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Am J Physiol Lung Cell Mol Physiol 292: L469-L475, 2007. First published October 20, 2006; doi:10.1152/ajplung.00297.2006
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Surfactant protein D regulates the cell surface expression of alveolar macrophage beta2-integrins

Albert P. Senft,1 Thomas R. Korfhagen,1 Jeffrey A. Whitsett,1 and Ann Marie LeVine2

1Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and 2Division of Pediatric Critical Care Medicine, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida

Submitted 7 August 2006 ; accepted in final form 17 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The beta2-integrin receptors (CD11a/CD18, CD11b/CD18, and CD11c/CD18) are expressed on the surface of alveolar macrophages and are important for the phagocytic clearance of pathogens. In the present study, we demonstrate that surfactant protein D (SP-D) modulates surface expression of CD11b and CD11c, but not CD11a or CD18, on alveolar macrophages. While cell surface receptors were reduced, CD11b and CD11c mRNAs were increased by SP-D deficiency. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D in the lung, were used to study the kinetics and reversibility of beta2-integrin receptors in response to changes in alveolar SP-D. Surface CD11b and CD11c were reduced on the alveolar macrophages within 3 days of SP-D deficiency and were restored with 3 days for CD11b and 7 days for CD11c of repletion of SP-D. SP-D deficiency caused a loss of cellular CD11b and CD11c content, indicating that the decrease in total cell content of the receptors was related to degradation rather than to redistribution of the receptor within the macrophage. CD11b and CD11c staining colocalized with Lamp-1 during SP-D deficiency, supporting the concept that reduced macrophage receptor levels resulted from increased lysosomal trafficking. Hydroxychloroquine, a lysomotropic agent, prevented the reduction of cellular and surface CD11b and CD11c. SP-D regulates surface CD11b and CD11c levels on the alveolar macrophage by modulating receptor trafficking, providing a mechanism by which SP-D mediates phagocytic activity in the alveolar macrophage.

SURFACTANT PROTEIN D (SP-D) is a member of the collectin family of innate defense polypeptides that includes surfactant protein A (SP-A), mannose-binding lectin (MBL), conglutinin, and CL-43 (25, 28, 31). Collectins form multimeric structures consisting of a collagenous NH2-terminal domain and a globular COOH-terminal carbohydrate-binding domain (33) that binds carbohydrate surfaces of many microorganisms, enhancing uptake and killing by phagocytic cells (36). In addition to the integral role that SP-D plays in removal of microorganisms from the lung, surfactant proteins modulate phagocyte (9, 13, 29, 32, 37) and lymphocyte (5–7) function, thereby, underscoring the role of surfactant proteins in regulation of innate and adaptive immune function.

SP-D gene-inactivated mice (SP-D–/–) are susceptible to various pulmonary pathogens. Phagocytosis of group B streptococcus (GBS), Haemophilus influenzae, influenza A virus, respiratory syncytial virus, and apoptotic cells by alveolar macrophages from the SP-D–/– mice is impaired (11, 23, 24, 26). Phagocytosis of pathogens and apoptotic cells by alveolar macrophages is facilitated through pattern recognition receptors. Recent studies demonstrate that collectins modulate phagocytic receptor levels on alveolar macrophages. Specifically, SP-A enhances the surface expression of scavenger receptor A on the alveolar macrophage through a casein kinase-2-dependent pathway (22). Similarly, both SP-A and SP-D increase cell surface levels of the mannose receptor on alveolar macrophages through a mechanism that does not involve new protein expression (4, 21). SP-D deficiency causes matrix metalloproteinase-12 (MMP-12)-dependent shedding of CD14 from the alveolar macrophage cell surface (34). Together these studies indicate that collectins are critical innate defense molecules that regulate alveolar macrophage function independently of, or in addition to, their direct opsonic effect.

