Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

Abstract

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.

Fig. 1.

Exacerbated lung dysfunction in SP-D^−/− mice on day 7 after allogeneic hematopoietic stem cell transplantation (HSCT). B6 SP-D^+/+ and SP-D^−/− were conditioned with cytoxan and total body irradiation and given bone marrow from B10.BR mice and inflammation-inducing donor spleen T cells. Mice were placed in a single-chamber plethysmograph and ventilated with delivered tidal volume of 100–350 μl. Effective tidal volume was measured and transpulmonary pressure was calculated using airway and intraesophageal pressures. Shown is lung compliance at a delivered tidal volume of 200 μl. Values are means ± SE for n = 3 different mice in each experimental group. *P < 0.05 compared with non-HSCT SP-D^+/+ mice. +P < 0.05 between SP-D^+/+ and SP-D^−/− mice after HSCT. Closed bars, SP-D^+/+; open bars, SP-D^−/−.

Fig. 2.

Wright-Giemsa stain of bronchoalveolar lavage fluid (BALF) cell pellets from nontransplanted (non-HSCT) SP-D^+/+ and SP-D^−/− mice, and on day 7 after allogeneic HSCT. Non-HSCT SP-D^−/− mice contained increased number of inflammatory cells and the presence of large foamy macrophages. After HSCT, there was further enhancement of the number of inflammatory cells and foamy appearance of alveolar macrophages in lungs of SP-D^−/− mice.

Fig. 3.

Absence of SP-D enhances MCP-1 and granulocyte/macrophage colony-stimulating factor (GM-CSF) BALF levels before and after allogeneic HSCT. Cytoxan/total body irradiation (Cy/TBI)-recipient SP-D^+/+ and SP-D^−/− B6 were infused with inflammation-inducing donor T cells. BALF was collected on day 7 after HSCT. TNF-α (A), IFN-γ (B), MCP-1 (C), and GM-CSF (D) were measured by sandwich ELISA. Values are means ± SE for n = 8–12 mice/group from 3 experiments. *P < 0.05 compared with non-HSCT SP-D^+/+ mice. +P < 0.05 between SP-D^+/+ and SP-D^−/− mice after HSCT. Closed bars, SP-D^+/+; open bars, SP-D^−/−.

Fig. 4.

Absence of SP-D enhances BALF·NO levels before and after allogeneic HSCT. Cy/TBI-recipient SP-D^+/+ and SP-D^−/− B6 were infused with inflammation-inducing donor T cells. BALF was collected on day 7 after HSCT. Nitrate was reduced to nitrite before measurement by the Griess reaction. Values are means ± SE for n = 8–12 mice/group from 3 experiments. *P < 0.05 compared with nontransplanted (non-HSCT) SP-D^+/+ mice. +P < 0.05 between SP-D^+/+ and SP-D^−/− mice after HSCT. Closed bars, SP-D^+/+; open bars, SP-D^−/−.

Fig. 5.

Immunoperoxidase staining of lung sections taken from non-HSCT (unmanipulated) wild-type B6 mice, and on day 7 after allogeneic HSCT from B6 and SP-D^−/− mice. Sections were incubated with nitrotyrosine antibody (NTAb) or with NTAb in the presence of 10 mM nitrotyrosine (NTblock). Increased staining of epithelium and inflammatory cells was observed after allogeneic HSCT. Shown is a representative experiment, which was repeated once (original magnification, ×100; resolution power, ×40 objective lens). NTAb binding was specific because it was completely blocked in the presence of excess antigen (NTblock).

Fig. 6.

Western blots of equal BALF volume (20 μl) obtained from nontransplanted (non-HSCT) and on day 7 after allogeneic HSCT from SP-D^+/+ and SP-D^−/− B6 mice. BALF SP-D was detected using anti-rabbit SP-D antibody. SP-D was undetectable in SP-D^−/− mice. BALF SP-A was detected using anti-rabbit SP-A antibody. SP-A protein levels were decreased in SP-D^−/− mice compared with SP-D^+/+ mice. Shown is a representative Western blot that was repeated twice.

Fig. 7.

The simultaneous absence of SP-A and SP-D increases permeability edema and BALF ^.NO levels. BALF collected from nontransplanted (non-HSCT) SP-A/D^−/− mice contained higher levels of nitrite plus nitrate compared with SP-D^−/− or wild-type mice. On day 7 after allogeneic HSCT, BALF recovered from SP-A/D^−/− mice contained higher levels of total protein (A) and nitrite plus nitrate levels (B) compared with SP-D^−/− or wild-type mice. Protein levels were determined using the bicinchoninic acid method with bovine serum albumin as the standard. Nitrate was reduced to nitrite before measurement by the Griess reaction. Values are means ± SE for n = 8–12 mice/group from 3 experiments. *P < 0.05 compared with nontransplanted (non-HSCT) SP-D^+/+ mice. +P < 0.05 between SP-D^+/+ and SP-D^−/− mice after HSCT. ^P < 0.05 comparing SP-A/D^−/− and SP-D^−/− mice. Closed bars, SP-D^+/+; hatched bars, SP-D^−/−; open bars, SP-A/D^−/−.

Fig. 8.

BALF levels of TNF-α, IFN-γ, and MCP-1 are increased in SP-A/D^−/− vs. SP-D^−/− and wild-type mice after allogeneic HSCT. Cy/TBI-recipient SP-D^+/+, SP-D^−/−, and SP-A/D^−/− B6 mice were infused with inflammation-inducing donor T cells. BALF was collected on day 7 after HSCT. TNF-α (A), IFN-γ (B), MCP-1 (C), and GM-CSF (D) were measured by sandwich ELISA. Values are means ± SE for n = 8–12 mice/group from 3 experiments. *P < 0.05 compared with nontransplanted (non-HSCT) SP-D^+/+ mice. +P < 0.05 between SP-D^+/+ and SP-D^−/− mice after HSCT. ^P < 0.05 comparing SP-A/D^−/− and SP-D^−/− mice. Closed bars, SP-D^+/+; hatched bars, SP-D^−/−; open bars, SP-A/D^−/−.