Published online before print July 1, 2003
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,1
* Department of Pathology, Vanderbilt University School of Medicine, and
Department of Veterans’ Affairs, Nashville, Tennessee
1Correspondence: VA Medical Center/Research Service, 1310 24th Ave. South, Nashville, TN 37212. E-mail: virginia.l.shepherd{at}vanderbilt.edu
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. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.
Key Words: innate immunity • Mycobacterium avium complex • nitric oxide
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Type II cells, nonciliated bronchiolar cells, and tracheobronchial gland cells in the lung produce SP-A [5
]. From in vitro studies, SP-A binds lipid, calcium, and carbohydrate [6
]. The lipid-binding characteristic of SP-A has been implicated in the role of the protein in surfactant homeostasis, and the carbohydrate-binding characteristic has been implicated in the host-defense properties of SP-A. A number of studies have shown that SP-A can bind to a variety of viral, fungal, and bacterial pathogens through its carbohydrate-binding domain [7
8
9
10
11
12
13
14
15
16
]. Recent studies using mice deficient in SP-A have further substantiated the importance of SP-A in host defense. These mice showed impaired clearance of Pseudomonas aeruginosa, group B Streptococcus, respiratory syncytial virus (RSV), adenovirus, and Mycoplasma pneumoniae [17
18
19
20
21
22
]. In vitro studies from our laboratory and others have shown that SP-A is involved in pulmonary host defense against mycobacterial species. First, we have reported previously that SP-A binds to BCG and enhances its entry into macrophages via a specific cell-surface receptor (SPR210) [3
]. Furthermore, this enhanced uptake is accompanied by increased production of the proinflammatory mediators, nitric oxide (NO) and tumor necrosis factor- (TNF-), which lead to increased pathogen killing [4
]. In addition, Pasula et al. [12
] demonstrated that SP-A promotes attachment of Mycobacterium tuberculosis to macrophages, and Gaynor et al. [23
] demonstrated that SP-A could mediate enhanced phagocytosis of M. tuberculosis by human macrophages.
The intracellular mechanisms and proinflammatory mediators involved in clearance of MAC by macrophages are only beginning to be elucidated. Differences between M. tuberculosis and MAC suggest that mechanisms of host-mediated destruction of these internalized mycobacteria may be quite different. Studies by Gomes et al. [24 ] reported that MAC and M. tuberculosis organisms occupy distinct phagocytic vacuoles inside the macrophage, suggesting that these two mycobacteria use different entry mechanisms and/or different intramacrophage survival schemes to evade killing by the host cells. Entry of M. tuberculosis and BCG as well as MAC into macrophages results in the production of NO and TNF. Although NO has been shown to be involved in BCG and M. tuberculosis killing by rodent macrophages [25 26 27 ], NO production does not appear to contribute to MAC killing [28 , 29 ], and mice lacking the inducible NO synthase (iNOS) gene actually show higher clearance rates following infection [30 ]. As we have previously demonstrated that SP-A enhances BCG killing through a NO-dependent pathway [4 ], in the current study, we examined the role of SP-A in MAC entry into macrophages, MAC-induced mediator production, and killing of internalized MAC by rat macrophages.
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Rat bone marrow macrophages (RBMM) were prepared as described previously [3
]. After 5 days in culture, mature RBMM were replated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO). The medium was changed to DMEM without serum 
12 h before infection with mycobacteria to limit interaction of the mycobacteria with complement components and subsequent ingestion through complement receptors. Removal of serum had no effect on cell viability.
SP-A was prepared by the butanol extraction method as described previously [6 ]. Briefly, 1–2 ml alveolar proteinosis fluid (a gift from Dr. Sam Hawgood, University of California, San Francisco, and Dr. Jo Rae Wright, Duke University, Durham, NC) in phosphate-buffered saline (PBS) was extracted with 25 ml 1-butanol and was then dried over nitrogen overnight. The dried protein was then suspended in HEPES buffer with 0.15 M NaCl and 20 mM n-octyl-ß-D-glucoside and centrifuged at 17,000 g, and the process was repeated. The final pellet was then suspended in 5 mM HEPES buffer with 1 mM EDTA (pH 7.5) and was incubated with polymyxin B-agarose beads (Sigma Chemical Co.). The mixture was dialyzed in the same buffer for 48 h with four buffer changes. The lipopolysaccharide content in the SP-A preparations was monitored after polymyxin treatment by the Limulus lysate assay (Associates of Cape Cod Inc., Falmouth, MA) and contained <0.05 endotoxin units/ml.
