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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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Complement opsonisation is required for monocyte phagocytosis of antibody-opsonised IE a Monocyte phagocytosis of CS2-IE (Unop) or CS2-IE opsonised with rabbit anti-human RBC antibody (OP) was determined using whole blood, whole blood reconstituted to its original volume with autologous heat-inactivated plasma (HI plasma) or with autologous plasma (plasma) as indicated. Phagocytosis by classical CD14hiCD16- (left hand panel), intermediate CD14hiCD16+ (middle panel) and non-classical CD14loCD16+ (right hand panel) monocytes were measured. Data represent mean (sem) of independent experiments using blood from three separate donors. Differences between conditions were assessed by one-way ANOVA using Tukey’s test for multiple comparisons. b Unopsonised CS2-IE or CS2-IE opsonised with rabbit anti-human RBC antibody were added to heparinised plasma for 0 and 30 minutes at 37 °C, stained with antiC3, and RBC analysed by flow cytometry. Histograms represent C3 staining at 0 time (dark grey histogram) or 30 minutes (light grey histogram). Right hand panel represents C3 staining of CS2-IE opsonised with rabbit anti-human RBC antibody after incubation in heparinised plasma for the indicated times at 4 °C (solid black circles) or 37 °C (open circles). c Compstatin (R&D Systems) was added to whole blood from stock solutions dissolved in PBS at the indicated final concentrations then phagocytosis of CS2-IE opsonised with rabbit anti-human RBC antibody by intermediate (open circles) or classical monocytes (solid black circles) determined (left hand panel) or non-classical monocytes (open squares) determined (right hand panel). The absolute values of phagocytosis by non-classical monocytes were very low and hence these data are plotted separately. Data represent mean (sem) of independent experiments using blood from three separate donors. ANOVA analysis of variance, IE infected erythrocytes, RBC red blood cells, sem standard error of the mean
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Fig2: Complement opsonisation is required for monocyte phagocytosis of antibody-opsonised IE a Monocyte phagocytosis of CS2-IE (Unop) or CS2-IE opsonised with rabbit anti-human RBC antibody (OP) was determined using whole blood, whole blood reconstituted to its original volume with autologous heat-inactivated plasma (HI plasma) or with autologous plasma (plasma) as indicated. Phagocytosis by classical CD14hiCD16- (left hand panel), intermediate CD14hiCD16+ (middle panel) and non-classical CD14loCD16+ (right hand panel) monocytes were measured. Data represent mean (sem) of independent experiments using blood from three separate donors. Differences between conditions were assessed by one-way ANOVA using Tukey’s test for multiple comparisons. b Unopsonised CS2-IE or CS2-IE opsonised with rabbit anti-human RBC antibody were added to heparinised plasma for 0 and 30 minutes at 37 °C, stained with antiC3, and RBC analysed by flow cytometry. Histograms represent C3 staining at 0 time (dark grey histogram) or 30 minutes (light grey histogram). Right hand panel represents C3 staining of CS2-IE opsonised with rabbit anti-human RBC antibody after incubation in heparinised plasma for the indicated times at 4 °C (solid black circles) or 37 °C (open circles). c Compstatin (R&D Systems) was added to whole blood from stock solutions dissolved in PBS at the indicated final concentrations then phagocytosis of CS2-IE opsonised with rabbit anti-human RBC antibody by intermediate (open circles) or classical monocytes (solid black circles) determined (left hand panel) or non-classical monocytes (open squares) determined (right hand panel). The absolute values of phagocytosis by non-classical monocytes were very low and hence these data are plotted separately. Data represent mean (sem) of independent experiments using blood from three separate donors. ANOVA analysis of variance, IE infected erythrocytes, RBC red blood cells, sem standard error of the mean

Mentions: Since the higher phagocytic activity by intermediate monocytes was observed only when IE were added to whole blood, but not to PBMC preparations, we reasoned that opsonins apart from IgG, such as complement components, might contribute to this activity. We, therefore, centrifuged heparinised whole blood, washed and reconstituted the cells to the original blood volume using either autologous serum or heat-inactivated autologous serum (collected in a separate serum tube at the same time as blood collection), then measured phagocytosis of CS2 IE. Phagocytosis was not affected by washing and reconstituting the blood cells in normal serum, but was abolished when heat-inactivated serum was used (Fig. 2a). These data suggest that complement opsonisation occurs during the 30 minute incubation of IE with whole blood and that this opsonisation is essential for efficient phagocytosis of IgG-opsonised IE by CD14hiCD16+ monocytes. To verify this, we added purified, IgG-opsonised CS2-IE to heparinised plasma for various times at 37 °C and measured deposition of C3b onto the opsonised IE by flow cytometry. There was minimal C3b bound to IE at 0 time (Fig. 2b, left and middle panels; dark grey histograms), or after 30 minutes in the absence of IgG opsonisation (Fig. 2b, left panel: light grey histogram), but considerable deposition onto IgG-opsonised IE after 30 minutes (Fig. 2b, middle panel: light grey histogram). C3b was deposited onto IE with a half time of 2.7 minutes (Fig. 2b, right panel). The fact that complement deposition required IgG opsonisation suggests that complement is fixed under these conditions primarily by the classical pathway. To determine the significance of complement to IE phagocytosis by monocytes in whole blood, we next examined the effect of an inhibitor of C3 activation, compstatin. Compstatin inhibited phagocytosis of IgG-opsonised IE by both CD14hiCD16- and CD14hiCD16+ monocytes (Fig. 2c) showing that even when opsonised with IgG, complement opsonisation is required for efficient phagocytosis of IE. In these experiments phagocytosis by CD14loCD16+ monocytes was very low and was thus excluded from this analysis.Fig. 2


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)

