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
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ABSTRACT |
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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)- 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-
-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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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%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 atAnimals
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(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)-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 (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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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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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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SP-A-Mediated Mycoplasmal Killing by
IFN--Activated AMs
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Involvement of · NO and SP-A in Mycoplasmal Killing
Incubation of activated AMs with mycoplasmas resulted in a significant (P = 0.006) increase in
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DISCUSSION |
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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- 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-
demonstrated no
appreciable change in mycoplasma CFUs (data not shown). IFN-
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--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- 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.
and
, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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