Polyamine-mediated Apoptosis of Alveolar Macrophages during Pneumocystis Pneumonia

Mark E Lasbury,Salim Merali,Pamela J Durant,Dennis Tschang,Chad A Ray,Chao-Hung Lee

doi:10.1074/jbc.m611686200

Abstract

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N1-acetylspermine, and N1-acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.

Highlights

Pneumocystis-infected lungs usually contain much alveolar exudate and numerous inflammatory cells in perivascular and peribronchiolar areas (1–3)
Alveolar Macrophage Number in a Rat and Two Mouse Models of Pneumocystis pneumonia (Pcp)—Previous studies show that the number of alveolar macrophages decreases by 60% as early as 7 days after the initiation of Pneumocystis infection in rats and remains low for the duration of the infection (21)
The results showed that polyaminedepleted Bronchoalveolar lavage (BAL) fluids from Dex-Pc rats lost their ability to induce apoptosis in alveolar macrophages as measured by caspase-3 activation (Fig. 5, top), suggesting that polyamines were a source of apoptotic stimulus in alveolar macrophages incubated with BAL fluids from Dex-Pc rats

Summary

The abbreviations used are

Pneumocystis pneumonia; BAL, bronchoalveolar lavage; ROS, reactive oxygen species; SSAT, spermine and spermidine acetyltransferase; APAO, acetylpolyamine oxidase; ALF, alveolar lining fluid; LPS, lipopolysaccharide; H2DCFDA, 2Ј,7Ј-dichlorohydrofluorescein diacetate; HPLC, high pressure liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BisTris, 2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; TUNEL, terminal dUTP nick-end labeling; Dex, dexamethasone; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; TNF, tumor necrosis factor. Polyamines are implicated in the initiation of apoptosis of several cell types, either through direct action (32) or via the reactive oxygen species (ROS) such as H2O2 (11) produced during the back-conversion of polyamines through the action of spermine and spermidine acetyltransferase (SSAT), spermine oxidase, and acetylpolyamine oxidase (APAO) (33). These factors upset the redox potential of the cytoplasm and cause mitochondrial membrane damage and leakage of pro-apoptotic factors. Depletion of polyamines from BAL fluids of animals with Pcp suppressed their ability to induce ROS production and apoptosis of alveolar macrophages

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION