Division of Pulmonary and Critical Care Medicine, Departments of 1 Anesthesiology and 2 Medicine, Division of Nephrology and Nephrology Research Training Center, Departments of 3 Genomics and Pathobiology and 4 Medicine, 6 Department of Physiology and Biophysics, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 5 Environmental Protection Agency, Research Triangle Park, North Carolina 27711-0001
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ABSTRACT |
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We investigated putative mechanisms by
which human surfactant protein A (SP-A) effects killing of
Klebsiella pneumoniae by human alveolar macrophages (AMs)
isolated from bronchoalveolar lavagates of patients with transplanted
lungs. Coincubation of AMs with human SP-A (25 µg/ml) and
Klebsiella resulted in a 68% decrease in total colony
forming units by 120 min compared with AMs infected with
Klebsiella in the absence of SP-A, and this SP-A-mediated
effect was abolished by preincubation with
NG-monomethyl-L-arginine. Incubation
of transplant AMs with SP-A increased intracellular Ca2+
concentration ([Ca2+]i) by 70% and nitrite
and nitrate (NOx) production by 45% (from 0.24 ± 0.02 to 1.3 ± 0.21 nmol · 106
AMs1 · h
1). Preincubation with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester inhibited the increase in
[Ca2+]i and abrogated the SP-A-mediated
Klebsiella phagocytosis and killing. In contrast, incubation
of AMs from normal volunteers with SP-A decreased both
[Ca2+]i and NOx production and
did not result in killing of Klebsiella. Significant killing
of Klebsiella was also seen in a cell-free system by
sustained production of peroxynitrite (>1 µM/min) at pH 5 but not at
pH 7.4. These findings indicate that SP-A mediates pathogen killing by
AMs from transplant lungs by stimulating phagocytosis and production of
reactive oxygen-nitrogen intermediates.
innate immunity; collectins; peroxynitrite; lung transplant; phagolysosome
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INTRODUCTION |
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KLEBSIELLA PNEUMONIAE is an important cause of gram-negative nosocomial bacteremia (56) because it accounts for 10-13% of ventilator-associated pneumonias (31) and is one of the top three complicating pathogens isolated from patients with acute respiratory distress syndrome (2). Innate pulmonary immunity against bacterial pathogens involves resident phagocytes and a number of inducible proteins and peptides produced by these leucocytes and the lung epithelial cells (10). As part of this innate immune system, surfactant protein A (SP-A) is synthesized by a variety of airway cells, is a member of the family of collagenous Ca2+-dependent lectins or collectins, and is important for the clearance of many important lung pathogens (26, 34, 35, 55, 61).
SP-A has been shown to bind to the capsular polysaccharide of Klebsiella and to enhance phagocytosis and killing of this pathogen by rat and guinea pig alveolar macrophages (AMs) (28). SP-A has also been reported to modulate the production of nitric oxide (NO) by a variety of cell types (5, 29), and recently SP-A stimulation of NO production and inducible nitric oxide synthase (iNOS) upregulation were related to cell state and mechanism of activation (52).
The importance of NO for the clearance of bacteria from the lung has
been well documented (4, 25), and the requirement of NO
specifically for Klebsiella clearance was previously
demonstrated utilizing a mouse model (56). Although NO
itself does not possess microbicidal activity, it may react with iron
or thiol groups on proteins forming iron-nitrosyl complexes that
inactivate enzymes important for bacterial DNA or mitochondrial
replication (15). Additionally, at high inspired NO
concentrations, autoxidation of NO may form toxic reactive species such
as NO), which is highly bactericidal (9,
25). To this extent, SP-A may enhance killing by stimulating the
production of superoxide and NO and/or enhancing phagocytosis, thus
exposing pathogens to intense localized production of reactive
oxygen-nitrogen intermediates in phagosomes.
