INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

AT THE INITIATION of inflammation, neutrophils are recruited from the bloodstream into extravascular tissue. The presence of an inflammatory stimulus in the tissue, such as bacteria or bacterial LPS, results in the localized generation of cytokines, leukotrienes, and other proinflammatory mediators that enter the vasculature and stimulate the surrounding tissue, causing changes in surface molecule expression and potentiation of proinflammatory mediator production. Circulating neutrophils respond to these stimuli by altering surface molecule expression resulting in neutrophil adhesion to the surrounding vascular endothelium. Gradients of proinflammatory signals then stimulate the migration of these neutrophils across the vascular endothelium and into the adjacent infected tissue (reviewed in Refs. 20, 21).

The directed migration or chemotaxis of neutrophils is essential to execution of an inflammatory response. A variety of molecules are known to stimulate neutrophil chemotaxis (7). These include microbial products such as the bacterial peptide formyl met-leu-phe (fMLP), activated complement (C5a), cytokines such as IL-8 and macrophage inhibitory protein 2 (MIP-2), and bioactive lipids such as leukotriene B₄. All of these chemoattractants bind G protein-coupled receptors on the surface of neutrophils. Ligand binding of these receptors stimulates signaling events that result in cytoskeletal rearrangements and cell migration.

Surfactant protein A (SP-A) is a large multimeric protein found in the airways and alveoli of the lungs. SP-A is a member of the collectin protein family that is characterized by NH₂-terminal collagen-like domains and COOH-terminal lectin, or carbohydrate-binding domains. These proteins are innate immune molecules, and SP-A's best-characterized immunoregulatory interaction is with the resident pulmonary phagocyte, the alveolar macrophage (30). SP-A stimulates a variety of macrophage responses such as chemotaxis, actin polymerization, and phagocytosis (25, 30, 31). SP-A also regulates responses involved in the initiation and potentiation of inflammation by decreasing proinflammatory tumor necrosis factor- production in response to LPS (14) and by accelerating the clearance of apoptotic neutrophils during the resolution of inflammation (17). In this way, SP-A is believed to help protect the delicate pulmonary tissue from potentially damaging effects of inflammation. Studies with mice made deficient for SP-A by homologous recombination support this hypothesis; these mice show a decreased ability to clear a pulmonary bacterial challenge and a large and persistent pulmonary infiltration of neutrophils (12).

In this study, we investigated the ability of SP-A to regulate neutrophil chemotaxis and found that although SP-A did not directly stimulate chemotaxis, it did regulate neutrophil chemotaxis toward known chemoattractants. This regulation was, however, dependent on the activation state of the neutrophil; peripheral neutrophils showed decreased chemotaxis in the presence of SP-A, and neutrophils isolated from inflamed lungs showed increased chemotaxis in the presence of SP-A. We examined the ability of homologous proteins to mediate neutrophil chemotaxis and explored the mechanism by which SP-A is differentially regulating the migration of the two different neutrophil populations. This study provides evidence that SP-A indeed regulates neutrophil migration most likely via its collagen-like domain through some internal regulatory mechanism that remains to be identified.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. Hetastarch was produced by Abbott Laboratories (North Chicago, IL). DPBS, Gey's balanced salt solution (GBSS), and HBSS were obtained from Gibco Laboratories (Grand Island, NY). Iodo-Beads, desalting gel columns, and bicinchoninic acid protein assays were all obtained from Pierce (Rockford, IL). Na¹²⁵I was purchased from DuPont-NEN (Boston, MA). Human serum C1q was purchased from Advanced Research Technologies (San Diego, CA). IgG purified from normal rat serum was purchased from Sigma Chemical (St. Louis, MO). The IgG was reconstituted from lyophilized powder in saline and used without further treatment. All other chemicals used were from Sigma Chemical, unless otherwise indicated.

Animals. Male Sprague-Dawley rats weighing ~200-400 g were obtained from Charles River (Raleigh, NC) or Taconic Farms (Germantown, NY).

Protein purification. SP-A was purified from the bronchoalveolar lavage (BAL) fluid of patients with alveolar proteinosis as previously described (14). Briefly, SP-A was extracted from lavage fluid with butanol and sequential solubilization in octylglucoside and 5 mM Tris, pH 7.4. The SP-A was treated with polymyxin B agarose beads to reduce endotoxin contamination. All SP-A preparations had <0.1 pg endotoxin/µg SP-A by the Limulus amebocyte lysate assay QCL-1000 (BioWhittaker, Walkersville, MD). SP-A was stored in 5 mM Tris, pH 7.4, at 20°C. Heat treatment of SP-A was carried out at 95°C for 10 min.

Iodination of SP-A. Purified SP-A was iodinated using N-chloro-benzenesulfonamide oxidizing agent immobilized on Pierce Iodo-Beads (26). Free Na¹²⁵I was separated from ¹²⁵I-labeled protein on D-salt exocellulose GF-5 desalting gel columns. Fractions that contained radioactivity that was >85% precipitable by trichloroacetic acid were analyzed for protein concentrations by bicinchoninic acid protein assay and stored at 4°C. Radiolabeled SP-A was used within 2 wk.

Neutrophil isolation. Peripheral neutrophils were isolated as previously described with minor modifications (23, 29). Briefly, rats were anesthetized by intramuscular injection of 0.1 mg acepromazine, 7 mg ketamine, and 29 mg xylazine per 250 g body wt, and the jugular was cannulated with a 23-gauge butterfly needle attached to a three-way stop cock. An exchange of 3-4 ml of 6% hetastarch solution with 100 U/ml heparin for 3-4 ml of blood was performed until 40 ml were exchanged per rat. The first 10 ml of blood were collected in a syringe containing 10 ml of the hetastarch solution. After the exchange, blood was collected until the rats died. Blood was allowed to settle 30-45 min at room temperature, and the red blood cell-depleted fraction was collected and centrifuged at 330 g for 10 min. Cells were resuspended in 2 ml of DPBS and underlayed with a five-step Percoll gradient. Step densities were 1.081, 1.085, 1.089, 1.093, and 1.097. Gradients were centrifuged at 500 g for 30 min at room temperature, and the fractions at the interfaces of 1.085-1.089 and 1.089-1.093 density layers were pooled, washed, and resuspended in the appropriate buffer. Neutrophil purities were 83 ± 3% as determined by hematoxylin differential stain (EM Science, Gibbstown, NJ). Other cell types present were 7 ± 1% eosinophils and 10 ± 3% mononuclear cells. Cells were 98 ± 1% viable as determined by trypan blue exclusion.

Chemotaxis assay. Chemotaxis assays were performed using a Neuro Probe 48-well microchemotaxis chamber (Cabin John, MD) (31). Lower wells were filled with the chemotactic test solution diluted in chemotaxis buffer (GBSS + 2% BSA). Poretics polyvinylpyrrolidone-free polycarbonate filters with 2-µm pores (Osmonics, Livermore, CA) were used. The assembly was incubated at 37°C, 5% CO₂ for 10 min before cell suspensions (2.5 × 10⁶ cells/ml chemotaxis buffer) were added. After a 30-min incubation at 37°C, 5% CO₂, the filter was stained by hematoxylin differential stain and analyzed by light microscopy for the number of cells that had migrated through the filter. Ten randomly selected oil immersion fields were counted at ×100 magnification. Chemotactic stimuli were analyzed in triplicate and averaged for each experiment.

Phagocytosis assay. Fluorescein beads were conjugated with BSA according to the manufacturer's directions in the Polysciences Carbodiimide kit (Warrington, PA). Neutrophils at 10⁶ cells/ml were coincubated 1:10 with BSA-coupled beads in a final assay volume of 0.5 ml PBS + 0.1% BSA. Incubations were carried out in BSA-coated tubes (coated for 1 h at 37°C) for 30 min at 37°C in the presence and absence of 25 µg/ml SP-A. Cells were then washed three times and fixed with 1% formaldehyde before analysis by flow cytometry.

Iodinated SP-A binding assay. To measure binding of iodinated SP-A, neutrophils were suspended at 5 × 10⁶ cells/ml in binding buffer (DPBS with 0.1% BSA and either 0.9 mM CaCl₂ + 0.5 mM MgCl₂ or 1 mM EDTA as indicated). Assay tubes were precoated with 1% BSA overnight at 4°C. Iodinated SP-A was coincubated with 2 × 10⁶ cells in 0.5 ml of buffer for 4 h at 4°C with gentle rotation. To minimize nonspecific absorption of radioactivity to the tubes, cells were washed once with cold binding buffer lacking BSA and transferred to new tubes. Cells were then washed twice more and lysed (50 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40). All washes were done at 4°C. Incorporated radioactivity was analyzed by gamma counting, and radioactive signal was normalized to total cellular protein recovered (quantitated by bicinchoninic acid protein assay). Background was determined by performing the assay in the absence of cells and detectable background counts were subtracted from the samples with cells.

