Introduction

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Pulmonary surfactant is a lipoprotein, synthesized and secreted by alveolar type II cells, which reduces the surface tension at the lung alveolar air-liquid interface (1). Surfactant is composed of phospholipids, cholesterol, and proteins; the most abundant surfactant-associated protein is surfactant protein A (SP-A) (1). The 34-36 kD SP-A protein is thought to facilitate the surface-tension-lowering properties of surfactant and to regulate surfactant phospholipid synthesis, secretion, and recycling (1). SP-A is a member of the collectin subgroup of C-type lectins and, therefore, plays a role in innate host defense mechanisms in the lung (2). Other collectins include mannose-binding protein, conglutinin, CL-43, and surfactant protein D (2).

Collectins are characterized by a collagen-like domain, and the ability to bind carbohydrates on the surface of bacterial and viral pathogens, and in this way act as an opsonin (2). They mimic C1q in the activation of the classical complement pathway and activate macrophages and other phagocytic cells (2). Mutations in one of the collectins, mannose-binding protein (MBP), have been associated with increased susceptibility to infectious disease (3). No diseases have been associated with genetic mutations in human SP-A, although differences in allelic frequencies have been reported for respiratory distress syndrome (4). Transgenic mice with deletions in the SP-A gene have normal lung function but are more susceptible to infection with group B Streptococcus pneumonia, Pseudomonas aeruginosa, and Mycoplasma pulmonis (5, 6). SP-A levels are reduced in bronchoalveolar lavage (BAL) fluid from patients with bacterial pneumonia and respiratory failure (7). SP-A levels are also reduced in BAL of patients with cystic fibrosis (CF) (8, 9).

SP-A binds endotoxin and may influence the phagocytosis of gram-negative bacteria in the lung (10). It enhances the phagocytosis of several lung pathogens, including Staphylococcus aureus, P. aeruginosa, Hemophilus influenza, Myobacterium tuberculosis, and Pneumocystis carinii (2). SP-A has been shown to act as an opsonin for bacterial and viral phagocytosis by binding to bacterial and viral sugars (2). SP-A has been shown to increase cytokine production in phagocytic cells, and increase neutrophil uptake and killing of lung pathogens (11, 12). Other investigators, however, have reported that SP-A can decrease cytokine production (13, 14). It has also been shown that the binding of SP-A to pathogens may inhibit their adherence to the airway epithelium (12).

P. aeruginosa is the most common infectious agent in CF, a genetic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and characterized by chronic infection of the pulmonary conducting airways (15). The CFTR gene encodes a chloride channel that is found on the apical plasma membrane of airway epithelial cells (15). Several investigators have reported the presence of high chloride concentrations, ranging from 100 mM to 182 mM, in the airway surface liquid (ASL) of patients with CF (16). Alternatively, other investigators have observed little or no differences in ASL chloride concentrations between patients with CF and normal control subjects (20, 21). Normal ranges of chloride concentration in ASL have been estimated at 96 mM to 125 mM (16). In the present study, we characterized and validated an in vitro model system for studying the effects of human SP-A on the uptake of an important human lung pathogen, P. aeruginosa, by a human macrophage-like cell line, THP-1 cells. We also describe the effects of temperature, SP-A concentration, lipopolysaccharide (LPS), mannose, and NaCl concentration on the ability of SP-A to facilitate the uptake of this human lung pathogen.

	Materials and Methods

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Isolation of SP-A

SP-A was isolated from human alveolar proteinosis material as previously described (22). Briefly, the alveolar proteinosis material was delipidated using isopropanol ether and 1-butanol extraction, precipitated with ethanol, centrifuged, and resuspended in 0.02 M K₂HPO₄ (pH 8.0), and then purified by Affigel-blue column chromatography (Bio-Rad, Hercules, CA). The purified SP-A was then dialyzed against distilled water for 48 h. The protein concentration was determined by the method of Lowry and coworkers (23). The purity of this protein was characterized by polyacrylamide gel electrophoresis followed by staining with Coomassie blue. The endotoxin content (endotoxin units, EU) of the purified SP-A was measured by a Limulus Amoebocyte Lysate test (Bio-Whittaker, Waltersville, MO). LPS was removed from 1 mg of SP-A using polymixin agarose (Sigma, St. Louis, MO). Briefly, the SP-A was mixed with a 1:1 vol of the polymixin agarose that had been resuspended in phosphate-buffered saline (PBS), pH 7.4. The mixture was incubated for 1 h at room temperature with constant mixing and then the agarose beads removed by centrifugation. The LPS content of the treated SP-A was measured as described above.

