Selective inhibition of inducible NO synthase activity in vivo reverses inflammatory abnormalities in surfactant protein D-deficient mice

Abstract

Surfactant protein D (SP-D)-deficient (SP-D-/-) mice exhibit early development of emphysema. Previously we have shown that SP-D deficiency results in increased production and activity of inducible NO synthase (iNOS). In this study, we examined whether treatment with the iNOS inhibitor 1400W could inhibit the inflammatory phenotype. Mice were treated with 1400W systemically for 7 wk from 3 wk of age. Treatment reduced total lung NO synthase activity to 14.7+/-6.1% of saline-treated 10-wk-old SP-D-/- littermates. Long-term administration of 1400W reduced lung inflammation and cellular infiltration; and significantly attenuated the increased levels of matrix metalloproteinases 2 and 9, chemokines (KC, TARC), and cytokines (IFN-gamma) seen in bronchoalveolar lavage (BAL) of SP-D-/- mice. Abrogation of these levels was associated with decreasing BAL chemotactic activity for RAW cells. Two weeks of treatment with 1400W reduced total lung NO synthase (NOS) activity to 12.7+/-6.3% of saline-treated SP-D-/- mice. Short-term iNOS inhibition resulted in attenuation of pulmonary inflammation within SP-D-/- mice as shown by decreases in total BAL cell count (63+/-6% of SP-D-/- control), macrophage size (>25 microm) within the BAL (62+/-10% of SP-D-/- control), and a percentage of BAL macrophages producing oxidants (76+/-9% of SP-D-/- control). These studies showed that s.c. delivery of 1400W can be achieved in vivo and can attenuate the inflammatory processes within SP-D deficiency. Our results represent the first report linking defects in the innate immune system in the lung with alterations in NO homeostasis.

FIGURE 1

The iNOS expression is up-regulated in an age-dependent manner within the lung tissue of SP-D^−/− mice. Lung sections of mice 6, 8, and 10 wk of age for WT (bottom) and SP-D^−/− (top) animals were stained with anti-iNOS Abs as described in Materials and Methods. The specificity of staining was confirmed using parallel sections stained with nonimmune IgG (at right). Representative images are shown for each time point (6, 8 and 10 wk). Three to four random images from different slides from multiple mice were analyzed under each condition. Original magnification was at ×200. Immunostains of lung sections reveal age-related increases of iNOS expression within the inflammatory cells and airway epithelia of SP-D^−/− mice (top) when compared with WT mice (bottom).

FIGURE 2

Treatment with 1400W from 3 wk of age resulted in significantly reduced total lung NOS activity in SP-D^−/− mice. WT and SP-D^−/− mice 3 wk of age were i.p. injected daily with saline or 1400W for 3 wk, followed by implantation of osmotic pumps for another 4 wk as described in Materials and Methods. After 7 wk of treatment, all mice were sacrificed, and lung homogenates from WT and SP-D^−/− mice were analyzed for NOS activity as described in Materials and Methods. Data are expressed as total picomoles per microgram of protein per hour. Values shown as mean ± SEM (n = 3–10 animals in each group). Animals at 6-wk-old (), 8-wk-old (), and 10-wk-old (█) time points are shown. #, p = 0.044 for significant difference from the corresponding WT level; and *, p = 0.022 from the corresponding saline level. Treatment of WT mice with saline or 1400W had no effect on lung NOS activity.

FIGURE 3

SP-D^−/− mice demonstrated significantly reduced lung inflammation following 1400W treatment. WT and SP-D^−/− mice were treated with saline or 1400W for 7 wk as described in Materials and Methods. At different time points (6, 8, and 10 wk) mice were sacrificed and inflation fixation for histopathology was performed as described in Materials and Methods. A, Representative H&E stains of lung sections from WT and SP-D^−/− mice receiving either saline (top panels) or 1400W (bottom panels) are shown at equal magnification of ×200. Saline-treated SP-D^−/− mice demonstrated age-related increases of inflammatory injury and accumulation of large alveolar macrophages (top) and reversal of these abnormalities after 1400W treatment (bottom). B, Intensity of inflammation in the lung tissue of WT or SP-D^−/− mice treated with saline or 1400W. Histologic sections of lungs from each of the mice obtained at 6, 8, 10 wk of age were scored for inflammatory changes as described in Materials and Methods. Animals at 6-wk-old (), 8-wk-old (), and 10-wk-old (█) time points are shown. Data represent median values, with 5–20 samples in each group. #, p < 0.05 for significant difference from the corresponding WT level; and *, p < 0.05 from the corresponding saline level.

