Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages

Judy M. Hickman-Davis1, J. Russell Lindsey1, S. Zhu2, and S. Matalon2,3,4

Departments of 1 Comparative Medicine, 2 Anesthesiology, 3 Physiology and Biophysics, and 4 Pediatrics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mycoplasma pneumoniae is a leading cause of pneumonia and exacerbates other respiratory diseases in humans. We investigated the potential role of surfactant protein (SP) A in antimycoplasmal defense using alveolar macrophages (AMs) from C57BL/6NCr (C57BL) mice, which are highly resistant to infections of Mycoplasma pulmonis. C57BL AMs, activated with interferon (IFN)-gamma and incubated with SP-A (25 µg/ml) at 37°C, produced significant amounts of nitric oxide (· NO; nitrate and nitrite production = 1.1 µM · h-1 · 105 AMs-1) and effected an 83% decrease in mycoplasma colony-forming units (CFUs) by 6 h postinfection. Preincubation of AMs with the inducible nitric oxide synthase inhibitor NG-monomethyl-L-arginine abolished · NO production and SP-A-mediated killing of mycoplasmas. No decrease in CFUs was seen when IFN-gamma -activated macrophages were infected with mycoplasmas in the absence of SP-A despite significant · NO production (nitrate and nitrite production = 0.6 µM · h-1 · 105 AMs-1). These results demonstrate that SP-A mediates killing of mycoplasmas by AMs, possibly through an · NO-dependent mechanism.

nitric oxide; peroxynitrite; pulmonary defenses; Mycoplasma pulmonis; opsonophagocytosis

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

AS A LEADING CAUSE of pneumonia, Mycoplasma pneumoniae accounts for up to 30% of all pneumonias in the general population (25) and exacerbates other respiratory conditions such as asthma (18) and chronic obstructive pulmonary disease (33). The mechanisms of intrapulmonary defense against mycoplasmas are poorly understood, but current evidence suggests that innate immune mechanisms have the major role in early antimycoplasmal defenses and that humoral immunity is particularly important in later stages of infection (8, 14, 16, 27).

M. pulmonis infection in mice provides an excellent animal model that reproduces the essential features of human respiratory mycoplasmosis (25). Mouse strains differ markedly in resistance to M. pulmonis (10), with C57BL/6NCr (C57BL) and C3H/HeNCr mice representing the extremes in response to this infection (10). Infection in C57BL mice is the best characterized animal model of resistance to mycoplasmal disease (3, 4, 10, 15, 37). C57BL mice have a 100-fold higher 50% lethal dose, 50% pneumonia dose, and 50% microscopic-lesion dose than C3H/HeNCr mice (10). Mechanical clearance of mycoplasmas does not differ between the two strains of mice (37). Significant intrapulmonary killing of M. pulmonis occurs in resistant C57BL mice within 4 h postinfection, although cellular infiltrates and specific antibodies do not appear until after 72 h (4, 8, 10, 37). Freshly isolated alveolar macrophages (AMs) from naive C57BL mice are unable to kill mycoplasmas when exposed to organisms in vitro, but the addition of concentrated lavage fluid from infected mice results in AM killing of mycoplasmas as early as 4 h postinfection (8), suggesting that this in vivo preconditioned lavage fluid contains nonspecific opsonin(s) and activation factor(s) that enable the AMs to kill the organism (8).

Differentiated AMs occupy an environment rich in phospholipids and surfactant protein (SP) A and SP-D, which have important roles in host defense (12, 28, 39, 47-49). SP-A is a 650-kDa glycoprotein that is produced by type II pneumocytes and Clara cells and is the most abundant protein in pulmonary surfactant (45). SP-A has been shown to bind to AMs in a highly specific manner (5, 28, 40, 45), effect the release of reactive oxygen species from AMs (48), stimulate chemotaxis of AMs (49), and enhance phagocytosis and killing of bacterial pathogens by AMs (29, 45, 47). We investigated the role of SP-A in mycoplasmal killing by AMs. Our results demonstrate that activated AMs kill mycoplasmas by a temperature-sensitive nitric oxide (· NO)-mediated mechanism and that binding of SP-A to AMs is a necessary step in this process.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Media and Chemicals

Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM) with L-arginine and 4.5 g/l glucose, and Hanks' balanced salt solution plus (HBSS+) containing Ca2+ and Mg2+ were from Cellgro (Atlanta, GA). Saline was obtained from Abbott Laboratories (Abbott Park, IL). HBSS (without Ca2+ and Mg2+), horse serum (HS), and fetal bovine serum (FBS) were from Life Technologies (GIBCO BRL, Grand Island, NY). BBL mycoplasma broth base was obtained from Becton Dickinson (Microbiology Systems, Cockeysville, MD). Diff Quik stain kits were obtained from Baxter Healthcare (McGaw Park, IL). Unless specified, all other chemicals were from Sigma (St. Louis, MO). Rabbit anti-human SP-A was a kind gift from Dr. D. S. Phelps (Pennsylvania State University, Hershey, PA).

Endotoxin Testing of Solutions and Reagents

All purchased solutions were certified endotoxin free (endotoxin content <0.5 endotoxin units/ml). HS and FBS were certified to contain <5% gamma -globulin and to be free of mycoplasmas, equine infectious anemia virus (HS), bacteriophages (FBS), and endotoxin. All sera were heat inactivated at 55°C for 30 min before use. Each lot of SP-A was tested with an amebocyte lysate assay at the University of Alabama at Birmingham Media Preparation Shared Facility and found to contain <= 2 pg (0.01 endotoxin units/ml) of detectable endotoxin at final cell culture concentrations.

Purification of Human Alveolar Proteinosis SP-A

SP-A was purified under sterile conditions from the bronchoalveolar lavage (BAL) fluid of patients with alveolar proteinosis using the n-butanol extraction method as previously described (21). Polyacrylamide gel electrophoresis and Western blot analysis of SP-A were carried out to ensure the purity of the SP-A preparations (22). The results indicate that SP-A was free from any significant contaminants such as albumin or immunoglobulins. SP-A was stored at -20°C in 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4. Aliquots were cultured for aerobic bacteria in BBL brain heart infusion broth (Becton Dickinson), and only culture-negative SP-A aliquots were used in experiments.

Animals

Pathogen-free 8- to 12-wk-old C57BL mice were obtained from the Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD. Mice were subsequently maintained in autoclaved Microisolator cages (Lab Products, Maywood, NJ) and provided with food (Agway, Syracuse, NY) and water ad libitum. Mice were monitored at University of Alabama at Birmingham and found to be negative for the presence of murine pathogens (15). For euthanasia, mice were anesthetized by intramuscular injection with ketamine (8.7 mg/100 g body wt; Aveco, Fort Dodge, IA) and xylazine (1.3 mg/100 g body wt; Haver, Shawnee, KS) and then exsanguinated by transection of the brachial artery.

Macrophage Isolation

BAL fluids were collected as described previously (8). Briefly, mice were anesthetized, and the proximal trachea was exposed surgically. A sterile 19-gauge intravenous catheter (Deseret Medical, Becton Dickinson) was inserted through the wall 5 mm into the lumen of the trachea. The lungs were lavaged in situ with four separate 1-ml washes of sterile saline. The lavagates from animals in each experiment were pooled and centrifuged to pellet the cellular fraction. Cells were resuspended in DMEM-BAH [DMEM containing 2% of an 8% solution of bovine serum albumin (BSA) and 2.5 g/l HEPES, pH 7.4] and counted using a hemacytometer and trypan blue, and 1 × 105 viable cells were aliquoted into sterile 12 × 45-mm glass vials or onto round 12-mm coverslips to be used for SP-A binding studies. Cells were >90% viable by trypan blue exclusion and >95% macrophages, as differentiated on cytospin preps using Diff Quik stain. Macrophage function was verified by phagocytosis of 0.41-µm latex beads (Serdyn Particle Technology Division, Indianapolis, IN) (8, 9).

Mycoplasmas

The virulent UAB CT strain of M. pulmonis was used in all experiments (7). Mycoplasmas were incubated at 37°C for 18 h before use to ensure that they were actively growing in the log phase. Mycoplasmas were washed three times in HBSS+ containing 0.1% BSA before addition to AM cultures. Mycoplasmas were tested for their ability to grow in this medium and found to be viable for up to 24 h without the addition of fresh medium. Colony-forming units (CFUs) in each inoculum were confirmed by enumeration after standard dilution, inoculation of agar plates, and incubation for 7 days at 37°C in room air with 95% humidity (9).