Stimulation of NO production by rat and mouse macrophages has become a standard part of cell activation protocols, while normal human monocytes remain refractile to a variety of stimuli (27). In contrast, AMs isolated from the lungs of patients with tuberculosis produce NO (53). Thus the mechanisms responsible for the production of NO by human AMs are complex and poorly understood. The interaction of SP-A with human AMs and its effects on NO production and pathogen killing may depend on preexposure of AMs to activating stimuli in the alveolar milieu. To address these issues, we isolated human AMs from bronchoalveolar lavagates from transplanted lungs and lungs of normal human volunteers and measured the ability of human SP-A to effect production of NO and killing of K. pneumoniae. Our results are the first to delineate the differential ability of SP-A to modulate innate immune responses of primary human AMs and to show that SP-A may modulate killing by enhancing phagocytosis in addition to NO production.
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METHODS |
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Materials. PBS, DMEM with L-arginine and 4.5 g/l of glucose, and Hanks' balanced salt solution (HBSS containing Ca2+ and Mg2+) were from Cellgro (Atlanta, GA). Diff Quik stain kits were obtained from Baxter Healthcare (McGraw Park, IL). 3-Morpholinosydnonimine (SIN-1), xanthine oxidase (XO), 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPANONOate), and 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-acetoxymethyl ester were from Calbiochem. Dihydrorhodamine (DHR)-123 and fura 2-acetoxymethyl ester came from Molecular Probes (Eugene, OR). Unless otherwise specified all other chemicals were from Sigma (St. Louis, MO).
Isolation of AMs.
Bronchoalveolar lavage (BAL) fluid was obtained either from lung
transplant patients undergoing routine surveillance lavage [Univ. of
Alabama at Birmingham (UAB)] or normal volunteers [Environmental Protection Agency (EPA)]. The BAL was centrifuged at 800 g for 10 min to pellet cells. Cells were resuspended in DMEM
supplemented with 1% L-glutamine, 2.5% HEPES, 0.2% low
endotoxin BSA, and penicillin/streptomycin antibiotic. Cells were
counted with a hemocytometer, and sample viability tested by trypan
blue exclusion was >90%. Samples that could not be maintained as
"sterile" in the presence of antibiotics and utilizing standard
primary tissue culture techniques were discarded. Furthermore, in order
for samples to be utilized, they were >90% AMs as determined by
examination of cytospin preparations stained with Diff Quik.
Examination of cell content of BAL from transplant patients has
demonstrated increased neutrophils during infection and increased
lymphocytes during rejection (16). For this reason,
samples containing >10% lymphocytes or neutrophils were not utilized.
Identification of infection or rejection by the UAB hospital as
reported in Table 1 was
determined according to different criterion than BAL cellular content,
i.e., rejection was identified according to standard histological
examination of transbronchial biopsy and infection by culture of BAL
fluid and examination of fluid for fungal hyphae.
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Purification of SP-A.
SP-A is purified sterilely from the BAL of patients with alveolar
proteinosis by N-butanol extraction as previously described (21). PAGE and Western blot analysis of SP-A were carried
out to ensure the purity of SP-A preparations (23). SP-A
was stored at 20°C in 5 mM HEPES, pH 7.4. Aliquots were cultured
for aerobic bacteria in BBL brain heart infusion broth
(Becton Dickinson, Franklin Lakes, NJ), and only
culture-negative aliquots were used in experiments.
Endotoxin testing. Each lot of SP-A was tested for endotoxin, and only batches of SP-A with <0.5 U/ml of endotoxin were used in experiments. PBS, DMEM, HBSS+, and saline were tested and certified to contain <0.5 U/ml of endotoxin. Low endotoxin BSA was used in all tissue culture experiments. Endotoxin testing was performed by the Media Preparation Shared Facility at UAB.
Measurement of NO
Pathogen.
For the purposes of these studies, we utilized K. pneumoniae
[American Type Culture Collection (ATCC) 43816, type 2]. Bacterial stocks were maintained at 80°C until used. For in vitro
experiments, stocks were thawed, inoculated into broth, and grown to
log phase. All bacteria were washed free of growth media before
infection studies. Colony forming units (CFUs) were determined by
enumeration after serial dilution and inoculation onto agar plates.
