Immunology Letters, 31 (1992) 189-198 Elsevier IMLET 01728

Development of a Langerhans cell phenotype from peripheral blood monocytes Gertrud Rossi 1, Nikolaus Heveker I , Bernhard Thiele 2, Hans Gelderblorn 3 and Falko Steinbach I llnstitut fiir Molekularbiologie und Biochemie, Freie Universitiit Berlin; 2Diagnostisches Zentrum, Berlin, and 3Robert.Koch lnstitut des Bundesgesundheitsamtes, Berlin, F.R.G. (Received 12 August 1991; accepted 15 September 1991)

1. Summary

Epidermal Langerhans cells (ELC) are definitively primed to differentiate into dendritic cells (DC). It is unknown at what stage of monocyte development this priming occurs. In a culture system characterized by low paracrine stimulation, i.e. Iscove's modified Dulbecco medium (IMDM) with 2°70 FCS, we tested the ability of peripheral blood monocytes to turn to the route of the LC-DC lineage. In this system monocytes did not develop significant yeast cell phagocytosis, although mannose receptors were available. However, they became strong stimulators of mannan specific T cell proliferation. Phenotype development was analysed by flow cytometry using the monoclonal antibodies OKT6 (CDla), IOT2 (HLA-DR), IOM2 (CDI4) and the ligand Man-BSA-FITC. CDla was the first marker which distinguished cultured monocytes from developing macrophages, obtained by addition of 807ohuman serum. Like cord blood Langerhans cells (CBLC) they internalized OKT6 in deep coated pits. They maintained a phenotype of monocyte derived Langerhans cells (MoLC) during eight days of in vitro culture, expressing CDla, mannose receptors and HLA-DR and decreasing CD14, if left in their own conditioned medium. MoLC could be converted into macrophages by adKey words: CD1 clustering; Mannose receptor; Langerhans cell differentiation; Autocrine stimulation Correspondence to: Dr. Gertrud Rossi, Institut fiir Molekularbiologie und Biochemie, Freie Universit~it Berlin, Arnimallee 22, D 1000 Berlin 33, F.R.G.

dition of human serum only within the first four days in vitro. Our data suggest that monocytes acquire an LC phenotype by autocrine stimulation. 2. Introduction

Epidermal Langerhans cells (ELC) bear a panel of monocyte markers such as Fc receptors, integrins, Leu M3 and serin esterase [1 - 3]. Although not phagocytic, their predominant function consists of processing intact antigens [4]. They share the expression of CDIa with cortical thymocytes and interdigitating cells (IDC) [5]. The LC phenotypes isolated from peripheral blood (PBLC) and infant cord blood (CBLC) [6, 7], which coexpress CD14 and CDla, are presumed to be LC precursors. During in vitro culture ELC rapidly mature into potent stimulatory dendritic cells (DC) [8], loosing most of their monocyte markers and endosomes [9, 10], together with their ability to process native exogenous antigens [11]. These data suggest that PBLC/ELC/DC form a distinct functional lineage, which apparently derives from a monocyte precursor. The question therefore arises, at what stage of differentiation they separate from the monocyte/macrophage lineage. In past years several methods have been used, to turn monocytes into the accessory cell direction, by culture in the absence of serum and exogenous proteins [12, 13], under nonadhering conditions [14], or by treatment with IFN7 [15]. However, the appearance of an LC phenotype has not yet been reported. In our studies PBM are cultured in conditions of

0165- 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

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low paracrine stimulation (2°7o FCS supplement). FACS analysis and EM show that they acquire a phenotype similar to CBLC and later to ELC. Functionally they differ from macrophages by low yeast phagocytosis and high efficiency of mannan presentation to autologous T cells. These functions are mediated by the mannose/fucose receptor expressed by LC and macrophages [16, 17], a differentiation marker for promonocytes and macrophages [18, 19], which on PBM is hidden [20]. Our results indicate that cultured monocytes acquire an LC phenotype by predominant autocrine stimulation and maintain this phenotype in the absence of T cell factors.