AM Binding of SP-A

Enzyme-linked immunosorbent assay. AMs (1 × 105 cells) were adhered to glass vials for 30 min at 37°C, and nonadherent cells were removed by washing. AMs were resuspended in HBSS+ containing 0.1% BSA and incubated with 0-25 µg/ml concentrations of SP-A at 37 or 4°C for 30 min or at 4°C for 2 h. AMs were washed three times with HBSS to remove excess BSA and media, lysed by sonication, and resuspended in 200 µl of sterile water. Concentrations of AM-associated SP-A were determined by indirect enzyme-linked immunosorbent assay (ELISA) using rabbit anti-human SP-A as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) as the secondary antibody. SP-A was quantified using a standard curve generated with SP-A diluted to 80 ng/ml applied to Immulon 2 ELISA plates (Dynatech Laboratories, Chantilly, VA), serially diluted, and allowed to bind for 1 h at 37°C. After the wells were blocked with BSA, they were incubated with each antibody for 1 h at 37°C. The plates were washed, the peroxidase reaction product was generated using O-phenylenediamine dihydrochloride (2.2 mM) as substrate, and optical density was measured at 490 nm (22, 42). Endogenous cellular SP-A levels were measured on samples receiving only HEPES buffer, pH 7.4. Total protein content of each sample was determined using the BCA protein assay microtiter plate protocol (Pierce Chemical, Rockford, IL), and results are expressed as nanograms of SP-A bound per microgram protein (40).

Immunofluorescence. AMs (1 × 105 cells) were adhered to round 12-mm glass coverslips in 24-well plates for 30 min at 37°C, and nonadherent cells were removed by washing. AMs were washed with HBSS+ containing 0.1% BSA and incubated with 0-100 µg/ml of SP-A at 37°C for 30 min. AMs were washed, fixed, and permeabilized with cold filtered methanol and blocked with 3% BSA in sterile PBS. AMs were reacted with rabbit anti-human SP-A as the primary antibody and anti-rabbit IgG conjugated to rhodamine B isothiocyanate as the secondary antibody. In control slides, AMs were incubated with equivalent amounts of nonimmune rabbit IgG, followed by anti-rabbit IgG conjugated to rhodamine B isothiocyanate as the secondary antibody. AMs were washed in PBS, mounted on glass slides in 90% glycerol, and viewed with an ausJena Sedival (Jenoptic Jena) inverted microscope. AM fluorescence images were captured by a Photometrics Series 250 cooled charge-coupled device camera system (Photometrics, Tucson, AZ) connected to a Power Macintosh 8100/80 computer (Apple, Cupertino, CA). IPLab Spectrum 2.5.5 image-analysis software (Signal Analytical, Vienna, VA) was used for image acquisition and analysis. Fluorescent images were captured from at least 12 fields selected randomly from each group while viewed through Nomarski optics. Video images were digitized into pixels, and mean pixel intensity was computed for individual cells (8-10 AMs/field). Background and autofluorescence values were subtracted digitally (30, 38).

Mycoplasmal Binding of SP-A

Siliconized microfuge tubes (Fisher Scientific, Pittsburgh, PA) were filled with HBSS containing 1% BSA and placed overnight at 4°C to block possible sites of nonspecific binding to plastic (45). Log-phase M. pulmonis UAB CT cells were resuspended to a concentration of 1 × 1010 mycoplasmas/100 µl in HBSS containing 1% BSA and either Ca2+ or ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.4. Mycoplasmal numbers were confirmed by serial dilution and culture. SP-A was mixed with the mycoplasmas for a final concentration of either 1 mM Ca2+ or 1 mM EGTA in the absence of Ca2+ and either 0-5 or 0-25 µg/ml SP-A. Samples were incubated for 30 min at room temperature and centrifuged to pellet mycoplasmas. The initial pellet was resuspended and transferred to a new microfuge tube, and a second aliquot was taken for enumeration of mycoplasmas. Pellets were washed twice more, resuspended in 200 µl of sterile water, and sonicated to disperse the mycoplasmas. Control samples were incubated with the appropriate buffers in the absence of mycoplasmas to quantify any remaining nonspecific binding of SP-A to plastic. Mycoplasma-associated SP-A was then measured by ELISA as described in Enzyme-linked immunosorbent assay. Nonspecific binding was subtracted at each concentration for all samples. Preliminary experiments were also performed with equimolar concentrations of Ca2+ and EGTA to assess the ability of EGTA to remove all available cation. Likewise, in the absence of mycoplasmas, SP-A aggregation was measured spectrophotometrically with increasing concentrations of either Ca2+ or EGTA in Ca2+-free 1% BSA buffer at 400-nm absorbance. To test the effect of excess carbohydrate on the binding of SP-A to mycoplasmas, 1 × 1010 M. pulmonis cells in HBSS with 1% BSA, 1 mM Ca2+, and 15 µg/ml SP-A were incubated with increasing concentrations of mannosyl-BSA (0-1 mM), with a molar ratio of mannose to albumin of ~20:1 (53).

