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Divisions of
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Pulmonary and Critical Care Medicine and
Infectious Diseases, Department of Medicine, Iowa City Veterans Affairs Medical Center and University of Iowa, Iowa City, IA 52242;
Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, and Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80206; and
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Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Cincinnati, Cincinnati, OH 45267.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pulmonary surfactant is a heterogeneous multimolecular complex, composed of phospholipids and associated proteins, that lines the alveolar space. The collectin proteins of surfactant, surfactant protein-A (SP-A) and surfactant protein-D (SP-D), are produced and secreted by alveolar type II epithelial cells and nonciliated bronchiolar (Clara) cells (2, 3). These proteins are calcium-dependent carbohydrate-binding proteins that contain an N-terminus with cysteines that participate in covalent oligomerization, a collagen-like region that imparts pronounced linear character to the molecules, and a highly conserved carbohydrate recognition domain (CRD) that is necessary for carbohydrate binding. SP-A and SP-D are highly homologous to the collectins mannose binding protein and bovine conglutinin, respectively (4, 5, 6). Both SP-D and SP-A have been implicated in innate immunity of the host (5, 7, 8, 9). The observation that mice homozygous for null alleles of SP-A are more susceptible to infection from group B streptococci supports this concept (10).
SP-A is an oligomeric glycoprotein composed of 28- to 35-kDa polypeptide subunits that form a complex of six trimers through covalent and noncovalent interactions (8). SP-A enhances phagocytosis through opsonization of microorganisms and/or up-regulation of phagocyte receptor function (11, 12, 13, 14, 15, 16) and may also induce reactive oxygen intermediate formation and chemotaxis by macrophages (17, 18). Receptors for SP-A on macrophages have been described (19, 20, 21), one of which has been implicated in the effect of SP-A on the increased uptake of Mycobacterium bovis-bacillus Calmette Guérin into rat bone marrow-derived macrophages (22). SP-A enhances the interaction between M.tb and macrophages (14, 23, 24). Previous work from our laboratory has demonstrated that SP-A, through a direct interaction with the macrophage, enhances macrophage phagocytosis of virulent M.tb potentially by up-regulating mannose receptor activity (14).
Mature monomeric SP-D is a 43-kDa polypeptide that oligomerizes to form
four trimers that are covalently associated at their N termini. The
resultant 
516-kDa dodecameric protein has a cruciform shape with the
CRDs arranged peripherally at the end of long stalks (9). SP-D binds to
carbohydrates such as maltose, glucose, and mannose but binds poorly to
amino sugars (25). The role of SP-D in the microbe-host cell
interaction is a growing area of interest. SP-D has been shown to bind
to certain bacteria, influenza virus, Cryptococcus neoformans,
Pneumocystis carinii, and Aspergillus fumigatus through
lectin interactions (26, 27, 28, 29, 30, 31). These interactions can result in
aggregation of the microorganism (26, 27, 29, 31, 32) and altered
interactions with host cells (26, 30, 31, 33).
Although SP-A and SP-D are members of the same protein family, they
differ significantly in size and tertiary structure, and their
recognition of microorganisms is not identical. Additionally, SP-A is
10-fold more abundant and is more tightly associated with surfactant
phospholipids than SP-D (34). Together, these differences suggest that
SP-A and SP-D may have different roles in the innate immune response of
the host, potentially at different sites in the lung.
The aim of the present study was to investigate the interaction between M.tb and SP-D and to determine the impact of this interaction on the adherence of M.tb to human macrophages. Our studies demonstrate that SP-D binds to M.tb, but not the avirulent Mycobacterium smegmatis, and that the M.tb cell wall lipoglycan lipoarabinomannan (LAM) serves as a major binding molecule for SP-D. The SP-D-M.tb interaction reduces the adherence of bacteria to human macrophages. Thus, these studies provide evidence for different roles of SP-A and SP-D in the innate immune response to M.tb in the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium with L-glutamine (RPMI) was
purchased from Life Technologies (Grand Island, NY). Middlebrook 7H9
broth was purchased from BBL Microbiology Systems (Becton Dickinson,
Cockeysville, MD). 7H11 agar was prepared with Bacto Middlebrook 7H10
agar (Difco Laboratories, Detroit, MI), oleic acid albumin dextrose
catalase enrichment medium (Difco), 0.1% casein enzymatic hydrolysate
(Sigma, St. Louis, MO), and glycerol (Difco), as described (35), and
dispensed into 100 x 15-mm bacteriologic petri dishes.
Auramine-rhodamine stain (Difco) was purchased. Formaldehyde solution
37% w/w and potassium permanganate were purchased from EM Science
(Gibbstown, NJ). Tran35S-label was purchased from
ICN (Costa Mesa, CA). Ficoll-sodium diatrizoate (Ficoll) was purchased
from Pharmacia Fine Chemical (Piscataway, NJ). Chromerge was purchased
from Fisher Scientific (Pittsburgh, PA ). 
