Mechanisms for impaired effector function in alveolar macrophages from marijuana and cocaine smokers

Abstract

Lung macrophages provide a first line of host defense against inhaled pathogens and their function is impaired in the lungs of inhaled substance abusers. In order to investigate the mechanism for this impairment, alveolar macrophages (AM) were recovered from nonsmokers (NS), regular tobacco smokers (TS), marijuana smokers (MS), or crack cocaine smokers (CS), and evaluated for their production of nitric oxide (NO) and the role of NO as an antimicrobial effector molecule. AM from NS and TS efficiently killed Staphylococcus aureus and their antibacterial activity correlated closely with the production of nitrite and the expression of mRNA encoding for inducible nitric oxide synthase (iNOS). In contrast, AM collected from MS and CS exhibited limited antimicrobial activity that was not affected by an inhibitor of iNOS, or associated with expression of iNOS. Treatment with either granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-γ restored the ability of these cells to produce NO and to kill bacteria. These findings confirm a significant role for NO as an antibacterial effector molecule used by normal human AM and suggest that this host defense mechanism is suppressed by habitual exposure to inhaled marijuana or crack cocaine in vivo.

Introduction

Alveolar macrophages (AM) are differentiated tissue macrophages that line the epithelial surface of the lung and provide a first line of host defense against inhaled pathogens Sibille and Reynolds, 1990, Reynolds, 1997. In their normal state, AM are highly phagocytic, exhibit potent antimicrobial activity, and secrete a variety of immunoregulatory cytokines that activate both innate and adaptive immune responses. However, their function can be altered when habitually exposed to inhaled marijuana or crack cocaine, as previously reported (Baldwin et al., 1997). AM isolated from marijuana smokers (MS) were less effective than AM recovered from normal nonsmokers (NS) or tobacco smokers (TS) in their ability to phagocytose bacteria, secrete inflammatory cytokines, or kill ingested Staphylococcus aureus (Baldwin et al., 1997). AM from long-term cocaine smokers (CS) also exhibited impaired antistaphylococcal activity (Baldwin et al., 1997). In normal AM, bacterial killing was reduced, but not eliminated, by the addition of an inhibitor of nitric oxide synthase (NOS), N^G-monomethyl-l-arginine monoacetate (NGMMA). This finding suggested that bacterial killing can be mediated by both nitric oxide (NO) dependent and independent pathways. However, cells recovered from the lungs of MS and CS were not affected by NGMMA, suggesting a deficiency in their ability to synthesize or use NO as an antibacterial effector molecule.

Nitric oxide is a signaling and effector molecule that mediates a diverse number of physiologic and pathophysiologic processes Mayer, 2000, Bogdan, 2000, Bogdan, 2001. NO was first suggested as an inducible host immune effector when mouse macrophages were observed to produce nitrite and nitrate in an l-arginine-dependent manner after stimulation with lipopolysaccharide (LPS) and cytokines (Stuehr and Marletta, 1987). Subsequently, activated macrophages from mice, rats, and a variety of larger mammals (goats, sheep, dog, and cattle) were all shown to produce NO Adler et al., 1996, Jungi et al., 1997, MacMicking et al., 1997. Inhibition of NO synthesis reduces their responses against fungi (Granger et al., 1990), bacteria Denis, 1991, Higginbotham et al., 1992, Nathan and Shiloh, 2000, parasites James and Glaven, 1989, Adams et al., 1990, Roach et al., 1991, and tumor cells Hibbs et al., 1987, Keller et al., 1990, Kwon et al., 1991, Xie et al., 1996, Pervin et al., 2001. Generation of high levels of NO by macrophages is mediated by expression of an inducible form of NOS (iNOS) (Stuehr and Nathan, 1989). Stimuli that induce expression of the iNOS gene within macrophages include infection by intracellular pathogens or dual stimulation by LPS in combination with cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or granulocyte/macrophage colony-stimulating factor (GM-CSF) (Ding et al., 1988).

High-output iNOS activity was initially characterized in rodent macrophages. As a result, most of the available data concerning production of NO and its role as an effector molecule have been derived from rodent models. In contrast, the role of NO in the human response to inflammatory stimuli has remained controversial Denis, 1994, Albina, 1995. In vitro stimulation of human monocyte-derived macrophages by LPS and cytokines activates antimicrobial activity, but with limited evidence for a role mediated by NO Thomassen and Kavuru, 2001, Zhang et al., 1996. While expression of iNOS has been observed in these studies, biochemical evidence of NO or NO-dependent antimicrobial activity has been difficult to detect (Weinberg, 1998). However, the response of human AM to pathogens may be different from that of cultured monocyte-derived macrophages. Biochemical evidence of NO, mRNA encoding for iNOS, and iNOS proteins has been detected in AM from patients with pulmonary fibrosis (Nozaki et al., 1997) infected with BCG in vitro, or from patients infected with tuberculosis in vivo Nicholson et al., 1996, Wang et al., 1998. Yet, in spite of this foregoing evidence, no study to date has provided a direct connection among iNOS gene expression, NO protein production, and NO-dependent antimicrobial activity in normal human AM.

The present study was designed to evaluate the role of NO as an effector molecule in the antibacterial activity of human AM and to test our hypothesis that chronic exposure to marijuana smoke or crack cocaine impairs this effector pathway. AM were retrieved from the lungs of NS, TS, MS, and CS, and cocultured with S. aureus in the presence or absence of NGMMA. Cocultures were analyzed simultaneously for antistaphylococcal activity, production of nitrite (as a marker of NO), and expression of mRNA encoding for iNOS.