Mycoplasmal Killing

AMs (1 × 105 cells) were adhered to glass vials for 30 min at 37°C, nonadherent AMs were washed away with excess DMEM-BAH, and adherent AMs were resuspended in DMEM-FHC (DMEM containing 15% FBS and 2.5 g/l HEPES, pH 7.4). Adherent cells were counted by a modification of the pronase and cetrimide assay (9) using a Coulter counter. AMs were activated with 100 U/ml mouse recombinant interferon (IFN)-gamma for 18 h at 37°C (6, 11), washed once, and resuspended in HBSS+ containing 0.1% BSA (32) and either 25 µg/ml SP-A or 5 mM HEPES. Samples were incubated at 37°C for 30 min and washed twice to remove SP-A not associated with cells (13). AMs were infected by addition of ~1 × 1010 viable M. pulmonis cells to each vial. Cultures were centrifuged and incubated at 37°C for 15 min to promote attachment of mycoplasmas to AMs. AMs were washed to remove unattached mycoplasmas and resuspended in HBSS+ containing 0.1% BSA. Vials processed immediately after washing were designated as time 0, and remaining vials were processed at 4, 6, and 8 h postinfection (8). Viable organisms at each time point were determined by quantitative culture (9). Mycoplasmal killing was defined as the difference between the log of mycoplasma CFUs in the original versus the control and experimental groups at each time point. AM quantitation was verified at each time point. Samples containing 25 µg/ml SP-A or HEPES buffer in HBSS+ were incubated with 1010 mycoplasmas in the absence of cells to determine the mycoplasmacidal effect of SP-A alone. In some samples, mycoplasmas were preopsonized with SP-A by mixing 1011 M. pulmonis cells with 0.1 mg/ml SP-A for 30 min at 37°C and then added to AMs as for the adherence of mycoplasmas to cells (above), with a final concentration of 25 µg/ml SP-A, 1010 mycoplasma CFUs, and 105 AMs/cell culture vial. In some experiments, after time 0, vials were incubated at 4°C for 6 h and cultured quantitatively to assess the role of phagocytosis in mycoplasmal killing. Positive control vials were incubated concomitantly at 37°C.

Inducible Nitric Oxide Synthase Inhibition

Killing assays were carried out as above with the exception that 1 mM NG-monomethyl-L-arginine (L-NMMA) was added to cultures 30 min before the addition of SP-A and subsequently maintained throughout each experiment. Control killing assays without L-NMMA were run simultaneously with time points of 0 and 6 h.

Nitrate/Nitrite

Culture media were collected after AM activation, after incubation of AMs with SP-A alone, and at each time point in the assay of SP-A-mediated killing by AMs. Media samples were pooled within groups and dried using a rotary evaporator. Samples were resuspended in sterile water, and nitrate (NO<SUP>−</SUP><SUB>3</SUB>) was converted to nitrite (NO<SUP>−</SUP><SUB>2</SUB>) with Escherichia coli reductase. Total NO<SUP>−</SUP><SUB>3</SUB>/NO<SUP>−</SUP><SUB>2</SUB> concentrations were determined by microplate assay. Briefly, 100 µl of each sample were incubated in duplicate with an equal volume of Greiss reagent [1% sulfanilamide-0.1% N-(1-naphthyl)ethylenediamine dihydrochloride] for 10 min at room temperature. Absorbance was measured at 550 nm, and NO<SUP>−</SUP><SUB>2</SUB> concentration was determined using an NaNO2 standard (6, 34).