Complement opsonisation is required for monocyte phagocytosis of antibody-opsonised IE a Monocyte phagocytosis of CS2-IE (Unop) or CS2-IE opsonised with rabbit anti-human RBC antibody (OP) was determined using whole blood, whole blood reconstituted to its original volume with autologous heat-inactivated plasma (HI plasma) or with autologous plasma (plasma) as indicated. Phagocytosis by classical CD14hiCD16- (left hand panel), intermediate CD14hiCD16+ (middle panel) and non-classical CD14loCD16+ (right hand panel) monocytes were measured. Data represent mean (sem) of independent experiments using blood from three separate donors. Differences between conditions were assessed by one-way ANOVA using Tukey’s test for multiple comparisons. b Unopsonised CS2-IE or CS2-IE opsonised with rabbit anti-human RBC antibody were added to heparinised plasma for 0 and 30 minutes at 37 °C, stained with antiC3, and RBC analysed by flow cytometry. Histograms represent C3 staining at 0 time (dark grey histogram) or 30 minutes (light grey histogram). Right hand panel represents C3 staining of CS2-IE opsonised with rabbit anti-human RBC antibody after incubation in heparinised plasma for the indicated times at 4 °C (solid black circles) or 37 °C (open circles). c Compstatin (R&D Systems) was added to whole blood from stock solutions dissolved in PBS at the indicated final concentrations then phagocytosis of CS2-IE opsonised with rabbit anti-human RBC antibody by intermediate (open circles) or classical monocytes (solid black circles) determined (left hand panel) or non-classical monocytes (open squares) determined (right hand panel). The absolute values of phagocytosis by non-classical monocytes were very low and hence these data are plotted separately. Data represent mean (sem) of independent experiments using blood from three separate donors. ANOVA analysis of variance, IE infected erythrocytes, RBC red blood cells, sem standard error of the mean
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Related In: Results  -  Collection

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Fig2: Complement opsonisation is required for monocyte phagocytosis of antibody-opsonised IE a Monocyte phagocytosis of CS2-IE (Unop) or CS2-IE opsonised with rabbit anti-human RBC antibody (OP) was determined using whole blood, whole blood reconstituted to its original volume with autologous heat-inactivated plasma (HI plasma) or with autologous plasma (plasma) as indicated. Phagocytosis by classical CD14hiCD16- (left hand panel), intermediate CD14hiCD16+ (middle panel) and non-classical CD14loCD16+ (right hand panel) monocytes were measured. Data represent mean (sem) of independent experiments using blood from three separate donors. Differences between conditions were assessed by one-way ANOVA using Tukey’s test for multiple comparisons. b Unopsonised CS2-IE or CS2-IE opsonised with rabbit anti-human RBC antibody were added to heparinised plasma for 0 and 30 minutes at 37 °C, stained with antiC3, and RBC analysed by flow cytometry. Histograms represent C3 staining at 0 time (dark grey histogram) or 30 minutes (light grey histogram). Right hand panel represents C3 staining of CS2-IE opsonised with rabbit anti-human RBC antibody after incubation in heparinised plasma for the indicated times at 4 °C (solid black circles) or 37 °C (open circles). c Compstatin (R&D Systems) was added to whole blood from stock solutions dissolved in PBS at the indicated final concentrations then phagocytosis of CS2-IE opsonised with rabbit anti-human RBC antibody by intermediate (open circles) or classical monocytes (solid black circles) determined (left hand panel) or non-classical monocytes (open squares) determined (right hand panel). The absolute values of phagocytosis by non-classical monocytes were very low and hence these data are plotted separately. Data represent mean (sem) of independent experiments using blood from three separate donors. ANOVA analysis of variance, IE infected erythrocytes, RBC red blood cells, sem standard error of the mean
Mentions: Since the higher phagocytic activity by intermediate monocytes was observed only when IE were added to whole blood, but not to PBMC preparations, we reasoned that opsonins apart from IgG, such as complement components, might contribute to this activity. We, therefore, centrifuged heparinised whole blood, washed and reconstituted the cells to the original blood volume using either autologous serum or heat-inactivated autologous serum (collected in a separate serum tube at the same time as blood collection), then measured phagocytosis of CS2 IE. Phagocytosis was not affected by washing and reconstituting the blood cells in normal serum, but was abolished when heat-inactivated serum was used (Fig. 2a). These data suggest that complement opsonisation occurs during the 30 minute incubation of IE with whole blood and that this opsonisation is essential for efficient phagocytosis of IgG-opsonised IE by CD14hiCD16+ monocytes. To verify this, we added purified, IgG-opsonised CS2-IE to heparinised plasma for various times at 37 °C and measured deposition of C3b onto the opsonised IE by flow cytometry. There was minimal C3b bound to IE at 0 time (Fig. 2b, left and middle panels; dark grey histograms), or after 30 minutes in the absence of IgG opsonisation (Fig. 2b, left panel: light grey histogram), but considerable deposition onto IgG-opsonised IE after 30 minutes (Fig. 2b, middle panel: light grey histogram). C3b was deposited onto IE with a half time of 2.7 minutes (Fig. 2b, right panel). The fact that complement deposition required IgG opsonisation suggests that complement is fixed under these conditions primarily by the classical pathway. To determine the significance of complement to IE phagocytosis by monocytes in whole blood, we next examined the effect of an inhibitor of C3 activation, compstatin. Compstatin inhibited phagocytosis of IgG-opsonised IE by both CD14hiCD16- and CD14hiCD16+ monocytes (Fig. 2c) showing that even when opsonised with IgG, complement opsonisation is required for efficient phagocytosis of IE. In these experiments phagocytosis by CD14loCD16+ monocytes was very low and was thus excluded from this analysis.Fig. 2

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