FIGURE 4

Inhibition of iNOS from 3 wk of age normalized the distribution of alveolar macrophage size in SP-D^−/− mice. A, Representative Diff-Quik staining of cytospins from WT and SP-D^−/− mice treated with either saline or 1400W. B, Quantitative analysis was done by identification and counting of macrophages in BAL cytospins followed by stratification according to size. Data are expressed as a percentage of macrophages >25 microns. Data shown are mean ± SEM (n = 50 macrophages/cytosine sample per 3–10 animals in each group). Animals at 6-wk-old (); 8-wk-old (), and 10-wkold (█) time points are shown. #, p < 0.005 for significant difference from the corresponding WT level; and *, p < 0.0001 from the corresponding saline level.

FIGURE 5

Continuous inhibition of iNOS at 3 wk of age attenuated chemokine, cytokine, and MMP protein expression levels and chemotactic function of BAL in SP-D^−/− mice. A 200 μl aliquot of cell-free BAL from saline- or 1400W-treated WT and SP-D^−/− mice was assessed for chemokine (A), cytokine (B), and MMP (C) analysis by a SearchLight Technology multiplex cytokine assay. Results are expressed as a percentage of saline-treated WT. Data shown are mean ± SEM (n = 6 in each group). WT saline-treated (◻), WT 1400W-treated (), SP-D^−/− saline-treated (), and SP-D^−/− 1400W-treated (█) mice are shown. #, p < 0.005 for significant difference from the corresponding WT level; and *, p < 0.0001 from the corresponding saline level. D, BAL from WT and SP-D^−/− mice was assessed for their ability to induce RAW 264.7 macrophage migration using a modified Boyden chamber, following treatment with saline or 1400W. Data are expressed as the number of migrated cells per field. Results shown are mean ± SEM (n = 5–9 in each group). Saline-treated () and 1400W-treated (█) samples are shown. #, p < 0.005 for significant difference from the corresponding WT level; and *, p < 0.005 from the corresponding saline level.

FIGURE 6

Treatment with 1400W from 8 wk of age resulted in significantly reduced total lung NOS activity in SP-D^−/− mice. WT and SP-D^−/− mice were implanted with osmotic pumps filled with saline or 1400W. After 2 wk of treatment, lung homogenates from WT and SP-D^−/− mice were analyzed for NOS activity as described in Materials and Methods. Data are expressed as total picomoles per microgram of protein per hour. Results shown are mean ± SEM (n = 3–10 animals in each group). Bars: gray, Saline-treated () and 1400W-treated (█) samples are shown. #, p = 0.028 for significant difference from the corresponding WT level; and *, p = 0.032 from the corresponding saline level.

FIGURE 7

Inhibition of iNOS from 8 wk of age partially reversed inflammation parameters in SP-D^−/− mice. WT and SP-D^−/− mice implanted with osmotic pumps filled with saline or 1400W were sacrificed after 2 wk of treatment. A, BAL cell counts were done using a UZ3 hemocytometer as described in Materials and Methods. Data are expressed as total BAL cell count × 1000. Results shown are mean ± SEM (n = 5–20 animals in each group). Saline-treated () and 1400W-treated (█) samples are shown. #, p < 0.0001 significant difference from the corresponding WT level; and *, p = 0.002 from the corresponding saline level. B, Diff-Quik staining of cytospins from SP-D^−/− mice and littermate WT controls 2 wk after treatment with either saline or 1400W. C, Quantitation of macrophage size was done manually as described in Materials and Methods. Data are expressed as a percentage of macrophages >25 microns. Results shown are mean ± SEM (n = 3–15 animals in each group). #, p < 0.0001 significant difference from the corresponding WT level; and *, p < 0.008 from the corresponding saline level. Saline-treated () and 1400W-treated (█) samples are shown. D, BAL cells isolated from WT or SP-D^−/− mice were incubated with DCF diacetate as described in Materials and Methods. Cells were harvested on ice and green fluorescence was measured by flow cytometry. Data are expressed as the percentage of macrophages producing oxidants. Results shown are mean ± SEM (n = 4 animals in each group). Saline-treated () and 1400W-treated (█) samples are shown. Two weeks of iNOS inhibition significantly reduced level of oxidants generated by macrophages in SP-D^−/− mice. *, p = 0.031 significant difference from the corresponding saline level.