-D-mannosidase
(exomannosidase) was purchased from V-labs (Covington, LA). Maltose,
mannose, glucose,
N-acetyl-D-galactosamine and
N-acetyl-D-glucosamine were purchased
from EY Laboratories (San Mateo, CA). LPS from Escherichia
coli 055:B5 strain was purchased from Sigma, suspended in
endotoxin-free deionized water at 1 mg/ml, and stored frozen at
-20°C.
Growth and preparation of bacteria
Lyophilized M.tb Erdman strain (ATCC 35801) was
obtained from American Type Culture Collection (ATCC, Manassas, VA),
reconstituted, and used as described (36). Briefly, for each
experiment, aliquots of frozen stock in 7H9 broth were thawed. The
bacteria were then cultured for 9–11 days on 7H11 agar, scraped from
the culture plate into microfuge tubes containing buffer or medium and
two 4-mm glass beads, pulse vortexed briefly (1–2 s), and allowed to
settle over 30 min. The upper bacterial suspension was then removed to
a second tube. After a second settling for 10 min, an aliquot from the
second tube was transferred to a third tube and allowed to settle for
an additional 10 min. The top portion of this aliquot was used in the
experiments. The concentration of bacteria (1–2 x
108 bacteria/ml) and the degree of clumping
(
10%) were determined by counting in a Petroff-Hausser chamber.
M. smegmatis strain MC26 was provided
by Dr. Steven Clegg (Department of Microbiology, University of Iowa).
M. smegmatis was prepared as above except that the bacteria
were grown for a period of 3–4 days and then harvested. After counting
in a Petroff-Hausser chamber, the M. smegmatis suspensions
were diluted to a concentration of 1–1.5 x
108 bacteria/ml and stored frozen at -70°C
until the day of the experiment.
Collectin proteins, lipoglycans, and Abs
Recombinant rat SP-D, expressed in Chinese hamster ovary (CHO)
cells, and recombinant rat SP-D deficient in hydroxylation of proline
residues expressed in Sf9 cells
(SP-Dhyp) were prepared and characterized as
described previously (37, 38). The experiments were performed using the
CHO cell-expressed SP-D except where otherwise stated. SP-A was
prepared from whole lung lavage fluid from a patient with alveolar
proteinosis as described previously (39). The endotoxin concentration
of the collectin preparations was determined by the Limulus
amebocyte lysate assay (BioWhittaker, Walkersville, MD) and found to be
0.82 pg/ng for CHO cell-expressed SP-D, 40.8 pg/ng for
SP-Dhyp, and 1.5 pg/ng for SP-A. To produce
anti-SP-D antiserum, a New Zealand White rabbit was immunized and
boosted with 50 µg purified SP-Dhyp in IFA over
a 3-wk interval. After 1 mo, serum was harvested. The anti-SP-D
antiserum did not react with M.tb or with SP-A by ELISA (not
shown) and reacted only with SP-D in alveolar lavage fluids, but not
with purified SP-A or SP-A in alveolar lavage fluids by Western
blotting (Fig. 1
). Lipoarabinomannan
(LAM), from the virulent Erdman strain of M.tb, and rabbit
anti-LAM Ab were prepared and characterized as described (40).
M. smegmatis-type LAM (AraLAM) was provided by Dr. Patrick
Brennan and colleagues (Colorado State University, National
Institute of Allergy and Infectious Diseases, National Institutes
of Health contract NO1-AI-25147).
2,2'-Azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS substrate)
and AHRP-conjugated goat anti-rabbit IgG (GRIgG-HRP) were
purchased from Bio-Rad (Hercules, CA).
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Normal human volunteers who were not cigarette smokers and were
purified protein derivative (PPD) negative underwent bronchoalveolar
lavage (BAL) with 200 ml of saline using a previously described
procedure (41) that has been approved by the University of Iowa
Institutional Review Board for human subjects. After removing cellular
material and passing through a 0.22-µm filter, the BAL was
concentrated (CBAL) 10-fold using a Centriprep-10 concentrator (W.R.
Grace, Danvers, MA). BAL, CBAL, and the low m.w. filtrate of the CBAL
were stored in 1-ml aliquots at -70°C until the day of use. Whole
lung lavage (WLL) fluid (the first 3 liters of an 21-liter total
lavage) was obtained from a patient with alveolar proteinosis, who was
undergoing therapeutic lavage, and stored at -70°C.
Isolation and preparation of human macrophages
Monolayers of monocyte-derived macrophages (MDM) were prepared from healthy PPD-negative human volunteers as described (42). Briefly, mononuclear cells were isolated from heparinized blood on Ficoll gradients and then were cultured in Teflon wells (Savillex, Minnetonka, MN) for 5 days in the presence of 20% autologous serum. On the day of each experiment, the cells were removed from the Teflon wells and washed, then resuspended in RPMI containing 10% autologous serum. The MDM were then plated onto Chromerge-cleaned glass coverslips or plastic coverslips in 24-well tissue culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) for 2 h at 37°C (1 x 105 MDM/coverslip). The wells were washed with RPMI and then used in the experiments.
