Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Surfactant protein (SP) A and SP-D are the pulmonary members of the collectin family, structurally related proteins involved in innate immune responses. Here, we have examined the abilities of SP-A, SP-D, mannose-binding protein (MBP), and the complement component C1q to stimulate actin-based cellular functions in rat alveolar macrophages and peripheral blood monocytes. Our goal in this study was to examine the cell specificity of the effects of the collectins to understand further the mechanisms by which SP-A and SP-D stimulate alveolar macrophages. We found that SP-A and SP-D have lung cell-specific effects at physiologically relevant concentrations; they stimulate directional actin polymerization and chemotaxis in alveolar macrophages but not in monocytes. Although C1q and MBP weakly stimulate the rearrangement of actin in both cell types, C1q is chemotactic only for peripheral blood monocytes and MBP does not stimulate chemotaxis of either cell type. Neither C1q nor MBP stimulates actin polymerization in alveolar macrophages. These results support the hypothesis that alveolar macrophages express receptors specific for the pulmonary collectins SP-A and SP-D and provide insight into the potential roles of collectins in the recruitment and maturation of mononuclear phagocytes in the lung.
lung; host defense; peripheral blood monocyte; collectins; chemotaxis
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) A and SP-D are the pulmonary members of a family of proteins called collectins, which also includes the serum proteins mannose-binding protein (MBP) and conglutinin (reviewed in Ref. 51). The collectins share a great deal of structural homology, including the common structural motifs of triple-helical collagen-like domains and globular trimeric carbohydrate-binding (lectin) head domains, as well as a similar multimeric arrangement in which trimeric subunits group together to form either octodecamers (SP-A and MBP) or dodecamers (SP-D and conglutinin). Structurally related to this family is the complement component C1q, the first protein of the classic complement cascade, which contains both collagen-like domains and globular head regions and which shares the octodecameric structure of SP-A and MBP but which is not a lectin.
The collectins and C1q have been implicated, in various ways, in the pathways of non-antibody-mediated host defense. SP-A and MBP can both act as opsonins, directly mediating the uptake of particles by phagocytic cells (41, 48). SP-A and SP-D have been shown to interact with various pathogens (29, 48). Additionally, SP-A and C1q both act as activation ligands, increasing the phagocytosis of immunoglobulin G (IgG)-opsonized particles (45). Because of their highly homologous structures and similar functions, it has been suggested that the collectins and C1q share a common cell surface receptor on monocytes and macrophages (22, 32).
SP-A has been shown to interact with alveolar macrophages in a saturable, specific, high-affinity manner consistent with the existence of a cell surface receptor for SP-A on alveolar macrophages (30, 33, 38). However, a study (22) was unable to show specific, saturable binding of SP-A to peripheral blood monocytes. It has been demonstrated that SP-A stimulates chemotaxis of alveolar macrophages (53) and that SP-A stimulates the phagocytosis of specific pulmonary pathogens by alveolar macrophages (48).
In the latter study, a significant difference was found between alveolar macrophages and peripheral blood monocytes. The observation by Geertsma et al. (22) that the stimulatory effect of SP-A on monocyte phagocytosis is eliminated by adherence of the cells to a C1q-coated surface was confirmed. In contrast, however, when C1q receptors sequestered, alveolar macrophages are still responsive to SP-A (48). This finding suggests that peripheral blood monocytes and alveolar macrophages express a different assortment of receptors for SP-A and C1q. These conclusions are based on the presumption that the adherence of cells to a ligand-coated surface clusters receptors for that ligand to the basal surface of the cell, making them unavailable to interact with free (or pathogen-bound) ligand (55). Indeed, that same study showed that SP-A was unable to stimulate phagocytosis in alveolar macrophages adhered to an SP-A-coated surface (48).
This study (48) also suggested that alveolar macrophages possess a receptor (or receptors) for SP-A not found on peripheral blood monocytes. This suggestion is consistent with recent findings about both the functions and maturation of alveolar macrophages. Alveolar macrophages are the immune cells primarily responsible for non-antibody-mediated host defense in the lung (21). Among the functions of alveolar macrophages are the metabolism of pulmonary SPs (54), regulation of lung inflammation and lymphocyte proliferation (20), and production of various cytokines and mediators of immune responses (reviewed in Ref. 21).
Alveolar macrophages arise from blood monocytes, which differentiate in the lung capillaries and tissues and migrate into the alveolar air space (4). Among the changes known to occur in this differentiation pathway are a change in the expression of cell surface adhesion molecules (39), the disappearance of high-affinity binding of the complement component C3 through the C3 receptor (12), and changes in the responses of the cells to various extracellular and intracellular signals including glucocorticoid-induced eicosanoid inhibition (17) and metabolism of endogenous arachidonic acid by 5-lipoxygenase (11). The exact stimuli necessary for the processes of recruitment and maturation are not known, although the lung-specific production of the chemoattractant SP-A has led some to postulate that SPs may be integral to them.