K. pneumoniae was grown in BBL brain heart infusion broth,
and nutrient agar plates (DIFCO; Becton Dickinson) incubated in room
air at 37°C for 18-24 h.
SP-A binding. Binding assays were performed as previously described (26). Briefly, siliconized microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA) were filled with HBSS containing 1% BSA for 24 h at 4°C to block nonspecific binding of SP-A to plastic (55). Log-phase K. pneumoniae were resuspended to a concentration of 1010 CFUs/100 µl in HBSS containing 1% BSA, pH 7.4. Bacterial numbers were confirmed by serial dilution and culture. Bacteria were mixed with SP-A in the presence of 1 mM Ca2+ or 1 mM EGTA in the absence of Ca2+ and incubated for 30 min at room temperature. Solutions were then centrifuged to pellet bacteria. Pellets were washed twice and resuspended in 200 µl of sterile water. Solutions were sonicated to disperse pellets. Control samples were incubated in the absence of bacteria to quantify nonspecific binding of SP-A to plastic. Bacteria-associated SP-A was then measured by ELISA.
SP-A ELISA. Concentrations of SP-A in samples were determined by indirect ELISA using a polyclonal rabbit anti-human SP-A (Dr. David Phelps, Univ. of Pennsylvania, Hershey, PA) as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G 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. The wells were blocked with BSA, incubated with each antibody for 1 h at 37°C, the reaction product generated using O-phenylenediamine dihydrochloride (2.2 mM) as substrate, and the optical density read at 490 nm (23). Results of the SP-A ELISA was expressed as nanograms of SP-A per total bacteria.
Cell culture assay. Bacterial in vitro killing assay. 5 × 105 AMs were allowed to adhere to plastic in 24-well tissue culture plates for 2 h, washed, and resuspended in DMEM supplemented as described previously. For all experiments, AMs were washed and resuspended in HBSS+ (containing Ca2+ and magnesium) and 0.1% low endotoxin BSA. AMs were then infected with K. pneumoniae in a concentration ratio of 10:1-100:1 bacteria per AM in the presence or absence of SP-A. After 30 min, infected AM cultures were washed to remove unattached bacteria and resuspended in HBSS+ and 0.1% low endotoxin BSA. AMs were scraped at 0, 1, and 2 h, and aliquots were cultured for determination of viable organisms by quantitative culture. In some experiments, 3-6 µg/ml of gentamycin sulfate was added after the 30-min incubation with SP-A to remove extracellular bacteria (57).
Generation of reactive oxygen and nitrogen species.
The aim of these experiments was to expose Klebsiella to a
variety of reactive oxygen and nitrogen species at pH 7.4 and 5, the
phagosomal pH. K. pneumoniae (108) were
suspended in 10 ml of HEPES (25 mM) and exposed to the following
reactants in autoclaved 130-ml centrifuge tubes: SIN-1 (0.5 or 1 mM),
pH 7.4; PAPANONOate (100 µM), pH 7.4 or 5; and 10 mU/ml XO,
500 µM xanthine, and 100 µM FeCl3-EDTA. Because SIN-1 does not decompose at pH 5, in a subsequent series of experiments we
generated ONOO at pH 5 by coincubating 10 milliunits/ml
XO, 500 µM xanthine, 100 µM FeCl3-EDTA, and 100 µM
PAPANOate. Tubes were agitated in a shaking water bath at 37°C
(25). Aliquots were taken at 0, 15, 30, 45, 60, and 90 min
for determination of CFUs. The generation of ONOO
by
SIN-1 was calculated from the oxidation of DHR-123 (22, 32). NO was generated with 100 µM PAPANONOate in 25 mM
HEPES buffer, pH 7.4 or pH 5. NO concentration was measured with an ISO-NO electrochemical probe (World precision Instruments, Sarasota, FL). Because the rate of PAPANONOate decomposition increases markedly at low pH, additional PAPANONOate was added every 10 min to maintain [NO] at a constant level based on the measured depletion of substrate at pH 5. Reactive oxygen species were generated by 10 mU/ml XO, 500 µM xanthine, and 100 µM FeCl3-EDTA in 25 mM HEPES
buffer. Based on calculated depletion of the substrate, additional
xanthine (500 µM) was also added every 15 min to maintain constant
substrate levels. Reactivity of XO at pH 5 was adjusted according to
the production of urate as measured spectrophotometrically at 400 nm.