For experiments with human alveolar macrophages (AM), healthy
PPD-negative donors underwent BAL as described above. The AM were
collected by centrifugation and washed in RPMI (14). Cell viability
(
93%) was assessed using Trypan blue exclusion, and the percentage
of macrophages present (90%) was determined using a differential
cell stain (Leukostat, Fisher Scientific, Pittsburgh, PA). The cells
were then resuspended in RPMI with 10% autologous serum and plated
onto Chromerge-cleaned glass coverslips at 1 x
105 AM/coverslip. After 2 h at 37°C in 5%
CO2 and air, the monolayers were washed with RPMI
and then used in the experiments. Monolayer integrity and density were
monitored by inverted phase-contrast microscopy throughout the
experiments.
Radiolabeled SP-D-binding experiments
SP-D expressed in CHO cells was metabolically labeled with
Tran35S-label (ICN) as described (38).
M.tb was incubated in 50 mM tris-HCl, 150 mM NaCl (pH 7.5)
(TBS) and 1 mg/ml human serum albumin (HSA) in preblocked microfuge
tubes (5% HSA in TBS overnight at 4°C) with various concentrations
of [35S]SP-D in the presence and absence of
Ca2+, excess unlabeled protein, EDTA, or
competing carbohydrates for 30 min at 37°C. At the end of the
incubation period, the pellets were washed three times with ice-cold
TBS containing 2 mM CaCl2 (TBS-C) and transferred
to scintillation fluid for liquid scintillation counting in a beta
counter (Beckman, Irvine, CA). The sp. act. of
[35S]SP-D ranged from 50 to 1500 cpm/ng. The
mean ± SD of the cpm bound to triplicate pellets was calculated,
and the amount in nanograms bound per 5 x
105 bacteria was determined by dividing the cpm
bound per pellet by the sp. act. of the protein. Counts obtained in the
absence of M.tb or the presence of 10 mM EDTA were
subtracted out in each experiment (30 cpm).
Bacterial ELISA
In experiments designed to determine the saturable binding of
SP-D to M.tb, bacterial suspensions (5 x
105 bacteria in 100 µl TBS) were dried in the
wells of a microtiter plate (Dynatech, Chantilly, VA) (36), followed by
exposure to ultraviolet light for 2 h to kill viable bacteria.
After blocking with TBS-C containing 3% BSA and 0.2% Triton X-100
overnight at 4°C, the wells were then incubated with increasing
concentrations of SP-D in blocking buffer in the presence or absence of
100 mM maltose for 60 min at room temperature. After washing away
unbound SP-D, the wells were incubated with a 1:20,000 dilution of
anti-SP-D antiserum in blocking buffer for 2 h at room
temperature, washed, and then incubated with a 1:3000 dilution of
GRIgG-HRP for 2 h at room temperature. After washing, ABTS
substrate (Bio-Rad) was added. The substrate development was stopped
after 20 min by 1% oxalic acid, and the absorbance of individual wells
was determined at 405 nm in a microplate spectrophotometer (Benchmark,
Bio-Rad). The mean ± SD of triplicate wells for each
concentration of SP-D was calculated. The background (0.15 absorbance
units) for each concentration of SP-D was defined as the absorbance of
wells incubated with SP-D in the absence of M.tb and was
subtracted out in each case. The absorbance of wells containing
M.tb that were incubated with primary and secondary Abs but
not SP-D was below background levels (0.05 absorbance units). In
experiments designed to examine the divalent cation requirements for
SP-D binding to M.tb, the wells were incubated with a fixed
concentration of SP-D in the presence or absence of 0.2 mM EDTA, and in
the presence or absence of 5 mM CaCl2,
MgCl2, MnCl2, or
SrCl2. SP-D binding was then detected by ELISA as
above.
In ELISA experiments to further characterize the binding of SP-D to
mycobacteria, M.tb or M. smegmatis (2.5 x
107) was incubated in TBS-C containing 1 mg/ml
HSA with SP-D, lavage fluids, or buffer control in the presence or
absence of EDTA, maltose, and N-acetylgalactosamine for
1 h at 37°C. The reaction volume was 200 µl. The bacteria were
extensively washed in ice-cold TBS-C, resuspended in 300 µl buffer,
pipetted into triplicate wells (100 µl/well) of a 96-well microtiter
plate (Becton Dickinson), and allowed to dry overnight. The plates were
then treated with UV light for 2 h. The wells were blocked in
TBS-C containing 5% BSA at room temperature for 2 h and then
sequentially incubated with primary and secondary Abs in TBS-C
containing 0.5% BSA and processed as described above. The mean ±
SD of the absorbance for triplicate wells of each condition was
calculated. The absorbance of buffer control wells (devoid of SP-D or
BAL) was subtracted out in each case. At concentrations of SP-D
5 µg/ml, the results from the two types of ELISAs were similar.