Quantitation of fMLP receptor expression. fMLP receptor expression was measured using the fluorescein-conjugated formyl peptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Molecular Probes, Eugene, OR) (1). Varying concentrations of peptide (1, 2, 5, 10, and 20 nM) were incubated with 2.5 × 10⁶ neutrophils/ml binding buffer (HBSS + 1% BSA + 0.1% azide) for 30 min on ice. Cells were then washed and resuspended in fixative (1% formaldehyde in DPBS) for analysis by flow cytometry. Cells were analyzed for the mean relative fluorescence as an indication of the relative quantity of formyl peptide bound. For measurement of the effect of SP-A on formyl peptide binding, 25 µg/ml SP-A were added to the binding reaction. For measurement of the effect of SP-A on fMLP receptor expression, cells were incubated in the chemotaxis buffer plus 25 µg/ml SP-A for 30 min at 37°C with gentle shaking. Cells were then washed with DPBS without calcium or magnesium + 1 mM EDTA + 1% BSA and resuspended in binding buffer, and formyl peptide binding was measured as described.

CD11b expression. Neutrophils at 2 × 10⁶ cells/ml HBSS + 0.1% BSA were incubated with FITC-conjugated CD11b (PharMingen, San Diego, CA) for 5 min at 37°C and then stimulated with SP-A (25 µg/ml), fMLP (0.1 mM), or SP-A + fMLP for 10 min at 37°C (3). Cells were immediately washed with 1 ml of cold HBSS + 0.1% BSA and suspended in fixative for analysis by flow cytometry.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SP-A does not directly stimulate neutrophil chemotaxis. In contrast to its effects on macrophage chemotaxis, SP-A did not directly stimulate peripheral or inflammatory BAL neutrophil migration above buffer alone (GBSS) at a range of concentrations tested (0.5-25 µg/ml) (Fig. 1). The bacterial peptide fMLP (10 nM) stimulated chemotaxis, and inflammatory BAL neutrophils were significantly more responsive to fMLP than peripheral neutrophils.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Effect of surfactant protein A (SP-A) on neutrophil chemotaxis. Rat peripheral and inflammatory bronchoalveolar lavage (BAL) neutrophils were allowed to migrate through a membrane with 2-µm pores toward buffer control [Gey's balanced salt solution (GBSS)], SP-A (0.5, 2, or 25 µg/ml), or formyl-met-leu-phe (fMLP; 10 nM) for 30 min. Membranes were then stained and scored for the average number of neutrophils that had migrated through the membrane per oil immersion field (OIF). Experiments were done in triplicate. Means ± SE; n = 3-10. * P < 0.005 vs. GBSS.

