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Divisions of
*
Pulmonary and Critical Care Medicine and
Infectious Diseases, Departments of Medicine and Microbiology, Interdisciplinary Immunology Program, Iowa City Veterans Affairs Medical Center and University of Iowa, Iowa City, IA 52242; and
Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
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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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Work from our laboratory demonstrates that SP-A increases the phagocytosis of M.tb by alveolar macrophages through a direct interaction with the macrophage, potentially by up-regulating macrophage mannose receptor activity (9). In contrast to SP-A, we have recently demonstrated that SP-D reduces the phagocytosis of M.tb due in part to an interaction between SP-D and the bacterium (10). These data provided evidence for distinct immune functions of SP-A and SP-D in the lung. SP-D binds to M.tb in a manner consistent with an interaction between the carbohydrate recognition domain (CRD) of the protein and the surface of the bacterium. SP-D binds to the terminal mannosyl units (mannose caps) of the M.tb surface lipoglycan, lipoarabinomannan (LAM).
The effects of microbial agglutination in vitro on the pathogenesis of
diseases due to respiratory pathogens are largely unknown. It is
possible that agglutination could serve to enhance mucociliary
clearance of bacteria and viruses, alter host cell function such as
cytokine release or reactive oxygen species formation, or modulate the
phagocytic interactions between host cell and microbe. The
concentration of SP-D in the airspace is estimated to be 
36–212
µg/ml in the rat (2), which is much higher than
concentrations required for M.tb aggregation in vitro
(10).
The purpose of this study was to determine the relationship between agglutination of M.tb and the subsequent uptake of bacteria into macrophages. We specifically tested the role of SP-D valency on mycobacterial agglutination and the subsequent effects of altered valency on modulation of macrophage uptake. Our findings reveal that collagen domain-dependent loss of valency abolishes microbial aggregation, but does not alter the inhibitory effect of SP-D on macrophage uptake of the pathogen.
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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 (BD Biosciences, Mountain View, CA). 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 Aldrich, St. Louis, MO), and glycerol (Difco), as described (11), 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). Ficoll-sodium diatrizoate (Ficoll) was purchased from Pharmacia Fine Chemical (Piscataway, NJ). Chromerge was purchased from Fisher (Pittsburgh, PA). Maltose and N-acetyl-D-galactosamine were purchased from EY Laboratories (San Mateo, CA).
Collectin proteins and Abs
Recombinant rat SP-D (SP-D) and the collagen domain deletion mutant of SP-D (CDM) were produced in Chinese hamster ovary cells (12, 13). Briefly, the cDNA for rat SP-D or the CDM mutant were ligated into the pEE14 vector using XbaI and HindIII sites. The plasmid vectors were then transfected into Chinese hamster ovary cells using lipofectamine. Appropriate clones were selected, and then the proteins were isolated from culture supernatants over a mannose-Sepharose matrix. The CDM protein contains the carbohydrate-recognition domain, neck domain, and the N terminus of SP-D, but lacks the collagen-like region and its associated N-linked sugar. The CDM mutant forms trimers under nonreducing conditions, and has an approximate mass of 67 kDa by gel chromatography (13). A rabbit anti-SP-D antiserum was prepared as described (10).
Growth and preparation of bacteria
Lyophilized M.tb Erdman strain (ATCC 35801) was
obtained from American Tissue Culture Collection (Manassas, VA),
reconstituted, and used as described (11). 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, and allowed to settle 10 min. An aliquot from the second
tube was then 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.
Bacteria prepared in this fashion are 
90% viable by CFU assay.
Isolation and preparation of human macrophages
Monolayers of monocyte-derived macrophages (MDM) were prepared from healthy purified protein derivative-negative human volunteers using a protocol that has been approved by the Internal Review Board, College of Medicine, University of Iowa (Iowa City, IA; 9709753) as described (14). Briefly, mononuclear cells were isolated from heparinized blood on Ficoll gradients, and were then cultured in Teflon wells (Savillex, Minnetonka, MN) for 5 days in the presence of 20% autologous serum. The cells were then removed from the Teflon wells and washed, then resuspended in RPMI containing 10% autologous serum. For M.tb uptake experiments, the MDM were then plated onto Chromerge-cleaned glass coverslips in 24-well tissue culture plates (Falcon; BD Labware, Mountain View, CA) for 2 h at 37°C (1 x 105 MDM/coverslip). The wells were washed with RPMI and then used in the experiments. For M.tb intracellular growth assays, the MDM were plated in the wells of 24-well tissue culture plates for 2 h, washed with RPMI, then incubated with RPMI containing 20% serum for an additional 7 days at 37°C.
