Dexamethasone blocks the systemic inflammation of alveolar hypoxia at several sites in the inflammatory cascade

Abstract

Alveolar hypoxia produces a rapid and widespread systemic inflammation in rats. The inflammation is initiated by the release into the circulation of monocyte chemoattractant protein-1 (MCP-1) from alveolar macrophages (AMO) activated by the low alveolar Po(2). Circulating MCP-1 induces mast cell (MC) degranulation with renin release and activation of the local renin-angiotensin system, leading to microvascular leukocyte recruitment and increased vascular permeability. We investigated the effect of dexamethasone, a synthetic anti-inflammatory glucocorticoid, on the development of the systemic inflammation of alveolar hypoxia and its site(s) of action in the inflammatory cascade. The inflammatory steps investigated were the activation of primary cultures of AMO by hypoxia, the degranulation of MCs by MCP-1 in the mesentery microcirculation of rats, and the effect of angiotensin II (ANG II) on the leukocyte/endothelial interface of the mesentery microcirculation. Dexamethasone prevented the mesentery inflammation in conscious rats breathing 10% O(2) for 4 h by acting in all key steps of the inflammatory cascade. Dexamethasone: 1) blocked the hypoxia-induced AMO activation and the release of MCP-1 and abolished the increase in plasma MCP-1 of conscious, hypoxic rats; 2) prevented the MCP-1-induced degranulation of mesentery perivascular MCs and reduced the number of peritoneal MCs, and 3) blocked the leukocyte-endothelial adherence and the extravasation of albumin induced by topical ANG II in the mesentery. The effect at each site was sufficient to prevent the AMO-initiated inflammation of hypoxia. These results may explain the effectiveness of dexamethasone in the treatment of the systemic effects of alveolar hypoxia.

Fig. 1.

Top: representative bright-field photomicrographs of mesentery postcapillary veins of an untreated rat breathing room air (A) and of an untreated rat (B) and a dexamethasone-treated rat (C) after 4 h of breathing 10% O₂ in the conscious state. Blue arrows indicate adherent or emigrated leukocytes, and red arrows indicate mast cells (MCs). The red color of MCs due to uptake of ruthenium red indicates degranulation. The large solid black circles are used to align the optical Doppler velocimeter and occasionally are moved to obtain a better image of the leukocyte-endothelial interface for photographs. The bar graphs below the bright-field images are average data (n = 5/group) of leukocyte-endothelial adherence (left), leukocyte emigration (center), and MC degranulation intensity (right). Bright-field data were obtained 30 min after the postsurgery stabilization period, with the animals in the hypoxia groups breathing 10% O₂ continuously. D–F: representative fluorescence images of mesenteric postcapillary venules of an untreated rat breathing room air (D), and of an untreated (E) and a dexamethasone-treated rat (F) after 4 h of breathing 10% O₂ in the conscious state. The graph below the fluorescence images depicts the time course of the extravascular-to-intravascular fluorescein isothiocyanate (FITC)-albumin fluorescence intensity ratio after iv injection of the dye in the three groups of rats. Data are mean ± SE of 5 rats in each group. *P < 0.05, 4 h hypoxia (Hx) untreated vs. normoxia (Nx) untreated. #P < 0.05, 4 h Hx Dexa vs. 4 h Hx untreated.

Fig. 2.

Effect of dexamethasone (Dexa) on the hypoxia-induced release of H₂O₂ (A) and monocyte chemoattractant protein (MCP)-1 (B) by primary cultures of alveolar macrophages (AMO). Cultures were equilibrated with 15% O₂-5%CO₂-balance N₂ (normoxia) and 5% O₂-5% CO₂-balance N₂ (hypoxia). Samples for supernatant MCP-1 concentration were obtained at the end of the normoxic period in the untreated group and at 15 min of hypoxia in the untreated, Dexa AMO, and Dexa rat groups. Dexa rats, AMO harvested from rats treated with dexamethasone; Dexa AMO, AMO from untreated rats to which dexamethasone (1 μM) was added to the supernatant 30 min before the experiment. Data are means ± SE of 5 primary cultures in each group. *P < 0.05 vs. corresponding average H₂O₂ concentration obtained during normoxia (A) and vs. untreated Nx sample (B).

Fig. 3.

Plasma MCP-1 concentration (top) and mean arterial blood pressure and heart rate (bottom) of conscious rats before and during 10% O₂ breathing. Data are means ± SE of 5 untreated and 5 dexamethasone-treated rats. *P < 0.05 vs. corresponding normoxic control.

Fig. 4.

Representative photomicrographs of mesentery postcapillary venules of untreated rats receiving vehicle (A) or MCP-1, 30 ng/ml (B). C: dexamethasone-treated rat receiving MCP-1. D: effect of MCP-1 on a rat pretreated with the MC stabilizer cromolyn 30 min before MCP-1. The images were obtained ∼30 min after topical application of MCP-1 (30 ng/ml) or vehicle on the mesentery. Red arrows point to MCs, and blue arrows point to adherent leukocytes (Leuko). MC degranulation in the untreated rat is evidenced by the red coloration produced by ruthenium red uptake. No MCs were identified in the image of the dexamethasone-treated rat. Cromolyn prevents the MC degranulation and leukocyte-endothelial adherence produced by topical MCP-1 applied after a 30-min control period. The data depicted in the bar graphs were obtained 30 min after administration of MCP-1 or vehicle. Data are means ± SE of 5 rats in each group. *P < 0.05, MCP-1 vs. vehicle. #P < 0.05, MCP-1 + Dexa vs. MCP-1. §P < 0.05, MCP-1 + cromolyn vs. MCP-1.

Fig. 5.

Effect of MCP-1 on the extravasation of FITC-albumin. The arrows indicate the times of iv injection of FITC-albumin and the topical administration of MCP-1 or vehicle. Data are means ± SE of 5 rats in each group. *P < 0.05, vehicle vs. MCP-1. #P < 0.05, MCP-1 + Dexa vs. MCP-1. §P < 0.05, MCP-1 + cromolyn vs. MCP-1.

Fig. 6.

Representative photomicrographs of mesentery postcapillary venules of an untreated rat given vehicle (A) or ANG II, 10 nM (B) and of a dexamethasone-treated rat given ANG II (C). Images were obtained ∼30 min after the topical administration of ANG II or vehicle. Vehicle and ANG II were applied after a 30-min control period. The red arrows point to MC, and the blue arrows point to adherent leukocytes. Notice the adherence of leukocytes in the absence of MC degranulation produced by ANG II in the untreated rat. The data depicted in the bar graph were obtained 30 min after application of ANG II or vehicle. Data are means ± SE of 5 rats/group. *P < 0.05, vehicle vs. ANG II. #P < 0.05, Dexa + ANG II vs. ANG II.

Fig. 7.

Effect of dexamethasone in the ANG II-induced increase in FITC-albumin extravasation. The vertical arrows indicate the times of injection of FITC-albumin and the topical administration of ANG II (10 nM) or vehicle. Data are means ± SE of 5 rats in each group. *P < 0.05, vehicle vs. ANG II. # P < 0.05, ANG II + Dexa vs. ANG II.