The goals of this study were to define the actin-based responses of alveolar macrophages to the collectins and C1q and to determine the cellular specificity of responsiveness to the same proteins. To do this, we looked at three receptor-mediated cellular responses likely to be involved in host defense functions: stimulation of filamentous actin (F-actin) formation in alveolar macrophages; stimulation of cytoskeletal rearrangement, specifically of directed actin-filled filopodia, in both alveolar macrophages and monocytes; and stimulation of directed cell migration, or chemotaxis, in both cell types. Directed actin rearrangement and the subsequent downstream stimulation of cell migration are functions that require cell surface receptors that stimulate specific downstream signaling pathways (reviewed in Ref. 44).
Preliminary reports of these studies were published as abstracts (46, 47).
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MATERIALS AND METHODS |
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Media and Chemicals
Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced salt solution, Gey's balanced salt solution (GBSS) and RPMI 1640 cell culture medium were from GIBCO BRL (Grand Island, NY). All other chemicals (except as noted) including C1q were from Sigma (St. Louis, MO). Bovine serum albumin (BSA; also from Sigma) for buffers in contact with isolated cells was from fraction V, fatty acid free, <0.1 ng/mg endotoxin, and cell culture tested.Purification of Human Alveolar Proteinosis SP-A
SP-A was purified from the bronchoalveolar lavage (BAL) fluid of patients with alveolar proteinosis (AP) as previously described (52). SP-A was stored in 5 mM Tris (Tris-buffered water), pH 7.4, atPurification of Rat SP-D
Rat SP-D was purified from the BAL fluid of silica-treated rats as previously described (15). Briefly, the rats were instilled intratracheally with silica and killed ~21 days later. At this time, their lungs were lavaged in situ with buffer containing 5 mM Tris, pH 7.4, and 150 mM NaCl at 37°C. After calcium was added to 5 mM, the lavage fluid was incubated overnight with maltose-Sepharose beads. The SP-D-bound beads were pelleted and put into a column, which was then washed with buffer containing 2 mM Tris, pH 7.8, 5 mM CaCl2, and 100 mM NaCl (maltose loading buffer). The SP-D was then eluted with buffer containing 5 mM Tris, pH 7.8, 2 mM EDTA, and 100 mM NaCl (maltose elution buffer). The measured level of endotoxin in the rat SP-D was 0.133 pg/µg.Purification of Recombinant Rat SP-D
Full-length rat recombinant SP-D (rSP-D) cDNA was provided by Dr. J. H. Fisher (University of Colorado, Denver) and has been previously described by Shimizu et al. (42). This construct was subcloned into the pEE14 vector, and the construct was transfected into Chinese hamster ovary (CHO) K1 cells (American Type Culture Collection, Manassas, VA). Consequent selection, production of a single SP-D-producing subclone, and production of rSP-D were done via a slight modification of the procedures of Crouch et al. (14). Changes to this protocol were as follows: transfected cells were originally grown inPurification of Rat MBP
MBP was purified from rat serum (Pel-Freeze, Rogers, AR) via a slight modification of the method of Kawasaki et al. (28). Briefly, the serum was centrifuged for 10 min at 2,060 g, diluted 1:1 with a 2× concentration of mannose loading buffer (final concentration, 50 mM Tris, pH 7.4, 25 mM CaCl2, and 1.25 M NaCl), and incubated overnight at 4°C on a rotator. The serum was then applied to a mannose-agarose column and washed with mannose loading buffer. The MBP was eluted with a buffer containing 50 mM Tris, pH 7.4, 2 mM EDTA, and 1.25 M NaCl (mannose elution buffer). MBP for actin polymerization studies was diluted in sizing column buffer (50 mM Tris, pH 7.4, 150 mM NaCl, and 10 mM EDTA) and further purified by gel-filtration chromatography with a Sephacryl S-300HR column (Pharmacia Biotech, Uppsala, Sweden). The MBP-containing fractions, which were also free of IgG and IgM (as analyzed by Western blot), were pooled, concentrated with a Centricon-30 (Amicon, Beverly, MA), and buffer exchanged into DPBS free of calcium or chelating agents.Preparation of Zymosan-Activated Serum
Zymosan-activated serum (ZAS) was prepared according to the method of Snyderman and Pike (43). One milliliter of lyophilized rat serum was resuspended in sterile deionized water and incubated at 37°C for 60 min with 1 mg of zymosan A in 0.1 ml of 0.9% NaCl. The solution was then heated to 56°C for 30 min. The ZAS was divided into aliquots and stored atAnimals
Male Sprague-Dawley rats (200-250 g) were obtained from Charles River Laboratories (Raleigh, NC).Isolation of Alveolar Macrophages and Peripheral Blood Monocytes
Rat alveolar macrophages were isolated as previously described (52). Briefly, rat lungs were lavaged six times with DPBS, pH 7.2, containing 0.2 mM EGTA and then twice with DPBS, pH 7.2, containing 1 mM CaCl2. The cells were pelleted at 228 g and resuspended in the appropriate medium. Alveolar macrophages comprised >95% of the cells obtained as measured by differential staining with the Hemacolor stain kit (EM Diagnostic Systems, Gibbstown, NJ).Peripheral blood monocytes were isolated as previously described (48), with a slight modification of the protocol of Boyum (6). Briefly, rat blood was collected via heart puncture and separated via centrifugation and dextran sedimentation to remove plasma and erythrocytes. The resulting leukocyte suspension was then centrifuged on a two-step Percoll gradient, and the mononuclear cell fraction was collected and resuspended in the appropriate buffer or medium. Monocytes comprised ~50-60% of the cells in this fraction (the remainder were primarily lymphocytes) as measured by differential staining with the Hemacolor stain kit. For assays involving adherent cells, the cells were adhered to glass slides (Nunc, Naperville, IL). After adherence to the slides, the monocytes were enriched to >85% of the total cells.
Stimulation of Actin Polymerization and Rearrangement
Microscopic analysis of F-actin structures. Alveolar macrophages or peripheral blood monocytes (2.5 × 105 cells/ml in RPMI 1640 medium) were allowed to adhere to eight-well glass cell culture slides (Nunc, Naperville, IL) for 3 (alveolar macrophages) or 1 h (peripheral blood monocytes) in a cell culture incubator (37°C and 5% CO2). After adherence, the medium was changed to remove nonadherent cells, and either AP SP-A, SP-D, C1q, or MBP was added at a final concentration of 25 µg/ml. Equal amounts of the appropriate protein storage buffer were added to the control wells. After 30 min of stimulation, the slides were washed once with DPBS containing 1 mM CaCl2, fixed for 10 min with 3.7% formaldehyde in DPBS, and washed twice with DPBS. Rhodamine-phalloidin (Molecular Probes, Eugene, OR) was then added to the cells as a 2 U/ml solution in DPBS containing 0.05% Triton X-100, and the slides were incubated for 10 min in the dark. Slides were then washed twice with DPBS, mounted with 1,4-diazabicyclo(2.2.2.)octane (Kodak, Rochester, NY), dissolved as a 25 mg/ml solution in 90% glycerol, 0.27 mM KCl, 0.15 mM KH2PO4, 13.7 mM NaCl, and 0.81 mM Na2HPO4, final pH 8.6, and sealed with clear nail polish (Maybelline, Memphis, TN). The cells were examined for rhodamine fluorescence with an epifluorescence microscope at ×1,000 magnification.Fluorometric analysis of total cellular F-actin. Total cellular F-actin was measured with a modified version of the fluorometric assay developed by Howard and Oresajo (26). Briefly, 7.5 × 105 cells were suspended 0.35 ml of RPMI 1640 medium and added to the appropriate protein or buffer in 125 µl of DPBS. The cells were stimulated for 3 min at 37°C, after which formaldehyde was added at a final concentration of 3.7%. The cells were fixed for 15 min, after which 5 units of fluorescein-phalloidin (Fl-Ph; Molecular Probes) in 5 mg/ml of lysophosphatidylcholine in DPBS were added (to final concentrations of 9 U Fl-Ph/ml and 90 µg lysophosphatidylcholine/ml). The cells were stained for 10 min, pelleted in a microcentrifuge to remove unbound Fl-Ph, and homogenized in 0.5 ml of methanol with a 21-gauge needle and syringe. The cells were incubated for at least 1 h in methanol to extract the Fl-Ph from the cells, cellular debris was pelleted in a microcentrifuge, and the fluorescence of 400 µl of each sample was measured in a Fluoro-Max fluorometer running dm3000F software (SPEX Industries, Edison, NJ).