Bacterial agglutination
M.tb was incubated with increasing concentrations of
SP-D or the control protein HSA in TBS-C in the presence or absence of
EDTA, maltose, and N-acetylgalactosamine for 1 h at
room temperature. Aliquots of the reaction mixture were dried on glass
coverslips, fixed in 10% formaldehyde, and stained with
auramine-rhodamine. Agglutination was assessed by examining 30
consecutive fields per coverslip under fluorescence microscopy at
1250x magnification.
Lectin blotting
M.tb. (2 x 107) and
purified Erdman strain LAM or AraLAM (2 or 3 µg each) were reduced
and denatured by boiling in 2% SDS and 10% 2-ME and resolved by 10%
SDS-PAGE (43) or 15% polyacrylamide, 0.96% bisacrylamide SDS-PAGE
(40). The gels were stained using the periodate silver method (44) or
transferred to nitrocellulose. After blocking nonspecific sites with
1% BSA and 0.05% Tween 20 in TBS-C, the nitrocellulose membranes were
incubated with 5 x 105 cpm/ml
[35S]SP-D in blocking buffer in the presence
and absence of 10 mM EDTA overnight at 4°C, washed extensively,
sprayed with Enhance (DuPont, Boston, MA), and exposed to x-ray film
(Eastman Kodak, Rochester, NY). Additional nitrocellulose membranes
were Western blotted with an anti-Erdman LAM primary Ab and
GRIgG-HRP secondary Ab (40). The Western blots were developed using
4-chloro-1-napthol (Bio-Rad) and hydrogen peroxide (Mallinckrodt,
Paris, KY).
Exomannosidase treatment of LAM
Erdman LAM and AraLAM (1 µg/well in carbonate-bicarbonate
buffer, pH 9.6) were adhered to wells of a microtiter plate (Dynatech)
overnight at 37°C. After washing, plate-bound LAM was incubated with
0.1 M sodium acetate buffer (pH 5.5), or with buffer containing 0.18
U/ml exomannosidase for 34 h at 37°C to remove the terminal
mannosyl oligosaccharides, as described previously (45). After blocking
with TBS-C containing 0.05% Tween 20, the wells were incubated with 4
µg/ml SP-Dhyp in blocking buffer for 1 h
at room temperature in the presence and absence of EDTA, maltose, or
N-acetylglucosamine. After washing, the wells were
sequentially incubated with anti-SP-D Ab or anti-LAM Ab for
2 h at room temperature, followed by GRIgG-HRP. Color
development with HRP substrate was stopped by 1% oxalic acid. The
absorbance in individual wells was determined in a microplate
spectrophotometer, and the mean ± SD of triplicate wells was
determined. Equivalent coating of wells with the LAMs was determined
using the Dig-Glycan Detection Kit (Boehringer Mannheim, Mannheim,
Germany). The LAMs were adhered to a microtiter plate as above and then
incubated with sodium metaperiodate and then
DIG-succinyl-
-amidocaproic acid hydrazide. After blocking, the wells
were then incubated with alkaline phosphatase-conjugated anti-DIG
Ab and developed with ABTS substrate.
M.tb adherence assay
M.tb was incubated in RPMI containing 20 mM HEPES,
and 1 mg/ml HSA (pH 7.2) (RHH) in the presence or absence of SP-D or
SP-A for 1 h at 4°C. Following the incubation period, the
M.tb/collectin mixture was added to MDM or AM monolayers on
glass coverslips and incubated for 2 h at 37°C in 5%
CO2 and air. After washing away nonadherent
bacteria, the coverslips were fixed in 10% formaldehyde, and bacteria
were stained with auramine-rhodamine. The macrophages were
counterstained using 5% potassium permanganate. The mean number (±SD)
of cell-associated bacteria per macrophage on three (three or nine in
the case of AM) coverslips per condition was determined by counting
100 consecutive macrophages per coverslip using phase-contrast and
fluorescence microscopy at 1250x magnification (42).
Additional adherence assays were conducted as above except that either the bacteria were washed before adding the mixture to coverslips, or the MDM monolayer was incubated with collectin, then washed before adding bacteria. Additionally, LPS was used in some experiments to determine the effect of the contaminating LPS of the collectins.
Electron microscopy
MDM monolayers on duplicate plastic coverslips were incubated with M.tb in the presence or absence of SP-D as described above. After 2 h at 37°C, the monolayers were washed with RPMI, then fixed in 2.5% gluteraldehyde, stained, and sectioned as described (42). The number of attached vs internalized bacteria per MDM cross-section and the percentage of MDM cross-sections that contained internalized bacteria were determined by counting greater than 20 consecutive MDM cross-sections in each monolayer under transmission electron microscopy.
