Pneumocystis Mediates Overexpression of Antizyme Inhibitor Resulting in Increased Polyamine Levels and Apoptosis in Alveolar Macrophages

Chung-Ping Liao,Mark E Lasbury,Shao-Hung Wang,Chen Zhang,Pamela J Durant,Yasuko Murakami,Senya Matsufuji,Chao-Hung Lee

doi:10.1074/jbc.m805787200

Abstract

Pneumocystis pneumonia (PcP) is the most common opportunistic disease in immunocompromised patients. Alveolar macrophages are responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PcP due to increased intracellular polyamine levels. In this study, the sources of polyamines and mechanisms of polyamine increase and polyamine-induced apoptosis were investigated. The level of ornithine decarboxylase (ODC) was elevated in alveolar macrophages, and the number of alveolar macrophages that took up exogenous polyamines was increased 20-fold during PcP. Monocytes, B lymphocytes, and CD8+ T lymphocytes that were recruited into the lung during PcP expressed high levels of ornithine decarboxylase, suggesting that these cells are sources of polyamines. Both protein and mRNA levels of antizyme inhibitor (AZI) were increased in alveolar macrophages during PcP. This AZI overexpression correlated with increased polyamine uptake by alveolar macrophages, because AZI expression knockdown decreased the polyamine uptake ability of these cells. AZI expression knockdown also decreased the apoptosis rate of alveolar macrophages. Pneumocystis organisms and zymosan A were found to induce AZI overexpression in alveolar macrophages, suggesting that beta-glucan, which is the major component of the Pneumocystis cell wall, induces AZI overexpression. The levels of mRNA, protein, and activity of polyamine oxidase were increased in alveolar macrophages during PcP, indicating that the H(2)O(2) generated during polyamine catabolism caused alveolar macrophages to undergo apoptosis. Taken together, results of this study indicate that Pneumocystis organisms induce AZI overexpression in alveolar macrophages, leading to increased polyamine synthesis and uptake and apoptosis rate of these cells.

Highlights

Pneumocystis is an opportunistic pathogen; it causes Pneumocystis pneumonia (PcP)3 in immunocompromised individuals, especially in patients with AIDS
Polyamine Synthesis in Alveolar Macrophages during PcP— To examine whether the increased polyamine levels in alveolar macrophages during PcP are due to increased polyamine synthesis, the expression levels of Ornithine decarboxylase (ODC), spermidine/spermine N1-acetyltransferase (SSAT), and SRM were determined by real-time RT-PCR and Western blotting
The results showed that the mRNA levels of ODC, SSAT, and SRM in alveolar macrophages from immunosuppressed (Dex) rats were 0.93, 1.54, and 1.21-fold, respectively, of those of Normal rats

Summary

The abbreviations used are

PcP, Pneumocystis pneumonia; TMP, trimethoprim; SMX, sulfamethoxazole; TNF, tumor necrosis factor; BALF, bronchoalveolar lavage fluid; ODC, ornithine decarboxylase; SRM, spermidine synthase; SMS, spermine synthase; SSAT, spermidine/spermine N1-acetyltransferase; PAO, polyamine oxidase; SMO, spermine oxidase; OAZ, ODC antizyme; Dex, dexamethasone; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; RT, reverse transcription; HRP, horseradish peroxidase; SPD, spermidine; PE, phycoerythrin; siRNA, small interference RNA; IHC, immunohistochemical; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ELISA, enzyme-linked immunosorbent assay; FAM, fluorophore 5-(and-6)-carboxyl fluorescein succinimidyl ester; AZI, antizyme inhibitor. We have found that bronchoalveolar lavage fluid (BALF) obtained from Pneumocystis-infected animals can induce normal macrophages to undergo apoptosis and that polyamine levels in BALF from animals with PcP are high [11] This BALF-induced alveolar macrophage apoptosis is diminished when the polyamines in the BALF are depleted and regained when polyamines are added back to the polyamine-depleted BALF [11]. Polyamine catabolism is activated in response to the increased intracellular polyamine levels to maintain the cellular polyamine homeostasis [14] This catabolism is mediated by spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO), and spermine oxidase (SMO). The oncoprotein c-Myc activates the expression of ODC, which produces polyamines to stimulate cell proliferation [22] Another oncoprotein, K-ras, inhibits polyamine catabolism by suppressing the transcription of SSAT, resulting in the accumulation of intracellular polyamines that cause increased cell proliferation and development of neoplasia [18]. The results showed that inflammatory cells present in the lung during PcP are sources of polyamines and that the increase in intracellular polyamine levels in alveolar macrophages are due to both de novo synthesis of polyamines and increased uptake of exogenous polyamines

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION