Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria

Drevets, Douglas A.; Campbell, Priscilla A.

doi:10.1016/0022-1759(91)90289-r

Cited by 125 publications

(92 citation statements)

References 20 publications

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“…Live gates were used on mouse PMNs (as distinguished by PE-labeled Rat IgG anti-Gr-1/Ly-6; Becton Dickinson). In experiments where ingested bacteria were quantified by FACS, Trypan Blue 21 or ethidium bromide 22 was used to quench extracellular FITC-labeled pneumococci, both of which resulted in similar and efficient quenching. TAT peptides and inhibitors, when used, were added together with IgG1 and pneumococci to isolated cell preparations without prior incubations.…”

Section: Phagocytosis Experimentsmentioning

confidence: 99%

FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis

Vidarsson

Stemerding

Stapleton

et al. 2006

Blood

167

164

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Here, we report that the MHC class I-related neonatal Fc receptor (FcRn) is expressed within azurophilic and specific granules of neutrophils and relocates to phagolysosomes on phagocytosis of IgGopsonized bacteria. We found FcRn to enhance phagocytosis in a pH-dependent manner which was independent of IgG recycling. IgG-opsonized bacteria were inefficiently phagocytosed by neutrophils from ␤2M knock-out or FcRn ␣-chain knock-out mice, which both lack expression of FcRn. Similarly, low phagocytic activity was also observed with mutated IgG (H435A), which is incapable of binding to FcRn, while retaining normal binding to classical leukocyte IntroductionPhagocytic cells express members of 3 classes of leukocyte IgG-Fc receptors, Fc␥RI, Fc␥RII, and Fc␥RIII. All share considerable structural and functional homology and recognize similar residues within the CH2 region of IgG. 1 Fc␥R activates phagocytes on interaction with IgGopsonized particles, involving immunoreceptor tyrosine-based activation motifs (ITAMs). This activation signal may possibly be downregulated by the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing Fc␥RIIb receptor on polymorphonuclear neutrophils (PMNs) and monocytes. 2 No other signaling motifs have been implicated in Fc␥R-mediated phagocytosis. Crosslinking of ITAM-bearing receptors (eg, T-cell receptor, Fc⑀RI) does not initiate phagocytosis, but it generally triggers fusion and release of granule contents into sealed immunologic synapses between effector cells and targets. In phagocytes these granules contain various components, including enzyme complexes that initiate the respiratory burst and phagosome acidification, as well as antimicrobial peptides and enzymes that serve to kill invading pathogens. 3,4 A distinct IgG receptor, the neonatal Fc␥R (FcRn), consisting of a unique ␣-chain and ␤2-microglobulin (␤2M), is a major histocompatibility class I (MHC-I) homolog. 5 FcRn is present in epithelial cells, placental syncytiotrophoblasts, as well as endothelial cells. In these cells, FcRn has been implicated in transport of IgG across mucosal cells, 5,6 from mother to fetus, 7 and regulation of IgG half-life, [8][9][10][11] respectively. This receptor has been found in human monocytes, 12 albeit that no function has been attributed to monocyte FcRn. FcRn does not bind IgG at physiologic pH (7.4). Only in the acidic environment of endocytic vacuoles (pH Յ 6.5), where histidine residues in the Fc-tail of IgG become protonated, can FcRn bind IgG with high affinity. Both ␤2M and the FcRn ␣-chain participate in IgG binding within the CH2-CH3 interface. 13 In this study we document expression of FcRn within PMNs. Furthermore, we observed FcRn translocation to nascent phagosomes, where FcRn facilitates IgG-mediated bacterial phagocytosis through signaling motifs found within the cytoplasmic tail. These results point to a novel role for FcRn in phagocyte biology. Materials and methods Recombinant antipneumococcal 6A/B antibodiesThe generation and functional characterization in vitro a...

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Section: Phagocytosis Experimentsmentioning

confidence: 99%

FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis

Vidarsson

Stemerding

Stapleton

et al. 2006

Blood

167

164

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“…The technique described by Devrets & Campbell (1991) and Bandı! n et al (1995) was used to differentiate adherent from intracellular bacteria.…”

Section: Bacteriamentioning

confidence: 99%

Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum

et al. 2000

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Photobacterium damselae subsp. piscicida is a fish pathogen which causes serious disease in commercial warmwater fish species. Because information on the initial stages of the infection is scarce, an investigation of the invasion ability of this pathogen was undertaken utilizing a fish epithelial cell line (epithelioma papillosum carpio, EPC), a virulent capsulated strain of P. damselae (MT1415), an avirulent non-capsulated strain of P. damselae (EPOY-8803-II) and Escherichia coli HB101 as a non-invasive control. P. damselae was found to be able to adhere to and invade fish epithelial cells and remain inside them for 6-9 h. There were no significant differences in invasiveness between the capsulated and non-capsulated strains. A kinetics study demonstrated that P. damselae invasiveness was more efficient at low m.o.i., reaching saturation at higher m.o.i., suggesting internalization may be receptor-mediated. Invasion efficiency (IE) was significantly higher than in the control E. coli HB101. Engulfment of bacteria was possibly by an endocytic process and was unaffected by killing the bacteria with UV light. However, heat-killed bacteria had significantly reduced invasion capability. Ultrastructural studies showed that inside the epithelial cells, the bacteria remained within large vacuoles for a few hours and no evidence of intracellular replication was found, by either fluorescence or electron microscopic studies. Normal sea bass serum slightly reduced the invasion capability of the MT1415 strain, but heat-inactivated normal serum had no effect. On the other hand, heat-inactivated fish antiserum raised against the same strain reduced the percentage of invaded epithelial cells by 50 %. As for other pathogens, an intracellular phase of P. damselae may be a mechanism to delay or avoid phagocytosis and host immune responses, favouring the spread of infection.

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“…Accordingly, several microscopic methods have been developed to accomplish this task (Heesemann and Laufs, 1985;Drevets and Campbell, 1991;Drevets and Elliott, 1995). Here, we present a novel staining procedure that takes advantage of bacteria simultaneously labelled with FITC and biotin.…”

Section: Discussionmentioning

confidence: 99%

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method

Agerer

Waeckerle

Hauck

2004

Journal of Microbiological Methods

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Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC -biotin -avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Grampositive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms. D

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Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria

Cited by 125 publications

References 20 publications

FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis

FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis

Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method

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