CD14 was assessed by flow cytometry on alveolar macrophage isolated from BAL 2 h after saline treatment or intratracheal infection with GBS or H. influenzae. BAL fluid from SP-D+/+ and SP-D–/– mice contained >90% macrophages after infection (data not shown). Significantly less CD14 was detected on alveolar macrophages from uninfected, GBS-, or H. influenzae-infected SP-D–/– mice compared with SP-D+/+ mice (Fig. 1A). Alveolar macrophage CD14 was reduced 2 h after infection with H. influenzae, and peaked at 24 h postinfection in both SP-D–/– and SP-D+/+ mice. CD14 was significantly decreased on alveolar macrophages from SP-D–/– mice at all time points examined (Fig. 1B). No difference in CD14 was observed on peritoneal macrophages from SP-D–/– and SP-D+/+ mice (mean fluorescence intensity = 11.7 ± 1.1 and 14.6 ± 1.1 respectively; n = 8, mean ± SEM).
FIGURE 1. CD14 is reduced on alveolar macrophages from SP-D–/– 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 uninfected mice). A, CD14 was significantly decreased on alveolar macrophages from uninfected, GBS-infected, and H. influenzae-infected SP-D–/– () compared with SP-D+/+ mice (). B, CD14 was significantly decreased on alveolar macrophages from SP-D–/– (?) compared with the SP-D+/+ mice () at all time points examined after infection with H. influenzae. Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice with similar treatment conditions.
Impaired CD14-mediated endocytosis of LPS by macrophages from SP-D–/– mice
Endocytosis of LPS was assessed by flow cytometry to determine whether decreased levels of surface CD14 were associated with impaired macrophage endocytic function. Endocytosis of LPS was reduced by 66% in alveolar macrophages isolated from SP-D–/– mice (Fig. 2A). LPS uptake was inhibited by an Ab against the CD14 receptor, indicating that endocytosis of the LPS was a CD14-dependent process (Fig. 2B). These data indicate that reduced surface expression of CD14 on alveolar macrophage from SP-D–/– mice contributes to impaired CD14-mediated endocytosis.
FIGURE 2. Impaired uptake of LPS by SP-D–/– alveolar macrophages. Flow cytometry analysis was performed on cultured alveolar macrophages treated with BODIPY-LPS to determine LPS uptake. A, Alveolar macrophages from SP-D–/– mice () endocytosed significantly less LPS than those from SP-D+/+ mice (). B, Pretreatment with the CD14 blocking Ab significantly inhibited LPS uptake in the SP-D+/+ mice indicating CD14 specificity of LPS endocytosis. Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ alveolar macrophages.
Impaired TNF- production by macrophages from SP-D–/– mice
LPS-induced TNF- production by alveolar macrophages from SP-D–/– mice was reduced (Fig. 3), indicating that reduced surface expression of CD14 on alveolar macrophage from SP-D–/– mice was associated with impaired CD14-TLR4 signaling.
FIGURE 3. Alveolar macrophages from SP-D–/– mice produce less 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 TNF- was measured in culture medium by ELISA. LPS-challenged alveolar macrophage from SP-D–/– mice () produced significantly less TNF- than those from SP-D+/+ mice (). No differences in TNF- levels were detected in unchallenged alveolar macrophage from SP-D–/– mice () and SP-D+/+ mice (). Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ alveolar macrophages.
Whole cell CD14 levels are reduced in alveolar macrophages from SP-D–/– mice
Whole cell CD14 levels were assessed by Western immunoblot analysis in uninfected and H. influenzae-infected mice to determine whether reduced surface expression of CD14 was caused by reduced cellular content of CD14. Significantly less CD14 was detected in alveolar macrophage lysates from both uninfected and H. influenzae-infected SP-D–/– mice (Fig. 4).
FIGURE 4. Cellular CD14 levels are reduced in alveolar macrophage from SP-D–/– mice. Immunoblot was used to compare CD14 levels from SP-D–/– and SP-D+/+ alveolar macrophages that were uninfected or infected with H. influenzae. CD14 levels were reduced in alveolar macrophages from uninfected and H. influenzae-infected SP-D–/– compared with SP-D+/+ mice. Each lane represents alveolar macrophage cell lysates from one animal.
SP-D does not influence CD14 mRNA in alveolar macrophages
Because cellular CD14 was reduced in alveolar macrophages from the SP-D–/– mice, CD14 mRNA was measured by real-time PCR analysis. Alveolar macrophage CD14 mRNA levels were similar in SP-D–/– and SP-D+/+ mice (CD14/L32 = 1.0 ± 0.2 and 1.0 ± 0.2, respectively; n = 8, mean ± SEM).
sCD14 is increased in the BAL fluid from SP-D–/– mice
To determine whether reduced macrophage CD14 levels were caused by receptor shedding, sCD14 levels were assessed by Western immunoblot analysis on BAL fluid. Significantly more sCD14 was detected in BAL fluid from SP-D–/– mice (Fig. 5).