Bacterial ELISA
In experiments designed to determine the saturable binding of
SP-D or CDM to M.tb, bacteria were suspended (1.5 x
107 bacteria/ml) in 50 mM Tris-HCl containing 150
mM NaCl, pH 7.5 (TBS). Aliquots (100 µl) of the bacterial suspension
or buffer control were then dried in the wells of a microtiter plate
(Immulon 1; Dynatec Laboratories, Chantilly, VA) (10, 11),
followed by exposure to UV light for 2 h to kill viable bacteria.
After blocking with TBS containing 2 mM CaCl2,
3% BSA, and 0.2% Triton X-100 overnight at 4°C, the wells were then
incubated with increasing concentrations of SP-D or CDM in blocking
buffer for 60 min at room temperature. After washing away unbound
collectin, the wells were incubated with a 1/10,000 dilution of
anti-SP-D antiserum in blocking buffer for 2 h at room
temperature, washed, and then incubated with a 1/3,000 dilution of
HRP-conjugated goat anti-rabbit IgG for 2 h at room
temperature. After washing, ABTS substrate (Bio-Rad, Hercules,
CA) 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 (A405) in a microplate
spectrophotometer (Benchmark; Bio-Rad). The mean ± SD of
duplicate wells for each concentration of collectin was calculated in
each experiment. The background (0.15 absorbance units) for each
concentration of collectin was defined as the absorbance of wells
incubated with collectin 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/or secondary Abs,
but not collectin was below background levels (0.05 absorbance
units). In experiments to determine the calcium-dependence or
carbohydrate-inhibition of SP-D or CDM binding to M.tb,
collectins were incubated at a fixed concentration in triplicate
microtiter wells in the presence or absence of 2 mM
CaCl2, 100 mM maltose, 100 mM
N-acetyl-D-galactosamine, or 10 mM
EDTA, and then processed as described above.
Agglutination assay
To determine whether increasing concentrations of SP-D or CDM caused agglutination of M.tb, we used fluorescence microscopy combined with ordinal scale counting. Bacterial suspensions (1 x 108/ml in TBS) were incubated in the presence of 2 mM CaCl2 with increasing concentrations of SP-D or CDM, and then allowed to dry on glass coverslips. After fixing with 10% formalin, the bacteria were stained with auramine-rhodamine, and examined under fluorescence microscopy at x1250. At least 300 sequential single bacteria and clumps (two or more bacteria in direct contact) were counted per coverslip. The ratio of percentage clumps in the experimental condition (SP-D or CDM) to the percentage clumps in the control condition, human serum albumin (HSA), was used as the agglutination index.
M.tb uptake by macrophages
M.tb was incubated in RPMI containing 20 mM HEPES,
and 1 mg/ml HSA (pH 7.2) in the presence or absence of increasing
concentrations of SP-D or CDM for 1 h at 4°C. Following the
incubation period, the M.tb/collectin mixture was added to
MDM monolayers on glass coverslips and incubated for 2 h at 37°C
in 5% CO2 and air. The multiplicity of infection
was 
10 bacteria/MDM. After nonadherent bacteria were washed away,
the coverslips were fixed in 10% formalin, 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 coverslips per condition was
determined by counting 100 consecutive macrophages per coverslip
using phase-contrast and fluorescence microscopy at x1250
magnification (14). Under these conditions for Erdman
M.tb, uptake by the macrophage is equivalent to phagocytosis
as assessed by transmission electron microscopy (10).