Determination of SP-A–MAC binding by enzyme-linked immunosorbent assay (ELISA)
MAC organisms (1x106) in Tris-buffered saline (TBS) were added to multiple wells of a flat-bottom 96-well Immulon 2HB plate (DYNEX Technologies, Chantilly, VA) and were allowed to dry overnight under a laminar flow hood. Dried MAC remained viable as demonstrated by growth on Middlebrook agar plates (data not shown). Wells containing MAC were then blocked with TBS containing 2 mM Ca2+, 3% milk, and 0.02% Triton X-100 for 24 h. The plate was blotted dry, and SP-A in blocking buffer at stated concentrations was added for 60 min at room temperature. The wells were then emptied and washed three times for 5 min each with TBS containing 2 mM Ca2+ with gentle rocking. Anti-human SP-A antibody (a gift from Dr. Jeffrey Whitsett, University of Cincinnati, OH) at a 1:10,000 dilution was then added to the wells, and the plate was incubated at room temperature for 2 h with gentle rocking. The wells were washed as described above, and an appropriate secondary antibody conjugated to horseradish peroxidase (HRP) at a 1:5000 dilution was added to the wells with incubation at room temperature for 2 h with gentle rocking. The plate was then washed for a final time, and 0.0006 mM 2,2'-azido-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) solution in a 0.1 M anhydrous citric acid solution (pH 4.35 and 3% H2O2) was added to the wells, followed by measurement of the absorbance at 450 nm. To quantify the amount of SP-A binding to MAC, a standard curve for SP-A binding was performed by drying known concentrations of SP-A to wells and performing the ELISA assay in the absence of mycobacteria. The amount of SP-A binding to MAC was then determined by comparing the absorbance of wells treated with MAC and SP-A with the generated standard curve. To determine specificity of binding, EDTA (10 mM) was added to the blocking buffer in place of the 2 mM Ca2+.
Measurement of MAC ingestion by rat macrophages
RBMM were plated at 2.5 x 105 cells per well in chamber slides (Laboratory-Tek; ICN Biochemicals, Inc., Aurora, OH) in DMEM without serum or antibiotics. MAC organisms were pelleted and resuspended in PBS. Clumps of mycobacteria were broken up by vortexing with a glass bead for 15 s. MAC organisms were incubated with SP-A at a ratio of 20 µg SP-A per 5 x 105 organisms for 30 min at 37°C. MAC or SP-A–MAC complexes were then added to the cells and cells plus additions, incubated at 37ºC for 4 h. The cells were washed twice with PBS and fixed in freshly prepared 10% formaldehyde. The cells were then stained for internalized mycobacteria using auramine-rhodamine staining (Difco Laboratories, Detroit, MI), according to the manufacturer’s directions. To quantify the number of mycobacteria internalized by cells, 100 cells in a random field for each treatment were counted using light microscopy, and then fluorescent mycobacteria associated with those cells were counted by fluorescent microscopy. To verify that the MAC detected by auramine-rhodamine staining were internalized and not simply bound to the surface of the macrophages, the following method was used. MAC (1x108 MAC/ml) were incubated for 1 h at 37°C with Cell Tracker (Molecular Probes, Eugene, OR) at a final concentration of 10 µM in PBS containing 0.02% Tween 80. The mycobacteria were washed and resuspended in PBS–Tween. RBMM (1x105) were cultured on glass coverslips in 24-well plates in serum- and antibiotic-free DMEM. The cells were then infected with Cell Tracker-labeled MAC, opsonized or not with 20 µg SP-A for 4 h. After infections, cells were washed with PBS, and a 100-µl 1:1 (vol:vol) mixture of DMEM and trypan blue was added to quench the fluorescence of any noningested bacteria. Cells were washed, and fresh DMEM was added. Three fields per coverslip with 100 cells per field were counted. Data are expressed as the percentage of cells containing fluorescent bacteria.
