Extracellular proton release by stimulated neutrophils.

Zwieten, Rob van; Wever, Ron; Hamers, Mic N.; Weening, R.S.; Roos, Dirk

doi:10.1172/jci110250

Cited by 80 publications

(44 citation statements)

References 13 publications

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“…Assuming that one proton is generated in the cytoplasm for each electron translocated by NADPH oxidase (37,38), a cell volume of 320 fL, and a cytoplasmic buffer capacity of 30 mM (3), pH i would decrease by 0.27 units/min in the absence of proton extrusion. The peak rate of acidification in the presence of Zn 2ϩ is 0.31-0.33 units/min (Figs.…”

Section: Discussionmentioning

confidence: 99%

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Morgan

Capasso

Musset

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

161

192

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Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH i in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na ؉ /H ؉ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH i decreased sharply, and recovery required both Na ؉ /H ؉ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn 2؉ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn 2؉ . Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis. innate immunity ͉ ion channels ͉ phagocyte ͉ respiratory burst ͉ SNARF W e have reinvestigated the regulation of cytoplasmic pH, pH i , in human neutrophils during phagocytosis. For 3 decades, pH i during the respiratory burst has been characterized as a small (0.05-0.1 unit) transient drop in pH i that is followed by larger (0.16-0.60 unit) prolonged alkalinization caused by Na ϩ /H ϩ antiport activity, whether the stimulus is fMLF (fMetLeuPhe, a chemotactic peptide), PMA (a PKC-activating phorbol ester), phospholipase C, or OPZ (opsonized zymosan) (1-7). In most studies, soluble stimuli were applied to populations of neutrophils. When phagocytosis was studied, the responses of many cells were averaged (5, 8). A powerful new imaging approach, confocal microscopy using the fluorescent pH indicator SNARF-1 enhanced by shifted excitation and emission ratioing (SEER) (9, 10) enabled us to examine the responses of individual cells with high spatial and temporal resolution. We report here that the behavior of individual human neutrophils during phagocytosis differs markedly from the prevailing view. The fundamental pH i response is triphasic: rapid and profound acidification followed by rapid but variably complete recovery, and after longer times, slow acidification that may reflect the onset of apoptosis. ResultsCells That Phagocytose Become Acidic. Human neutrophils were allowed to adhere to a g...

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Section: Discussionmentioning

confidence: 99%

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Morgan

Capasso

Musset

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

161

192

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“…The obvious impediment to this scenario is the neutral pH of the internal milieu. However, it is known that locally, acidic conditions occur, and that antimicrobial responses are amplified at sites of inflammation (43)(44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Membrane Permeabilization by Trypanosome Lytic Factor, a Cytolytic Human High Density Lipoprotein

Harrington

Howell

Hajduk

2009

Journal of Biological Chemistry

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Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membranedisrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobinrelated protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.

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“…Subsequently, mild acidosis (pH 6.5) can occur at the local site of infection or in a severe systemic infection, due to the massive infiltration of immune cells to the site of infection, which activates the respiratory burst (27,28). Several CLs containing a C-terminal Ca 2+ -dependent C-type carbohydrate recognition domain have been reported to be sensitive to pH shift, which causes conformational changes (29,30).…”

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confidence: 99%

Secreted M-Ficolin Anchors onto Monocyte Transmembrane G Protein-Coupled Receptor 43 and Cross Talks with Plasma C-Reactive Protein to Mediate Immune Signaling and Regulate Host Defense

Zhang

Yang

Ang³

et al. 2010

The Journal of Immunology

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Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin–GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin–GPCR43 complex. The collaboration among CRP–M-ficolin–GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin–CRP provides a powerful template for future design of potential immunomodulators.

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Extracellular proton release by stimulated neutrophils.

Cited by 80 publications

References 13 publications

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Membrane Permeabilization by Trypanosome Lytic Factor, a Cytolytic Human High Density Lipoprotein

Secreted M-Ficolin Anchors onto Monocyte Transmembrane G Protein-Coupled Receptor 43 and Cross Talks with Plasma C-Reactive Protein to Mediate Immune Signaling and Regulate Host Defense

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