Growing and Labeling Bacteria

A frozen glycerol stock of P. aeruginosa, non-mucoid strain PAO1, was obtained from the laboratory of Dr. Peter Greenberg, Department of Microbiology, University of Iowa (24). A mucoid strain of P. aeruginosa was obtained from a clinical isolate (a kind gift of Dr. Paul McCray, Department of Pediatrics, University of Iowa). The bacteria were grown in 50 ml Terrific Broth overnight in an environmental shaker. Quantitation of the bacteria was performed by measuring optical density at 600 nm. Bacteria were labeled with fluorescein isothiocyanate (FITC; Sigma) by washing twice in 1 M sodium carbonate buffer, pH 9.5, and then incubating with 0.1 mg/ml FITC in sodium carbonate buffer at 37°C for 1 h. All washes of the bacteria were performed by resuspension in buffer at 4°C followed by centrifugation for 5 min at 1,000 × g. Bacteria were washed twice in 1× PBS, once in RPMI 1640 media (Life Technologies, Inc., Rockville, MD) containing 2.5% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), and then resuspended in either the same media or in a salt solution (10 mM HEPES, 1 mM glucose, 5 mM KCl, 1.2 mM MgSO₄, 1.2 mM calcium gluconate, 2.4 mM K₂HPO₄, 0.4 mM KH₂PO₄, pH 7.4), supplemented with 10, 50, 100, 150, or 200 mM NaCl as indicated, with or without 1 to 50 µg/ml SP-A, and incubated at 37°C for 30 min.

THP-1 Cells

THP-1 cells, a monocytic/macrophage cell line (gift of Dr. Lois Geist, Dept. of Internal Medicine, University of Iowa) were grown in suspension in T-75 flasks in RPMI media supplemented with 10% FBS and 1% antibiotic/antimycotic mixture (Life Technologies), and passed at a ratio of 1:2 approximately every 2 d (25). Cells were treated with 1 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h before use in the bacterial uptake assay. The PMA treatment was used to differentiate the cells to exhibit a more macrophage-like phenotype (25). Cells were counted using a hemocytometer and phase contrast microscope. All washes of the THP-1 cells were performed by resuspending the cells at 4°C, followed by centrifugation for 5 min at 1,000 × g. PMA-treated macrophages were washed twice in RPMI 1640 containing 2.5% FBS, resuspended in media or in the salt solution described above, with or without 1 to 50 µg/ml SP-A as indicated, and incubated at 37°C for 30 min.

Phagocytosis Assay

After pretreatments with or without SP-A, followed in some cases by washing, the THP-1 cells and bacteria were combined in a total volume of 1 ml and incubated at 37°C for 45 min on a rocker. Living bacteria were used for all experiments. In all experiments, 1 × 10⁶ THP-1 cells and 1 × 10⁸ bacteria were used per incubation condition, resulting in a ratio of 100 bacteria to one THP-1 cell. The bacterial uptake assay was stopped and the free bacteria removed by washing the THP-1 cells with ice-cold PBS, then centrifuging at 750 × g. Fluorescence from extracellular adherent bacteria was then quenched by resuspending the washed cells in trypan blue solution (200 µg/ml in PBS). After 5 min, the cells were then fixed in 1 ml paraformaldehyde (2%) in PBS. Half of the fixed cells were used for analysis of cell-associated fluorescence in a FACScan (FACS, fluorescence-activated cell sorter; Becton Dickinson Immunometry Systems, San Jose, CA) as described below, and the remaining cells were examined using a fluorescence microscope. Some cells and bacteria were preincubated in 50 µg/ml bovine serum albumin (BSA) or in 1 to 2 EU/ml of LPS alone (Bio-Whittaker) or 1 EU of LPS plus SP-A. In some experiments, mannose (0 to 250 mM final concentration) was added to the cell-bacteria mixture along with 25 µg/ml SP-A. Phagocytosis assays were performed in RPMI supplemented with 2.5% FBS. For the experiments in which the concentration of NaCl was varied, cells and bacteria were incubated in the salt solution described above. The various salt solutions were made iso-osmolar using mannitol (Sigma). For the experiments in which the NaCl concentration was varied, macrophages and bacteria were not preincubated in SP-A. Instead, the SP-A was added directly to the cells and bacteria in the salt solution. To evaluate the possible aggregation of SP-A in response to increasing salt concentrations, SP-A (25 µg/ml) was resuspended in buffer containing various amounts of NaCl as described above and incubated for 45 min at 37°C, then the absorbance at 300 nm was determined with a spectrophotometer. The effects of mannitol (80 to 260 mM) on SP-A aggregation were also evaluated.

Flow Cytometry

A flow cytometer (Becton Dickson Immunometry Systems) interfaced with a computer was used to measure intracellular fluorescence in the THP-1 cells. Light transmission data from cells passing through a laser beam generated by a single air-cooled, argon ion laser (488 nm excitation) were collected by a forward scatter detector (FSC), side scatter detector (SSC), and a fluorescent detector. FSC indicates cell size, and SSC indicates the granularity of each cell. Fluorescence data, which indicated internalized bacteria, were collected on a log scale, with green fluorescence measured at 530 ± 30 nm. Data from ~ 10,000 events (cells) per condition were collected and analyzed using CellQuest software (Becton Dickson Immunometry Systems).

Statistics

Experiments were repeated three to nine times. Data were normalized by making the control, i.e., cells incubated with bacteria in the absence of SP-A, equal to 100%. Results were calculated as the number of fluorescent cells or the mean fluorescence per cell compared with those parameters in the control cells. Data analysis was performed using Student's t test and one- or two-way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test (26). Data are expressed as mean ± standard error (SE). All statistical analyses were performed using Sigma-Stat software (Jandel Scientific, Chicago, IL).

	Results

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Effect of Temperature

To evaluate the effects of temperature on the uptake of P. aeruginosa by the THP-1 cells, we performed the phagocytosis assay at 4°C and 37°C. The data in Figure 1 represent the relative percentage of cells (compared with controls) that contained fluorescence. There was a slight increase in the number of bacteria phagocytosed at 37°C when compared with the 4°C condition, as shown by an increase in the number of fluorescent cells, but the difference was not significant (Figure 1, white bars). When the bacteria and THP-1 cells were incubated at 4°C in the presence of 10 µg/ml SP-A, there was no change in the number of fluorescent cells when compared with the number of fluorescent cells detected in the absence of SP-A (Figure 1). However, when the phagocytosis assay was performed at 37°C, there was a significant, threefold increase in the uptake of bacteria by THP-1 cells incubated in the presence of 10 µg/ml SP-A when compared with the uptake of bacteria in THP-1 cells incubated in the absence of SP-A (Figure 1).

View larger version (10K): [in this window] [in a new window]   Figure 1.   Effect of temperature on the uptake of P. aeruginosa by THP-1 cells. THP-1 cells were incubated with FITC-labeled P. aeruginosa at 4°C or 37°C. Data are normalized to the number of fluorescent cells in the 4°C control condition, which was made equal to 100%. Open bars represent the control condition and the filled bars indicate the SP-A condition. Asterisk indicates a significant difference (P < 0.05) when compared with controls. Data are the mean of three to nine different experiments.

Effect of Washing Off SP-A

To determine if the effect of SP-A on bacterial uptake is mediated via interactions with the bacteria and/or the THP-1 cells, we preincubated THP-1 cells and bacteria with or without 10 µg/ml SP-A for 30 min at 37°C. Preincubation of the cells and bacteria with SP-A, followed by mixing of the two components without washing (i.e., continued incubation in the presence of 10 µg/ml SP-A), resulted in an ~ twofold increase (P < 0.05) in the uptake of bacteria by THP-1 cells when compared with the uptake of bacteria in the absence of SP-A (Figure 2). Preincubation of THP-1 cells and bacteria with 10 µg/ml SP-A, followed by washing both before they were mixed, resulted in no increase in bacterial uptake when compared with controls (Figure 2). When the bacteria alone were preincubated with 10 µg/ml SP-A, then washed, and mixed with untreated THP-1 cells, there was no significant increase in uptake when compared with the control condition (Figure 2). When the THP-1 cells alone were preincubated with 10 µg/ml SP-A, washed, then mixed with untreated bacteria, there was again no significant increase in bacterial uptake when compared with the control condition (Figure 2). Thus, the continued presence of SP-A during the uptake assay was necessary for enhanced phagocytosis.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Effect of incubation of bacteria and/or THP-1 cells with SP-A, followed by washing, on the uptake of FITC-labeled P. aeruginosa by THP-1 cells. THP-1 cells and P. aeruginosa were preincubated with or without SP-A at 37°C as indicated. In some cases, the SP-A-preincubated bacteria and/or THP-1 cells were washed prior to mixing. Asterisk indicates a significant difference (P < 0.05) when compared with controls (no SP-A, open bar). Data represented are the mean of 3 to 12 different experiments.