Ca2+ measurements. Cytosolic Ca2+ in AMs was measured with dual-excitation wavelength fluorescence microscopy using the fluorescent probe fura 2-acetoxymethyl ester. Fura 2 fluorescence was measured at an emission wavelength of 510 nm in response to excitation wavelengths of 340 and 380 nm and alternated at a rate of 25 Hz by a computer-controlled chopper assembly. AMs attached to glass coverslips in DMEM were loaded with 10 µM of dye dissolved in DMSO for 45 min at 37°C with gentle agitation every 10 min. AMs were washed and placed in normal Ringer solution for fluorescence measurements. Increases in cytosolic Ca2+ resulted in an increase in the fura 2 340-nm signal and a decrease in the 380-nm signal. The 340:380 ratios were converted into Ca2+ values using the equation of Grynkiewicz et al. (20). In situ calibration was accomplished by permeabilizing AMs with 5 µM ionomycin and measuring fluorescence at both wavelengths under Ca2+-free (2 mM EGTA) or saturating (1.5 mM CaCl2) Ca2+ conditions.
In additional experiments, the buffering action of the intracellular Ca2+ chelator BAPTA-acetoxymethyl ester on changes in AM [Ca2+]i was tested in paired AM samples. Baseline [Ca2+]i and the initial rate of elevation in [Ca2+]i in response to 1 µM ionomycin were measured in control AMs loaded with fura 2 and in the absence and presence of 2 µM BAPTA-acetoxymethyl ester.Labeling of bacteria with fluorescein.
Frozen stocks of K. pneumoniae were thawed and grown to log
phase. Bacteria were washed four times in sterile PBS and resuspended in 15 ml of 0.1 M NaCO3 buffer, pH 9.2. 5- (and-6-)Carboxyfluorescein succinymidyl ester (Molecular Probes) was
added to a final concentration of 100 µg/ml, and the suspension was
stirred at room temperature in the dark for 2 h. Labeled
Klebsiella were washed four times in sterile PBS to remove
unconjugated fluorophore, resuspended in 10% glycerol in sterile
H2O, and stored at 20°C.
Phagocytosis assay. Phagocytosis was assayed by flow cytometry as described previously (18, 49). Briefly, 5 × 105 AMs were suspended in DMEM, supplemented as described previously in the presence or absence of 2 µM BAPTA-acetoxymethyl ester, and incubated for 30 min at 37°C. AMs were infected with fluorescein-labeled K. pneumoniae at a ratio of 10 bacteria:1 AM in the presence or absence of 25 µg/ml SP-A and incubated for 1 h at 37°C with gentle shaking. Cells were centrifuged at 500 g for 10 min at 4°C to pellet, the supernatant was discarded, and 500 µl of ice-cold PBS plus 0.02% EDTA was added to stop phagocytosis. To identify extracellular bacteria, 10 µg/ml of ethidium bromide (EtBr) was added. EtBr immediately quenches the green fluorescence of extracellular bacteria by fluorescent resonance energy transfer but does not penetrate live cells. AMs with bound extracellular bacteria can therefore be distinguished from those containing phagocytosed bacteria because the former will fluoresce red and the latter will fluoresce green. Immediately after the addition of EtBr, samples were analyzed by flow cytometry.