Buffy coat monocytes, prepared by centrifugation on Ficoll-paque and adherence on polystyrene surfaces, were cultivated in IMDM with 2°70 FCS, for their differentiation into APC. For macrophage cultures the medium was supplemented with 8% of human serum, in addition to the 2°70 FCS.

3. Materials and Methods

3.4. Functional assays

3.1. Reagents p-Aminophenylmannopyranoside, fluorescein isothiocyanate monomer I, and Iscove's modified Dulbecco medium (IMDM) were obtained from Sigma (Munich, F.R.G.), bovine serum albumin (BSA) from Behringwerke (Marburg, F.R.G.). Gold/anti-mouse IgG was purchased from Janssen (Beerse, Belgium), phycoerythrin-labelled monoclonal antibodies IOT2 (HLA-DR) and IOM2 (CD14) from Dianova (Hamburg, F.R.G.) and FITC-labelled OKT6 (CDla) from Ortho (Heidelberg, F.R.G.).

Phagocytosis via the mannose receptor was tested with untreated C. albicans cells in serum-free IMDM for 30 min at 37 °C. Internalized yeast cells were detected by indirect immunofluorescence. Pinocytosis was assayed with the ligand Man-BSAFITC, for 20 rain at 37 °C, and visualized by fluorescence microscopy on a Leitz Aristoplan. Presentation of native or subtilisin digested mannan to autologous T cells was carried out as previously described [16]. MoLC or macrophages were primed with antigen on day 4 in vitro. T cells from previous assays were added 24 h later. After 48 h cells were pulsed with [3H]thymidine for 16 h.

3.2. Preparation o f ligand and antigen

3.5. Flow cytometry

Mannosyl-BSA (Man-BSA) was prepared according to the diazocoupling method of McBroom et al. [21] as previously described [16]. The binding rate was 96°70 as estimated by the Bradford micromethod [22] on the basis of ConA-bound to unbound BSA. The ligand was labelled with fluorescein isothiocyanate at + 4 °C overnight [16]. Specific binding of Man-BSA-FITC to the mannose receptor was controlled by spectrofluorometric inhibition assays with monomer sugars. Macrophages or MoLC (7 x 105 cells in each sample) were labelled in the presence of 25 mM of the inhibiting sugar for 30 min on ice. After washings with 10 mM Tris/145 mM NaC1, pH 7.6 the cells were dissolved in 950 #I of 1% SDS solution, and 50 #1 of 100 mM Tris, pH 8.3. The eluted fluorochrome

The differentiation of monocytes was followed by direct immunolabelling with the phycoerythrin conjugated mAbs IOT2 (HLA-DR) and IOM2 (CD14), in combination with the FITC-labelled mAb OKT6 (CD1) or the ligand Man-BSA-FITC, as a single histogram display or as a two-colour dot plot analysis. The cells were treated in test tubes with 1:10 diluted antibodies or with the ligand Man-BSA-FITC (1 mg/ml) for 30 min on ice, washed with PBS and fixed with 2.5% p-formaldehyde in PBS. Flow cytometric analysis was performed on a flow cytometer FACScan (Becton Dickinson), using "FACScan Research" as a software program.

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was measured on a spectrofluorometer Perkin Elmer 650-10S excitation at 492 rim, emission at 520 nm vs. an autofluorescence control. Mannan was prepared from Candida albicans as described [23]. 3.3. Monocyte differentiation

3.6. Immunoelectronmicroscopy Cells cultured for six days were incubated with OKT6, diluted to 1:10 for 30 min on ice, washed with PBS and stained with 5-nm gold/anti-mouse IgG for 30 min on ice. Fixation and epon embedding proceeded as described [23]. Electron micrographs were taken on a Zeiss EM 10 at 60 kV. 4. Results

