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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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Expression of Fcγ and complement receptors on monocyte subsets and the effect of blocking antibodies on phagocytosis by individual subsets. a Expression of Fcγ and complement receptors on monocytes was determined by whole blood staining. Black bars: C, white bars: IM, grey bars: NC. Bars represent mean (sem) of MFI using blood from nine separate donors (eight for CD32b). aCD11b refers to activated CD11b defined by the epitope recognised by the CBRM 1/5 monoclonal antibody. Differences between subsets were assessed using Wilcoxon’s matched pairs signed rank test. * p <0.05, ** p <0.01. b Whole blood was incubated for 30 minutes at 4 °C with the indicated concentrations of each blocking antibody before addition of CS2-IE and determination of phagocytosis. Representative dose response curves from four independent experiments, of inhibition of phagocytosis by closed black circles), IM monocytes (open circles) and NC monocytes (open squares) are shown in the upper panels and data (mean, sem) from experiments with whole blood from three separate donors conducted using 10 μg/mL of each blocking antibody are shown in the lower panels. c Effect of blocking LFA-1 (CD11a), CR3 (CD11b) and CR4 (CD11c) on phagocytosis of IgG opsonised CS2-IE. Upper panels show dose response of the indicated blocking antibodies and lower panels show aggregate data (mean, sem from n =3 independent experiments). Symbols are the same as in a and b. C classical, IM intermediate, N non-classical
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Fig4: Expression of Fcγ and complement receptors on monocyte subsets and the effect of blocking antibodies on phagocytosis by individual subsets. a Expression of Fcγ and complement receptors on monocytes was determined by whole blood staining. Black bars: C, white bars: IM, grey bars: NC. Bars represent mean (sem) of MFI using blood from nine separate donors (eight for CD32b). aCD11b refers to activated CD11b defined by the epitope recognised by the CBRM 1/5 monoclonal antibody. Differences between subsets were assessed using Wilcoxon’s matched pairs signed rank test. * p <0.05, ** p <0.01. b Whole blood was incubated for 30 minutes at 4 °C with the indicated concentrations of each blocking antibody before addition of CS2-IE and determination of phagocytosis. Representative dose response curves from four independent experiments, of inhibition of phagocytosis by closed black circles), IM monocytes (open circles) and NC monocytes (open squares) are shown in the upper panels and data (mean, sem) from experiments with whole blood from three separate donors conducted using 10 μg/mL of each blocking antibody are shown in the lower panels. c Effect of blocking LFA-1 (CD11a), CR3 (CD11b) and CR4 (CD11c) on phagocytosis of IgG opsonised CS2-IE. Upper panels show dose response of the indicated blocking antibodies and lower panels show aggregate data (mean, sem from n =3 independent experiments). Symbols are the same as in a and b. C classical, IM intermediate, N non-classical

Mentions: We next phenotyped monocytes from nine independent donors to determine how the subsets differ with respect to expression of receptors involved in binding and phagocytosis of complement and IgG-opsonised targets. Of the phagocytic Fcγ receptors, CD14hiCD16+ monocytes expressed significantly higher levels of Fcγ receptor IIa, CD32a, compared to the other subsets (Fig. 4a). The level of the inhibitory Fcγ receptor, CD32b, was also highest in this subset, although this receptor appeared to be expressed at much lower levels than CD32a. With respect to the phagocytic complement receptors, the CD14hiCD16+ monocytes expressed the highest levels of the α chain of CR4, CD11c. Of interest, however, was the observation that although CD14hiCD16+ monocytes expressed levels of CD11b (the α chain of CR3) that were intermediate between expression on the CD14hiCD16- and CD14loCD16+ subsets, they expressed the highest levels of activated CD11b suggesting that inside-out signaling required for CR3 activation was relatively stronger in this subset. CD14hiCD16+ monocytes also expressed the highest levels of the α chain (CD11a) of the adhesion molecule LFA-1. Since CD32a is the only Fcγ receptor expressed most highly on CD14hiCD16+ monocytes relative to other subsets we reasoned that it may have a unique role in phagocytosis of IgG-opsonised IE. We, therefore, used blocking antibodies to determine which Fcγ receptors are required for phagocytosis. Aliquots of whole blood were pre-incubated for 30 minutes with blocking antibodies specific for CD16, CD32a and CD64, then IgG-opsonised CS2 IE were added and phagocytosis measured after 30 minutes. The blocking antibody specific for CD16, 3G8, inhibited phagocytosis by CD14hiCD16+ and CD14loCD16+ monocytes by approximately 90 % at 10–20 μg/mL (Fig. 4b upper panel) but, as expected, had no effect on phagocytosis by CD14hiCD16- monocytes which do not express CD16. This inhibition was confirmed using whole blood from three individual donors incubated with 10 μg/mL blocking antibodies (Fig. 4b lower panel). In contrast, blocking antibodies specific for CD32a, IV.3, and for CD64, 10.1, had no effect on phagocytosis by any subset despite these receptors being expressed on all three subsets. Thus, CD16, but not CD32a or CD64 is necessary for phagocytosis of IgG-opsonised IE in whole blood. However, the fact that non-classical monocytes express CD16 but phagocytose IE poorly shows that CD16 expression is not sufficient. Since complement opsonisation was also necessary for phagocytosis of IgG-opsonised IE in whole blood, we investigated the effect of blocking antibodies to the phagocytic complement receptors CR1, CR3 and CR4 as well as antibodies to LFA-1. Antibodies to the α chain of CR3 (CD11b) and CR4 (CD11c) showed minimal inhibition at 10 μg/mL but blocked more efficiently at higher concentrations (Fig. 4c). Anti CD11a did not inhibit phagocytosis by any monocyte subset.Fig. 4


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)