Measurement of MAC growth by assessment of CFU
RBMM were plated at a concentration of 5 x 105 cells/well in 24-well tissue-culture dishes. MAC preincubated with or without SP-A (20 µg/5x105 MAC) were then added to cells for 4 h at 37ºC. The cells were washed twice with PBS, and fresh media containing serum was added. Prior to cell lysis and collection of viable mycobacteria, spent media was collected for NO and TNF determinations as described below. Middlebrook broth containing OADC and 0.083% sodium dodecyl sulfate was added to wells at the appropriate days to lyse the cells. The cell lysate was then transferred to microfuge tubes, and sterile 20% bovine serum albumin (BSA) was used to rinse the wells. The lysates containing BSA were then serially diluted and plated onto Middlebrook agar plates containing ampicillin at a concentration of 25 µg/ml to prevent growth of other bacterial species. The addition of ampicillin to the Middlebrook agar plates did not affect the viability of MAC and gave comparable results to MAC plated without the antibiotic (data not shown). Plates were then incubated at 37ºC, and mycobacterial growth was monitored for colonies, which were counted at 7–10 days postplating.
Assay of NO and TNF- production
Spent media from the colony-count assays described above were collected at the appropriate time points for assay of NO and TNF. In the cases where NO and TNF were measured within a 24-h period, MAC organisms were not washed from the cells. Previous studies in our laboratory have shown that mediator levels are comparable among cells that are treated with mycobacteria for 24 h or for 4 h with subsequent removal of bacteria (data not shown). NO was measured using the Griess reagent as follows: Medium (100 µl) was added to wells in a 96-well microtiter plate. Freshly prepared Griess reagent (1% sulfanilamine, 0.1% naphthylethylene diamide dihydrochloride, and 2.5% H3PO4; 100 µl) was added to the medium and incubated for 5–10 min at room temperature. The resulting absorbance was read at 540 nm. Nitrite concentrations were determined from a standard curve using sodium nitrite. ELISA measured TNF (BioSource International, Camarillo, CA).
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Figure 1. SP-A binds to M. avium organisms. MAC organisms (1x106) were dried in individual wells of a 96-well plate. Wells were blocked with buffer containing 3% milk and Ca2+ (2 mM) or EDTA (10 mM). SP-A in buffer was then added at increasing concentrations. The plate was incubated at room temperature for 60 min. The wells were washed extensively, and monoclonal anti-SP-A antibodies at a 1:10,000 dilution were added and allowed to incubate at room temperature for 1 h. The wells were again washed, and goat anti-mouse secondary antibodies conjugated to HRP at a 1:5000 dilution were added to the wells and allowed to incubate at room temperature for 2 h. The wells were washed a final time, and SP-A present in the wells was colorimetrically visualized by addition of ABTS solution and measurement of absorbance at 450 nm. The amount of SP-A bound was calculated from a standard curve prepared by drying known concentrations of SP-A to wells and performing the ELISA assay in the absence of mycobacteria. Results are the average +/- standard deviation for triplicate determinations (for SP-A–EDTA) and are representative of two separate experiments. SP-A + EDTA was performed twice in duplicate.
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Figure 2. SP-A enhances ingestion of M. avium by rat macrophages. (A) RBMM were plated at 2.5 x 105 cells per well in DMEM containing chamber slides. MAC with or without SP-A (20 µg per 5x105 MAC) were incubated at 37ºC for 30 min and then added to the cells. Cells and treatments were then incubated for 4 h at 37ºC, after which the cells were washed extensively with PBS and fixed in 10% formaldehyde. Internalized mycobacteria were visualized using auramine-rhodamine staining under fluorescence microscopy. One hundred cells in a random field were first counted by light microscopy, and then fluorescent mycobacteria associated with those cells were counted by fluorescent microscopy. (B) MAC (1x108 MAC/ml) were incubated for 1 h at 37°C with Cell Tracker (10 µM in PBS containing 0.02% Tween 80). The mycobacteria were washed, resuspended in PBS–Tween, opsonized for 30 min with SP-A or PBS, and added to 1 x 105 RBMM cultured on glass coverslips in 24-well plates in serum- and antibiotic-free DMEM. After a 4-h infection, cells were washed, and a 100-µl 1:1 (vol:vol) mixture of DMEM and trypan blue was added to quench the fluorescence of any noningested bacteria. Cells were washed, and fresh DMEM was added. Three fields per coverslip with 100 cells per field were counted. Data are expressed as the percentage of cells containing fluorescent bacteria. (A and B) *, P< 0.001, for MAC/SP-A compared with MAC alone by Student’s two-tailed t-test.
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1.5-fold, when MAC organisms were preincubated with SP-A. Although the enhancement of NO levels was comparable with our previous findings with SP-A–BCG infection (1.8-fold increase), SP-A-mediated enhancement of TNF production by MAC was significantly lower than production in the presence of BCG (3.5-fold). The differences found between these two mycobacteria may reflect their level of resistance to these mediators during macrophage infection.