Samples were analyzed with a FACScan flow cytometer (Becton Dickinson). The AM population was identified based on forward scatter (FSC) and side scatter (SSC) characteristics. Instrument settings and red/green fluorescence compensation were adjusted using appropriate unlabeled and single color control samples: AMs plus fluorescein-labeled Klebsiella and AMs plus EtBr. For each patient sample set, positive fluorescence was determined by gating on an unstained matched control AM population. Ten thousand events within the macrophage gate were analyzed per sample using histogram plots generated with CELLQuest analysis software (Becton Dickinson). Percentages of fluorescein- and EtBr-positive AM were determined together with mean channel fluorescence (MCF) of fluorescein- and EtBr-positive cells (an indication of mean number of phagocytosed/bound bacteria, respectively).Statistics.
All experiments were performed three times in duplicate with
n 2. Duplicate measurements were averaged and did
not contribute to n. For human sample data experiments,
n refers to total culture samples containing 5 × 105 cells with the total number of donors reported
separately for each experiment. Data were analyzed by ANOVA followed by
Tukey's multigroup comparison of the means for parametric data and by the Kruskal-Wallis ANOVA and Pearson's multigroup comparison of the
means for nonparametric data. Klebsiella CFUs were converted to common logarithms for statistical analysis. Flow cytometry data were
analyzed by paired Student's t-test. Results are expressed as means ± SE. P
0.05 was considered significant.
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RESULTS |
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SP-A- and NO-mediated killing of K. pneumoniae.
SP-A has been shown to regulate the uptake of bacteria to which it is
capable of binding (55), and there is a difference in the
ability of SP-A to bind different strains of K. pneumoniae (28). We found that K. pneumoniae ATCC 43816, type 2, bound to SP-A in a dose-dependent fashion and that this binding
was partially inhibited by the addition of EGTA (Fig.
1).
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NO and phagolysosomal involvement in bacterial killing.
To determine which specific reactive nitrogen species was involved in
Klebsiella killing, we exposed K. pneumoniae to
chemical generators of reactive species in the absence of AMs in 25 mM HEPES buffer. We utilized SIN-1 (1 mM or 500 µM), which is stable at
pH 5 but decomposes at pH 7.4, to release NO and
O.
One millimolar SIN-1 produces ~1 µM/min ONOO
in this
buffer at 37°C (25). In the presence of SIN-1,
Klebsiella CFUs continued to increase over the 90-min
incubation period (Fig. 3A). The
NO donor PAPANONOate (100 µM), which generates ~4-5 µM NO,
or PAPANONOate plus H2O2 (1 mM) also had no
effect on Klebsiella growth (Fig. 3B).
Combinations of xanthine (500 µM), XO (10 mU), and Fe-EDTA (10 µM),
which produce O
at pH 5 as measured by the oxidation of DHR-123.
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Calcium measurements.
Ca2+ release from intracellular stores is thought to play a
role in transcription, cell motility, and secretion. Previous studies have concluded that SP-A activates a phosphoinositide/Ca2+
signaling pathway in rat AMs that may be responsible for enhanced serum-independent phagocytosis of bacteria (42). We
measured changes in cytosolic Ca2+ levels within AMs from
transplant patients and normal volunteers in response to SP-A. Baseline
cytosolic Ca2+ levels were higher in AMs from normal
volunteers (110 ± 26, n = 9, 3 separate patient
samples) compared with transplant AMs (29 ± 10, n = 12, 8 separate transplant patient samples); means ± SE. In
response to SP-A, intracellular Ca2+ levels increased by
70% above baseline in eight out of eight batches of transplant AMs.
Examination of transplant patient demographics demonstrated that four
male and four female patient samples had been tested and that there
were no differences as to age, length of time from transplant, drug
therapy, or infection or rejection status (data not shown). In
contrast, SP-A decreased Ca2+ levels by 7% in AMs from
normal volunteers (P < .01; Fig.
4, A and B).
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Phagocytosis of Klebsiella by human AMs.
