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Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Corripio-Miyar Y, Hope J, McInnes CJ, Wattegedera SR, Jensen K, Pang Y, Entrican G, Glass EJ - Vet. Res. (2015)

Bottom Line: The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115.The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules.All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK. Yolanda.Corripio-Miyar@moredun.ac.uk.

ABSTRACT
Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

No MeSH data available.


Related in: MedlinePlus

Expression of CD14 and CD16 defines subpopulations of cells in bovine peripheral blood with differential expression. The expression of CD16 and CD14 was determined by flow cytometry in freshly isolated bovine PBMC. Live, single cells gated as in Figure 1, were analysed by staining for CD16 (A) or CD14 (B). To further characterise these populations, live single cells were then assessed for expression of CD16 and CD14 by double staining (C). Variable CD16 expression was observed. As shown in Figure 1 cells with CD16 fluorescence <400 were negative for expression. Cells with a CD16 fluorescence between 800 and 10 000 were NKp46+ NK cells (panel Q3). Cells with CD16 fluorescence above 10 000 (panel Q4) were identified as a discrete population of CD172a+ NKp46− CD16+CD14− cells (CD14−CD16++). CD14+ cells had fluorescence intensity >300 and expressed differential levels of CD16. Two CD14+ populations were defined by a diagonal gate (panels Q1 and Q2 [8]). Data shown are for one representative animal (A, B) and for six individual animals (C).
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Fig2: Expression of CD14 and CD16 defines subpopulations of cells in bovine peripheral blood with differential expression. The expression of CD16 and CD14 was determined by flow cytometry in freshly isolated bovine PBMC. Live, single cells gated as in Figure 1, were analysed by staining for CD16 (A) or CD14 (B). To further characterise these populations, live single cells were then assessed for expression of CD16 and CD14 by double staining (C). Variable CD16 expression was observed. As shown in Figure 1 cells with CD16 fluorescence <400 were negative for expression. Cells with a CD16 fluorescence between 800 and 10 000 were NKp46+ NK cells (panel Q3). Cells with CD16 fluorescence above 10 000 (panel Q4) were identified as a discrete population of CD172a+ NKp46− CD16+CD14− cells (CD14−CD16++). CD14+ cells had fluorescence intensity >300 and expressed differential levels of CD16. Two CD14+ populations were defined by a diagonal gate (panels Q1 and Q2 [8]). Data shown are for one representative animal (A, B) and for six individual animals (C).

Mentions: In order to identify myeloid cell populations in the peripheral blood of cattle, expression of CD14 and CD16 was analysed on PBMC (n = 6). After dead cell and doublet discrimination (Figure 1A and B), a number of CD16 positive sub-populations with different fluorescence intensities and complexity were evident (Figures 1F, 2A); whereas the expression of CD14 was more uniform with a major population observed (Figures 1G, 2B). In order to further define the nature of the CD14+ and CD16+ populations, PBMC were double-labelled with anti-CD14 and anti-CD16 conjugated antibodies, a method commonly used to identify monocyte subsets in human blood [2,7,9,42]. Although considerable variation in the staining patterns was observed across the six cattle studied (Figure 2C), there was evidence for the presence of a number of sub-populations of cells with differential CD14 and CD16 expression. Within the CD14 negative population there were cells with varying levels of CD16 expression. We demonstrated that CD14− cells expressing CD16 at MFI of ~800-10 000 (Figure 2C, panel Q3) were NK cells as these expressed NKp46 (Figure 1I) and were negative for CD172a (Figure 1J). Consequently, these cells were excluded from further analysis. A distinct population of cells also negative for CD14 and with a CD16 MFI of greater than 10 000 (CD14−CD16++; Figure 2C, panel Q4) was evident at relatively low proportion (0.9 ± 0.5%).Figure 2


Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Corripio-Miyar Y, Hope J, McInnes CJ, Wattegedera SR, Jensen K, Pang Y, Entrican G, Glass EJ - Vet. Res. (2015)

Expression of CD14 and CD16 defines subpopulations of cells in bovine peripheral blood with differential expression. The expression of CD16 and CD14 was determined by flow cytometry in freshly isolated bovine PBMC. Live, single cells gated as in Figure 1, were analysed by staining for CD16 (A) or CD14 (B). To further characterise these populations, live single cells were then assessed for expression of CD16 and CD14 by double staining (C). Variable CD16 expression was observed. As shown in Figure 1 cells with CD16 fluorescence <400 were negative for expression. Cells with a CD16 fluorescence between 800 and 10 000 were NKp46+ NK cells (panel Q3). Cells with CD16 fluorescence above 10 000 (panel Q4) were identified as a discrete population of CD172a+ NKp46− CD16+CD14− cells (CD14−CD16++). CD14+ cells had fluorescence intensity >300 and expressed differential levels of CD16. Two CD14+ populations were defined by a diagonal gate (panels Q1 and Q2 [8]). Data shown are for one representative animal (A, B) and for six individual animals (C).
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Fig2: Expression of CD14 and CD16 defines subpopulations of cells in bovine peripheral blood with differential expression. The expression of CD16 and CD14 was determined by flow cytometry in freshly isolated bovine PBMC. Live, single cells gated as in Figure 1, were analysed by staining for CD16 (A) or CD14 (B). To further characterise these populations, live single cells were then assessed for expression of CD16 and CD14 by double staining (C). Variable CD16 expression was observed. As shown in Figure 1 cells with CD16 fluorescence <400 were negative for expression. Cells with a CD16 fluorescence between 800 and 10 000 were NKp46+ NK cells (panel Q3). Cells with CD16 fluorescence above 10 000 (panel Q4) were identified as a discrete population of CD172a+ NKp46− CD16+CD14− cells (CD14−CD16++). CD14+ cells had fluorescence intensity >300 and expressed differential levels of CD16. Two CD14+ populations were defined by a diagonal gate (panels Q1 and Q2 [8]). Data shown are for one representative animal (A, B) and for six individual animals (C).
Mentions: In order to identify myeloid cell populations in the peripheral blood of cattle, expression of CD14 and CD16 was analysed on PBMC (n = 6). After dead cell and doublet discrimination (Figure 1A and B), a number of CD16 positive sub-populations with different fluorescence intensities and complexity were evident (Figures 1F, 2A); whereas the expression of CD14 was more uniform with a major population observed (Figures 1G, 2B). In order to further define the nature of the CD14+ and CD16+ populations, PBMC were double-labelled with anti-CD14 and anti-CD16 conjugated antibodies, a method commonly used to identify monocyte subsets in human blood [2,7,9,42]. Although considerable variation in the staining patterns was observed across the six cattle studied (Figure 2C), there was evidence for the presence of a number of sub-populations of cells with differential CD14 and CD16 expression. Within the CD14 negative population there were cells with varying levels of CD16 expression. We demonstrated that CD14− cells expressing CD16 at MFI of ~800-10 000 (Figure 2C, panel Q3) were NK cells as these expressed NKp46 (Figure 1I) and were negative for CD172a (Figure 1J). Consequently, these cells were excluded from further analysis. A distinct population of cells also negative for CD14 and with a CD16 MFI of greater than 10 000 (CD14−CD16++; Figure 2C, panel Q4) was evident at relatively low proportion (0.9 ± 0.5%).Figure 2

Bottom Line: The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115.The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules.All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK. Yolanda.Corripio-Miyar@moredun.ac.uk.

ABSTRACT
Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

No MeSH data available.


Related in: MedlinePlus