4.1. Growth conditions The viability of monocytes grown in IMDM with 207o FCS became progressively dependent on the presence of their own conditioned medium. A complete exchange of the culture medium later than on day 4 in vitro comported the loss of more than 50070 of the cells. Without medium exchange the cell number remained stable until day 8 in vitro. 4.2. Functions Most of the cells produced hydrolytic enzymes common to macrophages and LC such as unspecific esterase, acid phosphatase and ATPases (Gossrau et al., unpublished results). Unlike macrophages they did not develop significant yeast cell phagocytosis, although mannose receptors were expressed. Man-BSA-FITC was bound and patched, though to a lesser extent than on macrophages (Fig. 1, a and b). Binding of the ligand could be inhibited

with D-mannose, L-fucose and N-acetylglucosamine, the same sugars which were previously shown to inhibit the lectin activity of the mannose receptor protein isolated from macrophages [23]. Until day 4 of in vitro culture the veiled cells could still develop into macrophages when human serum (8070) was added to the culture medium, while in the later stage of differentiation this conversion failed (Table 1). Soluble mannan was processed and presented to autologous T cells from an immunized donor as previously shown [16]. Macrophages were weak stimulators of T cell proliferation. The peptide moiety of mannan seemed to be crucial in these assays, since subtilisin-digested mannan was not presented (Table 2). TABLE 1 MoLC lose their ability to convert into macrophages by addition of human serum within one week of in vitro culture. Buffy coat monocytes were cultured in IMDM with 2% FCS for differentiation into MoLC. Their ability to regain phagocytic activity by pretreatment with 8o70human serum (HUS) was tested on day 2 and on day 8 in vitro. Macrophages were obtained by continuous growth in the presence of 8% human serum. Time of in vitro culture

Day 2 Day 8

Phagocytosis of yeasts/cell 2°70 FCS

+8% HUS 1h

+8% HUS continuously

0.8 0.3

3.7 0.5

4.4 7.1

Fig. 1. Binding and clustering of MannosyI-BSA-F1TC by cultured monocytes, for 20 min at 37 °C, on day 6 in vitro. (A) Differentiated into MoLC in IMDM with 2o70 FCS. (B) Differentiated into macrophages in the presence of 8% human serum.

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TABLE 2

MoLe

MoLC function as antigen presenting cells. Presentation of mannan to autologous T cells. MoLC or macrophages were primed with native or subtilisin digested mannan on day 4 in vitro. T cells were added on day 5 in vitro. After 48 h cells were pulsed with [3H]thymidine (4 ttCi) for 16 h. APC

Antigen

[3H]Thymidine incorporation (cpm)

MoLC MoLC MoLC MO

native mannan protein-free mannan native mannan

2700 23900 4 t00 9600

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Phenotype development

F l o w c y t o m e t r i c analysis o f the e x p r e s s i o n o f C D 1 4 a n d the m a n n o s e r e c e p t o r as well as o f CD1 a n d H L A - D R was p e r f o r m e d every o t h e r d a y . We f o u n d a first d e v e l o p m e n t a l stage at 48 h o f in vitro culture. The cells p r e s e n t e d a single p h e n o t y p e , hom o g e n e o u s with respect to the expression o f the m a n n o s e receptor, C D I 4 a n d H L A - D R . CD1 labelling s h o w e d a wider d i s t r i b u t i o n f r o m w e a k to highly positive; nevertheless >90°70 o f the g a t e d m o n o c y t i c cells b o u n d O K T 6 (Fig. 2). By virtue o f their s i m i l a r i t y with C B L C , these cells were n a m e d "monocyte derived LC phenotype" (MoLC). The outgrowth of a CDl-positive population f r o m the a u t o f l u o r e s c e n c e p e a k was a c c o m p l i s h e d within 36 h. CD1 was not expressed o n the surface o f m a c r o p h a g e s o b t a i n e d b y a d d i t i o n o f 8 °7o h u m a n s e r u m (Fig. 3). F r o m d a y 4 o f in vitro c u l t u r e a divergent differe n t i a t i o n was o b s e r v e d . T h e h i s t o g r a m s p r e s e n t e d d o u b l e p e a k s i n s t e a d o f one p e a k (see Fig. 5), except for the m a n n o s e receptor, which was the m o s t stable m a r k e r in o u r e x p e r i m e n t s . Fig. 4 c o m p a r e s the d i s t r i b u t i o n o f M o L C in d o t p l o t analysis o f d o u b l e labelling on d a y 2 a n d d a y 8. H L A - D R sig-

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