Chemotaxis Assay
Directed migration (chemotaxis) of cells was performed as previously described (53). Briefly, 50 µl of cells (alveolar macrophages or peripheral blood monocytes) suspended at 2.5 × 106 cells/ml in GBSS containing 0.1% BSA were placed in the upper wells of a 48-well microchemotaxis chamber (Neuro Probe, Cabin John, MD). The concentration of glucose in GBSS was 5.5 mM. The lower chambers contained 28 µl of test solution, consisting of GBSS containing 0.1% BSA and either nothing (control); various concentrations of either SP-A, SP-D, C1q, or MBP; or, as a positive control, either 25% ZAS or 10Statistical Analysis
The response of cells to different treatments is expressed as percentage of the control (no-protein) response and was analyzed with a multiplicative model that results in a value ( ![]() |
RESULTS |
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Stimulation of Directional Actin Polymerization in Alveolar Macrophages by SP-A and SP-D
Human SP-A and rat SP-D, but not human C1q or rat MBP, stimulated the formation of directional actin structures in rat alveolar macrophages (Fig. 1). In a qualitative visual assay in which deviations from the untreated state were noted, only the lung collectins stimulated the formation of actin-filled structures in this manner. In this assay, the large majority of untreated macrophages (
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Stimulation of Actin Structure Formation in Peripheral Blood Monocytes by C1q and MBP
Human C1q and rat MBP, but not human SP-A or rat SP-D, stimulated the nondirectional formation of actin-filled cellular processes in peripheral blood monocytes (Fig. 2). Untreated monocytes exhibited a cortical distribution of F-actin similar to that seen in untreated alveolar macrophages (Fig. 2A). None of the proteins tested stimulated a directional rearrangement of F-actin such as that seen in alveolar macrophages stimulated with either SP-A or SP-D. C1q, added at 25 µg/ml, stimulated the formation of either filopodia (data not shown) or lamellopodia (Fig. 2D) in a response almost identical to that seen with alveolar macrophages. Similarly, the same concentration of MBP stimulated the formation of short F-actin-filled filopodia (Fig. 2E). Neither SP-A nor SP-D stimulated the rearrangement of F-actin in peripheral blood monocytes (Fig. 2, B and C, respectively).
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Quantification of SP-A and SP-D Stimulation of Actin Polymerization in Alveolar Macrophages
Human SP-A and rat SP-D, but not MBP or C1q, stimulated an increase in total cellular F-actin in alveolar macrophages (Table 1). After a 3-min stimulation with 25 µg of SP-A/ml, total F-actin levels were increased to 140 ± 8% of no-protein control levels. SP-D at 12.5 µg/ml increased F-actin to 134 ± 8% of the control level. Neither MBP (106 ± 10% of the control level) nor C1q (90 ± 13% of the control level) significantly changed total F-actin levels in alveolar macrophages.
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Concentration Range of SP-A and SP-D Stimulation of Actin Polymerization
Both SP-A and SP-D showed stimulation of F-actin polymerization in alveolar macrophages over a wide range of concentrations (Fig. 3). SP-A stimulated F-actin polymerization at a 3-min time point at concentrations as low as 0.5 µg SP-A/ml (120 ± 6% of no-protein control level). Actin polymerization was also significantly increased at 1 (119 ± 7% of the control level), 5 (131 ± 16%), and 10 µg (130 ± 22%) SP-A/ml. SP-D showed a concentration-dependent stimulation, with less than significant stimulation at 1 µg SP-D/ml (119 ± 15% of no-protein control level; P = 0.053) and significant stimulation at 5 (121 ± 8% of the control level) and 12.5 µg (134 ± 8%) SP-D/ml.
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Stimulation of Alveolar Macrophage Chemotaxis by SP-A and SP-D
Human SP-A and rSP-D, each at a concentration of 25 µg/ml, stimulated the chemotaxis of alveolar macrophages (Fig. 4A). Human C1q, at the same concentration, has no effect on their migration, and rat MBP significantly decreased migration compared with the control (buffer only) level. Migration of alveolar macrophages through a membrane with 5-µm pores was significantly increased to 445 ± 41% of the control level when 25 µg SP-A/ml were added to the lower chamber and 580 ± 85% of the control when 25 µg SP-D/ml were added (P < 0.05). In the presence of 25 µg MBP/ml in the lower chamber, migration was reduced to 14 ± 4% of the control level; C1q, at the same concentration, did not significantly change the level of migration (120 ± 8% of the control level).
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Concentration Dependence of SP-A and SP-D Stimulation of Alveolar Macrophage Chemotaxis
To confirm that both the protein concentrations used to examine chemotactic stimulation were maximally effective and the stimulation of chemotaxis by SP-A and SP-D occurred at physiologically relevant protein concentrations, the ability of SP-A and SP-D to stimulate alveolar macrophage chemotaxis over a wide range of concentrations (lower well concentrations from 5 ng/ml to 25 µg/ml) was tested. Results of these assays are shown in Table 2. Neither protein significantly stimulated alveolar macrophage migration at protein concentrations > 5 µg/ml, including concentrations as low as 5 ng/ml. In addition, both SP-A and SP-D stimulated significantly higher levels of alveolar macrophage migration at 25 µg/ml than at 5 µg/ml.