Statistics

All experiments contained five samples per group and were repeated at least twice to ensure reproducibility. Parametric culture data were analyzed by analysis of variance followed by Tukey's multigroup comparison for parametric data or by the Mann-Whitney U test with Bonferroni-adjusted probabilities for nonparametric data (43). Mycoplasma CFUs were converted first to common logarithms. Results are expressed as means ± SE. P values of 0.05 or less are considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Binding of SP-A to AMs

We chose to use a 30-min incubation time for SP-A at 37°C because of previous observations (50) indicating possible degradation of SP-A at this temperature with longer incubation times. Binding of SP-A to AMs, as measured by ELISA, was both temperature and concentration dependent at 4 and 37°C after 30 min of incubation, with significantly higher binding observed at 37°C (Fig. 1). Similar results were obtained by indirect immunocytochemistry. AMs exhibited bright, discrete intracellular fluorescence, suggesting endocytosis of SP-A (Fig. 2). There was much higher binding of SP-A to AMs at 4°C after 2 h compared with 30 min (Fig. 1).


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Fig. 1.   Binding of surfactant protein (SP) A to alveolar macrophages (AMs). AMs were adhered to glass and incubated with 0-25 µg/ml concentrations of SP-A at 4°C for 30 min and 2 h. Cells were washed with phosphate-buffered saline, resuspended with sterile water, and ruptured by sonication. Total AM-associated SP-A was measured by enzyme-linked immunosorbent assay (ELISA), and total cell protein/sample was determined as described in MATERIALS AND METHODS. Results are means ± SE from a total of 3 experiments, each performed in triplicate.


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Fig. 2.   Fluorescent images of AMs, representative of typical figures that were reproduced 3 times. AMs were adhered to glass coverslips and incubated with 0-100 µg/ml concentrations of SP-A at 37°C for 30 min. AMs were washed, fixed with methanol, stained using indirect immunocytochemistry, and mounted in glycerol. AM fluorescence images were captured and analyzed with IPLab Spectrum 2.5.5 image-analysis software. Background and autofluorescence were subtracted digitally. A: IgG control. B: 100 µg/ml concentration of SP-A. Similar results were obtained with SP-A concentrations ranging from 5 to 25 µg/ml. Original magnification, ×1,000. Average cell size, 12-30 µm.

Binding of SP-A to Mycoplasmas

SP-A bound to M. pulmonis in a concentration- and Ca2+-dependent manner. Mycoplasma-associated SP-A was detectable by indirect ELISA at concentrations of SP-A from 5 to 25 µg/ml, and binding was decreased 50-70% in the presence of 1 mM EGTA and in the absence of Ca2+ (Fig. 3). Preliminary binding experiments performed with equimolar concentrations of Ca2+ and EGTA indicated that EGTA at this concentration bound all available cation. Furthermore, whereas higher concentrations of Ca2+ (>1 mM) caused SP-A aggregation, high concentrations of EGTA (10 mM) had no effect on SP-A concentrations as measured by ELISA and spectrophotometry (data not shown). The addition of mannosyl-BSA (~20 µM to 2 mM mannose) had no effect on total binding of SP-A to mycoplasmas (data not shown).


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Fig. 3.   Binding of SP-A to Mycoplasma pulmonis. Late-log-phase M. pulmonis UAB CT cells (1010) were washed 3 times, resuspended in Hanks' balanced salt solution (HBSS) containing 1% bovine serum albumin with either 1 mM Ca2+ or 1 mM EGTA, and incubated with 0-25 µg/ml concentrations of SP-A at room temperature for 30 min. Mycoplasmas were washed with HBSS, resuspended with sterile water, and sonicated to disperse organisms. Total mycoplasma-associated SP-A was measured by indirect ELISA and expressed as total ng SP-A. Data are means ± SE from a total of 3 experiments, each performed in triplicate.