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Figure 3. SP-A enhances MAC-induced production of NO and TNF over 24 h. RBMM (5x105) in 24-well plates were treated with MAC (1:1 MAC/cell), MAC preopsonized with SP-A (20 µg) for 30 min, SP-A alone, or PBS. (A) NO released into the media was measured using the Greiss reagent. Baseline levels of NO (PBS-treated cells) were considered background and subtracted from the other levels. (B) ELISA measured TNF released into the media. Baseline levels of TNF (PBS-treated cells) were also subtracted from the other levels. Results +/- standard deviation are the average of triplicate determinations and are representative of three separate experiments. *, P < 0.001, for MAC/SP-A compared with MAC alone.
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Figure 4. SP-A enhances NO but not TNF production after 5 days of infection. Spent media was collected from cells infected for 0–5 days as described in Figure 5
. NO (A) and TNF (B) levels were measured as described in Figure 3
. *, P < 0.001, for MAC compared with MAC/SP-A.
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Figure 5. SP-A does not enhance clearance of MAC by RBMM. RBMM were incubated as described in Figure 2
. After 4 h of incubation, media was removed from all wells, cells were washed to remove extracellular mycobacteria, and fresh media containing serum was added. On days 0, 1, 3, 5, and 7, cells were lysed, and lysates containing viable mycobacteria were plated onto Middlebrook agar plates. Plates were incubated at 37ºC and monitored for colony growth at 7–10 days postplating. Results are the average of duplicate determinations within a single experiment and are representative of three separate experiments.
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Table 1. Inhibition of NO Production Does Not Significantly Alter M. avium Survival in Rat Macrophages
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In the present study, we show that SP-A binds to MAC and that there is a fivefold increase in uptake of these complexes by rat macrophages compared with MAC alone. Similar studies from our laboratory and others have demonstrated that SP-A binds to BCG and M. tuberculosis and that these complexes are taken up more avidly by macrophages [3 , 12 ]. However, the mechanisms involved in SP-A-mediated pathogen uptake are unclear. We have suggested that binding of SP-A to BCG redirects internalization through a specific SP-A receptor (SPR210), as anti-SPR210 antibodies block ingestion [3 ]. However, other groups have postulated that SP-A interacts with the macrophage, and this association leads to activation and/or increased expression of a mycobacterial receptor, which allows for increased uptake. For example, Gaynor et al. [23 ] reported that SP-A pretreatment of human macrophages led to increased surface expression of the macrophage mannose receptor, a major receptor for M. tuberculosis. In our studies, pretreatment with SP-A did not significantly alter BCG or MAC uptake (ref. [3 ]; data not shown), suggesting at least for BCG and MAC that SP-A appears to promote uptake in an opsonin-dependent manner.
The finding in the current study that SP-A enhances NO production by rat macrophages in response to MAC infection is consistent with our previous observations using BCG. SP-A has also been shown to increase NO in response to other lung pathogens including Klebsiella [8
] and Mycoplasma [18
]. Several groups have reported that BCG and M. tuberculosis are killed via NO-dependent mechanisms [25
26
27
]. Recent studies using inhibitors of iNOS and iNOS knockout mice further support a role for NO in the growth inhibition of these organisms. M. tuberculosis-infected wild-type mice treated with iNOS inhibitors and iNOS knockout mice showed increased mortality and increased pathogen loads [34
]. Our recent studies about SP-A-enhanced BCG clearance by macrophages through a NO-dependent mechanism further support a role for this mediator in mycobacterial clearance [4
]. In the current study, we found that although SP-A increased MAC-induced NO production, ingested MAC organisms were not killed. This is consistent with in vivo and in vitro findings from several groups, demonstrating that MAC survival is not altered by NO production. Appelberg and Orme [28
] reported that growth of MAC within bone marrow macrophages could be inhibited if the macrophages were treated with interferon-
(IFN-), but this restriction of growth was not reversed by the addition of iNOS inhibitors. Bermudez [35
] reported similar observations. Recent studies by Doherty and Sher [29
] and Gomes et al. [30
] showed that growth of MAC in mice with a disrupted iNOS gene was not increased, and they suggested a negative role of NO in MAC infection. In the latter study, Gomes et al. [30
] further found that iNOS-deficient mice were actually more efficient in clearing MAC.