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Effect of Competing Sugars on SP-A and SP-D Stimulation of Alveolar Macrophage Chemotaxis
To characterize further the mechanism by which SP-A and SP-D stimulate alveolar macrophage chemotaxis, we examined the ability of sugars that bind the lectin domains of the proteins to block their chemotactic effect (Fig. 5). Although a lower well concentration of 10 µg/ml of either SP-A or SP-D significantly stimulated alveolar macrophage chemotaxis to similar levels (262 ± 39 and 270 ± 66% of control migration, respectively), the presence of 10 mM mannose (in combination with SP-A) or maltose (in combination with SP-D) in both wells had no effect on this stimulation. Migration in the presence of both protein and sugar was 249 ± 45% of the control level for SP-A and mannose and 250 ± 85% of control migration for SP-D and maltose. Neither mannose nor maltose had any effect on migration in the absence of protein (96 ± 14 and 100 ± 7% of control levels, respectively).
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Stimulation of Peripheral Blood Monocyte Chemotaxis by C1q
Human C1q at 25 µg/ml stimulates the chemotaxis of peripheral blood monocytes (Fig. 4B). Neither SP-A, SP-D, nor MBP had a significant effect on monocyte chemotaxis at the same concentration. Migration of peripheral blood monocytes through a membrane with 2-µm pores was significantly increased above the control (buffer only) value when 25 µg C1q/ml were added to the lower chamber (318 ± 43% of control migration; P < 0.05). Monocyte migration was not significantly affected by the addition of 25 µg/ml of SP-A (138 ± 23% of the control level), SP-D (124 ± 75% of the control level), or MBP (116 ± 16% of the control level) to the lower chamber.In addition, neither SP-A nor SP-D had a significant effect on
peripheral blood monocyte migration over a wide range of
concentrations. In these assays, migration of peripheral blood
monocytes in response to SP-A and SP-D was not significantly different
from control (no-protein) migration at lower well concentrations of 5 µg/ml (with data expressed as means ± SD, 90 ± 12% of
control migration for SP-A and 77 ± 21% for SP-D), 0.5 µg/ml (71 ± 2% for SP-A and 71 ± 17% for SP-D), and 5 ng/ml (84 ± 2% for SP-A and 71 ± 47% for SP-D), in contrast to a previously
published report (16). In these same experiments,
108 M fMLP stimulated
peripheral blood monocyte migration to an average of 249% of the
control level.
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DISCUSSION |
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Our major goal in this study was to examine the responsiveness of alveolar macrophages and peripheral blood monocytes to SP-A and its structural homologues SP-D, MBP, and C1q. In narrowing our experimental scope, we focused on the actin cytoskeleton. Not only is the polymerization of actin highly regulated, but changes in actin polymerization and distribution are both receptor-mediated events and processes necessary for many of the host defense functions of phagocytes. Our results show that SP-A and SP-D have specific effects on directional actin-based functions of alveolar macrophages, a conclusion consistent with the hypothesis that these cells express functional receptors for the pulmonary collectins not found on peripheral blood monocytes, although inconsistent with the hypothesis that SP-A and SP-D play a direct role in monocyte recruitment to the lung.
In this study, we have shown for the first time that SP-A and SP-D stimulate the polymerization of actin in alveolar macrophages and that this stimulation is in a highly organized and directional manner. Furthermore, we have shown that SP-D stimulates the chemotaxis of alveolar macrophages and that only SP-A and SP-D, among the proteins tested, are capable of this stimulation. Finally, we have shown that C1q stimulates monocyte but not alveolar macrophage chemotaxis, suggesting that the two cell types respond in fundamentally different manners to the structural relatives SP-A and C1q.
