Surfactant Protein D Inhibition of Human Macrophage Uptake of Mycobacterium tuberculosis Is Independent of Bacterial Agglutination

Reagents

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 × 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 × 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 × 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 × 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 × 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 ×1250. 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 ×1250 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.

Characterization of anti-SP-D antiserum against SP-D and CDM

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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FIGURE 1.

Rabbit anti-rat SP-D antiserum recognition of SP-D and CDM is comparable. SP-D or CDM (100 ng each) were resolved by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose membrane, then blocked with PBS containing 5% BSA. After incubation with a 1/10,000 dilution of rabbit anti-rat SP-D antiserum, the membrane was washed, then incubated with a 1/5,000 dilution of HRP-conjugated goat anti-rabbit IgG. After washing, the Western blot was developed by ECL (Amersham, Buckinghamshire, U.K.). The m.w. (×1,000) are indicated on the left, and the positions of SP-D and CDM are indicated by arrows on the right (n = 3).

The binding of SP-D and CDM to M.tb

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 × 10⁻¹⁰ M for SP-D, 7.4 × 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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FIGURE 2.

The binding of SP-D and CDM to M.tb is saturable. Increasing concentrations of SP-D or CDM were incubated in wells of a 96-well microtiter plate that contained or did not contain (background) M.tb bacilli, followed by sequential incubation of primary and secondary Abs. After development, binding was determined by subtracting the background (SP-D incubated in wells that did not contain M.tb; <0.15 A₄₀₅ in all cases) from the total A₄₀₅. The A₄₀₅ for SP-D-negative wells that contained M.tb and were incubated with primary and/or secondary Abs was less than the background described above (≤0.05 A₄₀₅). Shown is the mean ± SEM of duplicate determinations from three independent experiments. Double reciprocal plot analyses of these data give approximate dissociation constants of 7.1 × 10⁻¹⁰ M for SP-D, and 7.4 × 10⁻⁹ M for CDM, assuming that the m.w. is 512,000 Da for SP-D, and 67,000 Da for CDM.

Because the CRDs of CDM and SP-D are identical, the binding of CDM to M.tb should also be carbohydrate inhibitable and calcium-dependent. To investigate this, CDM and SP-D binding to M.tb was compared in the presence of competing carbohydrates or EDTA. By ELISA (Fig. 3⇓), 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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FIGURE 3.

The binding of SP-D and CDM to M.tb is carbohydrate and EDTA inhibitable. SP-D or CDM at 2 μg/ml was incubated in wells of a 96-well microtiter plate containing M.tb in the presence or absence of maltose, EDTA, or N-acetyl-galactosamine, as indicated. Bound SP-D or CDM (total A₄₀₅ minus the background A₄₀₅) was detected by ELISA, as described in Fig. 2⇑. Shown is the mean ± SD of triplicate determinations from one experiment representative of three.

These data combined with our earlier studies provide strong evidence that the CRD of SP-D binds to M.tb. The avidity of this interaction is enhanced by the structure imparted by the collagen domain, likely due to the multivalent nature of SP-D.

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. 4⇓Aa). However, SP-D at concentrations ≥1 μg/ml caused agglutination of M.tb bacilli that was visibly apparent compared with the control condition (Fig. 4⇓Ab). In contrast, no agglutination above control was measured using CDM at any concentration.

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FIGURE 4.

CDM does not cause agglutination of M.tb. M.tb bacilli (1 × 10⁸/ml) were incubated in increasing concentrations of SP-D or CDM, or 1 mg/ml of HSA (control) for 1 h at room temperature, then dried on glass coverslips and fixed. The bacteria were then stained with auramine-rhodamine, and visualized under fluorescence microscopy at ×1250. A, Representative fields are shown (final magnification ×1100); a, HSA; b, SP-D 10 μg/ml; and c, CDM, 10 μg/ml. B, The bacteria were counted and scored for clumping under fluorescence microscopy at ×1250. The percentage clumping in the presence of each concentration of SP-D or CDM was divided by the percentage of clumping in the control condition (13.4 ± 1.3%, mean ± SD, n = 2) to derive the agglutination index. Shown is the mean of two independent experiments.

Uptake of M.tb by human macrophages in the presence of SP-D or CDM

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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FIGURE 5.

SP-D and CDM reduce the uptake of M.tb by human macrophages. M.tb suspensions (2 × 10⁷/ml) were incubated in increasing concentrations of SP-D or CDM for 1 h at 4°C. The collectin/M.tb mixtures were then incubated with human MDM for 2 h at 37°C. After washing away nonadherent bacteria, the monolayers were fixed in 10% formalin. The bacteria were stained with auramine-rhodamine, and visualized under phase-contrast and fluorescence microscopy. The number of bacteria per MDM was enumerated by counting ≥100 consecutive MDM per coverslip. The percent of control uptake was calculated by dividing the mean number of bacteria per MDM in the presence of collectin by the control value (mean number of bacteria/MDM in the absence of collectin) × 100. The control level of bacteria/MDM was 1.77 ± 0.35 (mean ± SEM, n = 7). Shown is the mean ± SEM of triplicate determinations from three to seven independent experiments. ∗, p < 0.05 vs control.

Intracellular growth of M.tb in macrophages in the presence of SP-D

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.