Phagosome-lysosome fusion is a calcium-independent event in macrophages.

Abstract

Abstract. Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome-granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several intracellular pathogens survive in macrophage phagosomes that do not fuse with lysosomes, we examined the regulation of phagosome-lysosome fusion in macrophages. Macrophages (MO) were treated with 12.5 IxM bis-(2-amino-S-methylphenoxy) ethane-N,N,N',N',-tetraacetic acid tetraacetoxymethylester (MAPT/AM), a cell-permeant calcium chelator which reduced resting cytoplasmic [Ca2+]; from 80 nM to ~<20 nM and completely blocked increases in [Ca2+]i in response to multiple stimuli, even in the presence of extracellular calcium. Subsequently, M~ phagocytosed serum-opsonized zymosan, staphylococci, or Mycobacterium bovis. Microbes were enumerated by 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining, and phagosome-lysosome fusion was scored using both lysosome-associated membrane protein (LAMP-l) as a membrane marker and rhodamine dextran as a content marker for lysosomes. Confirmation of phagosomelysosome fusion by electron microscopy validated the fluorescence microscopy findings. We found that phagosome-lysosome fusion in MO occurs normally at very low [Ca2+]i (~<20 nM). Kinetic analysis showed that in MO none of the steps leading from particle binding to eventual phagosome-lysosome fusion are regulated by [Ca2+]i in a rate-limiting way. Furthermore, confocal microscopy revealed no difference in the intensity of LAMP-1 immunofluorescence in phagolysosome membranes in calcium-buffered vs. control macrophages. We conclude that neither membrane recognition nor fusion events in the phagosomal pathway in macrophages are dependent on or regulated by calcium.AGOCYTOSIS is a tightly regulated process leading o the internalization of particulate ligands such as acteria and fungi. Its initiation depends on the engagement of specific plasma membrane receptors expressed on macrophages and neutrophils. In a manner analogous to that of hormones and growth factors, binding of particulate ligands to phagocytic receptors triggers intraceUular signals. One common intracellular signaling event that accompanies phagocytosis is an increase in in-

Localized Exocytosis of Primary (Lysosomal) Granules During Phagocytosis: Role of Ca2+-Dependent Tyrosine Phosphorylation and Microtubules

“…As reported before for specific granules (28), exocytosis of primary granules induced by opsonized particles was inhibited by chelation of Ca 2ϩ . This stresses the differences between neutrophils and macrophages, in which phagosome-lysosome fusion was recently demonstrated to be a Ca 2ϩ -independent process (29). Although essential for secretion, Ca 2ϩ does not appear to be the main determinant of the polarized exocytosis occurring during neutrophil phagocytosis of IgG-opsonized zymosan, as shown in the present study.…”

Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

“…The acidification was reduced but not eliminated by chelation of intracellular calcium or by depletion of intracellular calcium stores. In this regard, the transfected cells resemble neutrophils, in which prevention of calcium transients inhibits phago-lysosomal fusion (19), but they differ from macrophages, in which no effect was noted (37). It remains unclear whether these two closely related cell types utilize fundamentally different fusion mechanisms, or whether methodological differences account for the apparent discrepancies.…”

“…In neutrophils, fusion of phagosomes with lysosomal like granules that express CD63 is thought to be strictly dependent on elevated [Ca 2ϩ ] c (19). By contrast, phagosome to lysosome fusion was unaffected when [Ca 2ϩ ] c transients were prevented in macrophages (37). This discrepancy may reflect differences between neutrophils and macrophages in the regulation of phagosome to lysosome fusion and from the fact that multiple and different receptors are activated during phagocytosis.…”

Indirect Role for COPI in the Completion of Fcγ Receptor-mediated Phagocytosis

“…An increase in [Ca 2ϩ ] i mediated by activation of phospholipase C and release of Ca 2ϩ from intracellular stores is one of the earliest consequences of Fc receptor ligation (30,31). Although not essential for phagocytosis, this [Ca 2ϩ ] i transient is invariably associated with receptor clustering and activation in professional phagocytes and nonprofessional engineered phagocytes (32,33). In FcR-ldl cells maintained at 34°C, [Ca 2ϩ ] i spikes were similarly triggered by binding of IgG-opsonized particles (Fig.…”