Photomicrographs of cells incubated with fluorescent bacteria in the presence or absence of SP-A are shown in Figure 3. Only a few phagocytosed bacteria were observed in THP-1 cells incubated at 4°C in the absence of SP-A (Figure 3, panels A and B), and slightly more phagocytosed bacteria were detected in cells incubated at 37°C in the absence of SP-A (Figure 3, panels C and D). Upon the addition of 5 µg/ml SP-A, many more cells containing fluorescent bacteria were observed (Figure 3, panels E and F ), and a further increase in the proportion of fluorescent cells occurred in the presence of 10 µg/ml SP-A (Figure 3, panels G and H). Representative FACS profiles are depicted in Figure 4. There was an increase in the proportion of fluorescent THP-1 cells detected by FACS in the cell population that was co-incubated with fluorescent bacteria in the presence of 10 µg/ml SP-A at 37°C (Figure 4, panel B) when compared with control cells co-incubated with the bacteria in the absence of SP-A (Figure 4, panel A).

View larger version (70K): [in this window] [in a new window]   Figure 3.   Photomicrographs illustrating the phagocytosis of FITC-labeled P. aeruginosa by THP-1 cells. Panels A, C, E, and G: Nomarski micrographs of THP-1 cells with corresponding fluorescence photomicrographs; Panels B, D, F, and H: ingested fluorescent bacteria. THP-1 cells ingested very few fluorescent bacteria at 4°C when incubated in the absence of SP-A (Panels A and B). There was a slight increase in bacterial uptake when THP-1 cells were incubated at 37°C in the absence of SP-A (Panels C and D). Addition of 5 µg/ml SP-A increased phagocytosis of bacteria by THP-1 cells incubated at 37°C (Panels E and F ). There was a further increase in phagocytosis of fluorescent bacteria by THP-1 cells incubated in the presence of 10 µg/ml SP-A at 37°C (Panels G and H). The bar at the lower right corner indicates 25 µm.

View larger version (30K): [in this window] [in a new window]   Figure 4.   Representative FACS profiles documenting fluorescent bacterial uptake by THP-1 cells. Panel A: FACS profile of bacteria and THP-1 cells coincubated in the absence of SP-A. Panel B: FACS profile of bacteria and THP-1 cells coincubated in the presence of 10 µg/ml SP-A. Panels A and B, left: representative dot-plot of FITC fluorescence (y-axis) versus FSC (a measure of cell size, x-axis). Panels A and B, right: number of cells (y-axis) versus level of FITC fluorescence (x-axis). The gate (M1) was set such that the fluorescence of the cells to the left of the gate is considered background fluorescence. The data show increased bacterial uptake by THP-1 cells in the presence of SP-A.