M.tb intracellular growth assays in macrophages
Intracellular growth assays using CFU were performed as described (15). Briefly, 12-day-old MDMs were washed with RPMI, then incubated with M.tb in the presence or absence of 0.5 or 5.0 µg/ml SP-D (prepared as described above) at a multiplicity of infection of 0.5–1.0 bacteria/MDM for 2 h at 37°C. At the 2-h time point, the MDMs were washed with RPMI, then repleted with RPMI containing 10 mM HEPES (pH 7.2) and 1% autologous serum, and allowed to incubate for an additional 72 h. The supernatants and MDM monolayers were then processed for enumeration of CFU.
Statistics
For M.tb uptake and intracellular growth assays, the absolute level of uptake or growth by macrophages in each experiment varies among donors, although the pattern of experimental results is the same from donor to donor. To account for this variability, we normalized data to the internal control for each experiment. A ratio of experimental results to control (expressed as percent decrease) was obtained, and the mean ratio was then tested for a significant difference from one using t statistics. Statistical significance was defined as p < 0.05. This method has been verified by the Statistical Support Center, University of Iowa.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether a rabbit anti-rat SP-D antiserum
recognized SP-D and CDM, Western-blotting was performed (Fig. 1
). By densitometry (not shown), the
anti-SP-D antiserum recognition of recombinant rat SP-D and CDM was
comparable on a weight for weight basis. For a given weight, more
molecules of CDM are present because the m.w. of the two proteins
differ (512 kDa for SP-D, 66 kDa for CDM; Ref.
13).
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The binding of SP-D to M.tb is carbohydrate inhibitable
and calcium dependent, consistent with CRD-mediated binding of the
protein to carbohydrate structures on the surface of the bacteria
(10). However, the importance of the collagen-like region
of SP-D and its associated N-linked sugar in this
interaction is unknown. To determine whether the binding of SP-D to
M.tb is modulated by the collagen domain or
N-linked sugar, we used a previously characterized mutant
SP-D protein (CDM) that is deficient in these domains. The collagen
domain is necessary for complete oligomer (dodecamer) formation of
SP-D, but is not necessary for trimerization. Thus, the CDM protein
forms trimers but does not form dodecamers, and contains the identical
CRD of SP-D (13). Using the solid-phase bacterial ELISA
(Fig. 2), both CDM and SP-D bind to
M.tb in a saturable fashion, although the
A405 for a given concentration is less
for the CDM protein. Double reciprocal plot analyses of these data
reveal that the approximate dissociation constant of SP-D binding to
M.tb is 10-fold less than that for CDM (7.1 x
10-10 M for SP-D, 7.4 x
10-9 M for CDM), indicating that the avidity of
SP-D binding to M.tb is greater than its mutant
counterpart.
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), maltose and
EDTA inhibited the binding of CDM to M.tb, whereas the
binding in the presence of
N-acetyl-D-galactosamine was similar
to control. These data indicate that the binding of CDM to
M.tb is carbohydrate-inhibitable and calcium-dependent,
consistent with the known carbohydrate-binding properties of the CRD
of SP-D.
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Agglutination of M.tb by SP-D and CDM
The innate immune properties of SP-D are believed to be multiple.
One of these properties is agglutination of bacteria and viruses
(1). Previously, we determined that SP-D at concentrations
of 5 µg/ml agglutinated M.tb, whereas concentrations <1
µg/ml did not (10). The concentration of SP-D in
pulmonary surfactant is estimated to be 36–212 µg/ml
(2), a concentration that would be expected to cause
agglutination of the bacilli based on our previous data. We hypothesize
that the ability of SP-D to agglutinate M.tb is in part
determined by the multivalent binding properties of the protein. To
investigate this, we performed fluorescence microscopy experiments
comparing M.tb agglutination due to SP-D or CDM (Fig. 4, A and B).
Because of the hydrophobic nature of the M.tb surface, this
bacterium has some degree of inherent clumping that is observed in the
control condition (Fig. 4Aa). However, SP-D at
concentrations 1 µg/ml caused agglutination of M.tb
bacilli that was visibly apparent compared with the control condition
(Fig. 4Ab). In contrast, no agglutination above control was
measured using CDM at any concentration.
