Distinct responses of lung and liver macrophages to acute endotoxemia: role of toll-like receptor 4.

Exposure to excessive quantities of bacterial-derived lipopolysaccharide (LPS) is associated with injury to the lung and the liver. Macrophages are thought to play a key role in the pathogenic response to LPS by releasing proinflammatory/cytotoxic mediators. Macrophage responses to LPS are mediated in large part by toll-like receptor 4 (TLR4). In the present studies we used C3H/HeJ mice, which possess a mutated nonfunctional TLR4, to examine its role in lung and liver macrophage responses to acute endotoxemia induced by LPS administration. Treatment of control C3H/HeOuJ mice with LPS (3 mg/ml, i.p.) was associated with a significant increase in the number of macrophages in both the lung and the liver. This was most prominent after 48 h, and was preceded by expression of proliferating cell nuclear antigen (PCNA), suggesting that macrophage proliferation contributes to the response. In liver, but not lung macrophages, LPS administration resulted in a rapid (within 3 h) increase in mRNA expression of Mn superoxide dismutase (SOD) and heme oxygenase-1 (HO-1), key enzymes in antioxidant defense. In contrast, HO-1 protein expression decreased 3 h after LPS administration in liver macrophages, while in lung macrophages it increased. mRNA expression of enzymes mediating the biosynthesis of eicosanoids, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), but not 12/15-lipoxygenase (LOX), was upregulated in liver macrophages 3-24 h after LPS, with no effect on lung macrophages. However, COX-2 protein expression increased in both cell types. Loss of functional TLR4 significantly blunted the effects of LPS. Thus, no major changes were observed after LPS administration in the number of lung and liver macrophages recovered from TLR4 mutant mice, or on expression of PCNA. Increases in HO-1, MnSOD, COX-2 and PGES-1 mRNA expression in liver macrophages were also reduced in these mice. Conversely, in lung macrophages, loss of functional TLR4 resulted in increased expression of COX-2 protein and 12/15-LOX mRNA. These results demonstrate distinct lung and liver macrophage responses to acute endotoxemia are mediated, in part, by functional TLR4.

Ozone-induced lung injury and sterile inflammation. Role of toll-like receptor 4.

Inhalation of toxic doses of ozone is associated with a sterile inflammatory response characterized by an accumulation of macrophages in the lower lung which are activated to release cytotoxic/proinflammatory mediators that contribute to tissue injury. Toll-like receptor 4 (TLR4) is a pattern recognition receptor present on macrophages that has been implicated in sterile inflammatory responses. In the present studies we used TLR4 mutant C3H/HeJ mice to analyze the role of TLR4 in ozone-induced lung injury, oxidative stress and inflammation. Acute exposure of control C3H/HeOuJ mice to ozone (0.8ppm for 3h) resulted in increases in bronchoalveolar lavage (BAL) lipocalin 24p3 and 4-hydroxynonenal modified protein, markers of oxidative stress and lipid peroxidation. This was correlated with increases in BAL protein, as well as numbers of alveolar macrophages. Levels of surfactant protein-D, a pulmonary collectin known to regulate macrophage inflammatory responses, also increased in BAL following ozone inhalation. Ozone inhalation was associated with classical macrophage activation, as measured by increased NF-κB binding activity and expression of TNFα mRNA. The observation that these responses to ozone were not evident in TLR4 mutant C3H/HeJ mice demonstrates that functional TLR4 contributes to ozone-induced sterile inflammation and macrophage activation.

Activation of adherent vascular neutrophils in the lung during acute endotoxemia.

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5-2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-kappaB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells.

Activation of type II alveolar epithelial cells during acute endotoxemia.

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.

Acute endotoxemia prolongs the survival of rat lung neutrophils in response to 12-O-tetradecanoyl-phorbol 13-acetate.

Acute endotoxemia is associated with prolonged survival of adherent neutrophils in the lung vasculature. In the present studies, the effects of inflammatory mediators on signaling pathways regulating neutrophil survival were examined. We found that the protein kinase C activator, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not interferon-gamma (IFN-gamma), prolonged the survival of adherent vasculature lung neutrophils from endotoxemic rats, a response that was correlated with reduced apoptosis. Although endotoxin administration to rats induced the expression of the anti-apoptotic protein Mcl-1 in lung neutrophils, TPA had no effect on this response. Endotoxin administration also induced expression of total p38 and p44/42 mitogen activated protein kinases (MAPK) in neutrophils, as well as phosphatidyl inositol 3 kinase (PI3K) and its downstream target protein kinase B (PKB). Treatment of the cells with TPA increased p38 MAPK expression in cells from both control and endotoxin treated animals. Cells from endotoxin treated, but not control animals, were found to exhibit constitutive binding activity of nuclear factor kappa B (NF-kappaB) which was blocked by TPA. In contrast, constitutive CCAAT/enhancer binding protein (C/EBP) nuclear binding activity evident in neutrophils from control animals was reduced following endotoxin administration. Moreover, this response was independent of TPA. These data suggest that NF-kappaB plays a role in TPA-induced signaling leading to prolonged survival of adherent vascular neutrophils in the lung during acute endotoxemia.