Surfactant dysfunction in SP-A-/- and iNOS-/- mice with mycoplasma infection

Abstract

Surfactant dysfunction was studied in C57BL/6 (B6), B6.SP-A(-/-), and B6.iNOS(-/-) mice with pulmonary mycoplasma infection (10(7) colony-forming units). Cell-free bronchoalveolar lavage (BAL) from uninfected B6.SP-A(-/-) versus B6 mice had a reduced content of very large aggregates (VLA) and an increase in intermediate large aggregates (ILA), with no difference in total large aggregates (LA = VLA + ILA). However, LA from uninfected B6.SP-A(-/-) versus B6 mice contained less protein and were more sensitive to inhibition by serum albumin and lysophosphatidylcholine in pulsating bubble studies in vitro. Infection with Mycoplasma pulmonis caused significant lung injury and surfactant abnormalities in B6.SP-A(-/-), B6.iNOS(-/-), and B6 mice at 24, 48, 72 h after infection compared with uninfected mice of the same strain. Analyses of time-pooled data indicated that mycoplasma-infected B6.SP-A(-/-) and B6.iNOS(-/-) mice had significantly lower levels of LA and higher protein/phospholipid ratios in BAL compared with infected B6 mice. Infected B6.iNOS(-/-) versus B6 mice also had increased minimum surface tensions on the pulsating bubble and decreased levels of surfactant protein (SP)-B in BAL. These results indicate that pulmonary mycoplasma infection in vivo causes lung injury and surfactant abnormalities that are dependent in part on iNOS and SP-A. In addition, SP-A deficiency modifies surfactant aggregate content and lowers the inhibition resistance of LA surfactant in vitro compared with congenic normal mice.

Figure 1.

Content of different aggregate subfractions in surfactant from uninfected B6.SP-A^−/− and B6 mice. Surfactant aggregate fractions are: VLA sedimented by centrifugation at 1,500 × g for 10 min; ILA obtained when the supernatant after the 1,500 × g centrifugation was spun at 12,500 × g for 30 min; and smaller aggregates that did not sediment at 12,500 × g. *P < 0.05 and ^#P < 0.01 compared with B6 mice. Data are mean ± SEM for n = 3.

Figure 2.

Dynamic surface tension lowering of surfactant aggregates from uninfected B6.SP-A^−/− and B6 mice. Surface activity was measured on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression) at a uniform surfactant phospholipid concentration (1.0 or 2.0 mg/ml in 0.15 M NaCl + 2 mM CaCl₂). (A) Resuspended BAL. (B) Resuspended VLA pelleted from BAL by centrifugation at 1,500 × g for 10 min. (C) Resuspended ILA obtained from BAL by centrifugation of the VLA supernatant at 12,500 × g for 30 min. (D) Resuspended total large aggregates (LA = VLA + ILA). Data are mean ± SEM for n = 4–9.

Figure 2.

Dynamic surface tension lowering of surfactant aggregates from uninfected B6.SP-A^−/− and B6 mice. Surface activity was measured on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression) at a uniform surfactant phospholipid concentration (1.0 or 2.0 mg/ml in 0.15 M NaCl + 2 mM CaCl₂). (A) Resuspended BAL. (B) Resuspended VLA pelleted from BAL by centrifugation at 1,500 × g for 10 min. (C) Resuspended ILA obtained from BAL by centrifugation of the VLA supernatant at 12,500 × g for 30 min. (D) Resuspended total large aggregates (LA = VLA + ILA). Data are mean ± SEM for n = 4–9.

Figure 2.

Dynamic surface tension lowering of surfactant aggregates from uninfected B6.SP-A^−/− and B6 mice. Surface activity was measured on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression) at a uniform surfactant phospholipid concentration (1.0 or 2.0 mg/ml in 0.15 M NaCl + 2 mM CaCl₂). (A) Resuspended BAL. (B) Resuspended VLA pelleted from BAL by centrifugation at 1,500 × g for 10 min. (C) Resuspended ILA obtained from BAL by centrifugation of the VLA supernatant at 12,500 × g for 30 min. (D) Resuspended total large aggregates (LA = VLA + ILA). Data are mean ± SEM for n = 4–9.