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Nonagglutinating concentrations of SP-D reduce the phagocytosis of
M.tb by human macrophages due to an interaction between the
bacteria and the protein (10). However, phagocytosis of
M.tb in the presence of agglutinating concentrations of
SP-D, such as those likely to exist in the lung, could have different
effects. SP-D could reduce phagocytosis simply by masking phagocytic
receptor ligands on the surface of the bacteria, or if agglutination is
present, could reduce phagocytosis by presentation of large, poorly
phagocytosed particles. Alternatively, it is conceivable that
agglutination of the bacteria may serve to paradoxically enhance the
net uptake of M.tb by uptake of clumps of bacteria, albeit
possibly with fewer phagocytic events. We performed experiments to
determine whether agglutination of M.tb due to SP-D alters
the reduction in bacterial uptake by MDM (Fig. 5). SP-D reduced the uptake of
M.tb by MDMs over a range of concentrations including those
that do and do not lead to agglutination of bacteria. These data
suggest that agglutination of the bacilli by SP-D does not lead to
enhanced uptake by the macrophage. The CDM mutant is trimeric rather
than dodecameric like wild-type SP-D, and does not agglutinate
M.tb (Fig. 4
). Despite this, the CDM protein showed an
equivalent reduction in uptake of bacteria by MDMs when used at much
higher molar concentrations than the concentration of SP-D. Thus,
agglutination of M.tb by SP-D does not play a significant
role in the reduction of uptake of M.tb bacilli. These data,
along with the data in our previous study, provide evidence
that SP-D reduces the phagocytosis of M.tb by binding to
ligands on the surfaceof the bacteria, and thereby modulating
the bacteria-macrophage interaction.
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Successful infection of the human host by M.tb requires
multiplication of the bacilli within macrophages. Because SP-D reduces
the phagocytosis of M.tb by macrophages, we hypothesized
that SP-D would also reduce the intracellular growth of
M.tb. To investigate this, we performed CFU assays for
intracellular growth of M.tb in MDMs in the presence and
absence of SP-D (Table I). Agglutinating
and nonagglutinating concentrations of SP-D reduced the intracellular
growth of M.tb compared with the control condition (absence
of SP-D). These data provide additional evidence that SP-D reduces the
burden of M.tb infection within macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the CRDs of SP-D and CDM are identical, these data demonstrate that the binding between SP-D and M.tb occurs via the CRD of the protein and carbohydrates on the surface of the bacteria. The CRD of SP-D is relatively specific in its binding to carbohydrates, with the highest affinity binding occurring to di- and trisaccharides, and low-affinity binding to amino sugars (16). The surface of M.tb is composed of complex and simple carbohydrates, lipoglycans, glycolipids, and glycoproteins (17). LAM, the major lipoglycan on the surface of M.tb, has terminal dimannosyl units that are acceptor molecules for SP-D binding (10, 18).
The estimated dissociation constants of SP-D and CDM binding to
M.tb differ by 10-fold. This result is of interest
because the CRDs of the proteins are identical. The most likely
explanation for this result is that SP-D in its native
dodecameric form is multivalent with respect to trimeric
CRDs. Each trimeric CRD is functionally univalent with respect to
carbohydrate binding, but in dodecameric SP-D, four trimeric CRDs
linked by the rigid collagen-like region, are available for binding to
carbohydrates (19, 20, 21). In the cruciform configuration it
is likely that two trimeric CRDs can interact with a single bacterial
surface, thereby amplifying the affinity of a single CRD.
The presence of the collagen domain is necessary for SP-D to agglutinate M.tb bacilli, because the CDM protein does not have agglutinating properties, even at very high concentrations. Therefore, it is likely that in addition to imparting multivalency to SP-D, the long, rigid structure of the collagen domain also allows for greater bridging interactions between the bacteria (20, 21). These data are supported by other studies that indicate that the aggregating properties of collectins is determined by valency, carbohydrate affinity, and the spatial array of the CRDs (13, 22, 23, 24).