FIGURE 5. Increased sCD14 in BAL from SP-D–/– mice. Immunoblot was used to measure sCD14 in BAL fluid. sCD14 was increased in BAL fluid from SP-D–/– compared with SP-D+/+ mice. Each lane of the immunoblot was loaded with 20 μg of concentrated BAL fluid protein from an individual animal.
MMPs regulate sCD14 levels in SP-D–/– mice
MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– mice were used to determine the involvement of MMP-9 and MMP-12 in the production of sCD14. sCD14 was assessed by immunoblot analysis of BAL fluid from SP-D+/+, SP-D–/–, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice. sCD14 was significantly reduced in BAL fluid from MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– compared with SP-D–/– mice (Fig. 6A). However, loss of MMP-9 or MMP-12 in the SP-D–/– genetic background did not fully restore BAL sCD14 levels to those observed in SP-D+/+ mice (Fig. 6A). Immunoblot analysis for active forms of MMP-9 and MMP-12 was performed on BAL samples from SP-D–/–, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice to confirm that both MMP-9 and MMP-12 are involved in the formation of sCD14 in the SP-D–/– mice. MMP-9 was not detected in the BAL from the MMP-12–/–/SP-D–/– mice and MMP-12 was significantly reduced in the MMP-9–/–/SP-D–/– mice suggesting that MMP-12 was necessary for increased sCD14 observed in SP-D–/– mice and that MMP-9 played a role in the increased activity of MMP-12.
FIGURE 6. MMP-12 mediates SP-D-dependent changes in CD14. A, BAL sCD14 was determined by immunoblot analysis on samples from SP-D–/–, SP-D+/+, MMP-9–/–/SP-D–/–, and MMP-12–/–/SP-D–/– mice. sCD14 levels were reduced in BAL from MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– compared with SP-D–/– mice but remained increased when compared with SP-D+/+ mice. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal. B, The immunoblot shown is representative of three independent experiments that are summarized in the bar graph. Immunoreactive band densitometry data are expressed as mean ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice, #, p < 0.05 compared with SP-D–/– mice. C, Immunoblot analysis for active MMP-9 (92 kDa) and MMP-12 (22 kDa) was performed on BAL from SP-D–/–, MMP9–/–/SP-D–/–, and MMP12–/–/SP-D–/– mice. Active forms of MMP-9 and MMP-12 were detected in BAL from SP-D mice. MMP-9 was not detected in BAL from MMP-12–/–/SP-D–/– mice and MMP-12 was significantly reduced in the MMP-9–/–/SP-D–/– mice. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal.
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 was analyzed by Western immunoblot analysis. MMP-12 treatment of RAW 264.7 cells increased sCD14 in the cell culture medium (Fig. 7). The electrophoretic mobility of in vivo and in vitro generated sCD14 was compared by Western immunoblot analysis and was similar for the two samples (data not shown). This further supports the observation that MMP-12 is important for the formation of sCD14 in the SP-D–/– mouse lung.
FIGURE 7. MMP12 increases sCD14 in vitro. sCD14 in cell culture medium from sham- and MMP-12-treated RAW264.7 cells was compared by Western immunoblot analysis. MMP12 increased sCD14 in cell culture medium from RAW 264.7 cells. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated culture medium from one cell culture well. The immunoblot is representative of three independent experiments.
Kinetics of reduced surface and elevated sCD14 after loss of SP-D in vivo
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, conditionally expressing SP-D in the bronchiolar and respiratory epithelium under control of doxycycline (35), were used to determine the time course of decreased alveolar macrophage surface CD14 and increased BAL fluid sCD14 after the loss of SP-D. As previously reported (35), BAL SP-D concentration decreased rapidly after removal from doxycycline. Alveolar macrophage CD14 decreased (Fig. 8A) and sCD14 increased (Fig. 8B) within 3 days, findings that persisted thereafter. Immunoreactive bands for active MMP-12 were increased 36 h after the removal of doxycycline (Fig. 8B).
FIGURE 8. Conditional regulation of alveolar macrophage CD14 and sCD14 by SP-D. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D, were used to determine the kinetics of decreased membrane CD14 and increased BAL fluid sCD14 after the loss of SP-D. Alveolar macrophage CD14 was determined by flow cytometry analysis and BAL sCD14, SP-D and MMP-12 by immunoblot analysis. Alveolar macrophage CD14 decreased (A) while MMP-12 (22 kDa) and sCD14 increased (B) in CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice in association with the loss of SP-D after removal from doxycycline. Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid from an individual animal. The immunoblot shown is representative of three independent experiments. Flow cytometry data are expressed as mean ± SEM, n = 6; *, p < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with doxycycline.
