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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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Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16+ monocytes. CD56+ NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16− and CD16+(CD16mix) CD14+ monocytes. In some samples, the remaining CD16+ cells were depleted, leaving CD56−CD16− PBMCs. CD16mix or CD16− PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16− fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56−CD16mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16+ blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16+ monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16mix and CD16− monocytes were applied.
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fig6: Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16+ monocytes. CD56+ NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16− and CD16+(CD16mix) CD14+ monocytes. In some samples, the remaining CD16+ cells were depleted, leaving CD56−CD16− PBMCs. CD16mix or CD16− PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16− fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56−CD16mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16+ blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16+ monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16mix and CD16− monocytes were applied.

Mentions: In some depletion experiments, only the M-DC8+ subpopulation of CD14+CD16+ monocytes was removed. Removal of M-DC8+ cells only had no marked effect on the yield or phenotype of reverse-transmigrated cells (unpublished data), but when the depletion scheme eliminated all CD16+ monocytes (CD16 depleted), 47 ± 22% (P < 0.05; four experiments) to 66 ± 13% (P < 0.005; four experiments) fewer reverse transmigrated cells were recovered from unstimulated and zymosan-stimulated cultures, respectively (Fig. 6 A). These results are in agreement with the CFSE experiments in Fig. 3. Even after thorough depletion of CD16− monocytes, many reverse-transmigrated cells recovered from cultures receiving only the CD16+ monocytes expressed CD16 upon reverse transmigration, in contrast to the subendothelial monocyte-derived cells from the same cultures (Fig. 6 B). Thus, these cells appear to upregulate CD16 expression during reverse transmigration. When flow cytometry was conducted to analyze the maturation status of cells in the reverse-transmigrated fraction, the number of HLA-DR+CD86+ cells was 63 ± 14% (average of three experiments; P < 0.005) decreased per unit area of endothelial cell surface when blood CD16+ monocytes were depleted from the starting population (Fig. 6 C; cells shown in each group were recovered from an equivalent area of endothelial surface). Moreover, the residual DCs recovered after depletion of CD16+ monocytes expressed an order of magnitude less CD86 on the cell surface, indicating that these reverse-transmigrated, CD16− monocyte-derived cells were less mature than the reverse-transmigrated cells that develop in cultures that contained CD16+ blood monocytes.


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)

Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16+ monocytes. CD56+ NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16− and CD16+(CD16mix) CD14+ monocytes. In some samples, the remaining CD16+ cells were depleted, leaving CD56−CD16− PBMCs. CD16mix or CD16− PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16− fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56−CD16mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16+ blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16+ monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16mix and CD16− monocytes were applied.
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Related In: Results  -  Collection

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fig6: Evaluation of reverse transmigration and expression of CD16 after depletion of peripheral blood CD16+ monocytes. CD56+ NK cells were depleted from the starting PBMC fraction, leaving a fraction of PBMCs that included both CD16− and CD16+(CD16mix) CD14+ monocytes. In some samples, the remaining CD16+ cells were depleted, leaving CD56−CD16− PBMCs. CD16mix or CD16− PBMCs were applied to endothelial/collagen cultures at the same starting density. The number of reverse transmigrated cells in the CD16− fraction was evaluated after 2 d and compared in five independent experiments to the number of reverse transmigrated cells in the control CD16mix population of PBMCs (A). The relative recovery was calculated by setting equal to 1.0 the number of reverse transmigrated cells recovered per well of cultured endothelium after application of CD56−CD16mix PBMCs and then determining the fractional recovery in each experiment when CD16-depleted PBMCs were applied. (B) The possibility that CD16 might be upregulated on peripheral blood cells that originally lacked CD16 was tested by examining the expression of CD16 in reverse-transmigrated and subendothelial populations after full depletion of CD16+ blood cells. Flow cytometric evaluation of CD16 expression in reverse transmigrated (R/T) and subendothelial (S/E) cells that arose from CD16-depleted PBMCs is plotted as a histogram. (C) Phenotyping for CD86 and HLA-DR expression was conducted in reverse-transmigrated populations derived in the presence or absence of CD16+ monocytes. The dot plots shown in panel C represent the number and phenotype of cells recovered from equal numbers of zymosan-containing endothelial cell microtiter cultures to which equivalent densities of CD16mix and CD16− monocytes were applied.
Mentions: In some depletion experiments, only the M-DC8+ subpopulation of CD14+CD16+ monocytes was removed. Removal of M-DC8+ cells only had no marked effect on the yield or phenotype of reverse-transmigrated cells (unpublished data), but when the depletion scheme eliminated all CD16+ monocytes (CD16 depleted), 47 ± 22% (P < 0.05; four experiments) to 66 ± 13% (P < 0.005; four experiments) fewer reverse transmigrated cells were recovered from unstimulated and zymosan-stimulated cultures, respectively (Fig. 6 A). These results are in agreement with the CFSE experiments in Fig. 3. Even after thorough depletion of CD16− monocytes, many reverse-transmigrated cells recovered from cultures receiving only the CD16+ monocytes expressed CD16 upon reverse transmigration, in contrast to the subendothelial monocyte-derived cells from the same cultures (Fig. 6 B). Thus, these cells appear to upregulate CD16 expression during reverse transmigration. When flow cytometry was conducted to analyze the maturation status of cells in the reverse-transmigrated fraction, the number of HLA-DR+CD86+ cells was 63 ± 14% (average of three experiments; P < 0.005) decreased per unit area of endothelial cell surface when blood CD16+ monocytes were depleted from the starting population (Fig. 6 C; cells shown in each group were recovered from an equivalent area of endothelial surface). Moreover, the residual DCs recovered after depletion of CD16+ monocytes expressed an order of magnitude less CD86 on the cell surface, indicating that these reverse-transmigrated, CD16− monocyte-derived cells were less mature than the reverse-transmigrated cells that develop in cultures that contained CD16+ blood monocytes.

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
Related in: MedlinePlus