Non-adherent, low-density cells from human peripheral blood ... - NCBI

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Nov 27, 1985 - adherent by 16 hr, can be separated in the low-density interface on hypertonic ... Including both the 16 hr and 90 min non-adherent cells, over ...
Immunology 1986 57 595-603

Non-adherent, low-density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology STELLA C. KNIGHT, J. FARRANT, ANNETTE BRYANT, A. J. EDWARDS, S. BURMAN, A. LEVER, J. CLARKE & A. D. B. WEBSTER Clinical Research Centre, Harrow, Middlesex

Acceptedfor publication 27 November 1985

SUMMARY Dendritic cells (DC) from human peripheral blood, known to adhere transiently and to become nonadherent by 16 hr, can be separated in the low-density interface on hypertonic Metrizamide gradients. Many more low-density cells (5-8% of the mononuclear cells separated on Ficoll) were obtained from the population that was non-adherent after only 90 min. Over 95% of these low-density cells had veiled morphology. A proportion were monocytes by phenotypic and phagocytic properties. Onethird of the cells (on average) were DC on the basis of lack of monocyte phenotype and of potency as stimulators in the mixed lymphocyte reaction. Including both the 16 hr and 90 min non-adherent cells, over 2% of the mononuclear cells isolated from human peripheral blood may be DC.

MATERIALS AND METHODS Leucocytes from human peripheral blood samples Peripheral blood (50 ml) was taken from normal volunteers (laboratory personnel) into sterile glass universals containing glass beads and defibrinated by shaking. Each 15 ml was diluted with 5 ml of RPMI bicarbonate-buffered medium (Flow, Irvine, Ayrshire) with penicillin/streptomycin (100 IU/ml) and Lglutamine (2 mM). Diluted blood (10 ml) was layered onto 10 ml of Ficoll-paque (Pharmacia, Uppsala, Sweden). After centrifugation (650 g for 35 min), the leucocytes were removed from each interface and washed twice in the same medium. The cells were adjusted to 5 x 106/ml in medium with 10% heat-inactivated fetal calf serum (FCS). Each 5-ml aliquot was incubated at 370 for 90 min in a 5 cm diameter tissue culture grade petri-dish (Nunc, Roskilde, Denmark). The non-adherent cells were removed gently. The adherent cells were left on plastic with fresh medium for 16 hr and the cells that became detached after 16 hr were collected. The adherent cells were removed with a rubber policeman.

INTRODUCTION Since the first description of dendritic cells (DC) in the mouse (Steinman & Cohn, 1973), cells with similar properties have been found at a concentration of about 1% in many lymphatic organs (Steinman & Nussenzweig, 1980) and in human peripheral blood (Van Voorhis et al., 1982). These cells strongly express Ia (Spry et al., 1980; Steinman & Nussenzweig, 1980) and are potent stimulators of allogeneic cells in vitro in the mixed leucocyte reaction (MLR) (Steinman & Witmer, 1978; Klinkert, Labadie & Bowers, 1982; Kuntzcrow & Kunkel, 1982) and in vivo in the host-versus-graft reaction (Knight et al., 1983). DC are also powerful at presenting foreign antigens acquired on their cell surfaces, and initiate immune responses in vitro (Nussensweig et al., 1980; Balfour et al., 1981) and in vivo (Knight et al., 1985). In this paper, we first establish that the transiently adherent DC from the peripheral blood as described by Van Voorhis et al. (1982) could be separated using hypertonic gradients of Metrizamide (Knight et al., 1982, 1983). These gradients have already been used to obtain human DC from synovial fluids (Tyndall et al., 1983). We have also isolated low-density cells with veiled morphology from the cells that are non-adherent after 90 min. These non-adherent 'veiled' cells contain both monocyte-like cells and non-monocytic dendritic cells. We describe the morphological, phenotypic and functional properties of these cells and suggest that, when these non-adherent populations are considered, non-monocytic dendritic cells may exceed 2% of the mononuclear cells of human peripheral blood.

Preparation of low-density cells Low-density cells were obtained either from the Ficoll-separated cells taken before the depletion of adherent cells, from the non-adherent cells following the 90 min adherence step, or from the cells that became non-adherent overnight (Fig. 1). Each population of cells from a single donor was pooled into 10 ml of medium containing 10% FCS. Five ml of cell suspension were layered onto 2 ml of the Metrizamide gradient in I0-ml centrifuge tubes and spun at 650 g for 10 min. The Metrizamide gradient was prepared as previously described [i.e. 14-5 g Metrizamide (Nygaard, Oslo, Norway) dissolved in 100 ml RPMI (HEPES-buffered) (Flow) with penicillin/streptomycin

Correspondence: Dr S. C. Knight, Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ, U.K.