SP-A-Mediated Mycoplasmal Killing by IFN-gamma -Activated AMs

Preincubation of IFN-gamma -activated AMs with SP-A resulted in SP-A- and time-dependent mycoplasmal killing. SP-A significantly enhanced the killing of mycoplasmas by 4 h, and at 6 h there was a maximal decrease of 83% in total recoverable CFUs (P = 0.002). By 8 h there was a loss of significant SP-A-mediated killing and a concomitant increase in mycoplasma CFUs (Fig. 4A). The addition of fresh media and SP-A (25 µg/ml) at 5 h resulted in an 88% decrease in total recoverable CFUs at 8 h (P < 0.001) (Fig. 4B). The addition of an SP-A bolus (25 µg/ml) or 5 mM HEPES to mycoplasma cultures in the absence of AMs did not increase killing for up to 8 h (Fig. 4C). Furthermore, opsonization of mycoplasmas with SP-A before addition to activated AMs did not decrease mycoplasma CFUs (data not shown). Incubation of samples at 4°C abrogated the SP-A-mediated mycoplasmal killing effect at 6 h, indicating that phagocytosis by AMs is necessary for clearance of the organism (Fig. 5).


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Fig. 4.   SP-A-mediated killing of M. pulmonis by C57BL/6NCr (C57BL) AMs. A: AMs were cultured for 18 h with 100 U/ml interferon (IFN)-gamma , incubated with 25 µg/ml SP-A or 5 mM HEPES (no SP-A) at 37°C for 30 min, and then washed. AMs were infected with 1010 colony-forming units (CFUs) of M. pulmonis as described in MATERIALS AND METHODS and incubated at 37°C for 0, 4, 6, and 8 h. AMs were ruptured by sonication, and total remaining CFUs were determined by quantitative culture. Arrows, time points for addition of SP-A. B: same as A except an additional 25 µg/ml SP-A or 5 mM HEPES was added at 5 h postinfection. C: cultures without AMs containing 1010 CFUs of M. pulmonis and either 25 µg/ml SP-A or 5 mM HEPES. Results of quantitative cultures are means ± SE, with a total of 3 experiments and 12-15 data points/group. Because of inherent variability in CFUs between experiments, results were compared with control values obtained at the same time period. * Significant difference between control and experimental groups at each time point, P < 0.05.


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Fig. 5.   Effect of temperature on SP-A-mediated killing of M. pulmonis by C57BL AMs. AMs were cultured for 18 h with 100 U/ml IFN-gamma , incubated with 25 µg/ml SP-A or 5 mM HEPES at 37°C for 30 min, and then washed. AMs were infected with 1010 CFUs of M. pulmonis as described in MATERIALS AND METHODS and incubated at either 37 or 4°C for 0 and 6 h. AMs were ruptured by sonication, and total remaining CFUs were determined by quantitative culture. Data are means ± SE from a total of 2 experiments, with 9-16 data points/group. * Significant difference between control and experimental groups at each time point, P < 0.05.

Involvement of · NO and SP-A in Mycoplasmal Killing

Incubation of activated AMs with mycoplasmas resulted in a significant (P = 0.006) increase in NO<SUP>−</SUP><SUB>3</SUB>/NO<SUP>−</SUP><SUB>2</SUB> in the media by 6 h (Fig. 6A) but had no effect on total mycoplasma CFUs (Fig. 6B). In sharp contrast, incubation with SP-A enhanced NO<SUP>−</SUP><SUB>3</SUB>/NO<SUP>−</SUP><SUB>2</SUB> production by an additional 40% and resulted in a significant degree of mycoplasmal killing. AMs treated with L-NMMA did not produce NO<SUP>−</SUP><SUB>3</SUB>/NO<SUP>−</SUP><SUB>2</SUB> and did not kill mycoplasmas (Fig. 6, A and B).


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Fig. 6.   Effect of inducible nitric oxide synthase inhibition on SP-A-mediated killing of M. pulmonis by C57BL AMs. A: AMs were cultured for 18 h with 100 U/ml IFN-gamma , washed, and incubated with 1 mM NG-monomethyl-L-arginine (L-NMMA) at 37°C for 30 min. AMs were treated with SP-A (25 µg/ml) or HEPES (5 mM) as described in MATERIALS AND METHODS, infected with 1010 CFUs of M. pulmonis, and incubated at 37°C for 0 and 6 h. Positive control AM cultures lacking L-NMMA were processed at same time. Results of quantitative cultures are means ± SE from a total of 3 experiments with 9-12 data points/group. HEPES: activated AMs, 5 mM HEPES, and 1010 CFUs of M. pulmonis; SP-A 25 µg/ml: activated AMs, 25 µg/ml SP-A, and 1010 CFUs of M. pulmonis; L-NMMA 1 mM: activated AMs, 5 mM HEPES, 1 mM L-NMMA, and 1010 CFUs of M. pulmonis; L-NMMA/SP-A: activated AMs, 25 µg/ml SP-A, 1 mM L-NMMA, and 1010 CFUs of M. pulmonis. B: aliquots of media from AM cultures collected after 30 min with 25 µg/ml SP-A or 5 mM HEPES and after 6 h of infection with 1010 CFUs of M. pulmonis. Results are means ± SE as measured using Greiss reagent after reduction of nitrate to nitrite with Escherichia coli reductase. * Significant difference between control and experimental groups at each time point, P < 0.05. 