The beta2-integrins are heterodimeric receptors composed of one of three {alpha}-chains (CD11a, CD11b, or CD11c) and a common beta-chain (CD18). beta2-Integrins are important mediators of binding and phagocytosis of microbial pathogens including GBS (1, 17) as well as apoptotic cells (18). Functionally, the {alpha}-chain of the receptor confers specificity; CD11a functions only as an adhesion molecule, while CD11b and CD11c have overlapping functions for cell adhesion and phagocytosis. Cells of the monocyte/macrophage lineage express surface CD11a, CD11b, and CD11c; however, the number of each receptor changes as monocytes differentiate to macrophages. Alveolar macrophages express more CD11c and less CD11b than their monocytic precursors. Regardless of isoform specificity or cellular expression levels, the importance of beta2-integrins in phagocytic clearance is illustrated by the rare inherited immunodeficiency disease resulting from reduced leukocyte CD11/CD18 expression that is associated with recurrent and often fatal bacterial infections (2).

Although there is clear evidence that phagocytosis of GBS is impaired in SP-D–/– mice, mechanisms, other than pathogen opsonization, 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 beta2-integrin expression and function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. SP-D–/– mice were generated by targeted gene inactivation as previously described (20). 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 (38). Triple transgenic mice were fed doxycycline-containing food to induce the expression of the rSP-D protein. CCSP-rtTA+/(tetO)7-rSPD+/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 Cincinnati Children's Hospital Institutional Animal Care and Use Committee. Male and female mice 56–70 days old were used for this study.

Bronchoalveolar lavage. Mice were exsanguinated after a lethal intraperitoneal injection of pentobarbital sodium, and the lungs were lavaged three times with 1 ml of PBS. Bronchoalveolar lavage (BAL) cells were recovered by centrifugation at 800 g and then resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS, pH 7.4, containing 0.1% NaN3, and 1% BSA) for flow cytometric analysis. BAL fluid from SP-D+/+, SP-D–/–, and CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice contained >98% macrophages (data not shown).

Peritoneal lavage. Mice were killed by administration of a lethal dose of pentobarbital sodium. The peritoneal cavity was lavaged with 5 ml of PBS. Cells in the peritoneal lavage fluid were recovered by centrifugation at 800 g and resuspended in FACS buffer.

Flow cytometric analysis. Isolated macrophages were resuspended in 200 µl of FACS buffer and incubated with Fc block (rat anti-mouse CD16/32; BD Pharmingen, San Diego, CA) for 30 min at 4°C. Cells were incubated with phycoerythrin (PE)-conjugated anti-mouse CD11a, CD11b, CD11c, or CD18 (BD Pharmingen) for 1 h on ice and washed three times with FACS buffer. As controls, cells were assessed using primary isotype- and species-matched anti-mouse Igs. For some experiments, where total and extracellular expression of CD11b and CD11c was measured, macrophages were incubated with PE-conjugated anti-mouse CD11b or CD11c to determine surface expression, fixed and permeabilized using the Cytoperm Cytofix kit (BD Pharmingen), and one-half the population of cells was incubated with PE-conjugated anti-mouse CD11b or CD11c again to determine total cellular receptor levels. Cells were analyzed by single-color flow cytometry using a FACScan flow cytometer (BD Biosciences), and results were analyzed using CellQuest software on a Macintosh computer. Fluorescence data were collected using logarithmic amplification on 10,000 viable cells as determined by forward light scattering.

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 beta-mercaptoethanol in 25 mM sodium citrate buffer (GITC). Total RNA was isolated using the acidified guanidinium method (10), treated with DNase I (DNA-free; Ambion, Austin, TX), and reverse transcribed using a cDNA cycle kit (Invitrogen, Carlsbad, CA). PCR reaction mixes consisted of template, 0.5 µM each primer, 2.5 mM MgCl2, and 1x DNA Master SYBR Green I (Roche Molecular Biochemicals, Indianapolis, IN) 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, respectively, were as follows: L32 (257 bp), 5'-GTGAAGCCCAAGATCGTC-3', 5'-AGCAATCTCAGCACAGTAAG-3'; CD11a (190 bp), 5'-CTCCAGGAGGACAACTCAGC-3', 5'-CTAGTGTGGGCATGTTGTGG-3'; CD11b (195 bp), 5'-GCAGTCATCTTGAGGAACCGTGTC-3', 5'-GTTGGTATTGCCATCAGCGTCC-3'; and CD11c (181 bp), 5'-ATGTTGGAGGAAGCAAATGG-3', 5'-CCTGGGAATCCTATTGCAGA-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 (e.g., primer-dimers) 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, Ct) 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 r-squared value ≥0.985 were used to determine quantitation.