SP-A regulates neutrophil chemotaxis stimulated by chemoattractants. Although SP-A alone did not directly stimulate neutrophil migration, we analyzed its ability to affect neutrophil migration toward several different chemoattractants. We measured the effect of 25 µg/ml SP-A on neutrophil chemotaxis toward MIP-2, activated complement protein C3b in zymosan-activated serum (ZAS), and fMLP. SP-A significantly altered both peripheral and inflammatory BAL chemotaxis toward these chemoattractants. However, the effect was dependent on the activation state of the neutrophil. SP-A significantly inhibited peripheral neutrophil chemotaxis toward MIP-2 and fMLP and significantly enhanced inflammatory BAL chemotaxis toward each chemoattractant tested (Fig. 2). Significant inhibition of peripheral neutrophil chemotaxis toward ZAS was not observed at the concentration tested (5% ZAS) (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of SP-A on neutrophil chemotaxis toward different chemoattractants. Chemotaxis of rat peripheral and inflammatory BAL neutrophils toward buffer control (GBSS), macrophage inhibitory protein 2 (MIP-2; 50 ng/ml), zymosan-activated serum (ZAS; 5%), or fMLP (10 nM) in the absence and presence of SP-A (25 µg/ml) was measured for 30 min. Experiments were done in triplicate. Relative neutrophil chemotaxis = [(chemotaxis toward chemoattractant or control + SP-A)/(chemotaxis toward chemoattractant or control alone) × 100]. Means ± SE; n = 4-8. * P  0.006 vs. BAL neutrophil chemotaxis toward chemoattractant alone.  P  0.006 vs. peripheral neutrophil chemotaxis toward chemoattractant alone.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Effect of SP-A on human peripheral neutrophil chemotaxis. Chemotaxis of human peripheral neutrophils toward SP-A (25 µg/ml), IL-8 (1 nM), and SP-A + IL-8 was measured for 30 min and compared with neutrophil migration toward buffer alone. Experiments were done in triplicate. Data are expressed as % chemotaxis of migration toward buffer alone. Means ± SE; n = 3. * P < 0.04 vs. buffer.  P = 0.03 vs. IL-8.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of SP-A dose on neutrophil chemotaxis toward fMLP. Chemotaxis of peripheral and inflammatory BAL neutrophils toward 10 nM fMLP was measured in the presence of 0, 2, 10, 25, and 50 µg/ml SP-A for 30 min. Experiments were done in triplicate. Relative neutrophil chemotaxis = [(chemotaxis toward fMLP + SP-A)/(chemotaxis toward fMLP alone) × 100]. Means ± SE. BAL: n = 5; peripheral: 0 and 25 µg/ml, n = 7; and 2, 10, and 50 µg/ml, n = 3. * P < 0.005 vs. BAL neutrophil chemotaxis toward fMLP alone.  P < 0.005 vs. peripheral neutrophil chemotaxis toward fMLP alone.

SP-A regulates neutrophil phagocytosis. To explore whether SP-A may regulate another aspect of neutrophil function, neutrophil phagocytosis was examined. Peripheral and inflammatory BAL neutrophils were incubated with BSA-conjugated fluorescein beads in the presence and absence of 25 µg/ml SP-A. SP-A significantly reduced peripheral neutrophil phagocytosis to 70 ± 9% vs. control, whereas SP-A enhanced BAL neutrophil phagocytosis to 143 ± 35% vs. control (Fig. 5). Although enhancement of BAL neutrophil phagocytosis was not statistically significant, SP-A clearly had different effects on peripheral and BAL neutrophils.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   SP-A regulates neutrophil phagocytosis. Peripheral and inflammatory neutrophils were incubated 1:10 with fluorescein beads for 30 min in the absence and presence of 25 µg/ml SP-A. Cells were then washed, fixed, and analyzed by flow cytometery. Relative phagocytosis = (% neutrophil phagocytosis + SP-A)/(% neutrophil phagocytosis in the absence of SP-A). Means ± SE; n = 3-5. * P < 0.005 vs. phagocytosis in the absence of SP-A.

Specificity of SP-A's effect on neutrophil chemotaxis. To determine if SP-A's effect was specific, we examined the ability of other proteins to regulate neutrophil migration toward fMLP. IgG, which does not share any structural homology to SP-A but is a soluble immunoregulatory protein, did not alter peripheral or inflammatory BAL neutrophil chemotaxis toward fMLP (Table 1). MBL, a serum member of the collectin protein family, and complement protein C1q both share significant structural homology to SP-A. In contrast to SP-A, neither MBL nor C1q (at 25 µg/ml) significantly altered peripheral or inflammatory BAL neutrophil chemotaxis toward fMLP (Table 1). None of these proteins alone at 25 µg/ml significantly stimulated neutrophil chemotaxis (data not shown).

Binding of SP-A to peripheral vs. inflammatory BAL neutrophils. Because of the differential effect of SP-A on peripheral and inflammatory BAL neutrophil chemotaxis, we examined if there was a difference in binding of SP-A to the two neutrophil populations. Iodinated SP-A at 2, 5, and 10 µg/ml was incubated with the neutrophils for 4 h at 4°C. There was no apparent difference in binding of SP-A to peripheral and inflammatory BAL neutrophils (Fig. 6). Higher doses of SP-A were tested, but background levels of radioactivity (as assessed by radioactivity that pelleted in the absence of cells) became significant, suggesting that the iodinated protein was aggregating at concentrations equal to or greater than 25 µg/ml (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   SP-A binding to neutrophils. Iodinated SP-A (2, 5, 10 µg/ml) was incubated with peripheral and inflammatory BAL neutrophils for 4 h at 4°C. This incubation was carried out either in the presence of 0.9 mM CaCl2 + 0.5 mM MgCl2 (A) or in the presence of 1 mM EDTA (B). Cells were then washed, and the amount of radioactivity associated with the cells was determined and normalized to the total protein recovered. Data are expressed as ng SP-A recovered/µg total protein recovered. Means ± SE; n = 3-4.