Figure 2.

Dynamic surface tension lowering of surfactant aggregates from uninfected B6.SP-A^−/− and B6 mice. Surface activity was measured on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression) at a uniform surfactant phospholipid concentration (1.0 or 2.0 mg/ml in 0.15 M NaCl + 2 mM CaCl₂). (A) Resuspended BAL. (B) Resuspended VLA pelleted from BAL by centrifugation at 1,500 × g for 10 min. (C) Resuspended ILA obtained from BAL by centrifugation of the VLA supernatant at 12,500 × g for 30 min. (D) Resuspended total large aggregates (LA = VLA + ILA). Data are mean ± SEM for n = 4–9.

Figure 3.

Inhibition resistance to albumin for surfactant from uninfected B6.SP-A^−/− and B6 mice. (A) Surfactant concentration of 1 mg/ml phospholipid. (B) Surfactant concentration of 2 mg/ml phospholipid. Bovine serum albumin (BSA, 3.0 mg/ml) was added to resuspended BAL or LA and incubated for 30 min at 37°C before being studied on the bubble apparatus. Surface activity in the absence of BSA is shown earlier in Figures 2A and 2D. *P < 0.05 and ^#P < 0.01 compared with B6 mice at the same time of pulsation. Data are mean ± SEM for n = 4–9.

Figure 3.

Inhibition resistance to albumin for surfactant from uninfected B6.SP-A^−/− and B6 mice. (A) Surfactant concentration of 1 mg/ml phospholipid. (B) Surfactant concentration of 2 mg/ml phospholipid. Bovine serum albumin (BSA, 3.0 mg/ml) was added to resuspended BAL or LA and incubated for 30 min at 37°C before being studied on the bubble apparatus. Surface activity in the absence of BSA is shown earlier in Figures 2A and 2D. *P < 0.05 and ^#P < 0.01 compared with B6 mice at the same time of pulsation. Data are mean ± SEM for n = 4–9.

Figure 4.

Inhibition resistance to LPC for surfactant from uninfected B6.SP-A^−/− and B6 mice. (A) Surfactant concentration of 1 mg/ml phospholipid. (B) Surfactant concentration of 2 mg/ml phospholipid. C18:1 LPC (0.125 mg/ml) was added to resuspended cell-free BAL or LA and incubated for 30 min at 37°C before bubble measurements. Surface activity data in the absence of LPC are shown earlier in Figures 2A and 2D. *P < 0.05 and ^#P < 0.01 compared with B6 mice at the same time of pulsation. Data are mean ± SEM for n = 4–9.

Figure 4.

Inhibition resistance to LPC for surfactant from uninfected B6.SP-A^−/− and B6 mice. (A) Surfactant concentration of 1 mg/ml phospholipid. (B) Surfactant concentration of 2 mg/ml phospholipid. C18:1 LPC (0.125 mg/ml) was added to resuspended cell-free BAL or LA and incubated for 30 min at 37°C before bubble measurements. Surface activity data in the absence of LPC are shown earlier in Figures 2A and 2D. *P < 0.05 and ^#P < 0.01 compared with B6 mice at the same time of pulsation. Data are mean ± SEM for n = 4–9.

Figure 5.

Large aggregate content in BAL (as % of total phospholipids) for B6, B6.SP-A^−/−, and B6.iNOS^−/− mice with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu; shaded bars) or inoculated with sterile broth (solid bars) for 24–72 h. Data are mean ± SEM, with the numbers of mice analyzed in a given group shown within the bars. P values for infected versus noninfected for each mouse strain are shown below the x-axis (t tests), while P values among the various strains of infected mice are indicated in the graph (one-way ANOVA followed by post hoc Tukey's HSD test).