Hydroxychloroquine treatment of mice. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were treated with the lysomotropic drug hydroxychloroquine at a dose of 10 mg·kg–1·day–1 for 4 days before and 3 days following removal of doxycycline from the diet of the CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice. The dose of 10 mg·kg–1·day–1 was chosen based on its efficacy in ameliorating idiopathic pulmonary fibrosis without deleterious side effects to the patients (14).

Fluorescence microscopy. Alveolar macrophages were plated on poly-D-lysine-treated coverslips and allowed to adhere for 2 h and then gently washed two times with PBS to remove nonadherent cells. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, and stained with an FITC-conjugated monoclonal antibody against Lamp-1 (CD107a) and a PE-conjugated monoclonal antibody against CD11b or CD11c (all from BD Pharmingen) for 2 h at 37°C. Cells were washed three times with PBS and one time with double distilled H20 and mounted on slides with Vectashield mounting medium(Vector Laboratories, Burlingame, CA). Wide-field fluorescent images were acquired by using a Zeiss Axioplan 2 microscope equipped with an ApoTome system for optical sectioning and an AxioCam HRm camera (Carl Zeiss Microimaging, Thornwood, NY).

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 means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Reduced beta2-integrins on alveolar macrophages from SP-D–/– mice. Surface expression of CD11a, CD11b, CD11c, and CD18 was assessed by flow cytometric analysis on alveolar macrophages from SP-D–/– and SP-D+/+ mice. Previous studies demonstrated that alveolar macrophages from SP-D–/– mice exhibit altered morphology (20, 35), impaired phagocytosis (26), and reduced levels of the pattern recognition receptor, CD14 (34). Cell surface CD11b and CD11c were decreased by 58 and 54%, respectively, on alveolar macrophages from SP-D–/– compared with SP-D+/+ mice (Fig. 1). In contrast, the cell surface expression of CD11a on the alveolar macrophage was not influenced by the lack of SP-D (Fig. 1). Likewise, the cell surface expression of CD18 was similar on alveolar macrophages from SP-D+/+ (mean fluorescent intensity = 180 ± 24) and SP-D–/– mice (mean fluorescent intensity = 156 ± 16). CD11a, CD11b, and CD11c on peritoneal macrophages from SP-D–/– and SP-D+/+ mice were not different (data not shown).


Figure 1
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  		Fig. 1. CD11b and CD11c, but not CD11a, are reduced on alveolar macrophages from SP-D–/– mice. Flow cytometric analysis was used to measure levels of cell surface CD11a, CD11b, and CD11c on alveolar macrophages from SP-D–/– and SP-D+/+ mice. CD11b and CD11c were significantly decreased on alveolar macrophages from SP-D–/– compared with SP-D+/+ mice. CD11a was similar on alveolar macrophages from SP-D–/– and SP-D+/+ mice. Data are means ± SE; n = 6. SP-D, surfactant protein D. *P < 0.05 compared with SP-D+/+ mice.

 
SP-D influences alveolar macrophage CD11b and CD11c mRNA levels. beta2-Integrin mRNA was measured to determine whether decreased surface CD11b and CD11c on alveolar macrophages from the SP-D–/– mice resulted from reduced mRNA levels. Alveolar macrophage CD11b and CD11c mRNA levels were increased in SP-D–/– compared with SP-D+/+ mice while CD11a mRNA was unchanged (Fig. 2).


Figure 2
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  		Fig. 2. CD1lb and CD11c mRNA is increased in alveolar macrophages from SP-D–/– mice. Real-time PCR analysis was used to measure alveolar macrophage CD11a, CD11b, and CD11c mRNA levels from SP-D–/– and SP-D+/+ mice. Expression of CD11a, CD11b, and CD11c mRNA was normalized to mRNA levels of the ribosomal subunit L32. CD11b and CD11c mRNA was significantly increased in alveolar macrophages from SP-D–/– compared with SP-D+/+ mice. CD11a mRNA levels were similar in alveolar macrophages from SP-D–/– and SP-D+/+ mice. Data are means ± SE; n = 6. *P < 0.05 compared with SP-D+/+ mice.