Our previous and current data also demonstrate that SP-D does not enhance the interaction between M.tb and macrophages, but serves to reduce this interaction (10). Agglutination of bacteria does not appear to play a role in this observation, because the CDM protein does not agglutinate M.tb, but reduces the uptake of M.tb by human macrophages. Because SP-D binds to the terminal dimannosyl units of LAM that are ligands for the mannose receptor, these data suggest that SP-D reduces the uptake of M.tb by human macrophages by binding to the surface-exposed LAM and blocking its interaction with the mannose receptor.
Although agglutination of M.tb by SP-D was not required for the altered bacterial phagocytosis by the macrophage in this study, the possibility remains that agglutination may be an important host-defense mechanism. For instance, mucociliary clearance is important in several pulmonary infections (25). Agglutination of bacteria may enhance mucociliary clearance. In addition, postphagocytic events may be altered by aggregation of bacilli (26).
Phagocytosis of an organism is the net result of multiple interactions between the microorganism and host cell. These interactions depend upon ligation of phagocytic receptors by microbial ligands, or suitable opsonins. A microorganism that has many "natural" receptors on the surface of the host cell, may in fact enter the cell without the aid of opsonins, whereas other microorganisms that have few natural receptors may require binding by opsonins to engage phagocytic receptors. There is convincing evidence that M.tb is phagocytosed by macrophages via complement receptors and the macrophage mannose receptor in the absence of opsonins in vitro (11, 14, 27, 28). Therefore, SP-D may modulate the uptake of M.tb by altering the usual interaction between the ligands on the bacterial surface and receptors on the macrophage. The relative affinities of phagocytic receptors, opsonins, opsonin acceptor sites, and native microbial surface ligands for one another likely influence the ultimate outcome of pathogen interactions with the host. In support of these hypotheses, accumulating data indicate that the effects of the collectins, SP-A and SP-D, will be unique for different pathogens. SP-D enhances the phagocytosis of Asperigillus fumigatus conidia, and certain strains of Escherichia coli, Streptococcus pneumonia, and Staphylococcus aureus by neutrophils (22, 29). SP-D is an opsonin for Pseudomonas aeruginosa and Klebsiella pneumoniae by alveolar macrophages (30, 31). Thus, SP-D can either inhibit or enhance interactions between microbes and host cells, and as such, is not simply a nonspecific host defense protein.
Beyond cell association, the intracellular fate of organisms that are phagocytosed by macrophages will likely be influenced by surfactant proteins (7, 29, 32). Our data provide evidence that reduced uptake of M.tb is associated with reduced intracellular growth in macrophages.
Although there are similarities between SP-A and SP-D, these collectins appear to have unique host defense effects (33). The role that these proteins play in the defense of the host when both are available in the airspace is not known. It is likely that the precise role for each protein will be influenced by host-specific and microbe-specific factors. It is interesting to note that recent data suggest an association among several SP-A and SP-D alleles and the susceptibility to tuberculosis (34).
The human alveolar macrophage is a natural niche for virulent M.tb, whereas for many other pathogens, entry into this host cell is fatal for the pathogen. Those factors that enhance uptake of M.tb into macrophages, such as SP-A, are likely to be detrimental to the host. We speculate that reducing the alveolar macrophage uptake and growth of M.tb will be protective of the human host, and therefore that SP-D serves as a host defense protein against tuberculosis infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Scott Ferguson, Department of Internal Medicine, University of Iowa, C-33 GH200 Hawkins Drive, Iowa City, IA 52242. E-mail address: john-s-ferguson{at}uiowa.edu
3 Abbreviations used in this paper used in this paper: M.tb, Mycobacterium tuberculosis; SP-A, surfactant protein A; SP-D, surfactant protein D; CRD, carbohydrate recognition domain; LAM, lipoarabinomannan; HSA, human serum albumin; MDM, monocyte-derived macrophages; CDM, collagen domain deletion mutant of SP-D.
Received for publication March 9, 2001. Accepted for publication November 26, 2001.
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References |
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-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem. J. 318:505.
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 L. E. DesJardin, T. M. Kaufman, B. Potts, B. Kutzbach, H. Yi, and L. S. Schlesinger Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, Fc{gamma}RII and the mannose receptor Microbiology, October 1, 2002; 148(10): 3161 - 3171. [Abstract] [Full Text] [PDF]
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