Receptor-Mediated, Actin-Based Processes Are Important in the Host Defense Functions of Mononuclear Phagocytes
The assembly of actin monomers into a diverse array of submembrane structures is controlled by signals that originate at plasma membrane receptors (reviewed in Ref. 44). Common actin-based extensions from motile, phagocytic immune cells include long, thin extensions (filopodia), thick, sheetlike protrusions (pseudopodia or lamellopodia), membrane ruffles, pleats, and microvilli. The precise regulation of these actin-based structures is critical to many of the functions of alveolar macrophages, including motility, which is necessary for the cells to move to areas of infection (reviewed in Ref. 13), and to the type of pseudopod-based phagocytosis that is stimulated by SP-A (T. Schagat, M. J. Tino, and J. R. Wright, unpublished observations) and IgG (reviewed in Ref. 1). Furthermore, receptor-initiated signals that activate small G proteins, such as members of the rac and rho families, are required not only for redistribution of actin in the cell but also for secretion of many cytokines involved in the regulation of immune function and production of oxidants required for pathogen killing (reviewed in Ref. 23).Evidence for the Presence of SP-A and SP-D Receptors on Alveolar Macrophages Not Found on Peripheral Blood Monocytes
In this study, we have shown that SP-A and SP-D stimulate specific actin-based processes in alveolar macrophages and not in peripheral blood monocytes. Although this could be explained by the fact that these cell types differ in the expression of several intracellular signaling molecules, another possible explanation for this is that these effects are mediated by a receptor(s) that is expressed only after the differentiation of peripheral blood monocytes into alveolar macrophages. These findings are supported by the results of several studies examining SP-A binding to alveolar macrophages (30, 33, 38) and peripheral blood monocytes (22) that have shown that several of the characteristics of SP-A binding (including saturability and specificity) differ between these two cell types.Several proteins have been identified as putative receptors for SP-A and SP-D. Recent studies have identified a 210-kDa SP-A binding protein (210-kDa SP-A receptor) (9) and a 340-kDa SP-D binding protein (glycoprotein-340) (24). In the former study, Chroneos et al. (9) identified the 210-kDa SP-A receptor, an SP-A binding protein found on a wide variety of cells including bone marrow-derived macrophages, U-937 cells, alveolar macrophages, and alveolar type II epithelial cells. Antibodies against this receptor have been shown to block both SP-A-dependent inhibition of phospholipid secretion in 12-O-tetradecanoylphorbol-13-acetate-stimulated type II cells (9) and uptake of bacillus Calmette-Guérin by bone marrow-derived macrophages (50). Although this protein appears to be involved in SP-A-mediated uptake events, its widespread distribution cannot explain the differences in functional responses between alveolar macrophages and blood monocytes seen here. The interesting possibility exists that different cell surface receptors for SP-A are responsible for chemotactic and phagocytic cell functions. In the latter study, Holmskov et al. (24) have identified glycoprotein-340 as an SP-D binding protein present on alveolar macrophages, although its functions remain unknown. Although it does not seem to be present on alveolar type II epithelial cells, it has not been determined whether it is present on other leukocytes (24).
SP-A and SP-D Have Alveolar Macrophage-Specific Effects
Because actin-based responses can take many forms, it is especially important that an immune cell be able to direct its cytoskeletal redistribution in a directed manner. The clearance of pathogenic organisms and particulate matter from the lung is dependent on a swift, directed response that includes migration to the site of infection or contamination, extension of filopodia and pseudopodia to capture and surround the particles, and retraction of the plasma membrane to internalize them (49).We have shown here that only SP-A and SP-D, among the proteins tested, induce a directional actin-based response in alveolar macrophages. Not only do they stimulate an increase in total F-actin in the cells, but on stimulation with SP-A or SP-D alveolar macrophages, they assume a shape characteristic of a moving, responsive cell (reviewed in Ref. 19), with long filopodia extending from one end and an actin-filled, ruffled membrane at the other (Fig. 1). In particular, this shape is consistent with that of a motile leukocyte responding to a chemoattractant as seen in several studies (8, 10, 37) with both fluorescence microscopy and electron microscopy. Conversely, C1q and MBP induce a nondirectional actin response in alveolar macrophages, characterized by the uniform extension of filopodia and lamellopodia around the periphery of the cells.
These results correlate well with studies of directed cell migration (chemotaxis). We have shown that SP-A and SP-D, but not C1q or MBP, stimulate chemotaxis in alveolar macrophages. Neither protein affects either the distribution of F-actin in peripheral blood monocytes or, over a wide range of concentrations, the migration of those cells, suggesting that the effects on alveolar macrophages are due to a receptor(s) expressed differentially on that cell type. Two published reports (16, 31) have demonstrated that SP-D is chemotactic for neutrophils, suggesting the possibility that SP-D receptor(s) are expressed on different cells as well; in this study, a maximal chemotactic affect was seen at 5 ng/ml, well below even the average serum SP-D concentration in normal humans, which Honda et al. (25) measured to be 66 ng/ml.