Effect of SP-A on fMLP receptor expression. To determine if SP-A's ability to regulate neutrophil chemotaxis is due to an alteration in fMLP receptor expression, we measured receptor expression on the peripheral vs. inflammatory BAL neutrophils. Fluorescein-conjugated formyl peptide (formyl-Nle-Leu-Phe-Nle-Tyr-Lys) at 1, 2, 5, 10, and 20 nM was incubated with peripheral and inflammatory BAL neutrophils for 30 min on ice. Formyl peptide binding to neutrophils was dose dependent and saturated between 5 and 10 nM (Fig. 7). As has been reported for LPS-stimulated peripheral neutrophils (1), we found that fMLP receptor expression was greater for activated inflammatory BAL neutrophils than for peripheral neutrophils (Fig. 7). To determine if the presence of SP-A could alter peptide binding or receptor expression, we added SP-A to the binding reaction or pretreated the neutrophils with SP-A under the chemotaxis assay conditions (30 min at 37°C). Neither coincubation of the peptide with SP-A (Fig. 7) nor pretreatment of the neutrophils with SP-A (data not shown) altered formyl peptide binding to the peripheral or inflammatory BAL neutrophils.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Formylated peptide binding to neutrophils. Fluorescein-conjugated formylated peptide (0, 1, 2, 5, 10, and 20 nM) was incubated with peripheral and inflammatory BAL neutrophils for 30 min on ice in the absence or presence of 25 µg/ml SP-A. Cells were then washed and analyzed by flow cytometry. Data are expressed as the mean relative fluorescence of the labeled peptide that bound to the cells. Means ± SE; n = 3.

Effect of SP-A on fMLP-stimulated CD11b levels. Besides stimulating chemotaxis, fMLP also upregulates CD11b expression on the neutrophil surface by stimulating release of secondary and tertiary granules and secretory vesicles where this ₂-integrin is stored (4, 10). Consistent with the literature, we found that fMLP stimulated a significant increase in CD11b on the surface of peripheral neutrophils (Fig. 8). This was also observed for inflammatory BAL neutrophils, although the percent increase in levels was less in the BAL neutrophils than in the peripheral neutrophils (Fig. 8). This is likely due to the already high basal CD11b levels on the inflammatory BAL neutrophils (Fig. 8A). SP-A alone had no effect on levels of CD11b. However, SP-A subtly, yet significantly, reduced fMLP-stimulated CD11b levels in the peripheral neutrophils (Fig. 8B). SP-A had no significant effect on fMLP-stimulated CD11b expression on inflammatory BAL neutrophils.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of fMLP and SP-A on neutrophil CD11b expression. A: comparison of CD11b expression between peripheral and BAL neutrophils, either untreated (none) or treated with 10 nM fMLP. CD11b expression was detected by measuring FITC-anti-CD11b mAb binding. Data were collected by flow cytometry and expressed as mean relative fluorescence units (RFUs). B: relative effect of fMLP and SP-A treatment on neutrophil CD11b expression. Cells were incubated for 10 min before fixing at 37°C in the absence or presence of 25 µg/ml SP-A and/or 0.1 mM FMLP. Data are expressed as a percentage of fMLP-stimulated CD11b expression for each neutrophil population. Means ± SE; n = 4-5. * P < 0.005.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These studies describe for the first time SP-A's ability to alter neutrophil responsiveness to chemoattractants. We show that SP-A enhances chemotaxis of inflammatory BAL neutrophils toward fMLP, MIP-2, and ZAS, and SP-A reduces peripheral neutrophil chemotaxis toward fMLP and MIP-2. Because of the inherent limitations of in vitro chemotaxis assays (reviewed in Ref. 28), we also investigated the effects of SP-A on other neutrophil functions, including phagocytosis and expression of CD11b. Although the effects of SP-A were more pronounced for chemotaxis than for other functions, we found that SP-A differentially regulates a variety of peripheral and inflammatory neutrophil functions. These studies suggest that SP-A may play an important role in regulating neutrophil functions in the lung. It was surprising that SP-A did not directly stimulate neutrophil chemotaxis, as has been observed by alveolar macrophages. A comparison of SP-A binding to alveolar macrophages vs. neutrophils (Fig. 6) suggests that neutrophils have one-fifth the number of SP-A binding sites as has been reported for alveolar macrophages (15). This suggests that neutrophils may lack a receptor for SP-A chemotactic stimulation. One study reported the ability of SP-A to stimulate peripheral neutrophil chemotaxis (6). The ability of SP-A to stimulate in their assays may be due to subtle assay differences or differences in SP-A preparations. To rule out the possibility of the lack of effect being due to the species discrepancy between the SP-A and cells used, we also examined whether human SP-A could stimulate human peripheral neutrophil chemotaxis. No chemotaxis was observed at 5 µg/ml (data not shown) or 25 µg/ml SP-A (Fig. 3). The inability of SP-A to directly stimulate chemotaxis is supported by a study that reports that exogenous surfactant is unable to stimulate neutrophil chemotaxis (8). Importantly, it should be noted that the BAL neutrophils have been previously exposed to SP-A. Thus, any regulatory effects or the lack thereof that are a consequence of this exposure cannot be controlled for.

