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CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so.

Zhou J, Feng G, Beeson J, Hogarth PM, Rogerson SJ, Yan Y, Jaworowski A - BMC Med (2015)

Bottom Line: Ingestion of IE was confirmed by imaging flow cytometry.We show a special role for CD14(hi)CD16+ monocytes in phagocytosing opsonised P. falciparum IE and production of TNF.While ingestion was mediated by Fcγ receptor IIIa, this receptor was not sufficient to allow phagocytosis; despite opsonisation with antibody, phagocytosis of IE also required complement opsonisation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria, 3004, Australia. jingling@burnet.edu.au.

ABSTRACT

Background: With more than 600,000 deaths from malaria, mainly of children under five years old and caused by infection with Plasmodium falciparum, comes an urgent need for an effective anti-malaria vaccine. Limited details on the mechanisms of protective immunity are a barrier to vaccine development. Antibodies play an important role in immunity to malaria and monocytes are key effectors in antibody-mediated protection by phagocytosing antibody-opsonised infected erythrocytes (IE). Eliciting antibodies that enhance phagocytosis of IE is therefore an important potential component of an effective vaccine, requiring robust assays to determine the ability of elicited antibodies to stimulate this in vivo. The mechanisms by which monocytes ingest IE and the nature of the monocytes which do so are unknown.

Methods: Purified trophozoite-stage P. falciparum IE were stained with ethidium bromide, opsonised with anti-erythrocyte antibodies and incubated with fresh whole blood. Phagocytosis of IE and TNF production by individual monocyte subsets was measured by flow cytometry. Ingestion of IE was confirmed by imaging flow cytometry.

Results: CD14(hi)CD16+ monocytes phagocytosed antibody-opsonised IE and produced TNF more efficiently than CD14(hi)CD16- and CD14(lo)CD16+ monocytes. Blocking experiments showed that Fcγ receptor IIIa (CD16) but not Fcγ receptor IIa (CD32a) or Fcγ receptor I (CD64) was necessary for phagocytosis. CD14(hi)CD16+ monocytes ingested antibody-opsonised IE when peripheral blood mononuclear cells were reconstituted with autologous serum but not heat-inactivated autologous serum. Antibody-opsonised IE were rapidly opsonised with complement component C3 in serum (t1/2 = 2-3 minutes) and phagocytosis of antibody-opsonised IE was inhibited in a dose-dependent manner by an inhibitor of C3 activation, compstatin. Compared to other monocyte subsets, CD14(hi)CD16+ monocytes expressed the highest levels of complement receptor 4 (CD11c) and activated complement receptor 3 (CD11b) subunits.

Conclusions: We show a special role for CD14(hi)CD16+ monocytes in phagocytosing opsonised P. falciparum IE and production of TNF. While ingestion was mediated by Fcγ receptor IIIa, this receptor was not sufficient to allow phagocytosis; despite opsonisation with antibody, phagocytosis of IE also required complement opsonisation. Assays which measure the ability of vaccines to elicit a protective antibody response to P. falciparum should consider their ability to promote phagocytosis and fix complement.

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CD14hiCD16+ monocytes produce more TNF compared to other monocytes in response to IE. a Representative histograms showing intracellular TNF staining of monocytes four hours after addition of CS2-IE (IE, left hand panels) or CS2-IE opsonised with rabbit anti-human RBC antibody (IgG-IE, right hand panels). Grey histograms: 4 °C controls, red histograms: 37 °C. b Median (IQR) of intracellular TNF expression in classical (C; solid black circles), intermediate (IM; open circles) and non-classical (NC; open squares) monocytes from four independent experiments using blood from separate donors. IE infected erythrocytes, IQR interquartile range
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Fig3: CD14hiCD16+ monocytes produce more TNF compared to other monocytes in response to IE. a Representative histograms showing intracellular TNF staining of monocytes four hours after addition of CS2-IE (IE, left hand panels) or CS2-IE opsonised with rabbit anti-human RBC antibody (IgG-IE, right hand panels). Grey histograms: 4 °C controls, red histograms: 37 °C. b Median (IQR) of intracellular TNF expression in classical (C; solid black circles), intermediate (IM; open circles) and non-classical (NC; open squares) monocytes from four independent experiments using blood from separate donors. IE infected erythrocytes, IQR interquartile range

Mentions: CD14hiCD16+ monocytes respond to phagocytosis of bacterial pathogens by producing pro-inflammatory cytokines such as TNF [22]. Since this is thought to be required for both effective immunity to malaria and for immunopathogenesis we determined whether intermediate monocytes produce TNF in response to IE. Whole blood was incubated with unopsonised and opsonised CS2-IE for four hours, then intracellular TNF measured by flow cytometry for all three subsets. There was no TNF production in response to unopsonised IE (Fig. 3a, left hand panels). Both CD14hiCD16- and CD14hiCD16+ monocytes produced TNF following addition of opsonised parasites, with more of the CD14hiCD16+ subset producing TNF in agreement with their greater phagocytic potential, although this difference did not reach significance, whereas CD14loCD16+ monocytes produced very little (Fig. 3a, right hand panels and Fig. 3b).Fig. 3


CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so.