Our results differ significantly from those of Crouch et al. (16), who reported that SP-D is chemotactic for human mononuclear cell preparations. Potential explanations for the differences between our results and those of previous studies of SP-D stimulation of chemotaxis include our monitoring of proteins for the presence of endotoxin, as well as differences in the source (rats) and purity of our monocyte preparations. In the assays reported here, we used rat mononuclear cells and isolated rat SP-D; >50% of the cells in our mononuclear cell preparations were peripheral blood monocytes. In their study, Crouch et al. also used rat SP-D in their experiments, but in contrast to these studies, they used a total human mononuclear cell preparation and did not report the percentages of their cells that were monocytes and lymphocytes, cell types that might respond very differently in the chemotaxis assay.
A previous study (26) of the stimulation of actin polymerization in cells stimulated by chemoattractants such as fMLP and cAMP have shown effects on total F-actin levels in cells similar to the effects shown here (Table 1, Fig. 3). This polymerization of actin has been shown to be rapid and sustained; in Dictyostelium discoideum stimulated with cAMP, F-actin content increases significantly within 10 s of stimulation and stabilizes ~1.5-fold above unstimulated cells within 30 s of stimulation (18). Furthermore, Meyer and Howard (36) reported that differentiated HL-60 cells (a neutrophil-like promyelocytic leukemia cell line) stimulated with fMLP maintain levels of F-actin similar to those shown here, as well as high rates of locomotion, as long as 5 days after initial stimulation.
Role of C1q Receptors in the Stimulation of Cellular Functions by SP-A
To examine the possibility that SP-A stimulation is mediated by cell surface receptors for C1q, we examined the ability of C1q to stimulate chemotaxis and actin rearrangement in both cell types. C1q stimulated only nondirectional actin polymerization in both cell types and chemotaxis only in peripheral blood monocytes. These results are somewhat surprising given the observed correlation between directional actin polymerization and chemotaxis seen with chemoattractants such as SP-A, SP-D, and fMLP (37). They are also, however, inconsistent with the model of SP-A-cell interactions in which downstream effects of SP-A are mediated by a cell surface receptor that also binds C1q and that is present on both alveolar macrophages and peripheral blood monocytes.Our hypothesis does not exclude the possibility that several receptors, including receptors specific for the pulmonary collectins, exist, with various specificities for the collectins and C1q, and that these receptors are expressed on the many different types of cells on which these proteins have been found to have effects. If this were the case, it would be entirely possible that different receptors are responsible for the many functions shown to be mediated by this family of proteins, including such things as stimulation of immune functions such as phagocytosis in alveolar macrophages and peripheral blood monocytes (22, 34, 48) and chemotaxis in alveolar macrophages (53), activation of the phagocytosis of opsonized particles via the Fc and CR1 receptors on mononuclear phagocytes (45), mediation of cytokine production (35), mediation of lipid metabolism by alveolar epithelial type II cells (52, 54), stimulation of chemotaxis in neutrophils (16), and inhibition of T-lymphocyte proliferation (5).
SP-A and SP-D Stimulate Actin Polymerization at Physiologically Relevant Concentrations
We also set out to examine whether the actin polymerization and cell motility induced by SP-A and SP-D occurs at physiologically relevant concentrations. For chemotaxis to occur, cells must respond to a concentration gradient of protein that could extend to very low concentrations. Because of this, we tested a wide range of SP-A and SP-D concentrations for their ability to stimulate actin polymerization in alveolar macrophages.The exact ranges of SP-A and SP-D concentrations present in the alveolar epithelial lining are not known; estimates of their concentrations are averages based on levels in lavage fluid and do not account for the possibility that microenvironments could exist in which the local concentrations of protein are much higher (or lower) than the average. The average concentration of SP-A in rat pulmonary surfactant has been estimated to be 360 µg/ml (40). Estimates of the average concentration of SP-A in the epithelial lining fluid of normal human lungs are higher than this. Baughman et al. (3) calculated this concentration to be 831 µg SP-A/ml based on an estimated dilution factor of ~55-fold in their BAL technique. Although it has been found that 97-99% of SP-A exists bound to lipids (2) and it is possible that lipid-bound SP-A is unable to stimulate cellular processes, the average concentration of SP-A that exists free of lipids is calculated to be between 4 and 24 µg/ml.
On the basis of previously published estimates of the volume of hypophase fluid in the rat lung (40) and the total amount of SP-D estimated to be present there (15), the average SP-D concentration at the alveolar epithelial surface can be calculated to be ~3 µg/ml. More recent studies examining SP-D concentrations in the serum and BAL fluid of both normal humans and patients with various pulmonary diseases have led to increased estimates for the average SP-D concentrations. Honda et al. (25) found that serum levels of SP-D in normal humans averaged 66 ng/ml, with circulating SP-D levels reaching levels > 1 µg/ml in some patients with interstitial pneumonia with collagen disease, idiopathic pulmonary fibrosis, and AP. Furthermore, that study found that BAL fluid levels averaged 0.88 µg/ml. If a dilution factor of 55-fold in the lavage technique used (3) is assumed, the average SP-D concentration in the human lung lining fluid is then closer to 63 µg/ml. Although this estimate is likely to be quite high, SP-D levels even one-tenth this high are within the range of concentrations tested in our study.