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S. C. Knight et al. Defibrinated peripheral blood Ficol l-paque Mononuclear cells Petri-dish 90 min

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Figure 1. Separation of low-density cells.

and 10% FCS present (Knight et al., 1983)]. The low-density cells were taken from the Metrizamide interface, washed and counted. The cell pellet from the Metrizamide gradient (containing the lymphocytes) was also washed. It was essential to have serum (e.g. 10% FCS) present in the wash solutions after exposure of the cells to the Metrizamide gradient, otherwise cells were lost by osmotic lysis.

50,000 cells for each stained preparation. Dead cells were stained with propidium iodide. EA rosettes were done at 40 using sheep red blood cells exposed to a subagglutinating concentration of rabbit antibody to sheep red blood cells. Separation of the rosetting cells was on a Metrizamide gradient as already described, and the sheep red blood cells were lysed with a brief treatment with distilled water.

Phenotypic analysis Mononuclear cell suspensions (1 05-1 06 cells in 50 pl) prepared in Hanks' balanced salt solution containing sodium azide (0.02%) with 2% fetal calf serum (BSS) were treated with the appropriate mouse anti-human monoclonal antibody (5 Ml) on ice for 30 min. The cells were washed twice with 500 yl BSS and stained with goat anti-mouse immunoglobulin conjugated with fluorescein isothiocyanate (FITC) (Nordic, Maidenhead, Berks, 2 u1/ 106 cells/50 yl BSS) on ice for 30 min. Where the monoclonal antibody used was IgM, a monoclonal anti-mouse IgM conjugated with FITC (Becton-Dickinson, Mountain View, CA, 5 pl/ 106 cells/50 pl BSS) was substituted for the goat anti-mouse IgFITC. A control sample of cells incubated with the FITCconjugated antibody alone was always included. After two washes with BSS, the cells were resuspended in 50 pl BSS for analysis by fluorescence microscopy or in 500 pl BSS for analysis by the fluorescence-activated cell sorter (FACS). Cell suspensions labelled by indirect immunofluorescence were viewed using a fluorescence microscope equipped with Ploem-type epifluorescence illumination. One hundred cells were counted. Cell suspensions were passed through a fluorescence-activated cell sorter (FACS II. Becton-Dickinson) at a rate of 8001200 cells/second. All experiments were performed with the argon ion laser set at 300 mW and 488 nm, and the photomultiplier tube set at 750 V and a light scatter gain of 2. Subsequent mathematical calculations used the accumulated data from

Morphology For light microscopy, cytocentrifuge preparations were fixed in methanol and stained with Giemsa. Histochemical staining for non-specific esterase used alpha-naphthyl acetate as a substrate as described by Horwitz et al. (1977). For electron microscopy, cells were fixed in 3% glutaraldehyde in cacodylate buffer, attached on cover-glasses with polyL-lysine, post-fixed in osmium tetroxide, dehydrated and dried in a critical-point drying apparatus before viewing using a scanning electron microscope (Phillips SEM 500). Cells for transmission electron microscopy were processed in Eppendorf tubes. They were fixed, post-fixed and dehydrated as above before embedding in Spur resin and cutting on an ultramicrotome. They were then viewed on a JEOL 1200 EX electron microscope. Phagocytic function Zymosan (Koch-Light) was boiled in saline for 20 min, adjusted to 1 010/ml in saline and stored at 40 until use. An equal volume of filtered 1-2% nitroblue tetrazolium (NBT, Sigma, Poole, Dorset) was added 30 min before use. Chambers (1 cm square, 0-8 ml) were made from a silicone rubber framework attached to a large coverslip with silicone grease. Cells (0 1 ml) in complete medium at 106/ml were added to the wells with 0 1 ml of the zymosan/NBT mixture and the wells filled with medium. After 60 min at 37°, the sample was examined using an inverted phasecontrast microscope.

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Dendritic cells and monocytes 5' nucleotidase The levels of ecto-enzyme 5'-nucleotidase were determined by measuring the conversion of 3H-AMP to soluble 3H-adenosine as described by Van de Zeijst, Stewart & Schlesinger (1978). The levels were related to amount of protein estimated using the method of Lowry et al. (1951). Function as stimulators in MLR Hanging drop 20-yl cultures were done in Terasaki plates as previously described (O'Brien et al., 1979; Farrant et al., 1980) using RPMI-bicarbonate medium supplemented with penicillin/streptomycin (100 IU/ml) and L-glutamine (2 mM) together with 10% autologous (responder) serum. Low-density cells were washed, irradiated (2500 rads) and mixed with Ficoll-separated peripheral blood mononuclear cells as responders. DNA synthesis was measured on the appropriate harvest day by the uptake of [3H] thymidine ([3H]TDR, 2 hr pulse, 2 Ci/mmol, 0 1 ,ug total thymidine/ml; Farrant et al., 1980) using a Terasaki plate microharvester (O'Brien et al. 1979).

