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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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Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14+CD16−, CD14+CD16+, and CD14−CD16− cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.
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fig2: Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14+CD16−, CD14+CD16+, and CD14−CD16− cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.

Mentions: When the whole PBMC fraction is applied to the apical surface of cultured endothelial monolayers not pretreated with proinflammatory cytokines, very few B or T lymphocytes migrate beneath the endothelium, but a majority of monocytes (17, 18) and NK cells (19) will undergo transendothelial migration in less than 2 h of incubation. This step mimics the trafficking of these cells from the vascular compartment into subendothelial connective tissue, and in the absence of exogenous cytokine stimulation, may reflect populations of leukocytes that leave the blood in the steady-state. We conducted experiments to evaluate whether the CD16+ monocytes were among those that efficiently traversed the endothelium. We also examined the transmigration of the previously defined CD14−CD64−HLA-DR+ nonmonocytic DCs (1, 2), as their interaction with endothelium directly after isolation has not been reported. Flow cytometric cell sorting of this fraction of HLA-DR+ CD14− cells confirmed the differentiation of these cells into mature DCs after cultured in macrophage conditioned medium (unpublished data) as reported previously (1), indicating that the CD14 cells we tracked were indeed DC precursors. Both CD16− and CD16+ monocytes migrated efficiently across unstimulated endothelium (Fig. 2) , with more than two-thirds of each population entering the subendothelial collagen. On a relative basis, CD16+ monocytes migrated somewhat more efficiently than CD16− monocytes. Among the CD16+ cells that entered the collagen gel, 15 to 25% were M-DC8+ (unpublished data). Similar extents of transendothelial migration occurred in endothelial cultures in which zymosan, a preparation of yeast cell membranes, was embedded in the type I collagen matrix (unpublished data). In contrast, less than half of CD14−CD64−CD11c+HLA-DR+ DC precursors traversed the endothelium (Fig. 2).


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)

Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14+CD16−, CD14+CD16+, and CD14−CD16− cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.
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Related In: Results  -  Collection

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fig2: Transendothelial migration of blood DC precursors across unstimulated endothelium. The entire fraction of freshly isolated PBMCs were incubated with endothelial cell/collagen cultures for 1.5 h to permit transmigration. The apical surface of the cultures was washed to collect nonmigrated cells, and the migrated population was recovered from the subendothelial collagen using collagenase D. Cells considered for quantitative evaluation were large mononuclear cells (LMC) uniformly positive for HLA-DR and negative for the B cell marker CD19 or T cell marker CD3. The plot shows the percent distribution of three distinct populations: CD14+CD16−, CD14+CD16+, and CD14−CD16− cells. The total height of each bar represents the relative distribution of these populations in freshly isolated PBMCs. The filled portion of each bar indicates the fraction of each population that emigrated beneath the endothelium, and the open portion of each bar represents the portion of the population that was recovered in the nonmigrated fraction. These data are representative of results obtained using PBMCs from three different blood donors.
Mentions: When the whole PBMC fraction is applied to the apical surface of cultured endothelial monolayers not pretreated with proinflammatory cytokines, very few B or T lymphocytes migrate beneath the endothelium, but a majority of monocytes (17, 18) and NK cells (19) will undergo transendothelial migration in less than 2 h of incubation. This step mimics the trafficking of these cells from the vascular compartment into subendothelial connective tissue, and in the absence of exogenous cytokine stimulation, may reflect populations of leukocytes that leave the blood in the steady-state. We conducted experiments to evaluate whether the CD16+ monocytes were among those that efficiently traversed the endothelium. We also examined the transmigration of the previously defined CD14−CD64−HLA-DR+ nonmonocytic DCs (1, 2), as their interaction with endothelium directly after isolation has not been reported. Flow cytometric cell sorting of this fraction of HLA-DR+ CD14− cells confirmed the differentiation of these cells into mature DCs after cultured in macrophage conditioned medium (unpublished data) as reported previously (1), indicating that the CD14 cells we tracked were indeed DC precursors. Both CD16− and CD16+ monocytes migrated efficiently across unstimulated endothelium (Fig. 2) , with more than two-thirds of each population entering the subendothelial collagen. On a relative basis, CD16+ monocytes migrated somewhat more efficiently than CD16− monocytes. Among the CD16+ cells that entered the collagen gel, 15 to 25% were M-DC8+ (unpublished data). Similar extents of transendothelial migration occurred in endothelial cultures in which zymosan, a preparation of yeast cell membranes, was embedded in the type I collagen matrix (unpublished data). In contrast, less than half of CD14−CD64−CD11c+HLA-DR+ DC precursors traversed the endothelium (Fig. 2).

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