We have found that both SP-A and SP-D stimulate actin polymerization at every concentration tested, as low as 0.5 µg SP-A/ml and 1 µg SP-D/ml (although this lowest concentration was significant only to a P value of 0.053). The concentrations of SP-A at which actin polymerization took place are also within the range of the concentration at which Pison et al. (38) found cell binding to be one-half of its maximum, 4 µg SP-A/ml. Furthermore, we have shown that higher concentrations of both proteins stimulate greater actin polymerization, a result consistent with the finding that both SP-A and SP-D are chemotactic for alveolar macrophages.
Examinations of the concentration dependence of chemotactic stimulation also show that the effects of SP-A and SP-D on alveolar macrophages occur at physiologically relevant concentrations. Both proteins stimulate significant cell migration at concentrations as low as 5 µg/ml. Lower concentrations, including those below even average serum SP-D concentrations and including 5 ng SP-D/ml, which has previously been shown to be chemotactic for neutrophils and mononuclear cell mixtures (16, 31), did not stimulate alveolar macrophage chemotaxis in our hands. We conclude from these findings that the stimulation of both actin polymerization and cell migration we have observed occurs at concentrations likely to be encountered by alveolar macrophages in the lung, especially in areas to which it is vital that macrophages be recruited, such as areas of localized infection.
Role of SP-A and SP-D in the Recruitment and Maturation of Mononuclear Phagocytes
It is not known exactly which stimuli are necessary for the recruitment of monocytes into the lung and for their subsequent maturation into alveolar macrophages, although they are likely to include such small chemoattractants as monocyte chemoattractant protein-1 (7) and macrophage inflammatory protein-1We have shown that both SP-A and SP-D are chemotactic for alveolar macrophages and that neither affects the migration of unstimulated peripheral blood monocytes at concentrations likely to be found in the lung. These results suggest that neither protein has a direct role in the recruitment of resting monocytes into the lung tissue. These findings do not, however, exclude a role for SP-A and SP-D in the maturation of monocytes into alveolar macrophages or in the recruitment of activated monocytes or, at least, partially matured tissue macrophages into the alveolar air space. In fact, the development of the capability to respond in specific manners to SP-A and SP-D might be an important step in the maturation of alveolar macrophages from their precursor cells and might play a role in determining which cells migrate from the interstitial lung tissue into the alveoli.
The findings presented here, along with the results of many previous studies, have begun to shape a picture of the cellular specificity of the effects of SP-A and SP-D. In particular, these studies have shown that the pulmonary collectins have specific effects on alveolar macrophages, the primary immune cells resident in the lung. Several effects of SP-A on alveolar macrophages, including the stimulation of phagocytosis (48) and, as shown here, the stimulation of directed actin polymerization and chemotaxis, appear to be independent of cell surface receptors that bind C1q. Furthermore, it remains to be seen whether a role in macrophage maturation or recruitment will be identified for SP-A and SP-D. To complete this picture, functional receptors for the collectins and C1q must be conclusively identified and in vitro studies must be correlated with in vivo findings in both normal and genetically manipulated animals.
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ACKNOWLEDGEMENTS |
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We thank Elizabeth Ramsburg, Angela Swyers, and Daniel Zlogar for the purification of surfactant protein (SP) A and rat SP-D and for conducting endotoxin assays on SP-A samples; Dr. Qun Dong for making the recombinant SP-D construct, for the production of transfected cells producing recombinant SP-D, and for the purification of SP-D from silica-treated rats; and Sabrena Mervin-Blake for the purification of mannose-binding protein and recombinant SP-D. We also thank Dr. James Fisher for his generous gift of the complementary deoxyribonucleic acid for rat SP-D; Dr. Arturo De Lozanne for help in finding and modifying the protocol for F-actin quantification; Dr. Don Burdick and Heidi Ashih (Duke University Institute for Statistics and Decision Sciences, Durham, NC) for consultation on statistical analysis; and Dr. Paul Borron, Dr. William Mariencheck, Jeff Baker, and Trista Schagat for critical readings of the manuscript in its various forms.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-51134.
Address for reprint requests: J. R. Wright, Box 3709, Duke Univ. Medical Center, Durham, NC 27710.
Received 1 December 1997; accepted in final form 7 October 1998.
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