Treatment of the macrophage-like P388D.1 cells with bacterial lipopolysaccharide and interferon-γ causes long-term alterations in calcium metabolism

Abstract

The intracellular calcium concentrations ([Ca²⁺]_i) of P338D.1 macrophage-like cells, activated with interferon-γ (IFN-γ) and/or bacterial lipopolysaccharide (LPS) were determined using fura-2/AM and ratiometric imaging techniques. Treatment of macrophages with IFN-γ and LPS resulted in significant downward shift in [Ca²⁺]_i, 8, 16 and 24 h but not at 1 and 4 h after treatment. The decrease in [Ca²⁺]_i also occurred when macrophages were treated with LPS only, but not after exposure of the cells to recombinant IFN-γ, indicating that LPS was an essential signal in the observed changes in [Ca²⁺]_i of activated macrophages. The IFN-γ and/or LPS alteration in the [Ca²⁺]_i, paralleled the in vitro nitric oxide production of the activated macrophages, 8, 16 and 24 h after treatment. The decrease in the [Ca²⁺]_i may be caused by vigorous buffering and storing of Ca²⁺ by macrophages to below the normal resting quantities, following the reported transient increase in Ca²⁺ during the priming stage of macrophage activation. Thus, the downward shift in [Ca²⁺]_I may play a physiological role in the activation processes of macrophages for antimicrobial responses.

Introduction

The activation of macrophages for cytotoxic function is a two-stage receptor-mediated process requiring the exposure of macrophages to both a priming agent and a triggering agent [1], [2], [3]. The involvement of calcium as a second-messenger molecule during this activation process has yet to be satisfactorily ascertained. It is clear that certain inflammatory mediators such as platelet activating factor (PAF) and chemotactic factors induce transient biphasic increases in the intracellular calcium concentration ([Ca²⁺]_i) of macrophages within minutes of treatment [4], [5]. Although these factors induce chemotaxis and the production of inflammatory regulators by macrophages, they neither prime nor trigger macrophage cytotoxic functions. Early studies on macrophages treated with the potent triggering agent lipopolysaccharide (LPS) did not uncover immediate changes in intracellular Ca²⁺ levels [6], [7], [8]. In contrast, recent studies have shown that the perfusion of macrophages with LPS, induced transient increases in the [Ca²⁺]_i, within the first few minutes of application [9], [10], [11]. However, the increase in [Ca²⁺]_i of macrophages was found to be at least partially due to LPS cross-reacting with the PAF receptor [12]. Furthermore, the importance of such calcium transients in the induction of macrophage cytotoxic functions was called into question, when researchers showed that macrophages prevented from experiencing calcium transients were still capable of normal cytotoxic responses, such as production of tumour necrosis factor-α (TNF-α) and nitric oxide (NO) following treatment with LPS [12], [13].

It is clear that calcium plays some role in the priming stage of macrophage activation because treating macrophages with pharmacological agents that alter the intracellular calcium concentration, such as calcium ionophores or inhibitors of endoplasmic reticulum Ca²⁺–ATPase, primes the macrophages for subsequent full activation [13], [14], [15]. Although some cytotoxic functions of macrophages, such as the production of reactive oxygen intermediates, are induced within minutes of treatment of macrophages with activating agents [3], many cytotoxic functions, like the production of nitric oxide, are not induced until several hours after activator treatment [3], [16]. A long-term alteration in calcium metabolism reported in this study, may be responsible for induction or modulation of these delayed cytotoxic reactions. In this paper, we report experiments showing a lasting alteration in the [Ca²⁺]_i of macrophage-like P388D.1 cells treated with LPS and interferon-γ (IFN-γ).