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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+ intermediate monocytes phagocytose IE more efficiently than other monocytes. a Whole blood was incubated with EtBr-labelled CS2-IE for 30 minutes then uningested RBC removed by hypotonic lysis and washing. Cells were stained with anti-CD14 and CD16, monocytes gated using forward and side scatter then subsets defined as classical (C: CD14hiCD16-), intermediate (IM: CD14hiCD16+) and non-classical (NC: CD14loCD16+) as shown. Histograms show EtBr staining of the three subsets incubated at 37 °C (red histograms) or 4 °C (blue histograms) with unopsonised (IE, top) or opsonised (IgG-IE, bottom) IE. b Phagocytosis using blood from eight separate donors. Whole blood was incubated as in a with unopsonised CS2-IE (left hand panels; IE) or CS2-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. c Phagocytosis by monocyte subsets of IE opsonised with rabbit anti human RBC was measured using PBMC prepared from four separate donors (left hand panels). Phagocytosis of IE opsonised with pooled human immune serum was measured using PBMC prepared from six separate donors (right hand panels). d Phagocytosis of unopsonised CS2-IE (left hand panels; IE) and CS2-IE opsonised with pooled human immune serum (right hand panels; IgG-IE) was measured in a whole blood assay as in a using blood from nine separate donors. e Phagocytosis using blood from six separate donors. Whole blood was incubated as in a with unopsonised E8B-IE (left hand panels; IE) or E8B-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. Background phagocytosis measured at 4 °C was subtracted from all data points. The percent phagocytosis by intermediate (IM) monocytes was compared using pairwise comparisons in each case (b-e) with either that by classical (C) monocytes or non-classical (NC) monocytes, as indicated. Differences between groups were assessed using Wilcoxon matched pairs signed rank test: * p < .05, ** p <0.01. EtBr ethidium bromide, IE infected erythrocytes, PBMC peripheral blood monocytes; RBC red blood cells
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Fig1: CD14hiCD16+ intermediate monocytes phagocytose IE more efficiently than other monocytes. a Whole blood was incubated with EtBr-labelled CS2-IE for 30 minutes then uningested RBC removed by hypotonic lysis and washing. Cells were stained with anti-CD14 and CD16, monocytes gated using forward and side scatter then subsets defined as classical (C: CD14hiCD16-), intermediate (IM: CD14hiCD16+) and non-classical (NC: CD14loCD16+) as shown. Histograms show EtBr staining of the three subsets incubated at 37 °C (red histograms) or 4 °C (blue histograms) with unopsonised (IE, top) or opsonised (IgG-IE, bottom) IE. b Phagocytosis using blood from eight separate donors. Whole blood was incubated as in a with unopsonised CS2-IE (left hand panels; IE) or CS2-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. c Phagocytosis by monocyte subsets of IE opsonised with rabbit anti human RBC was measured using PBMC prepared from four separate donors (left hand panels). Phagocytosis of IE opsonised with pooled human immune serum was measured using PBMC prepared from six separate donors (right hand panels). d Phagocytosis of unopsonised CS2-IE (left hand panels; IE) and CS2-IE opsonised with pooled human immune serum (right hand panels; IgG-IE) was measured in a whole blood assay as in a using blood from nine separate donors. e Phagocytosis using blood from six separate donors. Whole blood was incubated as in a with unopsonised E8B-IE (left hand panels; IE) or E8B-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. Background phagocytosis measured at 4 °C was subtracted from all data points. The percent phagocytosis by intermediate (IM) monocytes was compared using pairwise comparisons in each case (b-e) with either that by classical (C) monocytes or non-classical (NC) monocytes, as indicated. Differences between groups were assessed using Wilcoxon matched pairs signed rank test: * p < .05, ** p <0.01. EtBr ethidium bromide, IE infected erythrocytes, PBMC peripheral blood monocytes; RBC red blood cells

Mentions: Whole blood obtained from an individual with no previous history of malaria infection was incubated with EtBr-stained CS2-IE, opsonised or not opsonised with rabbit anti human RBC IgG, and phagocytosis analysed by flow cytometry. The CS2 parasite isolate was chosen both for its relevance to pregnancy-associated malaria and because the lack of CD36 binding potentially minimises the level of non-opsonic phagocytosis. Events within the broad monocyte gate defined by forward and side scatter were analysed on a CD14 versus CD16 dot plot to define the three monocyte subsets CD14hiCD16- “classical”, CD14hiCD16+ “intermediate” and CD14loCD16+ “non-classical” monocytes (Fig. 1a). The extent of IE phagocytosis by each monocyte subset was determined from the intensity of EtBr fluorescence and compared to the 4 °C negative control. There was little or no phagocytosis of unopsonised IE by any monocyte subset (Fig. 1a, right, upper panels). Opsonisation with IgG increased phagocytosis of IE, particularly by intermediate monocytes (Fig. 1a, right lower panels). Surprisingly, we detected little IE ingestion in the CD14hiCD16- or the CD14loCD16+ subsets. CD14hiCD16+ monocytes showed much higher phagocytosis of both IgG-opsonised and non-opsonised IE with the CD14loCD16+ monocyte subset showing the least amount of activity (Fig. 1b). These differences were not due to generally higher phagocytic activity of CD14hiCD16+ compared to other monocyte subsets since when unopsonised Escherichia coli was used as a target the classical subset showed the highest degree of phagocytosis (Additional file 1: Figure S1). Nor was it due to a greater ability of CD14hiCD16+ monocytes to phagocytose particles of the size of erythrocytes (7 μm diameter) since when IE were incubated with isolated PBMC instead of whole blood the classical monocyte subset ingested IgG-opsonised IE to a similar extent (Fig. 1c). Since we used rabbit anti-human erythrocyte IgG to opsonise IE to high levels, we next confirmed that CD14hiCD16+ monocytes showed enhanced phagocytosis of IE opsonised with human IgG. IE were opsonised with a pool of immune sera from women with placental malaria who had a high titre of antibodies recognising the CS2 isolate; CD14hiCD16+ monocytes were again the only subset to substantially phagocytose parasites (Fig. 1d). CS2 is a P. falciparum line that expresses Var2CSA. To determine if the increased phagocytic capacity of CD14hiCD16+ monocytes was specific to this parasite strain, we incubated whole blood with E8B-IE. E8B is a malaria strain that expresses a mixture of var genes that, in contrast to CS2, promote binding to CD36 and ICAM-1 [33, 34]. CD14hiCD16+ monocytes were the only monocyte subset to efficiently ingest IgG-opsonised E8B-IE (Fig. 1e) indicating that the specificity for CD14hiCD16+ monocytes is independent of the PfEMP-1 type. More monocytes ingested IE when PBMC preparations were used in the phagocytosis assay compared to whole blood (Fig. 1c c.f 1d). This was true for both CD14hiCD16+ monocytes (median phagocytosis = 34.4 c.f. 10.4, p = 0.02) and for CD14hiCD16- monocytes (median phagocytosis = 47.9 c.f. 4.22, p = 0.0007).Fig. 1


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+ intermediate monocytes phagocytose IE more efficiently than other monocytes. a Whole blood was incubated with EtBr-labelled CS2-IE for 30 minutes then uningested RBC removed by hypotonic lysis and washing. Cells were stained with anti-CD14 and CD16, monocytes gated using forward and side scatter then subsets defined as classical (C: CD14hiCD16-), intermediate (IM: CD14hiCD16+) and non-classical (NC: CD14loCD16+) as shown. Histograms show EtBr staining of the three subsets incubated at 37 °C (red histograms) or 4 °C (blue histograms) with unopsonised (IE, top) or opsonised (IgG-IE, bottom) IE. b Phagocytosis using blood from eight separate donors. Whole blood was incubated as in a with unopsonised CS2-IE (left hand panels; IE) or CS2-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. c Phagocytosis by monocyte subsets of IE opsonised with rabbit anti human RBC was measured using PBMC prepared from four separate donors (left hand panels). Phagocytosis of IE opsonised with pooled human immune serum was measured using PBMC prepared from six separate donors (right hand panels). d Phagocytosis of unopsonised CS2-IE (left hand panels; IE) and CS2-IE opsonised with pooled human immune serum (right hand panels; IgG-IE) was measured in a whole blood assay as in a using blood from nine separate donors. e Phagocytosis using blood from six separate donors. Whole blood was incubated as in a with unopsonised E8B-IE (left hand panels; IE) or E8B-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. Background phagocytosis measured at 4 °C was subtracted from all data points. The percent phagocytosis by intermediate (IM) monocytes was compared using pairwise comparisons in each case (b-e) with either that by classical (C) monocytes or non-classical (NC) monocytes, as indicated. Differences between groups were assessed using Wilcoxon matched pairs signed rank test: * p < .05, ** p <0.01. EtBr ethidium bromide, IE infected erythrocytes, PBMC peripheral blood monocytes; RBC red blood cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: CD14hiCD16+ intermediate monocytes phagocytose IE more efficiently than other monocytes. a Whole blood was incubated with EtBr-labelled CS2-IE for 30 minutes then uningested RBC removed by hypotonic lysis and washing. Cells were stained with anti-CD14 and CD16, monocytes gated using forward and side scatter then subsets defined as classical (C: CD14hiCD16-), intermediate (IM: CD14hiCD16+) and non-classical (NC: CD14loCD16+) as shown. Histograms show EtBr staining of the three subsets incubated at 37 °C (red histograms) or 4 °C (blue histograms) with unopsonised (IE, top) or opsonised (IgG-IE, bottom) IE. b Phagocytosis using blood from eight separate donors. Whole blood was incubated as in a with unopsonised CS2-IE (left hand panels; IE) or CS2-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. c Phagocytosis by monocyte subsets of IE opsonised with rabbit anti human RBC was measured using PBMC prepared from four separate donors (left hand panels). Phagocytosis of IE opsonised with pooled human immune serum was measured using PBMC prepared from six separate donors (right hand panels). d Phagocytosis of unopsonised CS2-IE (left hand panels; IE) and CS2-IE opsonised with pooled human immune serum (right hand panels; IgG-IE) was measured in a whole blood assay as in a using blood from nine separate donors. e Phagocytosis using blood from six separate donors. Whole blood was incubated as in a with unopsonised E8B-IE (left hand panels; IE) or E8B-IE opsonised with rabbit anti-human RBC antibody (right hand panels; IgG-IE) as indicated. Background phagocytosis measured at 4 °C was subtracted from all data points. The percent phagocytosis by intermediate (IM) monocytes was compared using pairwise comparisons in each case (b-e) with either that by classical (C) monocytes or non-classical (NC) monocytes, as indicated. Differences between groups were assessed using Wilcoxon matched pairs signed rank test: * p < .05, ** p <0.01. EtBr ethidium bromide, IE infected erythrocytes, PBMC peripheral blood monocytes; RBC red blood cells
Mentions: Whole blood obtained from an individual with no previous history of malaria infection was incubated with EtBr-stained CS2-IE, opsonised or not opsonised with rabbit anti human RBC IgG, and phagocytosis analysed by flow cytometry. The CS2 parasite isolate was chosen both for its relevance to pregnancy-associated malaria and because the lack of CD36 binding potentially minimises the level of non-opsonic phagocytosis. Events within the broad monocyte gate defined by forward and side scatter were analysed on a CD14 versus CD16 dot plot to define the three monocyte subsets CD14hiCD16- “classical”, CD14hiCD16+ “intermediate” and CD14loCD16+ “non-classical” monocytes (Fig. 1a). The extent of IE phagocytosis by each monocyte subset was determined from the intensity of EtBr fluorescence and compared to the 4 °C negative control. There was little or no phagocytosis of unopsonised IE by any monocyte subset (Fig. 1a, right, upper panels). Opsonisation with IgG increased phagocytosis of IE, particularly by intermediate monocytes (Fig. 1a, right lower panels). Surprisingly, we detected little IE ingestion in the CD14hiCD16- or the CD14loCD16+ subsets. CD14hiCD16+ monocytes showed much higher phagocytosis of both IgG-opsonised and non-opsonised IE with the CD14loCD16+ monocyte subset showing the least amount of activity (Fig. 1b). These differences were not due to generally higher phagocytic activity of CD14hiCD16+ compared to other monocyte subsets since when unopsonised Escherichia coli was used as a target the classical subset showed the highest degree of phagocytosis (Additional file 1: Figure S1). Nor was it due to a greater ability of CD14hiCD16+ monocytes to phagocytose particles of the size of erythrocytes (7 μm diameter) since when IE were incubated with isolated PBMC instead of whole blood the classical monocyte subset ingested IgG-opsonised IE to a similar extent (Fig. 1c). Since we used rabbit anti-human erythrocyte IgG to opsonise IE to high levels, we next confirmed that CD14hiCD16+ monocytes showed enhanced phagocytosis of IE opsonised with human IgG. IE were opsonised with a pool of immune sera from women with placental malaria who had a high titre of antibodies recognising the CS2 isolate; CD14hiCD16+ monocytes were again the only subset to substantially phagocytose parasites (Fig. 1d). CS2 is a P. falciparum line that expresses Var2CSA. To determine if the increased phagocytic capacity of CD14hiCD16+ monocytes was specific to this parasite strain, we incubated whole blood with E8B-IE. E8B is a malaria strain that expresses a mixture of var genes that, in contrast to CS2, promote binding to CD36 and ICAM-1 [33, 34]. CD14hiCD16+ monocytes were the only monocyte subset to efficiently ingest IgG-opsonised E8B-IE (Fig. 1e) indicating that the specificity for CD14hiCD16+ monocytes is independent of the PfEMP-1 type. More monocytes ingested IE when PBMC preparations were used in the phagocytosis assay compared to whole blood (Fig. 1c c.f 1d). This was true for both CD14hiCD16+ monocytes (median phagocytosis = 34.4 c.f. 10.4, p = 0.02) and for CD14hiCD16- monocytes (median phagocytosis = 47.9 c.f. 4.22, p = 0.0007).Fig. 1

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