Activation of type II alveolar epithelial cells during acute endotoxemia

Abstract

Lung injury induced by acute endotoxemia is associated with increased generation of inflammatory mediators such as nitric oxide and eicosanoids, which have been implicated in the pathophysiological process. Although production of these mediators by alveolar macrophages (AM) has been characterized, the response of type II cells is unknown and was assessed in the present studies. Acute endotoxemia caused a rapid (within 1 h) and prolonged (up to 48 h) induction of nitric oxide synthase-2 (NOS-2) in type II cells but a delayed response in AM (12-24 h). In both cell types, this was associated with increased nitric oxide production. Although type II cells, and to a lesser extent AM, constitutively expressed cyclooxygenase-2, acute endotoxemia did not alter this activity. Endotoxin administration had no effect on mitogen-activated protein kinase or protein kinase B-alpha (PKB-alpha) expression. However, increases in phosphoinositide 3-kinase and phospho-PKB-alpha were observed in type II cells. The finding that this was delayed for 12-24 h suggests that these proteins do not play a significant role in the regulation of NOS-2 in this model. After endotoxin administration to rats, a rapid (within 1-2 h) activation of nuclear factor-kappaB was observed. This response was transient in type II cells but was sustained in AM. Interferon regulatory factor-1 (IRF-1) was also activated rapidly in type II cells. In contrast, IRF-1 activation was delayed in AM. These data demonstrate that type II cells, like AM, are highly responsive during acute endotoxemia and may contribute to pulmonary inflammation.

Fig. 1

Effects of acute endotoxemia on nitric oxide synthase (NOS)-2 and cyclooxygenase (Cox)-2 protein expression. Type II cells (T II) and alveolar macrophages (AM) were isolated from control animals (C) or 1–48 h after endotoxin administration. Cytoplasmic extracts were prepared from the cells immediately after isolation, and equal amounts were analyzed by Western blotting. One representative gel from three separate experiments is shown. The bands on the NOS-2 gel were scanned using Scanalytics (CSPI, Fairfax, VA). The relative densities for type II cells were as follows: C, 0.06; 1 h endotoxin (ETX), 6.47; 2 h ETX, 3.60; 12 h ETX, 4.44; 24 h ETX, 2.63; 48 h ETX, 6.53. The densities for AM were as follows: C, 0; 1 h ETX, 0; 2 h ETX, 0; 12 h ETX, 6.98; 24 h ETX, 3.59; 48 h ETX, 0.

Fig. 2

Effects of acute endotoxemia on nitric oxide production by type II cells (A) and alveolar macrophages (B). Cells isolated from control animals or 12, 24, or 48 h after endotoxin administration were incubated with medium, 100 ng/ml lipopolysaccharide (LPS), 10 U/ml inteferon (IFN)-γ, or 10 U/ml IFN-γ + 100 ng/ml LPS. Nitrite was quantified in culture supernatants 24 h later. Each point represents the mean ± SE of 3 experiments. *Significantly different (P < 0.01) from cells treated with medium, LPS, or IFN-γ.

Fig. 3

NOS-2 protein expression in type II cells and alveolar macrophages. Cells isolated from control animals (lanes 1, 3, 5, and 7) or 24 h after endotoxin administration (lanes 2, 4, 6, and 8) were treated with medium (lanes 1 and 2) or LPS + IFN-γ (lanes 3–8) in the absence (lanes 1, 2, 7, and 8) or presence of 1 μM (lanes 5 and 6) or 2.5 μM (lanes 3 and 4) pyrrolidine dithiocarbamate (PDTC). Cytoplasmic extracts were prepared 24 h later, and equal amounts of protein were analyzed by Western blotting. One representative gel from 3 separate experiments is shown. The bands on the gel were scanned using Scanalytics. The relative densities for type II cells were as follows: lane 1, 0.34; lane 2, 1.02; lane 3, 0.47; lane 4, 1.23; lane 5, 1.48; lane 6, 3.68; lane 7, 1.27; lane 8, 11.80. The densities for AM were as follows: lane 1, 0.13; lane 2, 0.56; lane 3, 0.05; lane 4, 1.39; lane 5, 0.88; lane 6, 3.22; lane 7, 2.41; lane 8, 3.92.

Fig. 4

Western blot analysis of type II cells and alveolar macrophages. Cells were isolated from control animals or 1–48 h after endotoxin administration. Cytoplasmic extracts were prepared immediately after isolation, and equal amounts were analyzed by Western blotting. One representative gel from 3 separate experiments is shown. PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B.

Fig. 5

Effects of acute endotoxemia on nuclear factor (NF)-κB nuclear binding activity. Type II cells and alveolar macrophages were isolated from control animals or 1–48 h after endotoxin administration. Nuclear extracts were prepared immediately after isolation, and equal amounts were analyzed for NF-κB binding activity by electrophoretic mobility shift assay (EMSA). Center: extracts prepared from cells collected 2 h after endotoxin administration (lane 1) were preincubated with antibodies to p50 (lane 2), p65 (lane 3), or 40-fold excess unlabeled NF-κB competitor probe (lane 4). One representative gel from 3 separate experiments is shown.

Fig. 6

Effects of acute endotoxemia on interferon regulatory factor (IRF)-1 nuclear binding activity. Type II cells and alveolar macrophages were isolated from control animals or 1–48 h after endotoxin administration. Nuclear extracts were prepared immediately after isolation, and equal amounts were analyzed for IRF-1 binding activity by EMSA. Center: extracts prepared from cells collected 24 h after endotoxin administration (lane 1) were preincubated with antibody to IRF-1 (lane 2) or 40-fold excess unlabeled IRF-1 oligonucleotide (lane 3). One representative gel from 3 separate experiments is shown.

Fig. 7

Dose-dependent inhibition of nitric oxide production by PDTC. Type II cells (A) and alveolar macrophages (B) isolated from control animals or 24 h after endotoxin (ETX) administration were cultured with LPS + IFN-γ with and without PDTC. Nitrite release was quantified in culture supernatants 24 h later. Each point represents the mean ± SE of 3 experiments.