Figure 6.

Protein values in BAL (μg/ml) for B6, B6.SP-A^−/−, and B6.iNOS^−/− mice with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu; shaded bars) or inoculated with sterile broth (solid bars) for 24–72 h. Data are mean ± SEM, with the numbers of mice analyzed in a given group shown within the bars. P values for infected versus noninfected for each mouse strain are shown below the x-axis (t tests), while P values among the various strains of infected mice are indicated in the graph (one-way ANOVA followed by post hoc Tukey's HSD test).

Figure 7.

Protein to phospholipid ratios in BAL (μg/ml) for B6, B6.SP-A^−/−, and B6.iNOS^−/− with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu; shaded bars) or inoculated with sterile broth (solid bars) for 24–72 h. Data are mean ± SEM, with the numbers of mice analyzed in a given group shown within the bars. P values for infected versus noninfected for each mouse strain are shown below the x-axis (t tests), while P values among the various strains of infected mice are indicated in the graph (one-way ANOVA followed by post hoc Tukey's HSD test).

Figure 8.

Minimum surface tension (mN/m) values for large aggregates (LA) from B6, B6.SP-A^−/−, and B6.iNOS^−/− mice with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu; shaded bars) or inoculated with sterile broth (solid bars) for 24–72 h and surface tension data at all three times were combined for statistical analysis. Minimum surface tension was measured for LA resuspended in 0.15 M NaCl + 2 mM CaCl₂ after 0.25 min (A) or 20 min (B) of pulsation on a bubble surfactometer (37°C, 20 cycles/min, 50% area compression, 1 mg/ml phospholipid). Values are mean ± 1 SEM, with the total number of mice shown inside each bar for the indicated group. P values for infected versus noninfected mice for a given strain are shown below the x-axis (t tests), while P values among various means of strains of infected mice are indicated in the graph (one-way ANOVA followed by post hoc Tukey's HSD test).

Figure 8.

Minimum surface tension (mN/m) values for large aggregates (LA) from B6, B6.SP-A^−/−, and B6.iNOS^−/− mice with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu; shaded bars) or inoculated with sterile broth (solid bars) for 24–72 h and surface tension data at all three times were combined for statistical analysis. Minimum surface tension was measured for LA resuspended in 0.15 M NaCl + 2 mM CaCl₂ after 0.25 min (A) or 20 min (B) of pulsation on a bubble surfactometer (37°C, 20 cycles/min, 50% area compression, 1 mg/ml phospholipid). Values are mean ± 1 SEM, with the total number of mice shown inside each bar for the indicated group. P values for infected versus noninfected mice for a given strain are shown below the x-axis (t tests), while P values among various means of strains of infected mice are indicated in the graph (one-way ANOVA followed by post hoc Tukey's HSD test).

Figure 9.

Western blotting detection of SP-B in BAL from B6, B6.SP-A^−/−, and B6.iNOS^−/− mice with and without mycoplasma infection. Mice were either infected with mycoplasmas (10⁷ cfu) or inoculated with sterile broth for 48 h. Five micrograms of total protein were separated under nonreducing conditions on 12% Bis-Tris gels. For immunodetection, the proteins in the gels were transferred onto a PVDF membrane and immunostained with the primary rabbit polyclonal anti–SP-B antibody (1:1,000 dilution) and horseradish peroxidase–conjugated goat anti-rabbit polyclonal anti-IgG. SP-B was then detected with the ECL Detection system. Upper panels: Each lane represents a Western blot from the indicated strain. Contr., inoculated with sterile broth; Myc., infected with mycoplasmas. Arrows and numbers on the left column indicate molecular weights (kD) based on molecular weight standards. Lower panels: Optical densities (absolute units) of digitized blots shown in the upper panels. Values are means ± 1 SEM; number of mice for each group: wild type =5; iNOS^−/− = 9; SP-A^−/− = 5. *P < 0.05 compared with the corresponding control value.