Zhou J, Feng G, Beeson J, Hogarth PM, Rogerson SJ, Yan Y, Jaworowski A - BMC Med (2015)

CD14hiCD16+ monocytes produce more TNF compared to other monocytes in response to IE. a Representative histograms showing intracellular TNF staining of monocytes four hours after addition of CS2-IE (IE, left hand panels) or CS2-IE opsonised with rabbit anti-human RBC antibody (IgG-IE, right hand panels). Grey histograms: 4 °C controls, red histograms: 37 °C. b Median (IQR) of intracellular TNF expression in classical (C; solid black circles), intermediate (IM; open circles) and non-classical (NC; open squares) monocytes from four independent experiments using blood from separate donors. IE infected erythrocytes, IQR interquartile range
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493812&req=5

Fig3: CD14hiCD16+ monocytes produce more TNF compared to other monocytes in response to IE. a Representative histograms showing intracellular TNF staining of monocytes four hours after addition of CS2-IE (IE, left hand panels) or CS2-IE opsonised with rabbit anti-human RBC antibody (IgG-IE, right hand panels). Grey histograms: 4 °C controls, red histograms: 37 °C. b Median (IQR) of intracellular TNF expression in classical (C; solid black circles), intermediate (IM; open circles) and non-classical (NC; open squares) monocytes from four independent experiments using blood from separate donors. IE infected erythrocytes, IQR interquartile range
Mentions: CD14hiCD16+ monocytes respond to phagocytosis of bacterial pathogens by producing pro-inflammatory cytokines such as TNF [22]. Since this is thought to be required for both effective immunity to malaria and for immunopathogenesis we determined whether intermediate monocytes produce TNF in response to IE. Whole blood was incubated with unopsonised and opsonised CS2-IE for four hours, then intracellular TNF measured by flow cytometry for all three subsets. There was no TNF production in response to unopsonised IE (Fig. 3a, left hand panels). Both CD14hiCD16- and CD14hiCD16+ monocytes produced TNF following addition of opsonised parasites, with more of the CD14hiCD16+ subset producing TNF in agreement with their greater phagocytic potential, although this difference did not reach significance, whereas CD14loCD16+ monocytes produced very little (Fig. 3a, right hand panels and Fig. 3b).Fig. 3

Bottom Line: Ingestion of IE was confirmed by imaging flow cytometry.We show a special role for CD14(hi)CD16+ monocytes in phagocytosing opsonised P. falciparum IE and production of TNF.While ingestion was mediated by Fcγ receptor IIIa, this receptor was not sufficient to allow phagocytosis; despite opsonisation with antibody, phagocytosis of IE also required complement opsonisation.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biomedical Research, Burnet Institute, Melbourne, Victoria, 3004, Australia. jingling@burnet.edu.au.

ABSTRACT

Background: With more than 600,000 deaths from malaria, mainly of children under five years old and caused by infection with Plasmodium falciparum, comes an urgent need for an effective anti-malaria vaccine. Limited details on the mechanisms of protective immunity are a barrier to vaccine development. Antibodies play an important role in immunity to malaria and monocytes are key effectors in antibody-mediated protection by phagocytosing antibody-opsonised infected erythrocytes (IE). Eliciting antibodies that enhance phagocytosis of IE is therefore an important potential component of an effective vaccine, requiring robust assays to determine the ability of elicited antibodies to stimulate this in vivo. The mechanisms by which monocytes ingest IE and the nature of the monocytes which do so are unknown.

Methods: Purified trophozoite-stage P. falciparum IE were stained with ethidium bromide, opsonised with anti-erythrocyte antibodies and incubated with fresh whole blood. Phagocytosis of IE and TNF production by individual monocyte subsets was measured by flow cytometry. Ingestion of IE was confirmed by imaging flow cytometry.

Results: CD14(hi)CD16+ monocytes phagocytosed antibody-opsonised IE and produced TNF more efficiently than CD14(hi)CD16- and CD14(lo)CD16+ monocytes. Blocking experiments showed that Fcγ receptor IIIa (CD16) but not Fcγ receptor IIa (CD32a) or Fcγ receptor I (CD64) was necessary for phagocytosis. CD14(hi)CD16+ monocytes ingested antibody-opsonised IE when peripheral blood mononuclear cells were reconstituted with autologous serum but not heat-inactivated autologous serum. Antibody-opsonised IE were rapidly opsonised with complement component C3 in serum (t1/2 = 2-3 minutes) and phagocytosis of antibody-opsonised IE was inhibited in a dose-dependent manner by an inhibitor of C3 activation, compstatin. Compared to other monocyte subsets, CD14(hi)CD16+ monocytes expressed the highest levels of complement receptor 4 (CD11c) and activated complement receptor 3 (CD11b) subunits.

Conclusions: We show a special role for CD14(hi)CD16+ monocytes in phagocytosing opsonised P. falciparum IE and production of TNF. While ingestion was mediated by Fcγ receptor IIIa, this receptor was not sufficient to allow phagocytosis; despite opsonisation with antibody, phagocytosis of IE also required complement opsonisation. Assays which measure the ability of vaccines to elicit a protective antibody response to P. falciparum should consider their ability to promote phagocytosis and fix complement.

Show MeSH
Related in: MedlinePlus