 
Surface CD11b and CD11c levels are rapidly modified by expression of SP-D. Because SP-D deficiency causes a chronic inflammatory condition in the mouse lung (35), it is unclear whether the reduction of alveolar macrophage surface CD11b and CD11c is directly regulated by SP-D or is secondary to chronic inflammation. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, which conditionally express SP-D in the respiratory epithelium, were used to determine the kinetics of CD11b and CD11c regulation by SP-D. Flow cytometric analysis was used to assess the levels of CD11b and CD11c on alveolar macrophages. Both CD11b and CD11c were reduced on the alveolar macrophages 3 days after conditional loss of SP-D in the CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice (Fig. 3). Surface expression of CD11b was restored by reexpression of SP-D for 3 days (Fig. 3). Expression of SP-D for 7 days was required to restore surface CD11c expression to normal levels (Fig. 3).


Figure 3
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  		Fig. 3. CD11b and CD11c on alveolar macrophages are influenced by the conditional expression of SP-D. Cell surface expression of CD11b and CD11c was determined by flow cytometric analysis on alveolar macrophages from CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice. CD11b and CD11c were decreased on alveolar macrophage 3 days (d) after the conditional loss of SP-D (–Dox 3d) compared with wildtype (+Dox). Restoration of SP-D expression for 3 days (–Dox 3d, +Dox 3d) was sufficient to increase alveolar macrophage CD11b to wildtype (+Dox) levels. For CD11c, restoration of SP-D expression for 7 days (–Dox 3d, +Dox 7d) was necessary to restore surface expression to wildtype (+Dox) levels. Flow cytometric analysis data are expressed as means ± SE; n = 6. *P < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice always treated with doxycycline (+Dox).

 
Rapid regulation of alveolar macrophage CD11b and CD11c mRNA during SP-D depletion. Real-time PCR analysis was used to measure the mRNA levels of alveolar macrophage CD11b and CD11c during SP-D depletion. Alveolar macrophage CD11b and CD11c mRNA levels were increased 3 days following the loss of SP-D (Fig. 4).


Figure 4
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  		Fig. 4. CD11b and CD11c mRNA increases rapidly in alveolar macrophages following the conditional loss of SP-D. Real-time PCR analysis was used to measure CD11b and CD11c mRNA levels in alveolar macrophages from CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice. Expression of CD11b and CD11c mRNA was normalized to alveolar macrophage L32 mRNA levels. CD11b and CD11c mRNA was significantly increased in alveolar macrophages in association with the loss of SP-D (open bars) following termination of doxycycline treatment (–Dox). Data are means ± SE; n = 6. *P < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with doxycycline.

 
Increased lysosomal localization of CD11b and CD11c following the loss of SP-D. Flow cytometric analysis was used to assess cell surface and total cellular levels of CD11b and CD11c on alveolar macrophages during depletion of SP-D. Loss of SP-D resulted in reduced total cellular and cell surface levels of alveolar macrophage CD11b and CD11c (Fig. 5). SP-D did not influence intracellular levels of CD11b or CD11c in alveolar macrophages (Fig. 5). These results suggest that loss of SP-D does not promote redistribution of CD11b and CD11c from the cell surface to an intracellular storage vesicle but caused their degradation.


Figure 5
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  		Fig. 5. Loss of SP-D results in decreased cell surface levels but not increased intracellular stores of alveolar macrophage CD11b and CD11c. Total cellular, cell surface, and intracellular levels of alveolar macrophage CD11b and CD11c were determined by flow cytometric analysis. Total cellular and cell surface levels of alveolar macrophage CD11b and CD11c were decreased, while no increase in intracellular levels of either receptor was observed in CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice in association with the loss of SP-D following termination of doxycycline treatment. Flow cytometric analysis data are expressed as means ± SE; n = 6. *P < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with doxycycline.