Statistics
For each experiment, a ratio of experimental results to control (expressed as fold increase or percentage decrease) was obtained, and the mean ratio was then tested for a significant difference from 1 using t statistics. Statistical significance was defined as a p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the specific interaction between SP-D and
M.tb, and to characterize this interaction,
SP-D-M.tb-binding studies were conducted in the presence
and absence of various inhibitors. SP-D bound to M.tb in a
specific and saturable manner. Ten-fold excess unlabeled SP-D inhibited
2 µg/ml [35S]SP-D binding by 63% ± 8% (mean ± SEM,
n = 3). [35S]SP-D binding to
M.tb in suspension was concentration dependent and appeared
to be saturable at low concentrations of SP-D; however, at higher
concentrations, bacterial aggregation (see below) limited the analysis.
Thus, saturable binding was determined by ELISA with M.tb
adsorbed to the plastic wells to avoid the confounding variable of
bacterial agglutination. The binding of SP-D to M.tb was
found to be saturable in a range between 2–4 µg/ml (Fig. 2). A double reciprocal plot of these
data gives an approximate dissociation constant of 7.15 x
10-10 M, assuming that the molecular mass of
SP-D is 516,000 daltons and that the reaction represents a simple
bimolecular interaction. These data provide strong evidence that SP-D
binds to M.tb in a specific manner and that there is a
finite number of binding sites available on the bacteria for
interactions with SP-D.
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). By
ELISA, SP-D binding was undetectable (<0.05 A405
nm) in the presence of 0.2 mM EDTA and 5 mM
MgCl2, MnCl2, or
SrCl2. However, SP-D binding in the presence of 5
mM CaCl2 and 0.2 mM EDTA was similar to SP-D
binding in the presence of 5 mM CaCl2 and the
absence of EDTA. These data provide evidence that the binding of SP-D
to M.tb is cation dependent and exhibits a specific
requirement for calcium.
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). Maltose, glucose, and mannose were
strong inhibitors of SP-D binding. The concentrations of these
carbohydrates required to reduce the binding of SP-D to M.tb
by 50% were derived from nonlinear regression curves of the data shown
in Fig. 3
and were found to be 0.97 mM for maltose, 1.21 mM for
glucose, and 4.61 mM for mannose. N-acetylgalactosamine did
not inhibit SP-D binding at the concentrations tested. These data are
consistent with the published carbohydrate specificity of the CRD of
SP-D (25). These data provide evidence that the binding occurs between
carbohydrate moieties on the surface of M.tb and the CRD
of SP-D.
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Since SP-D is located in alveolar surfactant and M.tb
gains entry to the human host via the airspace, we examined the ability
of human SP-D in surfactant-containing pulmonary lavage fluid to bind
to M.tb. The binding of SP-D contained in BAL fluid, CBAL
fluid, and WLL fluid from a patient with pulmonary alveolar proteinosis
was assessed by ELISA (Table I).
Significant binding of SP-D was detected from BAL, CBAL, and WLL
compared with buffer control. There was no binding detected using a low
m.w. filtrate of CBAL. These results support the concept that SP-D
contained in alveolar lining fluids can bind to M.tb.
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SP-D has been found to agglutinate certain strains of E.
coli bacteria, influenza virus, Cryptococcus
neoformans, and A. fumigatus, and this is believed to
be an important host defense property of the collectin (27, 29, 31, 33). We examined for SP-D-mediated agglutination of M.tb
using a fluorescence microscopy assay (Fig. 5). Aggregation of some M.tb
bacilli was seen under control conditions, consistent with the tendency
of this microorganism to clump. However, at concentrations of SP-D
5 µg/ml, larger and more numerous aggregates were readily apparent
in all fields visualized. EDTA and maltose but not
N-acetylgalactosamine inhibited the agglutinating effect of
SP-D. These results indicate that calcium-dependent lectin interactions
of SP-D with M.tb lead to agglutination of the bacteria.
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We performed [35S]SP-D lectin-blotting
studies to identify specific molecules on M.tb that serve as
binding molecules for the protein (Fig. 6A, panel a,
lane 1). The [35S]SP-D was incubated
with reduced, SDS solubilized M.tb preparations separated by
SDS-PAGE and immobilized on nitrocellulose. SP-D binding was
predominantly to a region of the blot that corresponds with the known
electrophoretic mobility and appearance of LAM (46), a major cell
wall-associated lipoglycan of M.tb. There was also a small
degree of SP-D binding to a lower molecular size M.tb
component consistent with the location of lipomannan, another
mannosylated lipoglycan (47). Based on these results, we next examined
whether SP-D binds to purified M.tb LAM. SP-D bound to
immobilized purified Erdman LAM in the presence of calcium (Fig. 6A, panel a, lane 2; and Fig. 6B, panel b, lane 1), but not in the
presence of 10 mM EDTA (Fig. 6B, panel c).
