Erythrocytes Induce Proinflammatory Endothelial Activation in Hypoxia

Abstract

Although exposure to ambient hypoxia is known to cause proinflammatory vascular responses, the mechanisms initiating these responses are not understood. We tested the hypothesis that in systemic hypoxia, erythrocyte-derived H 2 O 2 induces proinflammatory gene transcription in vascular endothelium. We exposed mice or isolated, perfused murine lungs to 4 hours of hypoxia (8% O 2 ). Leukocyte counts increased in the bronchoalveolar lavage. The expression of leukocyte adhesion receptors, reactive oxygen species, and protein tyrosine phosphorylation increased in freshly recovered lung endothelial cells (FLECs). These effects were inhibited by extracellular catalase and by the removal of erythrocytes, indicating that the responses were attributable to erythrocyte-derived H 2 O 2 . Concomitant nuclear translocation of the p65 subunit of NF-kB and hypoxiainducible factor-1a stabilization in FLECs occurred only in the presence of erythrocytes. Hemoglobin binding to the erythrocyte membrane protein, band 3, induced the release of H 2 O 2 from erythrocytes and the p65 translocation in FLECs. These data indicate for the first time, to our knowledge, that erythrocytes are responsible for endothelial transcriptional responses in hypoxia.Keywords: hypoxia; erythrocytes; endothelium; lung; inflammation Systemic hypoxia, which is characterized by a decrease in the partial pressure of oxygen in blood (PO 2 ), results from a lack of oxygen in inhaled breath, or from impaired blood oxygenation because of lung disease. The circulatory response to a decrease of PO 2 is best characterized by pulmonary arterial vasoconstriction, a protective strategy that redistributes the pulmonary blood flow from hypoxic to well-ventilated regions of the lung. An additional vascular effect may involve a hypoxia-induced innate immune response, characterized by leukocyte activation and tissue injury (1). However, this effect remains controversial. Support for the immune effect derives from evidence in animal models that ambient hypoxia causes lung injury (2-5), systemic inflammation (4, 5), and vascular leakage (2, 6). A 4-hour exposure to 8% O 2 in mice activates NF-kB in astrocytes and hepatocytes (7), suggesting that hypoxia-induced proinflammatory gene transcription occurs in these cells.The evidence opposing the hypoxic immune response comes from lung lymph flow studies in adult sheep. According to these studies, ambient hypoxia does not increase lung microvascular permeability to proteins, and it does not cause pulmonary edema (8, 9). The clinical evidence for the hypoxic immune effect is mixed, and is based on studies of high-altitude pulmonary edema (HAPE), a form of lung injury that follows exposure to hypoxia at high altitude. Radioactive tracer studies in patients with HAPE confirm the sheep data insofar as lung microvascular permeability does not increase (10). Several studies indicate that the bronchoalveolar lavage (BAL) of patients with HAPE is enriched in leukocytes, proteins, and proinflammatory factors, indicating that HAPE causes lu...

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