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of this study was to investigate the potential role of SP-A in early, innate antimycoplasmal defenses. Specifically, we wished to characterize the interactions of SP-A with resistant C57BL AMs and UAB CT strain of M. pulmonis and to quantitatively assess SP-A-mediated killing of this mycoplasma by AMs in vitro. Human SP-A was used in these studies because of the difficulties in obtaining sufficient quantities of mouse SP-A and because human SP-A has been shown to affect the functions of AMs from many other species (24, 32, 35, 36, 40).

Previous studies of AMs from C57BL mice have shown that opsonization by rabbit anti-M. pulmonis serum can induce killing of this mycoplasma in vitro (9). In the absence of immune serum, organisms attached to AM cell surfaces and continued to multiply (44). Previous studies also had shown that the concentrated noncellular portion of lavages from M. pulmonis-infected C57BL mice, although unable to kill mycoplasmas alone, could initiate killing of mycoplasmas in vitro when introduced into AM cultures (8). We hypothesized that nonspecific opsonin(s) in the lavage fluid plus activated AMs are the essential components for this mycoplasmacidal effect (8).

We found that human SP-A binds to C57BL mouse AMs in a concentration-, time-, and temperature-dependent manner. AM-associated SP-A increased directly with SP-A concentration after 30 min of incubation at 4°C, reaching values that were ~20-60% higher after 2 h. After 2 h of incubation at 4°C, AM-associated SP-A reached saturation at the concentration of 15 µg/ml, consistent with receptor-mediated binding. Although there was significant AM association of SP-A at 4°C after 30 min, the levels were 30-75% higher at 37°C for all concentrations of SP-A. Our results also showed that SP-A binding almost certainly involved endocytosis of the protein at 37°C because high levels of cell-associated SP-A were demonstrated by immunofluorescence assay (23, 38). These results differ from those of a previously published report (40) on SP-A binding to AMs because we found a significant increase in AM-associated SP-A at 37°C compared with 4°C. One reason for this difference could be that SP-A was degraded more rapidly at the cell surface or after internalization at 37°C (50). Also, the previously reported temperature-dependent time course was obtained over a period of 6 h, with a single low concentration (0.5 µg/ml) of SP-A (40), whereas our incubation time was only 30 min, with much higher SP-A concentrations (0-25 µg/ml), in accordance with our in vitro studies of mycoplasma-infected AMs.

Ca2+ (1 mM) was included in all of our cell binding assays because previous studies have shown that binding of SP-A to AMs is partially Ca2+ dependent (20, 35, 36, 40). Concentration- and Ca2+-dependent binding of SP-A to mycoplasmas is consistent with reports that SP-A is capable of stimulating phagocytosis of those bacteria to which it binds (45). The inability of SP-A to bind mycoplasmas at low concentrations is not surprising, considering that mycoplasmas lack cell walls and are considered the smallest and simplest of the self-replicating organisms (41). The partial Ca2+ dependence of SP-A-mycoplasma binding, as well as the inability of mannosyl-BSA to compete with SP-A for binding to mycoplasmas, is consistent with binding to the collagen-like domain of the SP-A protein, a region thought to have a lower Ca2+ affinity than the carbohydrate domain (20). All buffers and media were initially Ca2+ free, with Ca2+ (1 mM) or EGTA (1 mM) being added to ensure either the presence or absence of Ca2+. There was significant aggregation of SP-A when incubated with higher concentrations of Ca2+ (>1 mM) in the absence of mycoplasmas; however, EGTA had no effect on SP-A aggregation even at high concentrations (10 mM) (data not shown). The lack of any nonspecific effect by EGTA suggests that binding in the presence of EGTA was true binding.