SP-A has been shown to enhance phagocytosis of a variety of bacteria by
rat AMs (36, 37, 45, 55). To determine the contribution of
SP-A and intracellular Ca2+ changes to phagocytosis of
Klebsiella by human AMs, we loaded paired samples of
transplant patient AMs with 2 µM BAPTA-acetoxymethyl ester and
infected them with fluorescein-labeled Klebsiella in the
presence or absence of 25 µg/ml of SP-A. To discriminate
between adherent and internalized Klebsiella, 10 µg/ml of
EtBr was added to samples immediately before analysis in the flow
cytometer. EtBr transforms fluorescein-green fluorescence into red
fluorescence by resonance energy transfer between the two fluorochromes
(49, 54). However, EtBr does not enter living cells;
therefore, ingested fluorescein-labeled Klebsiella retain
their green fluorescence while extracellular Klebsiella
fluoresce red. SP-A significantly increased phagocytosis of
fluorescein-labeled Klebsiella by 32% compared with
controls in the absence of SP-A (P < 0.01), while BAPTA-acetoxymethyl ester decreased phagocytosis by 22% compared with
controls in the absence of BAPTA-acetoxymethyl ester (P = 0.01). The addition of SP-A to BAPTA-acetoxymethyl ester-treated samples protected against BAPTA-acetoxymethyl ester inhibition of
phagocytosis but did not increase phagocytosis above control untreated
levels (Fig. 7,
A-C).
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SP-A and NO production by human AMs.
We exposed AMs from transplant patients to human interferon (IFN)-
(100-1,000 U/ml), rough (J5) and smooth (055:B5) forms of
lipopolysaccharide (LPS; 10-200 ng), BSA (1 mg/ml), SP-A (25 µg/ml), and combinations of LPS and IFN-
as well as live K. pneumoniae and measured NO
1 · 106
AMs
1, and in the second group (n = 7),
NOx increased by ~0.3
nmol · h
1 · 106
AMs
1 compared with the corresponding media control (Fig.
8). IFN-
, LPS, and BSA had no effect
on NOx levels. On the other hand, coincubation of AMs with
Klebsiella resulted in a significant increase in
NOx in all samples tested (1.31 ± 0.652 nmol · h
1 · 106
AMs
1; n = 16, 7 separate transplant patient
samples, Fig. 9A).
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DISCUSSION |
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The major findings of this study are 1) AMs from transplant patients are stimulated by SP-A or Klebsiella to produce NOx; 2) killing of Klebsiella by AMs from transplant patients requires SP-A and NOx; 3) incubation of AMs from transplant patients with SP-A increased [Ca2+]i and phagocytosis; 4) abrogation of the increase in [Ca2+]i by preincubation with BAPTA-acetoxymethyl ester decreases phagocytosis and eliminates killing of Klebsiella; 5) neither SP-A nor Klebsiella increases NOx or [Ca2+]i in AMs from normal volunteers; and 6) AMs from normal volunteers incubated with SP-A do not kill Klebsiella.
The ability of human AMs to produce NO and effect bacterial killing has been alluded to in a number of clinical studies (1, 33, 41, 47, 59); however, the majority of NO bacterial pathogenesis studies has relied on information obtained by utilizing rodent models (15, 39, 40). Although those studies have provided a vast amount of information regarding the role of NO in bacterial pathogenesis, caution must be exercised when extrapolating these results to human cells because iNOS expression and production of NO by AMs differ dramatically among species (27). Likewise, information regarding the immunomodulatory functions of SP-A in the regulation of NO production (5, 29) and in bacterial clearance (25, 28, 34, 35, 55) relies heavily on rat and mouse models.
We found that SP-A binding to K. pneumoniae ATCC 43816, serotype 2, was Ca2+ dependent. Previous reports have indicated that other strains of Klebsiella bind to SP-A much more tightly than serotype 2 (28); however, Klebsiella serotype 2 is one of the most virulent of the Klebsiella strains and has been utilized in a variety of pathogenesis studies (7, 48, 56), making it a logical choice for this series of experiments.
Consistent with in vitro studies utilizing rat AMs (28), in the presence of SP-A, transplant AMs effected a 50% decrease in Klebsiella CFUs by 60 min and a 68% decrease in CFUs by 120 min. SP-A was unable to stimulate Klebsiella killing by AMs from normal volunteers, a fact that was not surprising, considering the inability of SP-A to stimulate NO production in these cells.
Previous studies in mice utilized the NOS inhibitor NG-nitro-L-arginine methyl ester to inhibit the clearance of respiratory K. pneumoniae infection (56). The addition of the iNOS inhibitor L-NMMA to infected transplant AM cultures abrogated SP-A-mediated Klebsiella killing, confirming the importance of reactive oxygen-nitrogen intermediates for this effect. The fact that Klebsiella alone significantly upregulated NO production in AMs from transplant patients demonstrates that live bacteria are more effective in generating a response from these cells than isolated LPS. However, despite significant NO production in the absence of SP-A, the addition of SP-A stimulated a significant increase in Klebsiella killing, which indicates that SP-A may be additionally stimulating phagocytosis. These data differ from a previous report in which human AMs from patients with pulmonary fibrosis were shown to kill the intracellular pathogen bacillus Calmette-Guerin by a NO-mediated mechanism in the absence of any immunomodulatory agent such as SP-A (41).
In the absence of AMs, Klebsiella was only susceptible to
killing by generation of O (3), which
has been shown to be extremely bactericidal (9, 11, 12,
25). At pH 5, most ONOO
is in the protonated form
of peroxynitrous acid (ONOOH), which has been demonstrated to cross
membranes and thereby damage intracellular components by passive
diffusion (13). Klebsiella have prominent capsules of complex polysaccharides that form thick bundles of fibrillous structures covering the bacterial surface and protecting the
bacterium from phagocytosis and the bactericidal effects of complement
(46). While the microenvironment of the phagolysosome allows for the concentration of such toxic radicals as
ONOO
(12), the low pH may also allow ONOOH
to penetrate the thick capsule of Klebsiella and thus damage
intracellular lipids, proteins, or DNA (15, 43). Previous
studies have demonstrated that SP-A activates a
phosphoinositide/Ca2+ signaling pathway in rat AMs, leading
to enhanced serum-independent phagocytosis of bacteria
(42). Alternatively, translocation of p47phox
for the respiratory burst production of superoxide is Ca2+
dependent (60), which may provide an additional mechanism
by which Ca2+ is important for pathogen killing. Consistent
with rat studies, SP-A stimulated a significant increase in
intracellular Ca2+ levels in AMs from transplant patients;
however, SP-A decreased intracellular Ca2+ levels in AMs
from normal volunteers. These differences in Ca2+
production between transplant patients and normal volunteers in
response to SP-A were not consistent with the differential effects of
SP-A for NO production since all transplant patient AMs reacted
similarly with an increase in [Ca2+]i in
response to SP-A. Ca2+ is known to be important for NO
production by the constitutive form of NOS; however, studies with mouse
AMs have indicated that this contribution to overall NO production by
the constitutive form of NOS is very slight (~0.12
nm · h
1 · 105
AMs
1) (57). Inhibition of
intracellular Ca2+ changes abrogated SP-A-mediated
Klebsiella killing by transplant AMs, demonstrating the
requirement for Ca2+ and implicating the importance of
phagocytosis and/or superoxide production for this effect.
We utilized flow cytometry to determine the ability of SP-A to stimulate phagocytosis of Klebsiella. We found that SP-A increased the total number of transplant patient AMs that phagocytose Klebsiella by 32% above untreated AMs. This method also allowed us to determine that SP-A did not increase the total amount of surface-bound Klebsiella per AM (MCF for red-positive AMs), which suggests that phagocytosis is the rate-limiting step, i.e., adherent bacteria are more likely to be phagocytosed in the presence of SP-A. Moreover, we did not see an increase in MCF for fluorescein-positive AMs (expected if Klebsiella accumulate within the cell) in the presence of SP-A, which suggests that once Klebsiella are phagocytosed, they are rapidly degraded. In contrast, preincubation of AMs from transplant patients with BAPTA-acetoxymethyl ester decreased Klebsiella phagocytosis without affecting adherence. This finding supports the premise that SP-A-stimulated intracellular Ca2+ fluxes are important for phagocytosis of Klebsiella.
