A skin homing molecule defines the langerhans cell progenitor in human peripheral blood - PubMed (original) (raw)

A skin homing molecule defines the langerhans cell progenitor in human peripheral blood

D Strunk et al. J Exp Med. 1997.

Free PMC article

Abstract

We have recently described a system for the generation of dendritic cells (DC) and Langerhans cells (LC) from defined CD34+ precursors purified from peripheral blood of healthy adult volunteers. This study has now been extended by the characterization of two distinct subpopulations of CD34+ cells in normal human peripheral blood as defined by the expression of the skin homing receptor cutaneous lymphocyte-associated antigen (CLA). CD34+/CLA+ cells from normal peripheral blood were found to be CD71LOW/CD11a+/CD11b+/CD49d+/CD45RA+ whereas CD34+/CLA- cells displayed the CD71+/CD11aLOW/CD11bLOW/CD49d(+)/ CD45RA(LOW) phenotype. To determine the differentiation pathways of these two cell populations, CD34+ cells were sorted into CLA+ and CLA- fractions, stimulated with GM-CSF and TNF-alpha in vitro, and then were cultured for 10 to 18 d. Similar to unfractionated CD34+ cells, the progeny of both cell populations contained sizable numbers (12-22%) of dendritically shaped, CD1a+/HLA-DR cells. In addition to differences in their motility, the two dendritic cell populations generated differed from each other by the expression of LC-specific structures. Only the precursors expressing the skin homing receptor were found to differentiate into LC as evidenced by the presence of Birbeck granules. In contrast, CLA precursor cells generated a CD1a+ DC population devoid of Birbeck granule-containing LC. Provided that comparable mechanisms as found in this study are also operative in vivo, we postulate that the topographic organization of the DC system is already determined, at least in part, at the progenitor level.

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Figures

Figure 1

Figure 1

Distinct phenotype of CLA+ and CLA− PBPC in normal peripheral blood. Using three-color flow cytometry, freshly isolated PBPC from healthy volunteers were analyzed for the distribution of the antigens shown on the y-axis. Reactivity was measured for gated CLA+/ CD34+ (black bars) and CLA-/CD34+ (hatched bars) PBPC. Results of one experiment are displayed as mean fluorescence intensity (MFI). Two additional experiments with cells from different donors yielded similar results. The lack of anti-CD1a-reactivity was used as a negative control.

Figure 2

Figure 2

Immunohistochemical analysis of CLA+ and CLA− PBPC progeny. CLA+ and CLA− PBPC were cultured for two weeks in the presence of GM-CSF/TNF-α and then subjected to Lag immunolabeling using the APAAP procedure. Clustered (asterisks) as well as single (arrows) Lag+ cells (red) were detectable within the CLA+/CD34+ progeny (A). No Lag reactivity was found within the progeny of CLA− PBPC (B). Original magnification, ×150.

Figure 3

Figure 3

Detection of Birbeck granules in CD1a+ cells derived from CLA+ progenitors. Representative ultramorphology of a dendritically shaped CD1a+ LC that was generated from CLA+ PBPC during 16 d of culture in the presence of GM-CSF and TNF-α. Numerous trilaminar Birbeck granules (arrowheads) can be easily seen in the perinuclear area. Original magnification, ×83,500.

Figure 4

Figure 4

MLR-stimulatory capacity of cells derived from CLA+ and CLA− PBPC. 3.3 × 103 cells generated in a 2-wk culture in GM-CSF/ TNF–containing medium from CLA+ (circles) and CLA− (diamonds) PBPC and, for comparison, monocytes from the same donor (squares) were tested for their capacity to stimulate 105 allogeneic T cells. Cells were pulsed with [3H]thymidine for 16 h after 4, 5, or 6 d of coculture. The [3H]thymidine incorporation rate is expressed as mean cpm values ± SD of triplicate cultures. 105 mitomycin C–treated APC or purified T cells gave <500 cpm. One representative experiment out of three performed is shown.

Figure 5

Figure 5

A subpopulation of cells derived from CLA+/CD34+ PBPC exhibits a high motility in culture. Time-lapse microphotography was performed with cells derived from CLA+ PBPC after 3 wk of culture in the presence of GM-CSF/TNF-α. Pictures of cells migrating within the culture well were taken every minute. One (arrowhead) of the two dendritically shaped cells shown (A, time point zero) covers a distance being multiples of its own diameter within 3 min (B). Original magnification, ×150.

Figure 5

Figure 5

A subpopulation of cells derived from CLA+/CD34+ PBPC exhibits a high motility in culture. Time-lapse microphotography was performed with cells derived from CLA+ PBPC after 3 wk of culture in the presence of GM-CSF/TNF-α. Pictures of cells migrating within the culture well were taken every minute. One (arrowhead) of the two dendritically shaped cells shown (A, time point zero) covers a distance being multiples of its own diameter within 3 min (B). Original magnification, ×150.

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