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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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Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16− monocytes. PBMCs were depleted of CD16+ cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.
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fig8: Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16− monocytes. PBMCs were depleted of CD16+ cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.

Mentions: The origin of CD16+ monocytes and their relationship to CD16− monocytes are unknown. TGFβ1, IL-10, and M-CSF have been previously shown to induce expression of CD16 on monocytes (13, 22–24), and neutralizing endogenous TGFβ with anti-TGFβ antibody prevents “spontaneous” up-regulation of CD16 by cultured monocytes (13). However, whether this cytokine induced other characteristic features of CD16+ monocytes was not studied. We depleted PBMCs of CD16+ cells and then cultured the remaining CD16− monocytes under nonadherent conditions in TGFβ1 or neutralizing anti-TGFβ antibody (to prevent spontaneous induction) for up to 3 d. Within an overnight period of culture, TGFβ1 treatment induced CD16 (Fig. 8 A), as expected from previous reports (13). During this time period, some other features of blood CD16+ monocytes (as shown in Fig. 1) became apparent, such as the slightly higher expression of CD86 and increased HLA-DP that typify CD16+ monocytes (Fig. 8A, compare with Fig. 1). Other markers that distinguish CD16+ monocytes from their CD16- counterparts took longer to become apparent. After 3 d of culture in TGFβ1, levels of CD14 were reduced by one-half to one log, just as observed on circulating CD16+ monocytes. Moreover, the slight increase in HLA-DR and somewhat decreased levels of CD64 that are observed in CD16+ blood monocytes were also acquired during culture with TGFβ1. Although occasionally observed, the M-DC8 antigen was not consistently up-regulated in response to TGFβ1. The Langerhans cell marker E-cadherin was not expressed on these TGFβ1-treated monocytes (unpublished data), even though TGFβ1 is known to induce E-cadherin on monocytes that are additionally cocultured in GM-CSF and IL-4. In contrast to our results with TGFβ1, culturing CD16− monocytes with IL-10 was accompanied by down-regulation of HLA-DR and CD86, and culturing in M-CSF failed to induce CD16 within the 3 d period tested (unpublished data).


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)

Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16− monocytes. PBMCs were depleted of CD16+ cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.
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Related In: Results  -  Collection

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fig8: Effect of TGFβ1 on the phenotype, migratory ability, and survival of CD16− monocytes. PBMCs were depleted of CD16+ cells using miniMACS magnetic selection. Then remaining cells were cultured for up to 3 d in TGFβ1 (bold lines) or anti-TGFβ (thin lines) to block activity of endogenous TGFβ. (A) The cell surface phenotype of these cultured cells was assessed by flow cytometry. Control mAb staining is depicted as a dashed line in top left histogram. (B) Monocytes cultured overnight in TGFβ1 or anti-TGFβ were applied to endothelial monolayers grown on collagen gels lacking zymosan and apical-to-basal transendothelial migration was quantified after a 1.5 incubation. (C) Reverse transmigration was quantified at 48 h as the percent of cells that originally migrated across the endothelium in each condition and then later retraversed the endothelium in ablumenal-to-lumenal direction. (D) Percentage of live cells in the reverse transmigrated populations derived from TGFβ1 or anti-TGFβ treated monocytes incubated with endothelial cultures lacking or containing zymosan within the subendothelium was assessed by trypan blue exclusion.
Mentions: The origin of CD16+ monocytes and their relationship to CD16− monocytes are unknown. TGFβ1, IL-10, and M-CSF have been previously shown to induce expression of CD16 on monocytes (13, 22–24), and neutralizing endogenous TGFβ with anti-TGFβ antibody prevents “spontaneous” up-regulation of CD16 by cultured monocytes (13). However, whether this cytokine induced other characteristic features of CD16+ monocytes was not studied. We depleted PBMCs of CD16+ cells and then cultured the remaining CD16− monocytes under nonadherent conditions in TGFβ1 or neutralizing anti-TGFβ antibody (to prevent spontaneous induction) for up to 3 d. Within an overnight period of culture, TGFβ1 treatment induced CD16 (Fig. 8 A), as expected from previous reports (13). During this time period, some other features of blood CD16+ monocytes (as shown in Fig. 1) became apparent, such as the slightly higher expression of CD86 and increased HLA-DP that typify CD16+ monocytes (Fig. 8A, compare with Fig. 1). Other markers that distinguish CD16+ monocytes from their CD16- counterparts took longer to become apparent. After 3 d of culture in TGFβ1, levels of CD14 were reduced by one-half to one log, just as observed on circulating CD16+ monocytes. Moreover, the slight increase in HLA-DR and somewhat decreased levels of CD64 that are observed in CD16+ blood monocytes were also acquired during culture with TGFβ1. Although occasionally observed, the M-DC8 antigen was not consistently up-regulated in response to TGFβ1. The Langerhans cell marker E-cadherin was not expressed on these TGFβ1-treated monocytes (unpublished data), even though TGFβ1 is known to induce E-cadherin on monocytes that are additionally cocultured in GM-CSF and IL-4. In contrast to our results with TGFβ1, culturing CD16− monocytes with IL-10 was accompanied by down-regulation of HLA-DR and CD86, and culturing in M-CSF failed to induce CD16 within the 3 d period tested (unpublished data).

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