Role of Nuclear Factor-kappa B in the Activation of Alveolar Macrophages by Fungal Beta-Glucans
Pneumocystis carinii (P. carinii) is a fungal pathogen that causes pneumonia in individuals with compromised immunity [1]. Beta- glucans (β-glucans) are complex carbohydrates which represent major structural components of both fungal and plant cell walls.
In addition to providing support in fungal cell walls, β-glucans also mediate important inflammatory responses through interactions with cognate glucan receptors on alveolar macrophagcs. We have recently demonstrated that particulate, endotoxin-free beta-glucan from the opportunistic fungal pathogen P. carinii, as well as the phylogenetically related non-pathogenic fungus Saccharomyces cerevisiae (S. cerevisiae), stimulate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein-2 release from alveolar macrophages [2]. Thus, β-glucan components of fungal cell walls participate directly in the inflammatory process that occurs during the course of fungal pneumonia. The signal transduction mechanisms mediating alveolar macrophage activation and release of cytokines through stimulation with fungal β-glucan are not known. Recent studies indicate that the soluble β-glucan, Betafectin, activates the transcription factor nuclear factor kappa-B (NF-κB) in a murine monocytic cell line [3]. We hypothesized that activation of alveolar macrophages by insoluble particulate fungal β-glucan similarly occurs through activation of NF-κB.
MATERIAL AND METHODS
We tested the effect of Pyrrolidine Dithiocarbamate (PDTC) and tepoxalin, pharmacological inhibitors of NF-κB, on β-glucan induced TNF-alpha release from rat alveolar macrophages. Normal rat alveolar macrophages were obtained from healthy rats using whole lung lavage. Alveolar macrophages (2 × 105) were stimulated with varying concentrations of S. cerevisiaeβ-glucan (200 μg/ml). Following 12 hours of incubation, TNF-alpha protein in the supernatant was measured.
To determine the levels of NF-κB protein subunits in alveolar macrophages following stimulation with β-glucan, we performed immunoblotting on cytosolic and nuclear extracts prepared from the alveolar macrophage cell line, NR8383 cells stimulated with S. cerevisiaeβ-glucan. Cells (5 × 106) were stimulated glucan (200 μg/ml) for 15–20 minutes, and cytosolic and nuclear lysates were prepared. Cytosolic IKBα levels and nuclear p65 levels were detected by immunoblotting.
To further evaluate if nuclear translocation of NF-κB occurs following stimulation of macrophages with β-glucan, electro-mobility shift assay (EMSA) was performed on nuclear lysates prepared from NR8383 cells stimulated with S. cerevisiaeβ-glucan (200 μg/ml) at different time points (0–60 minutes). Negative controls (unstimulated cells), positive controls (lipopolysaccharide stimulated controls) and sample EMSA were performed on 5μg of nuclear protein using a commercially available, 32P labeled murine NF-κB oligo-probe. Parallel competitive inhibition experiments were performed using unlabelled NF-κB and SP-1 oligo-probes.
RESULTS AND DISCUSSION
The NF-κB inhibitors PDTC and tepoxalin significantly reduce S. cerevisiaeβ-glucan induced TNF-alpha release from alveolar macrophages. Maximally, PDTC (lOOμM) inhibited β-glucan induced macrophage release of TNF-alpha by 79±3.2%, whereas tepoxalin (30μM) inhibited TNF-alpha release by 54±3% (p<0.01 for both PDTC and tepoxalin compared to control).
Upon activation of NF-κB, cytosolic levels of IKBα are depleted while p65 translocates into the nucleus where it binds to the promoter regions of inflammatory genes [4]. The levels of macrophage IKBα and p65 protein upon activation with fungal β-glucan (200μg/ml of S. cerevisiaeβ-glucan for 20 minutes) as determined by immunoblotting demonstrated significant decrease in cytosolic IKBα levels and a corresponding increase in nuclear p65 protein levels following stimulation with β-glucan.
EMSA demonstrated the presence of NF-κB in nuclear extracts within 15 minutes of stimulation with β-glucan and maximal activation at 60 minutes following stimulation, in contrast to lipopolysaccharide stimulated NF-κB activation which peaked at 15 minutes. The observed NF-κB signal was competitively inhibited by equimolar concentrations of cold NF-κB probe but not inhibited by equimolar amounts of SP-1 probe. These studies confirm that activation of NF-κB occurs as a result of activation of alveolar macrophages with fungal β-glucan and also demonstrate that the kinetics of NF-κB activation are different from those observed by lipopolysaccharide. This indicates that although similar to lipopolysaccharide in the ability to induce pro-inflammatory cytokine and chemokine release from alveolar macrophages, β-glucans employ different signal transduction pathways in alveolar macrophage activation.
These data provide important insight into the signaling pathways involved in alveolar macrophage activation by fungal β-glucans. In view of the prominent role that inflammation plays in the pathogenesis of P. carinii and other fungal pneumonias, elucidation of the mechanisms involved in macrophage activation during these infections is necessary to enable development of therapeutic agents that facilitate clearance of infection by the host and limit lung injury due to unrestrained inflammation in the host lung.
ACKNOWLEDGMENTS
Supported by NIH grants RO1-HL-62150, RO1-HL-55934, and ROl-HL-57125 to A.H.L.