SP-A stimulated a significant increase in NO production by AMs isolated from 10 out of the 20 transplant patients tested. BALs were performed only on the transplanted lung, which raises the question of the source of the AMs utilized in these studies, i.e., donor vs. recipient. Previous studies have utilized microsatellite technology (30) and human leukocyte antigen immunocytochemistry (44) to determine that transplant recipients rapidly repopulate the transplanted organ with host cells; however, some donor cells may remain in excess of 6 mo posttransplant. The average length of time post transplant for responder AMs was 11.4 ± 4 mo for females and 6.5 mo for males, while the time post transplant for nonresponder AMs was 29.7 ± 10.3 mo for females and 10.8 ± 5.9 mo for males. These data indicate that donor cells may be contributing to any of the responses to SP-A, either positive or negative, except for the nonresponder female patients. Additionally, 8 out of 10 of the transplant responder patients were female, a finding that correlates well with previous reports of increased NO production by AMs from female rats (51).
Previous studies also have attempted to identify the emergence of an inflammatory phenotype for AMs from transplant patients (19); however, inflammatory surface antigen was only weakly expressed on AMs from patients exhibiting normal cellular content (>90% AMs), as were all the samples utilized for these studies. It should be considered that the inability of transplant AMs to respond to LPS may be attributed to the lack of LPS-binding protein because all of the AMs were cultured in the absence of serum. Serum proteins interfere with SP-A interaction with AMs, and, therefore, serum was omitted from all cell activation protocols.
AMs isolated from normal volunteers produced significantly lower
baseline levels of NO compared with transplant AMs, and exposure to
SP-A significantly decreased NO production further. SP-A has been shown
to differentially regulate NO production by rat AMs (52),
and, recently, heterogeneous response patterns of NO production were
reported in IFN--stimulated AMs from lung cancer patients (14). Our results indicate that SP-A can stimulate human
AMs to produce completely different responses dependent on the cell source. The ability of SP-A to differentially regulate NO production and yet uniformly regulate Klebsiella killing indicates that
while SP-A and NO are both necessary for the killing of this pathogen, SP-A may not be the primary trigger for NO production involved in
Klebsiella killing.
These data further support the hypothesis that the ability of SP-A to modulate cell responses is dependent on the activation state of the cell (52) and that the cellular environment is of extreme importance when attempting to understand cellular responses to external stimuli (e.g., bacteria). However, these data also seem to present a dilemma. AMs from transplant patients produce significantly larger amounts of NO and kill Klebsiella in vitro and yet transplant patients are immunosuppressed and extremely susceptible to infections. Several explanations exist for this apparent dichotomy: 1) AMs isolated from immunosuppressed patients and cultured in the absence of drugs recover quickly (17); 2) transplanted organs are by definition foreign and, therefore, inflammatory for the host; and 3) constant inflammation caused by the transplanted organ in vivo will not only activate AMs but may stimulate the production of excess reactive oxygen-nitrogen species by a variety of cell types, causing damage to the epithelial lining, SP-A, and the innate immune response. Nitration of SP-A in vitro abrogates its ability to mediate binding and phagocytosis of Pneumocystis carinii (61), and nitrated SP-A has been isolated from the edema fluid of patients with Adult Respiratory Distress Syndrome (62). Although NO defends the host against infectious agents, its effects are nonspecific, and overproduction of NO may be cytotoxic not only for microbes but also for the cells and tissues that produce it (24).
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Russell Lindsey for intellectual input and Christine Miskall and Julie Gibbs-Erwin for excellent technical support.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants RR-00149 (to J. M. Hickman-Davis) and HL-31197 and HL-51173 (to S. Matalon).
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 S. 19th St., THT 940, Birmingham, AL 35294 (E-mail Sadis.Matalon{at}ccc.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 2, 2001;10.1152/ajplung.00216.2001
Received 12 June 2001; accepted in final form 22 October 2001.
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