RESULTS Metrizamide separation of dendritic cells (DC) from non-adherent cells after 16 hr incubation Van Voorhis et al. (1982) obtained low-density DC from human blood using BSA gradients. The cell preparations they applied to the BSA gradients were Ficoll-separated cells that were adherent after 60 min but non-adherent after a further 16 hr. We first investigated whether hypertonic Metrizamide gradients could be used instead of the BSA gradients. Ficoll-separated peripheral blood mononuclear cells were incubated for 90 min on petri-dishes and the non-adherent cells discarded. The remaining cells were incubated for a further 16 hr with fresh medium and the non-adherent cells (16 hr cells) were collected and separated on Metrizamide. The cells at the interface, averaging 1% of the total mononuclear fraction, were 95% large,

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Figure 2. Fluorescence profile (with log amplification) from the FACS of (a) transiently adherent low-density cells (non-adherent after 16 hr) and (b) initially non-adherent (90 min) low-density cells. The dashed line shows control second-layer fluorescent antibody alone, and continuous lines show profile with anti-Leu M3. In each sample, about half of the cells were negative for M3 staining and were therefore possible dendritic cells.

Separation of low-density cells non-adherent after 90 min Table 1 shows that when Ficoll-separated cells from 45 ml of blood were incubated for 90 min at 370 and the non-adherent cells separated on Metrizamide, the mean yield of low density cells was 3-1 x 106 cells (5.8% of the original number of Ficollseparated mononuclear leucocytes). Direct application to the Metrizamide of freshly isolated Ficoll-separated cells (without any incubation to remove adherent cells) gave only 4 3 x 105 lowdensity cells. The 90 min adherence step thus resulted in a sevenfold increase in the numbers of large low-density cells isolated (P=0 0001). This increase took place despite the removal (on average) of 7-8 x 106 adherent macrophages which could be recovered from the petri-dish after the 90 min incubation. Subsequent studies on the non-adherent low-density cells were done on those cells obtained after a 90 min incubation.

Table 1. Yield of LDC when cells separated on Ficoll were incubated for 90 min on Nunc petri-dishes and the non-adherent cells applied to the Metrizamide gradient (volume of blood donation =45 ml)

Post-Ficoll yield (x 106) x 106 Donor JF JF JF JF AL AL AL MT MT MT MT AE AE

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HLA DR-positive mononuclear cells containing fewer than 5% small cells including lymphocytes. Phenotypically, two populations of these large cells were present: one population lacked the monocyte marker Leu-M3 (range 3-48%), e.g. Fig. 2a shows a fluorescence profile of a sample of large cells labelled for the monocyte marker Leu-M3, where 48% of the cells were negative. The function of these Metrizamide separated cells was assessed by their ability to stimulate allogeneic MLR. Between 75 and 150 of the 16 hr low-density cells caused significant stimulation when added to 50,000 allogeneic lymphocytes. Details are discussed in the results section on MLR.

BL AB DH

52(26)* 45 53 70 50 45 75 56 56(36)* 64 57 35(7)* 72 54(27)* 50 58

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4-0 3.9 1-0 1-2 2-8 35 3.5 25 4-0 1.1 28 16 30 2-5

7.5 56 2-0 2-7 3.7 6-3 9.7 3.9 7-0 15 7 3-9 6-2 6-0

4.3 Mean 5 8 SD 3-3 n 16

* Only the number of cells given in parentheses (x 106) were applied to the adherence step and subsequent metrizamide gradient.

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Figure 3. Scanning electron microscope pictures of low-density cells (a, b, c) obtained from peripheral blood after a 90 min adherence depletion of macrophages. The bar lines indicate 1 gm. Magnification x 4375.

(c)^-... Morphology of 90 min non-adherent low-density cells By light microscopy, the low-density cells from the Metrizamide gradients in the non-adherent populations were large mononuclear cells. Scanning electron microscopy showed that they contained 96% of cells with a 'veiled' appearance (for example, see Fig. 3). Transmission electron microscopy showed that 95% of the cells had long 'dendritic' processes (Fig. 4a, b, c). Most cells were 9-11 gm in diameter, with a high ratio of nucleus to cytoplasm, but 5% of the cells had a diameter of 14-15 pm and a higher

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