A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

Abstract

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Figures

Figure 1

Effect of anti-MDR-1 mAbs on transendothelial migration of MP. (A) Monocytes were allowed to migrate across endothelial monolayers with or without addition of anti-MDR-1 mAb MRK16, or mAb to the β2 integrin (IB4) or CD31 (hec 7). Cultures were incubated for 1.5 hr. (B) In experiments to examine their subsequent basal-to-apical transmigration, monocytes were allowed to accumulate beneath the endothelium in the absence of added mAb. Then monolayers were washed, and individual wells received aliquots of culture medium with or without added mAbs [all IgG2a; MRK16 used as F(ab′)2 fragments] at the indicated concentrations. In some samples, PBMC were preincubated with MRK16 or isotype-matched control mAb hec 1 against cadherin 5 (Pre-MRK16 and Pre-Hec 1) before addition to the endothelium. After 48 hr, cultures were fixed for analysis. Data are plotted relative to control levels of reverse transmigration (no mAb added), in which 50% of the subendothelial MP retraverse the endothelium in 48 hr. Bars represent means ± SD from 3–10 experiments.

Figure 2

Effect of anti-MDR-1 mAb on emigration of DC and T lymphocytes from skin explants. Explants of human skin were floated in culture medium without added mAb (n = 10) or in medium containing anti-MDR-1 mAbs MRK16 (n = 10) or UIC2 (n = 3), anti-cadherin 5 mAb hec 1 (n = 5), anti-CD31 mAb hec 7 (n = 5), anti-MHC I mAb W6/32 (n = 3), anti-CD18 mAb IB4 (n = 2), verapamil (n = 5), or the vehicle control for verapamil 0.03% methanol (Vehicle, n = 3). After 3 days of incubation, DC and T lymphocytes that appeared in the culture medium were collected and counted as previously described (7). Each condition was tested in triplicate; n = number of experiments in which condition was included. The number of emigrated cells recovered from individual control explants was typically 5 × 105. To compare data from different experiments, the mean number of emigrated cells in the absence of added mAb in each experiment was set equal to 1.0, and relative values were obtained for the remaining data.

Figure 3

Expression of MDR-1 in situ by DC. (A) Epidermis derived from explants cultured with anti-MDR-1 mAb MRK16 were fixed, and Cy3-conjugated anti-mouse IgG was added to detect expression of MDR-1 (red). Addition of Cy3-labeled detection antibody to skin incubated in the presence of nonbinding control mAb (hec1) showed no staining (not shown). The section shown was cut parallel to the epidermal basement membrane. (B) Immunostaining of same sample using fluorescein isothiocyanate-conjugated anti-MHC II mAb (green). (C) Doubly exposed frame to examine colocalization (yellow) of MDR-1 and MHCII. (Bar is 10 μm.)

Figure 4

Expression of functional MDR-1 by emigrated DC. (A-C) MDR-1 expression in emigrated skin cells was examined by flow cytometry by using double staining with anti-MDR-1 mAb and the DC lineage marker MHC II (A and B) or the T lymphocyte lineage marker CD3 (C). Quadrants were marked based on the level of fluorescence intensity observed in samples stained with negative control mAbs. (D) For studies measuring efflux, emigrated cells were loaded with the dye DiOC2 (dashed line, no dye added) and cultured at 37°C for 90 min without addition of mAb (filled profile), in the presence of UIC2 mAb (bold line), or kept at 4°C for this duration (thin line). Analysis of efflux in the DC fraction of emigrants was assessed by double labeling with phycoerythrin-conjugated anti-MHC II mAb for flow cytometry. Figure shown was gated on MHC II+ cells. (E) Membranes prepared from purified, emigrated DC, PBMC, or HUVEC were immunoblotted with anti-MDR-1 mAb C219. Numbers to the left of bands are molecular weight markers.

Figure 5

Retention of DC in the epidermis after treatment with MDR-1 antagonists. (A) Epidermal sheets were stained with anti-MHC II mAb to enumerate DC before the onset of culture or after 3 days of culture in the absence of mAb (no mAb, n = 7), or in the presence of anti-cadherin 5 mAb hec 1 (n = 3); anti-MHC I mAb W6/32 (n = 3); anti-CD31 mAb hec 7 (n = 3); anti-MDR-1 mAb MRK16 (n = 6); anti-MDR-1 mAb UIC2 (n = 1); verapamil (n = 2); or the vehicle control for verapamil 0.03% methanol (Vehicle, n = 1). n = number of experiments in which each condition was examined. DC were counted from en face examinations of epidermal sheets in 16–20 high-power fields per experiment. Percent reduction in DC density was calculated by comparing the number of DC in cultured explants to the mean number present in a portion of the same skin sample before culture (typically 75 cells/field). (B–D) Photomicrographs show the distribution of DC within the epidermis before culturing of explants (B), after 3 days of culture under control conditions (no mAb) (C), and after 3 days of culture in the presence of MRK16 (D). (Bar is 50 μm.)

Figure 6

Effect of anti-MDR-1 mAb on the maturation of DC. The levels of MHC II expressed by epidermal DC were analyzed in gated epidermal suspensions by flow cytometry before the onset of culture (Day 0) or after 3 days of culture in the absence of mAb or in the presence of MRK16. Single cell suspensions of the epidermis were prepared by digestion with dispase followed by trypsin. Keratinocytes and other skin cells were excluded from the analysis by setting a gate to include only MHC II+ cells.