The finding that SP-A differentially regulates peripheral and inflammatory BAL neutrophil chemotaxis toward fMLP, MIP-2, and ZAS is consistent with previous reports that exudated cells differ in many respects from peripheral blood cells (5, 16, 18, 27). The findings are also consistent with previous reports that SP-A has differential effects on activated vs. resting macrophages (9, 22). Neutrophils are rarely found in the alveolar space of the noninflamed lung, but when neutrophils are recruited during inflammation, migration into the alveolar space and in contact with SP-A would give them the capacity to respond more rapidly to chemoattractants that are present during inflammation. A role for SP-A in regulating neutrophil migration is supported by data obtained from studies with SP-A knockout mice, which show that once pulmonary inflammation is initiated, there is a large persistent infiltration of neutrophils into the pulmonary space (12) in the lungs of the knockout mice. However, it is important to note that the increased neutrophil migration in these models may be due to factors other than the absence of SP-A, including differences in cytokine levels and regulation of production of oxidant species.

Extracellular matrix proteins have been reported to affect leukocyte trafficking. Fibrin degradation products have been implicated in stimulating neutrophil chemotaxis, and chemotaxis was enhanced by the presence of LPS (11). Also, collagen has been reported to stimulate neutrophil chemotaxis (19). However, it has been reported that extracellular matrix produced by cultured endothelial cells has no neutrophil chemotactic activity yet did inhibit neutrophil activation by fMLP (13).

It does not seem likely that SP-A is altering chemoattractant interactions at the neutrophil plasma membrane. FITC-formyl peptide Nle-Leu-Phe-Nle-Tyr-Lys is a marker for fMLP receptors (1), and the presence of SP-A did not affect peptide binding (Fig. 7). Also, when neutrophils were pretreated with SP-A under chemotaxis assay conditions and receptor expression was examined, there was no difference in peptide binding (data not shown). All of the attractants tested stimulate neutrophils through G protein-coupled receptors. A phenomenon of these receptors is desensitization. During desensitization, after the ligand binds the receptor, the receptor is internalized, which leads to a reduced ability of the cells to respond to subsequent chemoattractant stimulation (reviewed in Ref. 2). An exciting possible implication of the data reported here is that SP-A may be altering this desensitization process. Future studies may determine if this is the case.

Overall, these studies add to the growing body of data describing SP-A's immunoregulatory role in the pulmonary tissue. These data show that this regulation is not only dependent on the type of leukocyte examined but also the activation state of the leukocyte. The ability of SP-A to differentially regulate different leukocyte populations supports the model that SP-A plays a significant role in tipping the balance of inflammation in favor of the beneficial vs. the damaging effects. In this way, SP-A is helping protect the delicate pulmonary epithelium while facilitating pathogen clearance.