Monocyte chemoattractant protein-1 released from alveolar macrophages mediates the systemic inflammation of acute alveolar hypoxia

Abstract

Alveolar hypoxia produces rapid systemic inflammation in rats. Several lines of evidence suggest that the inflammation is not initiated by low systemic tissue partial pressure of oxygen (Po(2)) but by a mediator released into the circulation by hypoxic alveolar macrophages. The mediator activates tissue mast cells to initiate inflammation. Monocyte chemoattractant protein-1/Chemokine (C-C motif) ligand 2 (MCP-1/CCL2) is rapidly released by hypoxic alveolar macrophages. This study investigated whether MCP-1 is the mediator of the systemic inflammation of alveolar hypoxia. Experiments in rats and in alveolar macrophages and peritoneal mast cells led to several results. (1) Alveolar hypoxia (10% O(2) breathing, 60 minutes) produced a rapid (5-minute) increase in plasma MCP-1 concentrations in conscious intact rats but not in alveolar macrophage-depleted rats. (2) Degranulation occurred when mast cells were immersed in the plasma of hypoxic intact rats but not in the plasma of alveolar macrophage-depleted rats. (3) MCP-1 added to normoxic rat plasma and the supernatant of normoxic alveolar macrophages produced a concentration-dependent degranulation of immersed mast cells. (4) MCP-1 applied to the mesentery of normoxic intact rats replicated the inflammation of alveolar hypoxia. (5) The CCR2b receptor antagonist RS-102895 prevented the mesenteric inflammation of alveolar hypoxia in intact rats. Additional data suggest that a cofactor constitutively generated in alveolar macrophages and present in normoxic body fluids is necessary for MCP-1 to activate mast cells at biologically relevant concentrations. We conclude that alveolar macrophage-borne MCP-1 is a key agent in the initiation of the systemic inflammation of alveolar hypoxia.

Figure 1.

(A) Plasma monocyte chemoattractant protein–1 (MCP-1) concentrations in conscious alveolar macrophage (AMØ)–depleted (solid circles) and intact (open circles) rats before and during 1 hour of breathing 10% O₂. AMØ were depleted by tracheal instillation of clodronate-containing liposomes 4 days before the experiment. Intact rats received PBS-containing liposomes. Bars are 1 SE on either side of the mean, n = 5 rats per group for each data point. ***P < 0.01 versus corresponding normoxic control rats. ^§P < 0.01 versus corresponding value in intact rats. (B) Percentage of degranulated mast cells (MCs) after immersion in rat plasma. Peritoneal MCs (0.4 × 10⁶ cells) from normoxic, intact rats were immersed in 0.4 ml of plasma obtained from rats depicted in A at times indicated in horizontal axis. Solid bars, AMØ-depleted rats; gray bars, intact rats. ***P < 0.01 versus corresponding normoxic control rats. ^§P < 0.01 versus corresponding value in intact rats.

Figure 2.

Peritoneal MCs from normoxic, intact rats (0.4 × 10⁶ cells) were immersed in 0.4 ml of solutions containing increasing concentrations of MCP-1. MCs were immersed in normoxic AMØ supernatant (green), in normoxic plasma (blue), and in serum-free Dulbecco's minimum essential medium (DMEM) culture medium (black). Data represent means ± SE of three experiments per group for each data point. Red diamonds represent data obtained by immersing MCs in plasma drawn from AMØ-depleted rats (open diamonds) or intact rats (solid diamonds) breathing 10% O₂, as depicted in Figure 1.

Figure 3.

Representative microphotographs of postcapillary mesenteric venules of intact rats. Large black dots are used to align the optical Doppler velocimeter used to measure red cell velocity, and are occasionally moved from the vessel center to obtain a better view of the leukocyte–endothelial interface for photographs. Left and center: Photographs were taken before and after 30 minutes of breathing 10% O₂, respectively. Red arrows indicate MCs, and green arrows indicate adherent leukocytes. Hypoxia induces the degranulation of MCs, as shown by the uptake of ruthenium red, and increased adherence of leukocytes to the endothelium. Right: Venule of an intact rat pretreated with the CCR2b receptor antagonist RS-102895 (10 μM, applied topically). This photograph was obtained at 30 minutes of 10% O₂ breathing. In contrast with 30 minutes of breathing 10% O₂, pretreatment with RS-120895 prevented the degranulation of MCs and the leukocyte–endothelial adherence of alveolar hypoxia. Numbers below photographs represent mean ± SE of 5 rats in each group. LEA, leukocyte-endothelial adherence, leukocytes/100 μm, MCDI, mast cell degranulation intensity, arbitrary units. * P < 0.01 versus corresponding normoxic control rats. A complete set of the data for this series of experiments is included in the supplement.

Figure 4.

Left: Mesenteric postcapillary venule immediately before and 30 minutes after topical application of MCP-1 (30 ng/ml dissolved in serum-free DMEM). Red arrows, MCs; green arrows, adherent leukocytes. Right: Mesenteric venule 30 minutes after application of MCP-1 in a rat pretreated with RS-102895 (10 μM). Data represent the mean ± SE of 5 rats per group. *P < 0.01 versus corresponding control rats. A complete set of data for this series of experiments is included in the supplement.

Figure 5.

Left: Postcapillary mesenteric venule of an AMØ-depleted rat 30 minutes after topical application of 30 ng/ml of MCP-1 dissolved in serum-free DMEM. Right: Postcapillary mesenteric venule of an AMØ-depleted rat 30 minutes after topical application of 30 ng/ml of MCP-1 dissolved in plasma from intact rats. Data represent the mean ± SE of 5 rats in each group *P < 0.01 versus corresponding control rats. A complete set of data for this series of experiments is included in the supplement.