Expression of Fcγ and complement receptors on monocyte subsets and the effect of blocking antibodies on phagocytosis by individual subsets. a Expression of Fcγ and complement receptors on monocytes was determined by whole blood staining. Black bars: C, white bars: IM, grey bars: NC. Bars represent mean (sem) of MFI using blood from nine separate donors (eight for CD32b). aCD11b refers to activated CD11b defined by the epitope recognised by the CBRM 1/5 monoclonal antibody. Differences between subsets were assessed using Wilcoxon’s matched pairs signed rank test. * p <0.05, ** p <0.01. b Whole blood was incubated for 30 minutes at 4 °C with the indicated concentrations of each blocking antibody before addition of CS2-IE and determination of phagocytosis. Representative dose response curves from four independent experiments, of inhibition of phagocytosis by closed black circles), IM monocytes (open circles) and NC monocytes (open squares) are shown in the upper panels and data (mean, sem) from experiments with whole blood from three separate donors conducted using 10 μg/mL of each blocking antibody are shown in the lower panels. c Effect of blocking LFA-1 (CD11a), CR3 (CD11b) and CR4 (CD11c) on phagocytosis of IgG opsonised CS2-IE. Upper panels show dose response of the indicated blocking antibodies and lower panels show aggregate data (mean, sem from n =3 independent experiments). Symbols are the same as in a and b. C classical, IM intermediate, N non-classical
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Fig4: Expression of Fcγ and complement receptors on monocyte subsets and the effect of blocking antibodies on phagocytosis by individual subsets. a Expression of Fcγ and complement receptors on monocytes was determined by whole blood staining. Black bars: C, white bars: IM, grey bars: NC. Bars represent mean (sem) of MFI using blood from nine separate donors (eight for CD32b). aCD11b refers to activated CD11b defined by the epitope recognised by the CBRM 1/5 monoclonal antibody. Differences between subsets were assessed using Wilcoxon’s matched pairs signed rank test. * p <0.05, ** p <0.01. b Whole blood was incubated for 30 minutes at 4 °C with the indicated concentrations of each blocking antibody before addition of CS2-IE and determination of phagocytosis. Representative dose response curves from four independent experiments, of inhibition of phagocytosis by closed black circles), IM monocytes (open circles) and NC monocytes (open squares) are shown in the upper panels and data (mean, sem) from experiments with whole blood from three separate donors conducted using 10 μg/mL of each blocking antibody are shown in the lower panels. c Effect of blocking LFA-1 (CD11a), CR3 (CD11b) and CR4 (CD11c) on phagocytosis of IgG opsonised CS2-IE. Upper panels show dose response of the indicated blocking antibodies and lower panels show aggregate data (mean, sem from n =3 independent experiments). Symbols are the same as in a and b. C classical, IM intermediate, N non-classical
Mentions: We next phenotyped monocytes from nine independent donors to determine how the subsets differ with respect to expression of receptors involved in binding and phagocytosis of complement and IgG-opsonised targets. Of the phagocytic Fcγ receptors, CD14hiCD16+ monocytes expressed significantly higher levels of Fcγ receptor IIa, CD32a, compared to the other subsets (Fig. 4a). The level of the inhibitory Fcγ receptor, CD32b, was also highest in this subset, although this receptor appeared to be expressed at much lower levels than CD32a. With respect to the phagocytic complement receptors, the CD14hiCD16+ monocytes expressed the highest levels of the α chain of CR4, CD11c. Of interest, however, was the observation that although CD14hiCD16+ monocytes expressed levels of CD11b (the α chain of CR3) that were intermediate between expression on the CD14hiCD16- and CD14loCD16+ subsets, they expressed the highest levels of activated CD11b suggesting that inside-out signaling required for CR3 activation was relatively stronger in this subset. CD14hiCD16+ monocytes also expressed the highest levels of the α chain (CD11a) of the adhesion molecule LFA-1. Since CD32a is the only Fcγ receptor expressed most highly on CD14hiCD16+ monocytes relative to other subsets we reasoned that it may have a unique role in phagocytosis of IgG-opsonised IE. We, therefore, used blocking antibodies to determine which Fcγ receptors are required for phagocytosis. Aliquots of whole blood were pre-incubated for 30 minutes with blocking antibodies specific for CD16, CD32a and CD64, then IgG-opsonised CS2 IE were added and phagocytosis measured after 30 minutes. The blocking antibody specific for CD16, 3G8, inhibited phagocytosis by CD14hiCD16+ and CD14loCD16+ monocytes by approximately 90 % at 10–20 μg/mL (Fig. 4b upper panel) but, as expected, had no effect on phagocytosis by CD14hiCD16- monocytes which do not express CD16. This inhibition was confirmed using whole blood from three individual donors incubated with 10 μg/mL blocking antibodies (Fig. 4b lower panel). In contrast, blocking antibodies specific for CD32a, IV.3, and for CD64, 10.1, had no effect on phagocytosis by any subset despite these receptors being expressed on all three subsets. Thus, CD16, but not CD32a or CD64 is necessary for phagocytosis of IgG-opsonised IE in whole blood. However, the fact that non-classical monocytes express CD16 but phagocytose IE poorly shows that CD16 expression is not sufficient. Since complement opsonisation was also necessary for phagocytosis of IgG-opsonised IE in whole blood, we investigated the effect of blocking antibodies to the phagocytic complement receptors CR1, CR3 and CR4 as well as antibodies to LFA-1. Antibodies to the α chain of CR3 (CD11b) and CR4 (CD11c) showed minimal inhibition at 10 μg/mL but blocked more efficiently at higher concentrations (Fig. 4c). Anti CD11a did not inhibit phagocytosis by any monocyte subset.Fig. 4

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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Related in: MedlinePlus