Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide

Abstract

In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.

FIG. 1.

Influence of serum and mouse surfactant on the binding of FITC-LPS to BMC. BMC (5 × 10⁵ cells) from C3H/HeOU mice, pretreated (18 h, 37°C) with LPS from S. enterica serovar Choleraesuis (10 ng/ml) in serum-free CM, were reincubated (18 h, 4°C) with FITC-LPS (0.2 μg/ml) in medium alone (A) or in medium containing 8% FCS (B), mouse surfactant (800 ng of proteins per ml) (C), or mouse SP-C (800 ng of proteins per ml) (D). Histograms represent fluorescence analyzed by FACS on the gated granulocyte population of viable (propidium iodide-negative) cells. The mean fluorescence of the fluorescent population is indicated in each panel. Data are from one representative experiment.

FIG. 2.

Comparison of the influence of different mouse surfactant proteins on the binding of FITC-LPS to BMC. BMC (5 × 10⁵ cells) from C3H/HeOU mice, pretreated (18 h, 37°C) with LPS from S. enterica serovar Choleraesuis (10 ng/ml) in serum-free CM, were reincubated (18 h, 4°C) with FITC-LPS (0.2 μg/ml) in the presence of different protein components (800 ng of proteins per ml) isolated from mouse surfactant. Crude mouse surfactant (800 ng of proteins per ml) and FCS (8%) were also used for comparison. Histograms represent the percentage of fluorescent cells (A) and the mean fluorescence (B) determined by FACS on the gated granulocyte population of viable (propidium iodide-negative) cells. Data are the arithmetic mean of duplicates, in one representative experiment. a.f.u., arbitrary fluorescence units.

FIG. 3.

Interaction of ¹²⁵I-labeled CD14 and LPS with mouse SP-C. Polypropylene plates were coated with various amounts (0 to 300 pmol) (A) or with a fixed amount (300 pmol) (B) of mouse SP-C by evaporation of solutions of the peptide in chloroform-methanol (1:1 [vol]). A solution (100 μl) of ¹²⁵I-CD14 (30,000 cpm) in 0.15 M NaCl containing BSA (600 μg/ml) alone (A) or preincubated (2 h, 20°C) with various concentrations of S. enterica serovar Minnesota Re595 LPS (B) was added to the SP-C-coated wells. After the wells were incubated for 2 h at 20°C and washed three times, the bound radioactivity was measured. The broken line represents the nonspecific binding of ¹²⁵I-CD14 to the surface of uncoated wells. The results are the means ± standard deviations of three wells from one representative experiment.

FIG. 4.

Sequences of natural and synthetic analogs of SP-C used. Palmitoyl residues (Palm) are indicated.

FIG. 5.

Interaction of ¹²⁵I-labeled CD14 and LPS with the synthetic analog SP-C(LKS). Polypropylene plates were coated with various amounts (0 to 300 pmol) (A) of SP-C(LKS) or tripalmitoyl pentapeptide or with a fixed amount (300 pmol) (B) of SP-C(LKS) by evaporation of solutions of the peptides in chloroform-methanol (1:1 [vol]). The plates were preincubated (2 h, 20°C) with 90 μl of BSA (667 μg/ml in 0.15 M NaCl) in the absence (A) or presence (B) of various concentrations of S. enterica serovar Minnesota Re595 LPS. ¹²⁵I-CD14 (30,000 cpm; 10 μl in 0.15 M NaCl) was then added, and the plates were reincubated for 2 h at 20°C. After the wells were washed five times with saline, bound radioactivity was measured. The broken line represents the nonspecific binding of ¹²⁵I-CD14 to the surface of uncoated wells. The results are the means ± standard deviations of three wells from one representative experiment.

FIG. 6.

Interaction of ¹²⁵I-labeled CD14 and LPS with different synthetic analogs of SP-C. Solutions of ¹²⁵I-CD14 (30,000 cpm in 100 μl of 0.15 M NaCl containing 60 μg of BSA) were preincubated in glass tubes (2 h, 20°C) in the absence or presence of S. enterica serovar Minnesota Re595 LPS (50 nmol). The mixtures were then added to polypropylene wells coated with 300 pmol of different SP-C analogs, and the plates were reincubated for 2 h at 20°C. After the wells were washed five times with saline, bound radioactivity was measured. The results are the means ± standard deviations of three wells from one representative experiment.

FIG. 7.

Influence of SP-C(LKS) on the interaction between ¹²⁵I-labeled CD14 and LPS. Polystyrene plates were coated with LPS by incubation (18 h, 37°C) with suspensions (150 μl) of the S. enterica serovar Minnesota Re595 LPS (20 μg/ml in Tris buffer [pH 9.6]). The plastic surface was then saturated with BSA (1 mg/ml, 1 h, 37°C) and washed. A solution (100 μl) of ¹²⁵I-CD14 (30,000 cpm in 0.15 M NaCl containing 600 μg of BSA per ml), alone or preincubated (2 h, 20°C) with various concentrations of SP-C(LKS), was added to uncoated or LPS-coated wells. After the wells were incubated for 2 h at 20°C and washed five times, the bound radioactivity was measured. Specific binding of ¹²⁵I-CD14 was the calculated difference of radioactive material bound to LPS-coated and uncoated wells. The results are the mean ± standard deviations of three wells from one representative experiment.