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B, panel
b, lane 2). These data indicate that M.tb
LAM is a major SP-D-binding molecule on the surface of M.tb
and suggest that SP-D binds specifically to LAM exposed on pathogenic
but not avirulent mycobacteria. SP-D binds to the terminal mannosyl units of M.tb LAM
M.tb LAM and AraLAM differ with respect to the terminal
-(1
2)-linked mannosyl oligosaccharides ("mannose caps") that
are present on M.tb LAM (46, 48). Based on the known
affinity of SP-D for mannose and the observation that SP-D binds to
M.tb LAM but not AraLAM, we hypothesized that SP-D binds to
the mannose caps of M.tb LAM. To test this hypothesis, we
used an ELISA to examine the binding of SP-D to immobilized, intact LAM
in the absence and presence of various carbohydrates, and to
M.tb LAM devoid of the mannosyl caps (Fig. 7). SP-D bound to immobilized Erdman LAM,
consistent with the lectin-blotting studies. Maltose markedly inhibited
SP-D binding to LAM, whereas the inhibition observed with
N-acetylglucosamine was much less, consistent with the known
carbohydrate-binding specificity of the CRD. The maltose-inhibitable
SP-D binding was abolished by treatment of M.tb LAM with
exomannosidase, an enzyme that removes -(12)-linked oligomannosyl
units. Binding of SP-D to AraLAM was minimal compared with Erdman LAM,
and the small amount of maltose-inhibitable binding observed was not
influenced by treatment of AraLAM with exomannosidase. These data
provide evidence that SP-D binds to the terminal mannosyl
oligosaccharides of M.tb LAM. The adsorption of Erdman LAM
and AraLAM to the plates was comparable, based on the detection of
carbohydrate by the Digoxigenin-Glycan Assay. The absorbance at 405 nm
was .212 ± .038 for Erdman LAM and .217 ± .003 for AraLAM
(mean ± SEM, triplicate wells, n = 2). Additional
control experiments using a specific polyclonal Ab to LAM demonstrated
that exomannosidase treatment did not reduce the amount of LAM bound to
the microtiter plates (data not shown).
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All mycobacteria have carbohydrates associated with their outer
surface. However, it is likely that these complex carbohydrates differ
with respect to the amount expressed on the surface and the specific
residues included in the overall structure of the complex carbohydrate.
To determine whether SP-D binds to M. smegmatis, an
avirulent species of mycobacteria and the source for AraLAM, we used a
bacterial ELISA. By comparison with binding to M.tb, SP-D
bound minimally to M. smegmatis (Table II). In like fashion, by lectin blotting
using 2 x 107 M. smegmatis, no
binding of [35S]SP-D was observed (data not
shown). These results suggest that the binding of SP-D to
M.tb is dependent upon specific carbohydrate structures
exposed on the surface of M.tb that are not present on the
surface of M. smegmatis. Inasmuch as these results resemble
those obtained with M.tb LAM and AraLAM, they further
support the concept that M.tb LAM serves as a major binding
molecule for SP-D in the context of whole bacteria.
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SP-D has been reported to be an opsonin for A.
fumigatus (31) and influenza virus (33). We have previously
demonstrated that SP-A increases the phagocytosis of Erdman
M.tb by human macrophages (14). Since we determined that
SP-D binds to M.tb LAM, we hypothesized that this binding
would interfere with the recognition of LAM by the macrophage mannose
receptor and thereby reduce M.tb adherence to macrophages.
Alternatively, SP-D binding to M.tb could provide an
alternative route of entry for the bacterium by serving as an opsonin
that is recognized by a collectin receptor on the macrophage (49). To
examine these possibilities, we performed fluorescence microscopy
experiments to assess the adherence of M.tb to MDM in the
presence and absence of SP-D (Fig. 8 and
Table III). SP-D at a subagglutinating
concentration (0.5 µg/ml) did not demonstrate opsonic activity. In
fact, it reduced the number of bacteria that adhered to macrophages
(Fig. 8). In experiments in which an SP-D/M.tb mixture was
added to MDM, SP-D caused a 37.3% ± 3.3% (mean ± SEM,
n = 8, p < 0.001) reduction in the
adherence of bacteria compared with control. This effect was retained
if the SP-D/M.tb mixture was washed before incubation with
MDM (37.7% ± 1.8% reduced adherence, mean ± SEM,
n = 4), but lost if the MDM were incubated with SP-D
and washed before adding bacteria (Table III). These results indicate
that the reduced adherence is dependent on the SP-D-M.tb
interaction (SP-D binding to M.tb), and not due to an
SP-D-MDM interaction. In contrast, SP-A increased the adherence of
M.tb with macrophages (Fig. 8). Washing away excess
collectin from the monolayers before adding bacteria did not attenuate
the increased M.tb adherence (Table III). These results
provide evidence that an interaction between SP-A and the macrophage
plays an important role in the increased adherence of M.tb,
consistent with our previous observations (14). To control for LPS
contamination in our collectin preparations, LPS was included in some
experiments at the concentration found in the collectin preparations.
There was no effect by LPS alone on the adherence of M.tb to
macrophages (Fig. 8).