Reversibility of sCD14 by SP-D in vivo
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were used to determine whether the elevated BAL sCD14 after loss of SP-D was reversible with replacement of SP-D. sCD14 was increased in the BAL fluid 3 days after conditional loss of SP-D. Surprisingly, sCD14 remained increased when expression of SP-D was restored for 5 days (Fig. 9). Thus the loss of SP-D increased MMP-12 activity in the lung. However, restoration of SP-D for 5 days was not sufficient to reduce the increase in MMP-12, perhaps indicating a generalized inflammation in the lung during this time.
FIGURE 9. Conditional replacement of SP-D does not reduce BAL sCD14. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were used to used to determine whether increased BAL fluid sCD14 associated with lack of SP-D was reversed by restoration of SP-D. BAL sCD14 and MMP-12 were assessed by Western immunoblot analysis. The active forms of MMP-12 and sCD14 were increased in the BAL 3 days after the conditional removal of SP-D (–Dox 3d) compared with wild-type (+Dox). Restoration of SP-D for 5 days (–Dox 3d, +Dox 5d) did not reduce the levels of BAL sCD14 nor did it reduce the active form of MMP-12 (22 kDa) in the BAL (when compared with –Dox 3d). Each lane of the immunoblot was loaded with 20 μg of protein from concentrated BAL fluid of an individual animal.
Decreased cell surface expression of CD14 on alveolar neutrophils from SP-D–/– mice
Cell surface expression of CD14 was assessed by flow cytometry on neutrophils isolated from blood and BAL fluid from mice intratracheally infected with K. pneumoniae for 6 h. Significantly less CD14 was detected on alveolar neutrophils from SP-D–/– mice compared with SP-D+/+ mice (Fig. 10A). In contrast, CD14 was similar on blood neutrophils from SP-D–/– and SP-D+/+ mice (Fig. 10B). These results indicate that SP-D also has a tissue-specific effect on neutrophil CD14 expression.
FIGURE 10. CD14 is reduced on alveolar neutrophils from SP-D–/– mice. Intratracheal infection with K. pneumonia was used to cause neutrophil influx into the lung. Flow cytometry analysis was used to measure CD14 on alveolar and blood neutrophils. A, Blood neutrophil CD14 was similar for SP-D–/– () and SP-D+/+ mice (). B, CD14 was significantly decreased on alveolar neutrophils from SP-D–/– () compared with SP-D+/+ mice (). Data are means ± SEM, n = 6; *, p < 0.05 compared with SP-D+/+ mice with similar treatment conditions.
Discussion
CD14 in association with TLR4 and MD-2 comprise a tripartite receptor that mediates cellular responses to LPS and other pathogen recognition motifs (13). Studies were undertaken to examine the possible contributions of SP-D to the regulation of CD14 expression by alveolar macrophages. Loss of SP-D from the lung resulted in a rapid loss of CD14 from the alveolar macrophage cell surface and an accumulation of sCD14 in BAL. Accumulation of sCD14 was dependent, in part, on MMP-12 and appears not to be readily reversible with the reintroduction of SP-D into the lung. Reduced CD14 on the surface of the alveolar macrophage resulted in a functional impairment of LPS uptake and LPS-induced cytokine production. The present study identifies a novel mechanism by which SP-D influences CD14 levels on alveolar macrophages that in turn, regulates responses to LPS.
In the current study, sCD14 was significantly increased in BAL fluid from the SP-D–/– mice. Cleavage of CD14 from the cell surface is an important physiologic event that serves to down-modulate monocyte-macrophage activation (22, 23, 24). Previous studies demonstrated that collagenase (30), neutrophil elastase (23), and cathepsin G (26) cleave CD14 from the cell surface. Metalloproteases, including MMP-12, function as sheddases for a variety of cell surface receptors (30, 31, 32, 33). Because previous studies indicated that MMP-2, -9, and -12 were increased in the SP-D–/– mice (6), the present study sought to determine whether these metalloproteases were involved in the elevated sCD14 observed in BAL fluid from SP-D–/– mice. Loss of MMP-9 or MMP-12 in the SP-D–/– genetic background did not correct the foamy macrophage infiltrate or emphysema characteristic of the SP-D–/– mice (38). However, in the present study, targeted ablation of MMP-9 or MMP-12 significantly reduced BAL sCD14 levels in SP-D–/– mice (38). MMP-9 was not present in the MMP-12–/–/SP-D–/– mice. This observation is in agreement with findings by Lanone et al. (39) in which MMP-12 was required for optimal accumulation of MMP-9 after IL-13 stimulation. Surprisingly, MMP-12 was greatly reduced in the MMP-9–/–/SP-D–/– mice, suggesting that MMP-9 influences the accumulation of active MMP-12 in the SP-D–/– mouse lung. In addition, increased sCD14 in the cell culture medium from RAW 264.7 cells treated with active MMP-12 supports the concept that MMP-12 cleaves CD14 from the alveolar macrophage cell surface. MMP-12-dependent cleavage of CD14 accounts for formation of 60% of the sCD14 in BAL fluid from SP-D–/– mice. Mechanisms explaining sCD14 formation that was not MMP-12 dependent in the MMP-9–/–/SP-D–/– and MMP-12–/–/SP-D–/– mice have not been elucidated. However, cleavage of CD14 by MMP-2, a collagenase, is likely since previous studies have demonstrated that MMP-2 is increased in the SP-D–/– mice (6, 40) and collagenase reduced the level of CD14 on the macrophage cell surface (30).
CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D in the bronchiolar and respiratory epithelium (35), were used to understand the sequence of events after the loss of SP-D that lead to reduced alveolar macrophage surface CD14, elevated BAL sCD14, and appearance of the active form of MMP-12 in the BAL. In support of the concept that MMP-12 mediates production of a large portion of sCD14 found in the BAL of the SP-D–/– mice, increased levels of BAL MMP-12 and sCD14 as well as reduced surface expression of alveolar macrophage CD14 was observed three days after loss of SP-D. Thus changes in alveolar macrophages seen in SP-D–/– mice occur rapidly after the loss of SP-D. A possible explanation for the rapid changes is based upon recent findings that SP-D binds to CD14 via its carbohydrate recognition domain, inhibiting CD14-LPS interactions (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 restoration of SP-D expression. Surprisingly, sCD14 did not decrease five days after SP-D expression was restored. Interestingly, MMP-12 also remained elevated five days after SP-D expression was restored. This supports the concept that MMP-12 is integral is the proteolytic cleavage of CD14 in SP-D–/– mice. Although it is not clear why MMP-12 remained elevated, the observation is similar to that by Yoshida et al. (40) in which addition of mouse SP-D in vitro did not reduce MMP production by alveolar macrophages from SP-D–/– mice. Together the findings support the concept that activation of MMPs after the loss of SP-D occurs by initiation of a complex signaling pathway resulting in the formation of secondary mediators and that SP-D is not directly able to inhibit the secondary mediators activating the MMPs.
Decreased surface CD14 on macrophages from SP-D–/– mice was associated with decreased LPS-stimulated TNF- production. Macrophages from CD14–/– mice are also deficient in LPS-induced IL-6 and TNF- production (41) supporting the concept that reduced CD14 resulted in impaired LPS-induced cytokine production in the current study. The lack of TNF- production from SP-D–/– macrophage appears contradictory to previous in vivo studies that demonstrated elevated TNF-, IL-6, and IL-1 in H. influenzae-infected SP-D–/– mice (7). Interestingly, respiratory epithelial cells express TLRs (42) and when treated with LPS and sCD14 elicited IL-6 and IL-8 production (20). This suggests that the respiratory epithelium may be an important source of cytokines in SP-D–/– mice after bacterial challenge.
Phagocytic uptake of LPS was also impaired in alveolar macrophages from SP-D–/– mice. The reduced LPS uptake suggests that SP-D–/– mice are impaired in their ability to clear LPS from the lung; however, it is unlikely that phagocytic defect observed previously in SP-D–/– mice (7) is due solely to reduced CD14 because CD14-deficient macrophages were not impaired in their ability to phagocytose Escherichia coli (41).
The increased MMP-12, sCD14, and reduced LPS-induced 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 addition, human alveolar macrophages in culture produced significantly less TNF- and IL-6 after cigarette smoke exposure (46). This suggests MMP-12 may mediate reduction of alveolar macrophage 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 clearance and inflammatory processes that are important for host defense 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 macrophage responses to LPS.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Robert M. Senior for generously providing the MMP-9–/– mice. We thank Jaymi Semona, Victor LaFay, and Lindsey Malone for their assistance with animal husbandry. We thank Katy Davis and Theresa Richardson for their assistance in generating MMP/SP-D double knockouts.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants RO1HL71522 (to 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.).
2 Address correspondence and reprint requests to Dr. Ann Marie LeVine, Divisions of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: annmarie.levine{at}cchmc.org
3 Abbreviations used in this paper: SP-D, surfactant protein D; TLR4, toll-like receptor 4; MD-2, myeloid differentiation protein 2; MMP, matrix metalloprotease, GBS, group B streptococcus; BAL, bronchoalveolar lavage; sCD14, soluble CD14.
Received for publication November 11, 2004. Accepted for publication February 1, 2005.
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