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The CD16(+) (FcgammaRIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting.

Randolph GJ, Sanchez-Schmitz G, Liebman RM, Schäkel K - J. Exp. Med. (2002)

Bottom Line: These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels.CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix.We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

View Article: PubMed Central - PubMed

Affiliation: The Carl C. Icahn Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA. gwendalyn.randolph@mssm.edu

ABSTRACT
Much remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcgamma receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16(+) monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16(-) monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16(+) monocytes were inducible in CD16(-) monocytes by preincubation with TGFbeta1. We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

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Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14hi (b, c) and CD14med (d) expressing cells. Some of the CD14hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16+ cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were <300. This pattern of responses was observed in 2 independent analyses.
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fig7: Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14hi (b, c) and CD14med (d) expressing cells. Some of the CD14hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16+ cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were <300. This pattern of responses was observed in 2 independent analyses.

Mentions: The negative effects of anti-CD16 mAb on cell survival obviated experiments in which we sorted CD16+ monocytes using anti-CD16 mAb and then added them to endothelial cultures to assess differentiation and antigen-presenting capacity after encounter with a potent maturation stimulus such as subendothelial zymosan. Instead, we approached comparing the antigen-presenting cell function of these subsets after reverse transmigration using cell isolation techniques that did not leave residual anti-CD16 mAb on CD16+ cells. We reasoned that if CD16+ monocytes contributed significantly to the majority of mature antigen-presenting, reverse-transmigrated cells that their absence would result in reduced T cell proliferative responses. However, it was also important to be sure that any T cell proliferation observed did not arise from stimulation by the relatively rare CD14−CD11c+ DCs (1, 2). Thus, we separated monocyte subsets based on their level of CD14 staining (CD14− cells were discarded), as most CD16+ monocytes express lower levels of CD14 (CD14med) than CD16− monocytes (CD14hi). As there is some breadth in the spectrum of CD14 that is expressed by CD16+ monocytes, this approach did not achieve absolute purity of the two subsets, but did result in populations that were greatly enriched for one or the other subset (Fig. 7) . Some of the sorted CD14hi cells were finally depleted completely of CD16+ monocytes using anti-CD16 magnetic beads. All populations were then applied separately to endothelial monolayers containing zymosan in the subendothelial matrix. The presence of CD16+ monocytes in the population applied was correlated with recovery of reverse-transmigrated cells that exhibited robust capacity to induce T cell proliferation (Fig. 7). Mixing reverse-transmigrated cells from CD16-depleted cultures with reverse-transmigrated cells derived from a population highly enriched in CD16+ monocytes led to an overall inhibitory effect, consistent with the possibility that reverse-transmigrated cells derived from CD16− monocytes differentiated into presentation-suppressive macrophages (21) in the absence of CD16+ monocytes (Fig. 7). The presence of relatively few CD16+ monocytes amongst total monocytes added to endothelial cultures curbed this inhibitory effect (Fig. 7, compare d and b to d and c), raising the possibility that CD16+ cells may not only become DCs themselves but that they may also promote differentiation of reverse-transmigrated cells derived from CD16− monocytes toward a DC-like phenotype (Figs. 6 and 7).


The CD16(+) (FcgammaRIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting.

Randolph GJ, Sanchez-Schmitz G, Liebman RM, Schäkel K - J. Exp. Med. (2002)

Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14hi (b, c) and CD14med (d) expressing cells. Some of the CD14hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16+ cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were <300. This pattern of responses was observed in 2 independent analyses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig7: Capacity of reverse-transmigrated cells derived from monocyte subsets to stimulate allogeneic T cell proliferation. Monocytes were sorted according to their level of expression of CD14. First lymphocytes were removed from the PBMC fraction to generate a “presort” population of monocytes (a). Monocytes were then sorted into CD14hi (b, c) and CD14med (d) expressing cells. Some of the CD14hi monocytes were then treated with anti-CD16 miniMACs beads to remove any remaining CD16+ cells (c). These populations were added to endothelial cultures in which zymosan was present in the subendothelial matrix, and then live reverse transmigrated cells collected 2 d later were tested for their ability to induce allogeneic T cell proliferation at a ratio of 1 antigen-presenting cell: 80 T cells. In some conditions, a 1:1 mix of some reverse-transmigrated populations (d + b, or d + c) were used as APCs. Background counts for T cells cultured alone were <300. This pattern of responses was observed in 2 independent analyses.
Mentions: The negative effects of anti-CD16 mAb on cell survival obviated experiments in which we sorted CD16+ monocytes using anti-CD16 mAb and then added them to endothelial cultures to assess differentiation and antigen-presenting capacity after encounter with a potent maturation stimulus such as subendothelial zymosan. Instead, we approached comparing the antigen-presenting cell function of these subsets after reverse transmigration using cell isolation techniques that did not leave residual anti-CD16 mAb on CD16+ cells. We reasoned that if CD16+ monocytes contributed significantly to the majority of mature antigen-presenting, reverse-transmigrated cells that their absence would result in reduced T cell proliferative responses. However, it was also important to be sure that any T cell proliferation observed did not arise from stimulation by the relatively rare CD14−CD11c+ DCs (1, 2). Thus, we separated monocyte subsets based on their level of CD14 staining (CD14− cells were discarded), as most CD16+ monocytes express lower levels of CD14 (CD14med) than CD16− monocytes (CD14hi). As there is some breadth in the spectrum of CD14 that is expressed by CD16+ monocytes, this approach did not achieve absolute purity of the two subsets, but did result in populations that were greatly enriched for one or the other subset (Fig. 7) . Some of the sorted CD14hi cells were finally depleted completely of CD16+ monocytes using anti-CD16 magnetic beads. All populations were then applied separately to endothelial monolayers containing zymosan in the subendothelial matrix. The presence of CD16+ monocytes in the population applied was correlated with recovery of reverse-transmigrated cells that exhibited robust capacity to induce T cell proliferation (Fig. 7). Mixing reverse-transmigrated cells from CD16-depleted cultures with reverse-transmigrated cells derived from a population highly enriched in CD16+ monocytes led to an overall inhibitory effect, consistent with the possibility that reverse-transmigrated cells derived from CD16− monocytes differentiated into presentation-suppressive macrophages (21) in the absence of CD16+ monocytes (Fig. 7). The presence of relatively few CD16+ monocytes amongst total monocytes added to endothelial cultures curbed this inhibitory effect (Fig. 7, compare d and b to d and c), raising the possibility that CD16+ cells may not only become DCs themselves but that they may also promote differentiation of reverse-transmigrated cells derived from CD16− monocytes toward a DC-like phenotype (Figs. 6 and 7).

Bottom Line: These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels.CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix.We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

View Article: PubMed Central - PubMed

Affiliation: The Carl C. Icahn Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA. gwendalyn.randolph@mssm.edu

ABSTRACT
Much remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcgamma receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16(+) monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16(-) monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16(+) monocytes were inducible in CD16(-) monocytes by preincubation with TGFbeta1. We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

Show MeSH