 
Lysosomal localization of CD11b and CD11c was assessed by colocalizing CD11b and CD11c with the lysosomal marker Lamp-1. Increased colocalization of both CD11b and CD11c with Lamp-1 was observed in alveolar macrophages from CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice 3 days after loss of SP-D (Fig. 6). Together, the data suggest that SP-D deficiency causes lysosomal trafficking and degradation of CD11b and CD11c.


Figure 6
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  		Fig. 6. Increased lysosomal trafficking of alveolar macrophage CD11b and CD11c following the loss of SP-D. Immunofluorescence confocal microscopy was performed on alveolar macrophages to determine colocalization of CD11b (A) or CD11c (B) (red) with the lysosomal marker Lamp-1 (green). Alveolar macrophages were plated on coverslips, stained with an antibody directed against CD11b or CD11c, and Lamp-1, and examined by fluorescence confocal microscopy. Alveolar macrophages from CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice, where conditional expression of SP-D was lost for 3 days, had increased colocalization (yellow) of both CD11b and CD11c with LAMP-1. The presented data represent 3 experiments having similar results.

 
Hydroxychloroquine inhibits SP-D-dependent loss of alveolar macrophage CD11b and CD11c. CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice were treated with the lysomotropic agent hydroxychloroquine to determine whether the reduction of CD11b and CD11c caused by SP-D depletion was mediated by lysosomal degradation. Flow cytometric analysis was used to assess cell surface and total cellular expression of CD11b and CD11c on alveolar macrophages from mice where SP-D was depleted for 3 days. Hydroxychloroquine treatment increased total, extracellular, and intracellular levels of SP-D-deficient alveolar macrophage CD11b and CD11c (Fig. 7), supporting the concept that SP-D deficiency causes increased lysosomal trafficking and degradation of alveolar macrophage CD11b and CD11c.


Figure 7
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  		Fig. 7. Hydroxychloroquine inhibits the SP-D-dependent loss of alveolar macrophage CD11b and CD11c. The cellular pools of alveolar macrophage CD11b and CD11c were determined by flow cytometric analysis following treatment with the lysomotropic agent hydroxychloroquine (HCQ). CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with HCQ had increased total cellular, cell surface, and intracellular levels of alveolar macrophage CD11b and CD11c following the conditional loss of SP-D. Flow cytometric analysis data are expressed as means ± SE; n = 6. *P < 0.05 compared with conditional CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice treated with doxycycline.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
beta2-Integrins are important receptors for phagocytic clearance of bacteria (1) and apoptotic cells (18). Because phagocytosis of GBS and apoptotic cells is impaired in alveolar macrophages from SP-D–/– mice, these studies examined the possible involvement of SP-D in the regulation of alveolar macrophage beta2-integrin expression. Loss of SP-D from the lung resulted in a rapid loss of both CD11b and CD11c from the alveolar macrophage cell surface and was reversible with the reexpression of SP-D in the lung. Reduction of alveolar macrophage CD11b and CD11c cell surface expression was inhibited by hydroxychloroquine, suggesting that CD11b and CD11c are subject to increased lysosomal trafficking and degradation in the absence of SP-D. The present study identifies a novel mechanism by which SP-D influences alveolar macrophage CD11b and CD11c expression.

beta2-Integrin receptors are common molecules on the cell surface mononuclear phagocytes; however, expression of {alpha}-chains is differentially regulated on monocytes compared with macrophages. The differentiation of a monocyte to an alveolar macrophage results in a programmed change in the relative expression pattern of CD11a, CD11b, and CD11c on the cell surface. During the differentiation process, the cell surface abundance of CD11b on alveolar macrophages declines while CD11c increases. The maturation process results in a change in the relative expression pattern of CD11a > CD11b > CD11c on monocytes to that of CD11c > CD11a > CD11b on alveolar macrophages (3). Because the SP-D–/– mice have an elevated number of phagocytes in the lung (35) with an impaired phagocytic ability (26), we hypothesized that the loss of SP-D results in the selection of macrophages that have not fully differentiated and are functionally impaired. Results from the current study show that alveolar macrophages from SP-D–/– mice exhibit reduced levels of CD11b and CD11c; however, the macrophages have a relative surface expression pattern of CD11c > CD11a > CD11b, a finding consistent with beta2-integrin surface expression on normal alveolar macrophages.

