Malnutrition-induced macrophage apoptosis

Abstract

Background: Human and murine studies suggest protein-calorie malnutrition (PCM) results in significant host immunosuppression resulting in increased morbidity and mortality. Apoptosis has been implicated as an important mediator in the immunosuppression observed in several disease states. This study was designed to characterize macrophage apoptosis in a murine model of PCM and investigate components that regulate the apoptotic process, such as protein kinase C (PKC) and Bcl-2 activity.

Methods: Swiss-Webster mice (n = 50) were randomly assigned to receive either a control (24% protein) or a PCM diet (0% protein) for 7 days. Peritoneal macrophages were harvested and detection of apoptosis was performed by terminal deoxy-transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and propidium iodide DNA staining under baseline and pro-apoptotic conditions. Pro-apoptotic conditions included cells treated with tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL), interferon-gamma (IFN-gamma) (10 ng/mL), and a combination of both agents. In addition, levels of PKC activity and expression of Bcl-2 and p53 protein were measured.

Results: Peritoneal macrophages from PCM mice had a significantly greater amount of apoptosis at baseline and under stimulated conditions compared with controls. Levels of PCM apoptosis were elevated at baseline by TUNEL staining compared with macrophages from the control group (16.5% +/- 1.4%, versus 4.5% +/- 1.1%, P <.01). In addition, peritoneal macrophages from the malnourished animals were significantly more susceptible to the apoptotic effect of TNF-alpha and the effects of INF-gamma (27.3% +/- 2.1% and 31% +/- 1.4%) compared with control mice (5.5% +/- 0.7% and 7.2% +/- 0.5%, P <.01), respectively. Again, an increase in the baseline apoptosis rate was demonstrated in peritoneal macrophages from PCM mice compared with control fed mice (13.2% +/- 4.4% versus 4.3% +/- 3.1%, P <.01) as measured by propidium iodide staining. The combination of agents, TNF-alpha and INF-gamma, resulted in an additive apoptotic effect in the malnourished host compared with the control animals (43.4% +/- 4.7% versus 10.5% +/- 2.2%, P <.01), respectively. Furthermore, there was a significant decrease in the mean total PKC activity in the malnourished macrophages compared with results in controls (110,000 +/- 8000 versus 60,000 +/- 4000 cpm, P <.01). Similar changes were also observed in PKC cytosolic and membrane activity between both groups. In addition, Bcl-2 protein expression was significantly decreased in PCM animals compared with control animals.

Conclusions: Thus, peritoneal macrophages from PCM mice exhibit significantly greater levels of apoptosis at baseline and when stimulated with pro-apoptotic agents compared with controls. The propensity of macrophages from PCM mice to undergo apoptosis may be attributable in part to decreased PKC activity and Bcl-2 protein expression. These findings may help to explain the associated immune dysfunction observed in malnutrition.