Dose-Dependent Effects of SP-A on Bacterial Uptake

To compare the effects of different concentrations of SP-A on the uptake of P. aeruginosa by THP-1 cells, we performed phagocytosis assays on cells and bacteria using 1 to 50 µg/ml SP-A. We observed a dose-dependent increase in the uptake of bacteria by THP-1 cells incubated in the presence of SP-A (Figure 5A). There was a ~ 1.5-fold increase in the number of fluorescent cells in the 5 µg/ml SP-A condition when compared with controls; the effect was not statistically significant (Figure 5A). However, at 10, 25, and 50 µg/ml, SP-A caused a significant increase (1.7- to 3.5-fold, P < 0.05) in the number of fluorescent THP-1 cells (Figure 5A). We also observed that along with an increase in the number of cells ingesting bacteria, there was a similar increase in the number of bacteria phagocytosed by the THP-1 cells. This is shown in Figure 5B as an increase in mean fluorescence per cell with increasing amounts of SP-A. There was a significant increase in the mean bacterial fluorescence per cell upon the addition of 10, 25, and 50 µg/ml SP-A. One microgram per milliliter and 5 µg/ml SP-A did not increase the mean fluorescence per cell. Because albumin is a major protein component in the alveolar surface lining fluid, some cells and bacteria were preincubated with 50 µg/ml BSA instead of SP-A. BSA had no effect on the uptake of bacteria by THP-1 cells when compared with control cells incubated without SP-A (Figure 5, A and B).

View larger version (15K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 5.   The dose-dependent effects of SP-A on the uptake of P. aeruginosa by THP-1 cells. THP-1 cells and FITC-labeled P. aeruginosa were preincubated with or without various concentrations of SP-A or BSA (50 µg/ml) at 37°C, as indicated. Panel A: there was a dose-dependent effect of SP-A to increase the number of cells that contained fluorescent bacteria, with significant increases at 10, 25, and 50 µg/ml SP-A. 50 µg/ml BSA did not have an effect on the uptake of bacteria by THP-1 cells. Asterisk indicates a significant difference (P < 0.05) when compared with control cells (no SP-A, open bar). Data represented are the mean of three to nine different experiments. Panel B: there was a dose-dependent increase in the mean fluorescence (i.e., number of ingested fluorescent bacteria) per cell with increasing amounts of SP-A, with significant increases observed at 10, 25, and 50 µg/ml SP-A. 50 µg/ml BSA did not have an effect on the number of bacteria ingested by THP-1 cells. Asterisk indicates a significant difference (P < 0.05) when compared with control cells (no SP-A, open bar). Data represented are the mean of three to nine different experiments.

SP-A binds LPS (10). We determined that the LPS content of the purified human SP-A used in our experiments was ~ 2.2 pg/µg SP-A protein. Therefore, at 50 µg/ml SP-A, there is ~ 1.2 EU/ml LPS associated with the SP-A. We incubated the cells and bacteria in 1 and 2 EU/ml LPS to control for any nonspecific uptake of bacteria caused by the LPS present in our SP-A protein preparation. There was no effect of LPS on the uptake of bacteria by the THP-1 cells in the absence of SP-A (Figure 6A). The addition of 1 EU LPS to the SP-A had no effect on SP-A-mediated uptake of bacteria (Figure 6A). In addition, the removal of LPS from the SP-A with polymixin agarose had no significant effect on its biologic activity (Figure 6A). The LPS content of the polymixin-treated SP-A was ~ 90% less than that of the untreated SP-A. SP-A binds mannose via its lectin domain (2). Therefore, we also examined the effects of mannose (0 to 250 mM final concentration) added to SP-A (25 µg/ml) to determine if SP-A was binding to the bacteria via its carbohydrate recognition domain, as has been previously suggested (2). Mannose blocked the stimulatory effects of SP-A on the uptake of bacteria by THP-1 cells in a dose-dependent manner, with significant effects at the 100 and 250 mM concentrations (P < 0.05, Figure 6B).

View larger version (18K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 6.   The effects of LPS or mannose on the SP-A-mediated uptake of P. aeruginosa by THP-1 cells. Panel A: SP-A alone, SP-A with 1 EU of LPS added, and SP-A with the LPS removed all mediated a significant increase in the uptake of fluorescent bacteria when compared with the control condition. There was no significant difference observed between the SP-A alone, SP-A with 1 EU of LPS added, and SP-A with LPS removed groups. LPS, added alone at concentrations of 1 and 2 EU, had no effect on the uptake of the fluorescent bacteria in the absence of SP-A. Data were obtained from three independent experiments. Panel B: there was a dose-dependent effect of mannose to decrease the amount of fluorescent bacteria taken up by the THP-1 cells in the presence of 25 µg/ml SP-A (ANOVA, P < 0.05). Mannose at concentrations between 10 and 50 mM did not significantly affect the number of fluorescent cells. However, at the highest concentrations tested (100 and 250 mM), there was a significant decrease in the uptake of fluorescent bacteria. Data were obtained from three independent experiments.

Effect of Salt on Uptake

To describe the effects of different NaCl concentrations on the ability of SP-A to increase the uptake of bacteria by THP-1 cells, we varied the NaCl concentration of a HEPES-phosphate buffer by adding either 10, 50, 100, 150, or 200 mM NaCl to the buffer. Osmolality was kept constant by adjustment with mannitol. The cells and bacteria were incubated in the various solutions with or without 25 µg/ml SP-A (Figure 7). At 10 mM NaCl, SP-A had no effect on the uptake of bacteria by THP-1 cells, when compared with controls incubated without added SP-A. Incubation with 50 mM NaCl increased the stimulatory effect of SP-A on phagocytosis, but the effect was not significant. There was an ~ twofold increase (P < 0.05) in the effect of SP-A on bacterial uptake in the presence of 100 and 150 mM NaCl when compared with controls incubated in the same NaCl concentrations without added SP-A. SP-A in the presence of 200 mM NaCl did not have an effect on bacterial uptake when compared with controls incubated in the absence of SP-A. In three independent experiments, bacterial growth assays revealed that a 45-min exposure to the different NaCl concentrations had no effect on bacterial viability (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 7.   Effect of NaCl concentration on the phagocytosis of FITC-labeled P. aeruginosa by THP-1 cells. THP-1 cells and fluorescent bacteria were co-incubated in a HEPES-phosphate buffer with varying amounts of NaCl, as indicated, with or without 25 µg/ml SP-A. There was no effect of SP-A on the uptake of bacteria in the presence of low NaCl concentrations (10 mM, 50 mM) and at a relatively high concentration (200 mM NaCl). However, there was a significant effect of SP-A to increase bacterial uptake when the cells were incubated in 100 mM and 150 mM NaCl. Asterisk indicates a significant difference (P < 0.05) when compared with corresponding controls (no SP-A, open bars). Data are the mean of three to six different experiments.

Haagsman and coworkers reported that increased aggregation of human SP-A occurs at pH 7.0 at 25°C as a function of NaCl concentration, but that minimal SP-A aggregation occurs at 37°C (27). We evaluated the aggregation of human SP-A (25 µg/ml) at 37°C at NaCl concentrations from 0 to 200 mM, the range used in our studies. We observed no evidence of SP-A aggregation in three independent experiments (data not shown). Mannitol, likewise, had no effect on SP-A aggregation (data not shown).

Effect on Mucoid versus Nonmucoid Strains of Pseudomonas

Mucoid strains of P. aeruginosa are more commonly found in patients with CF than nonmucoid strains (15). In three experiments, we found that SP-A (25 µg/ml) increased the uptake of mucoid and nonmucoid strains of P. aeruginosa equally well (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study we describe a model for studying the effects of human SP-A on the uptake of a common pulmonary pathogen, P. aeruginosa, by a human monocytic/macrophage cell line, THP-1 cells. The results of FACS and fluorescent microscopy indicate that human alveolar proteinosis SP-A enhances the phagocytosis of the bacteria by these cells.

THP-1 cells have been used as a model for the human macrophage in a number of studies of phagocytosis because they are easily available and more homogeneous than freshly isolated alveolar or peritoneal macrophages. They can be treated with either vitamin D or phorbol ester to differentiate from a monocyte-like phenotype to cells which exhibit macrophage-like properties (25). Kremlev and colleagues used this cell line to study the regulation of cytokine production (TNF- and IL-1) by SP-A and showed that the differentiated THP-1 cells respond to SP-A in a manner similar to human alveolar macrophages (11). In addition, Stokes and coworkers have reported that the uptake of Mycobacterium tuberculosis by monocyte-derived macrophages and differentiated THP-1 cells is similar (25).

Our studies indicate that human alveolar proteinosis SP-A increases the uptake of P. aeruginosa by THP-1 cells. This SP-A-mediated increase in phagocytosis is temperature-dependent because an SP-A-mediated increase in bacterial uptake was observed only at 37°C and not at 4°C. We also observed a dose-dependent increase in the uptake of bacteria with increasing concentration of SP-A. This included an increase in the number of THP-1 cells phagocytosing the bacteria, as well as an increase in the number of bacteria ingested per cell. SP-A may weakly bind P. aeruginosa and THP-1 cells because it was easily washed off, and the continued presence of SP-A during the phagocytosis assay was essential for SP-A-enhanced phagocytosis.

Studies in SP-A knockout mice infected with P. aeruginosa revealed that these mice had higher bacterial loads, fewer phagocytosed bacteria in alveolar macrophages, and an increased inflammatory response when compared with wild-type mice (5). A study in 1992 by Manz-Keinke and colleagues showed that SP-A enhances serum-independent phagocytosis of P. aeruginosa by rat alveolar macrophages (28). It has been reported that the effect of SP-A on macrophage uptake of P. aeruginosa occurs via an effect of SP-A on the macrophages and not via an effect on the bacteria (28, 29). This difference in observations from our results may be the result of the use of rat alveolar macrophages in the cited studies versus the use of a human cell line in our studies. In any case, experiments in which the SP-A is present during bacterial contact with phagocytic cells most closely resembles conditions that may occur in vivo. Under these conditions, we obtained a highly reproducible enhancement of bacterial uptake by THP-1 cells in the presence of SP-A.

It has been reported previously that human SP-A does not bind, aggregate, or enhance the phagocytosis of P. aeruginosa (30, 31). In these studies, both mucoid and nonmucoid strains of bacteria were used; however, the bacteria were heat-killed in both cases (30, 31). When the P. aeruginosa were not heat-killed, in a subsequent study, human SP-A enhanced the phagocytosis of a live, mucoid strain by rat alveolar macrophages (32). The investigators in this latter study also reported that preincubation of the mucoid strain with human SP-A, followed by washing, resulted in increased phagocytosis by the rat alveolar macrophages.

SP-A is thought to facilitate the uptake of bacteria via its carbohydrate recognition domain (2). Mariencheck and coworkers observed a decrease in the binding of SP-A to P. aeruginosa in the presence of 100 mM mannose (32). In the present study, we also observed an effect of mannose on the SP-A-mediated uptake of P. aeruginosa by THP-1 cells. These results suggest that the lectin domain of the SP-A molecule is involved in phagocytosis of these bacteria. We also observed no increase in bacterial uptake by THP-1 cells in the presence of either BSA or LPS. These results suggest that the stimulation of bacterial uptake is specific to the SP-A protein itself, because another lung alveolar protein had no effect and the LPS content of the SP-A could not account for our results.

An intriguing finding in our study was that the SP-A- mediated increase in uptake of P. aeruginosa by THP-1 cells occurred in the presence of relatively high NaCl concentrations. Our initial studies concerning the effects of SP-A on bacterial uptake by THP-1 cells were performed in RPMI 1640, a medium which contains ~ 100 mM NaCl. It has been previously reported that high concentrations of chloride ions in ASL inhibit the activity of antimicrobial factors such as lysozyme and -defensins (33, 34). Some investigators have reported that normal ASL has a relatively low NaCl concentration (~ 60 to 80 mM) and that the ASL in patients with CF has elevated sodium and chloride concentrations (~ 100  to 150 mM) (16). However, other investigators have not observed a difference in sodium and chloride concentrations in ASL of CF vs. non-CF patients (20, 21). A recent study showed that high Cl levels compromise neutrophil bactericidal function against P. aeruginosa (35). Yet another report suggests that ionic composition does not effect neutrophil killing of P. aeruginosa (36). Travis and colleagues have described the antimicrobial activity of several factors including lysozyme, lactoferrin, and secretory leukoproteinase inhibitor 1 (SLP1) in killing pathogens found in cystic fibrosis patients, including P. aeruginosa (24). These investigators examined the effects of high sodium chloride concentrations (150 mM) on the antimicrobial function of BAL fluids, nasal lavage fluids, and the antimicrobial factors described above. They consistently observed a decrease in bacterial killing in the presence of high sodium chloride concentrations (24). Our study with SP-A resulted in the opposite finding, indicating that in a case where other antimicrobial factors lose their ability to function in host defense, SP-A may be even more important in host defense against pathogens. To our knowledge, this is the first report of the effect of sodium chloride concentration on the antimicrobial function of SP-A. SP-A levels may prove to be of clinical relevance in immunocompromised CF lungs, which are known to have low levels of SP-A (8, 9).