Mechanisms involved in MAC killing are not entirely clear. Several classical macrophage-dependent antimicrobial pathways have been studied in an attempt to delineate the mechanisms underlying MAC killing. First, TNF has been implicated in M. tuberculosis host defense and may also play a role in the immune response to MAC infection [36 ]. MAC infection has been shown to stimulate TNF production by various cell types with varying consequences, including down-regulation of the expression of the p75 TNF receptor, as well as a temporal decrease in secreted, bioactive TNF [37 , 38 ]. Studies by Bermudez and Young [39 , 40 ] have implicated TNF as important in macrophage-mediated MAC killing. Recent work involving mice deficient in the p55 TNF receptor demonstrated that TNF did not affect survival of MAC but did alter the progression of chronic pathologic states [41 ]. In our study, we actually found decreased TNF production in the presence of SP-A after 5 days of infection. It is possible that this reduction in TNF is linked to the balance of cytokines produced by the infected macrophage, i.e., an increase in levels of cytokines that would inhibit release of TNF. This is supported by our finding of enhanced IL-10 production by SP-A complex–MAC (data not shown) and by the recent report that MAC-induced production of IL-10 by human macrophages reduces levels of released TNF [42 , 43 ].
Second, although we have not examined the effect of SP-A on MAC-induced superoxide production, it has been reported that most strains of MAC are not killed by superoxide anion or hydrogen peroxide [40 , 44 ]. However, more recent reports of MAC disease in chronic granulomatous disease patients [45 , 46 ] and the increased susceptibility of mice lacking the gp47-phox gene to MAC infection [47 ] suggest that reactive oxygen intermediates may play a role in early defense against mycobacterial disease.
Finally, recent studies by Appelberg and Sarmento [48 ] and Medina et al. [49 ] suggest that MAC virulence may be linked to expression of Nramp1 in tissue macrophages. The Nramp1 protein has a number of effects in macrophages, including a role as an iron transporter. Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space. A role for iron in MAC survival is further supported by the observation that MAC phagosomes in activated macrophages are not associated with the transferrin receptor, and MAC growth is inhibited [50 ]. The effect of SP-A on iron transport or Nramp1 expression has not been examined.
The differences in the susceptibilities of MAC and BCG to acidity may provide further insight into their different fates when associated with SP-A. Recent studies have demonstrated that MAC and M. tuberculosis were able to inhibit fusion of mycobacterium-containing phagosomes with lysosomes [51 ]. However, MAC and M. tuberculosis are differentially susceptible to the acidic environment of the mature phagolysosome. Using colocalization studies, Gomes et al. [24 ] showed that MAC was able to grow inside Coxiella burnetii-containing, preformed acidic vacuoles, and M. tuberculosis was killed. Cell-free studies have also demonstrated the relative resistance of MAC but not of M. tuberculosis to an acidic pH [52 ]. It is possible that SP-A redirects mycobacteria to these acidic compartments, where M. tuberculosis organisms, but not MAC, are effectively killed.
In the current study, we have described the role of SP-A in the interaction of MAC with resident, nonactivated macrophages. In the absence of macrophage-activating agents, macrophages take up MAC followed by unrestricted intramacrophage replication. It would appear that the effect of SP-A on initial infection is to actually enhance uptake and survival of MAC organisms. As the infection progresses, the surrounding macrophages become activated by IFN- or other cell-derived cytokines. At this point, the effect of SP-A on MAC interaction with activated macrophages is not known. Pasula et al. [53
] have recently reported that SP-A decreased NO production and increased the survival of M. tuberculosis internalized by IFN--activated macrophages, suggesting that in later stages of infection, SP-A may actually contribute to mycobacterial survival. In the present study, we have shown that in the case of MAC infection, SP-A may promote survival, even at the onset of mycobacterial challenge. Therefore, although SP-A may play a major role in limiting initial challenge of M. tuberculosis and BCG as well as other pulmonary pathogens, including Klebsiella, group B streptococcus, mycoplasma, adenovirus, RSV, and Pseudomonas, SP-A does not contribute to MAC destruction and may actually enhance safe entry and replication of MAC in lung macrophages.
Received January 17, 2003; revised March 31, 2003; accepted April 1, 2003.
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is required in the protective immune response against Mycobacterium tuberculosis in mice Immunity 2,561-572[CrossRef][Medline] activity J. Infect. Dis. 180,1230-1237[CrossRef][Medline]This article has been cited by other articles:
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