Macrophage Internalization of Fungal (cid:2) -Glucans Is Not Necessary for Initiation of Related Inﬂammatory Responses

Cell wall (cid:1) -glucans are highly conserved structural components of fungi that potently trigger inﬂammatory responses in an infected host. Identiﬁcation of molecular mechanisms responsible for internalization and signaling of fungal (cid:1) -glucans should enhance our understanding of innate immune responses to fungi. In this study, we demonstrated that internalization of fungal (cid:1) -glucan particles requires actin polymerization but not participation of components of caveolar uptake mechanisms. Using ﬂuorescence microscopy, we observed that uptake of 5-([4,6-dichlorotriazin-2-yl] amino)-ﬂuorescein hydrochloride-Celite complex-labeled Saccharomyces cerevisiae (cid:1) -glucan by RAW macrophages was substantially reduced in the presence of cytochalasin D, which antagonizes actin-mediated internalization pathways, but not by treatment with nystatin, which blocks caveolar uptake. Interestingly, (cid:1) -glucan-induced NF- (cid:2) B translocation, which is necessary for inﬂammatory activation, and tumor necrosis factor alpha production were both normal in the presence of cytochalasin D, despite defective internalization of (cid:1) -glucan particles following actin disruption. Dectin-1, a major (cid:1) -glucan receptor on macrophages, colocalized to phagocytic cups on macrophages and exhibited tyrosine phosphorylation after challenge with (cid:1) -glucan particles. Dectin-1 localization and other membrane markers were not affected by treatment with cytochalasin D. Furthermore, dectin-1 receptors rather than Toll-like receptor 2 receptors were shown to be necessary for both efﬁcient internalization of (cid:1) -glucan particles and cytokine release in response to the fungal cell wall component. Phagocytosis assays and inhibitor treatments. To evaluate the mechanisms of macrophage internalization of (cid:2) -glucan particles, we performed phagocytosis assays in the absence and presence of inhibitors of various uptake pathways. Our previous studies revealed parallel responses of fungal (cid:2) -glucans interacting with native alveolar macrophages and with the RAW murine macrophage cell line (14). RAW 264.7 macrophage cells, thus, provide an efﬁcient model system to study interactions of fungal cell wall components with macrophages. In addition, in our experience RAW cells exhibit somewhat less autoﬂuorescence than native macrophages, making them useful for ﬂuorescence microscopy studies. Further- more, the RAW cell line exhibits substantially better transfection efﬁciency than native alveolar macrophages. For these reasons, we used these cells for most experiments in this study. RAW macrophages were pretreated with cytochalasin D (2 (cid:5) M) or nystatin (5 (cid:5) g/ml) for 30 min and throughout subsequent incubation with DTAF-labeled (cid:2) -glucan. After various times, particle uptake was termi-nated by washing cells with ice-cold PBS, and unbound particles were removed by acid stripping (Hanks balanced saline solution buffered to pH 3.5), followed by ﬁxation with 2% paraformaldehyde. Macrophage uptake of DTAF-glucan was visualized by ﬂuorescence microscopy. XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assays re- vealed no adverse effect of these uptake inhibitors on RAW cell viability at the concentrations employed (7). To evaluate the potential role of the dectin-1 receptor in mediating internalization and signaling from puriﬁed (cid:2) -glucan particles, we treated cells with either monoclonal antibody m2A11 or glucan phosphate (3). macrophages on ice