Activation of AMs with IFN-gamma was done in all mycoplasmal killing assays because preliminary experiments with rabbit anti-M. pulmonis immune sera showed that cell activation was a necessity for mycoplasmal killing. Initial experiments carried out in the absence of IFN-gamma demonstrated no appreciable change in mycoplasma CFUs (data not shown). IFN-gamma has been found to be significantly increased in the lungs of C57BL mice as early as 24 h postinfection (15). This is consistent with previous work on the role of natural killer cells in mycoplasmal infections. Natural killer cells have been shown to be important not for the clearance of mycoplasmas but in the activation of AMs (26).

Our data showed that the killing of mycoplasmas was SP-A and time dependent and that the SP-A-mediated mycoplasmacidal effect was lost when, presumably, SP-A became depleted in the media (50). Collectively, our results suggest that SP-A mediates the killing of mycoplasmas by AMs through 1) the induction of phagocytosis and 2) the production of reactive oxygen nitrogen species (17, 28, 45, 47, 48).

The role of SP-A in mycoplasmal killing by AMs probably involves specific interaction between SP-A and the AM rather than a mere function as a nonspecific opsonin for the following reasons: 1) "opsonization" of M. pulmonis with SP-A before the addition to IFN-gamma -activated AM cultures did not result in significant killing; 2) SP-A was adhered to AMs, and the excess SP-A was removed before infection with M. pulmonis (thus removing any free SP-A available for binding to mycoplasmas before they associated with AMs); and 3) mycoplasmas did not preferentially bind to SP-A-treated AMs as shown by equal numbers of organisms present after the initial interaction among either AMs, SP-A, and mycoplasmas or AMs, HEPES, and mycoplasmas (time 0) (13, 45, 47). The removal of excess SP-A from AM cultures before infection with mycoplasmas was not necessary for the mycoplasmacidal effect to occur. However, preliminary experiments yielded better results when this step was incorporated into the culture protocol.

SP-A is also known to effect the release of reactive oxygen species (47, 48), another mechanism that could account for the SP-A-mediated mycoplasmacidal activity of AMs. In the present study, the addition of the inducible nitric oxide synthase inhibitor L-NMMA to AM cultures abrogated the SP-A-mediated mycoplasmacidal activity, indicating that · NO is involved in mycoplasmal killing. · NO production after IFN-gamma stimulation of macrophages has been shown to play an important role in defense against intracellular and extracellular pathogens (1, 11, 19, 31, 46, 51). Peroxynitrite, a strong oxidant formed by AMs as a reaction product of superoxide and · NO, has also been shown to be highly bactericidal (2, 52) and could have an important role in mycoplasmal killing. NO<SUP>−</SUP><SUB>3</SUB> and NO<SUP>−</SUP><SUB>2</SUB>, the decomposition products of · NO, were markedly decreased in the media of cultures containing L-NMMA, further implicating · NO as a possible factor involved in SP-A-mediated mycoplasmal killing. However, it should be emphasized that SP-A-induced phagocytosis was necessary for killing to occur, since no killing was observed in the absence of SP-A or when AMs were incubated at 4°C.

In summary, we have demonstrated in vitro that SP-A binds specifically to AMs in culture and mediates killing of mycoplasmas by activated AMs and that · NO and/or its toxic metabolites are involved in mycoplasmal killing. SP-A-mediated killing of mycoplasmas by AMs may be the primary mechanism of innate host defense against mycoplasmal infection in the lungs.

    ACKNOWLEDGEMENTS

We thank Carpentanta Myles, Julie Gibbs-Erwin, Marilyn Shackelford, Jane Hosmer, and Kathy Hutchens for technical support; Dr. Denise Shaw for endotoxin testing; and Nancy Penney for editorial assistance.

    FOOTNOTES

This work was supported by National Institutes of Health Grants RR-11105 (to J. R. Lindsey) and HL-31197 and HL-51173 (to S. Matalon), funds from the Veterans Administration Research Service (to J. R. Lindsey), and Grant N00014-97-1-0309 from the Office of Naval Research (to S. Matalon).

This research was accomplished by J. M. Hickman-Davis in partial fulfillment of requirements for a PhD in Molecular and Cellular Pathology.

Address for reprint requests: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., Birmingham, AL 35233-6810.

Received 2 June 1997; accepted in final form 24 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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