Alveolar macrophage surface expression of CD11b and CD11c was significantly reduced 3 days after the conditional loss of SP-D, and this was readily reversible. The observation that the receptor changes were reversible is consistent with previous observations in CCSP-rtTA+/(tetO)7-rSPD+/SP-D–/– mice where abnormalities in alveolar macrophage morphology and lipid content were also readily reversed by restoration of SP-D in the adult mice (38). It is also important to note that it is unlikely that the changes to beta2-integrin surface expression observed in these studies are due to the abnormalities in surfactant phospholipid pools observed previously by Ikegami et al. (19), since phospholipid changes were not observed until 2 wk after the loss of SP-D.

In addition to cell surface changes, migration of monocytes from the blood to the lung promotes cellular differentiation to alveolar macrophages by initiating a specific gene transcription pattern that results in a cell type-specific cell surface receptor profile. During the monocyte-to-macrophage differentiation process, CD11b mRNA expression is repressed by ZBP-89 binding to response elements in the promoter of the CD11b gene (30). Likewise, CD11c mRNA expression is regulated by transcription factors binding to activator protein-1 and C/EBP elements within the promoter region during the differentiation from monocyte to macrophage (12). Given the changes observed in receptor expression on the alveolar macrophage cell surface, we sought to determine whether the reduction in CD11b and CD11c could be explained by decreased mRNA expression. The results from this study indicate that transcriptional repression cannot account for the observed decreases in CD11b and CD11c receptor levels, as mRNA levels for both genes were elevated with the loss of SP-D from the lung.

Cell surface receptors are routinely endocytosed and then enter a complex sorting pathway where they may be stored, destroyed, or recycled back to the cell surface (27). On the basis of the fact that neutrophil CD11b is stored in granules and released to the cell surface on stimulation, our initial hypothesis was that loss of SP-D promotes the intracellular localization of CD11b and CD11c in the alveolar macrophage. The flow cytometric analysis data from this study do not support this concept and are consistent with reports that macrophages do not store CD11b for release (3). Instead, the current study suggests that SP-D prevents CD11b and CD11c from being targeted for lysosomal degradation. The current study also shows that SP-D influences cell surface expression of CD11b and CD11c, two functionally similar receptors, but not CD11a. The differential regulation is likely due to differences in receptor substrate specificity and also due to trafficking differences conferred by the cytoplasmic tail of the {alpha}-chain of the receptor (8). The collectins SP-A and MBL bind the {alpha}2beta1-integrin receptor on the I-domain of the {alpha}-chain (15). Ligand binding to the I-domain of the integrin {alpha}-chain correlates with a higher-affinity form of the receptor and promotes clustering of laterally associated integrins (16). It is presently unclear what effect {alpha}-chain I-domain binding has on beta2-integrin receptor trafficking. Interestingly, SP-D–/– mice also have reduced levels of SP-A (19, 20). We propose that the pulmonary collectins SP-A and SP-D directly or indirectly modulate CD11b and CD11c receptor affinity and thereby prevent receptor degradation.

In summary, SP-D is an important regulator of microbial clearance and inflammatory processes that are essential for host defense and pulmonary homeostasis. The current study demonstrates that SP-D regulates beta2-integrin receptor recycling and degradation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-71522 (A. M. LeVine), HL-58795 (T. R. Korfhagen), and HL-63329 (J. A. Whitsett) and American Lung Association Research Training Fellowship (A. P. Senft) and Parker B. Francis Fellowship (A. P. Senft).


   ACKNOWLEDGMENTS
 
We thank Jaymi Semona and Victor LaFay, Jr., for assistance with animal husbandry.

Present address of A. P. Senft: Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108 (e-mail: asenft@lrri.org).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. LeVine, Pediatric Critical Care Medicine, Univ. of Florida College of Medicine, 1600 SW Archer Rd., Gainesville, FL 32610 (e-mail: levineam{at}peds.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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