Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses

Abstract

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-α, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF-α in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.

Introduction

An association between moldy microbial growth in buildings and respiratory symptoms in the inhabitants has been shown in several epidemiological studies. A variety of symptoms are related to inflammatory reactions e.g. irritation, respiratory infections, wheeze, bronchitis and even asthma (Platt et al., 1989, Waegemaekers et al., 1989, Brunekreef, 1992, Spengler et al., 1994). This suggests that inflammatory responses toward specific structural components or metabolic products of microbes present in the moldy buildings may contribute to the observed respiratory symptoms. However, the identification of the most potent microbes and specific cellular mechanisms responsible for the adverse health effects are not yet known.

Recent studies have shown that environmental mycobacteria can be isolated from indoor air and building materials of moldy houses (Andersson et al., 1997). Environmental mycobacteria include both potentially pathogenic and saprophytic species, which share the unique feature of all mycobacteria; a thick, hydrophobic and lipid-rich cell wall, also containing serologically active polysaccharides (Falkinham, 1996, Chatterjee and Khoo, 1998, Goodfellow and Magee, 1998). The cell wall structure enables mycobacteria to survive and even proliferate in extreme environments, such as in areas contaminated with xenobiotics or inside of host macrophages. The pathogenicity of mycobacteria such as the notorious Mycobacterium tuberculosis and Mycobacterium leprae is based on the immunological responses they elicit in the host, beginning with an ineffective elimination of the bacteria, and ending with the tissue-damaging hypersensitivity reactions (Clark-Curtiss, 1998). Some environmental mycobacteria have also been recognized as effective immunostimulators in murine models (Bradbury and Moreno, 1993; Denis and Ghadirian, 1994, Falcone et al., 1994, Sarmento and Appelberg, 1995). Mycobacteria present in moldy buildings are of special interest, because they are inhalable particles and have potential to stimulate a cascade of cytokines, leading to recruitment and activation of inflammatory cells.

Proinflammatory cytokines (e.g. IL-6, IL-1 and TNF-α) are known to initiate the expression of inducible nitric oxide synthase (iNOS) (Moncada and Higgs, 1993), whereas anti-inflammatory cytokines (e.g. IL-10) tend to suppress the inflammatory responses (Marshall et al., 1997). In a wide variety of cells in the respiratory tract, iNOS catalyzes the production of nitric oxide (NO) using l-arginine as a source. The role of nitric oxide as an immune defense molecule and important mediator in airway diseases together with reactive oxygen species (ROS) has been confirmed by an increasing amount of information during this decade (Fridovitch, 1995, Singh and Evans, 1997).

In our previous studies we have shown that other gram positive bacteria Streptomyces sp., typically present in moldy houses, induce the production of inflammatory mediators (cytokines, NO and ROS) in murine macrophages. In contrast, bacterial strains belonging to normal microbial flora of indoor air do not cause similar responses (Hirvonen et al., 1997a, Hirvonen et al., 1997b, Hirvonen et al., 1997c). Thus, the role of environmental mycobacteria as immunostimulators clearly needs further clarification, since exposure to these bacteria in moldy houses could be one of the etiological factors leading to the respiratory symptoms observed in the inhabitants.

In the present study we investigated whether five strains of environmental mycobacteria, isolated from indoor air in moldy buildings during demolition work, are able to stimulate mouse macrophages (RAW264.7) to produce inflammatory mediators (i.e. TNF-α, IL-6, IL-1, IL-10, iNOS, NO, ROS), and to cause cytotoxicity in these cells.