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To determine whether the reduced bacterial adherence due to SP-D correlated with decreased phagocytosis, we performed a transmission electron microscopy experiment to distinguish attached from internalized bacteria. No attached bacteria were seen in the consecutive MDM sections examined in all groups. SP-D reduced the number of internalized bacteria per MDM cross-section (control: 1.64 and 2.24 bacteria/section on two coverslips; SP-D: 0.20 and 0.78 bacteria/section and two coverslips), and the percentage of MDM cross-sections on two coverslips with internalized bacteria (control: 34.0 and 28.6%; SP-D: 8.7 and 6.6%). These results combined with the fluorescence microscopy data demonstrate that SP-D reduces the phagocytosis of M.tb by MDM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The surface of M.tb is composed primarily of carbohydrates and lipid (51). Since SP-D has been described to bind to other pathogens (27, 29, 30, 31, 52) and is felt to play an important role in the innate immune response in the lung, we investigated the potential for SP-D to bind to M.tb and the influence of this interaction on the adherence of M.tb to human macrophages. In this report, we show that the binding of SP-D to M.tb is specific, that SP-D in human BAL fluids binds to M.tb, that SP-D binds to LAM on the surface of Erdman M.tb via the mannose caps on Erdman LAM, and that the SP-D-M.tb interaction reduces the adherence of M.tb to human macrophages. Furthermore, we show that SP-D binds minimally to the avirulent mycobacterial species, M. smegmatis.
Our data provide evidence that the binding of SP-D to M.tb is specific. Saturable binding was demonstrated in a range of concentrations of SP-D that are likely to be below the actual concentration of SP-D in the normal airspace, estimated to be 50–90 µg/ml (53, 54). Saturable binding indicates that there is a finite number of binding sites available for SP-D on the surface of M.tb. We were unable to perform Scatchard analysis using radiolabeled protein and M.tb in a suspension system since higher concentrations of SP-D caused aggregation of SP-D and agglutination of M.tb. However, the estimated dissociation constant derived from our solid-phase assay (7.15 x 10-10 M) indicates that SP-D binds with high avidity to the surface of M.tb. This type of analysis is limited by the fact that SP-D is an oligomeric protein with the capability for multivalent binding.
We found that the binding of SP-D to M.tb is calcium dependent and carbohydrate inhibitable. These data indicate that SP-D binds to the surface of the bacteria via its CRD. However, it is possible that mycobacterial lectins may participate in some of the observed binding, presumably by binding to the N-linked sugar of SP-D (55, 56, 57). Although we cannot completely exclude that a mycobacterial lectin binds to SP-D, calcium-dependent mycobacterial lectins have not been described. Furthermore, the carbohydrate inhibition profile that we observed is consistent with the published carbohydrate specificity of SP-D (25). Thus, it is likely that the majority of the binding of SP-D to M.tb observed in our studies is via the CRD of the protein.
Our lectin-blotting studies indicate that LAM from the virulent Erdman
strain of M.tb is a major binding molecule for SP-D. LAM is
a complex lipoglycan from the cell wall of many mycobacteria and is
believed to be an important virulence determinant for M.tb
(58, 59, 60, 61, 62, 63). LAM serves as a ligand for the mannose receptor on
macrophages and mediates the phagocytosis of virulent M.tb
strains (40, 42, 45). The fatty acid tail of LAM serves as an anchor,
leaving the carbohydrate portion of the molecule exposed on the surface
(51, 64, 65). This carbohydrate portion is composed of an
-(16)-linked mannan core with short branching -(12)-linked
mannose side chains, followed by a large branching arabinofuran
segment. For the described M.tb LAMs, these arabinofuran
branches are 40% to 70% "capped" with approximately one to three
-(12)-linked mannosyl units. The presence of these mannose caps
on M.tb LAM contrasts sharply with AraLAM, which lacks
mannose capping and is 10% capped with inositol phosphate (48).
This structural difference is believed to be responsible for the
observation that M.tb LAM stimulates much less TNF-
production by macrophages than AraLAM (66). The mannose cap structure
on M.tb LAM also mediates the binding of LAM to the mannose
receptor (45).
Three lines of evidence indicate that SP-D binds to the mannose caps of
M.tb LAM. First, removal of the caps with exomannosidase
abolished maltose-inhibitable binding of SP-D to LAM. Exomannosidase
cleaves mannose units that are joined in the -(12) position, the
structure of the mannose caps. Second, when compared with Erdman LAM,
SP-D bound only minimally to AraLAM, which does not contain mannose
caps. The small degree of maltose-inhibitable binding to AraLAM (Figs. 6B and 7) may be the result of SP-D binding to the inositol
phosphate caps since SP-D can bind to inositol and phosphatidylinositol
(38). Third, exomannosidase did not affect the binding of SP-D to
AraLAM. This result suggests that the mannan core region and its
branching mannose side chains are not involved in the interaction
between SP-D and LAM.
Other carbohydrates present on the surface of M.tb may also participate in the binding of SP-D. We observed a small degree of binding to a region of the M.tb lectin blot consistent with lipomannan, another lipoglycan of M.tb. Free complex carbohydrates such as mannan, arabinomannan, and glucan, which have been described on the surface of M.tb (67), may potentially interact with SP-D. Our methods are less likely to detect these binding interactions, however, since some of these carbohydrates may not be resolved by SDS-PAGE.
We were unable to detect significant binding of SP-D to M. smegmatis, the source of AraLAM. There are at least three possibilities to account for this observation. First, it is possible that SP-D may bind only to the mannose-capped LAM of M.tb, which is lacking on the surface of M. smegmatis. Second, the avirulent M. smegmatis may have less surface-exposed carbohydrate for SP-D binding compared with M.tb (68). Along this line, we have observed much smaller amounts of LAM from solubilized M. smegmatis compared with Erdman M.tb by Western blotting (our unpublished observations). Third, there may be subtle differences in the spatial presentation of the exposed carbohydrates between the two species that result in reduced binding to M. smegmatis. The marked difference between SP-D binding to pathogenic and nonpathogenic mycobacteria invites speculation that the human host has developed a mechanism to identify virulent bacteria that possess unique carbohydrate surface structures. These possibilities warrant further study.
Agglutination of microorganisms is thought to be an important innate host defense mechanism. Agglutinated bacilli may be cleared more readily from the respiratory tract by the mucociliary system or may have altered interactions with host cells (13, 27). We were unable to assess macroscopic agglutination using spectrophotometry because of the self-aggregating property of M.tb, which did not allow us to observe additional macroscopic agglutinating effects of the collectin above control (our unpublished observations). Our data demonstrate, however, that SP-D causes microscopic agglutination of M.tb. The effect of microscopic agglutination on the host response to pathogens has not been determined. For M.tb, it is possible that microscopic agglutination may lead to altered phagolysosomal fusion (69) or entry of the bacterium into the host cell.
The determinants of entry of M.tb into macrophages is an active area of investigation. These bacteria survive in the intracellular environment of the macrophage whereas many other pathogens are killed. Within this context, mechanisms that enhance entry of M.tb into macrophages may be detrimental to the host, whereas mechanisms that reduce entry may be protective, at least for the initial exposure of the host to the bacterium. Molecules that regulate innate immunologic responses play an important role in this interaction. We have previously reported that SP-A enhances the phagocytosis of M.tb by human MDM and alveolar macrophages via up-regulation of macrophage mannose receptor activity (14). Others have also reported a potential opsonin effect of SP-A for M.tb (22, 23, 24).
The studies on SP-A in this report are consistent with our prior report (14). In contrast, subagglutinating concentrations of SP-D reduce macrophage adherence and phagocytosis of M.tb. Thus, the functions of SP-A and SP-D appear to differ. One prior study demonstrated that SP-A mediated phagocytosis of Gram-negative bacteria by alveolar macrophages, but SP-D did not (13). SP-D has been shown to bind to AM (70, 71). A putative receptor on human macrophages has been described recently (49), raising the possibility that there is a pathway for SP-D-mediated uptake of microorganisms. However, microorganisms differ with respect to their predominant mechanism of entry into host cells. Altering these mechanisms by the addition of a collectin, Ab, or complement is likely to change these pathways in unique ways. M.tb is phagocytosed via complement receptors and the mannose receptor found on macrophages (72). On the basis of the results of our studies, we speculate that SP-D inhibits the LAM-mannose receptor interaction. This effect of SP-D may override any potential enhancing effect of a SP-D-macrophage interaction for M.tb uptake. Further studies are needed to examine this possibility.
In summary, we found that SP-D binds to M.tb in a specific manner. This binding is likely mediated by the terminal mannosyl oligosaccharides of LAM and the CRD of the protein. The presence of SP-D reduces the adherence of M.tb to human macrophages, whereas SP-A leads to enhanced adherence. These studies indicate that SP-D and SP-A influence the M.tb-mononuclear phagocyte interaction in the lung and are consistent with the hypothesis that these two collectins serve different roles in innate immunologic responses to respiratory pathogens. Ongoing studies are addressing the net effect of combinations of surfactant proteins in the presence and absence of lipid.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Larry S. Schlesinger, SW 54 GH, Division of Infectious Diseases, Department of Internal Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: M.tb, Mycobacterium tuberculosis; SP-A, surfactant protein A; SP-D, surfactant protein D; CRD, carbohydrate recognition domain; LAM, lipoarabinomannan; AraLAM, LAM from Mycobacterium smegmatis; CHO, Chinese hamster ovary; ABTS substrate, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid); GRIgG-HRP, HRP-conjugated goat anti-rabbit IgG; BAL, bronchoalveolar lavage; CBAL, concentrated BAL; WLL, whole lung lavage; HSA, human serum albumin; MDM, monocyte-derived macrophages; A405 nm, absorbance at 405 nm; PPD, purified protein derivative; RPMI, RPMI 1640 medium with L-glutamine; AM, alveolar macrophage.
Received for publication September 25, 1998. Accepted for publication April 8, 1999.
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References |
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interferon-mediated activation of macrophages. Infect. Immun. 56:1232.. J. Immunol. 150:1886.[Abstract]
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