Biotechnd. Rog. 1881, 7, 391-396
38 1
Isolation of Human B-Cell Subpopulations for Pharmacological Studied Johann Bauer'J and Klaus G. E. Stiinkelt Max-Planck-Institutfur Biochemie, 8033 Martinsried, Germany, and Institut fur Immunologie und Onkologie, Bayer AG, 5600 Wuppertal, Germany
Three groups of human peripheral blood B-lymphocytes were separated from each other by countercurrent centrifugal elutriation and free-flow electrophoresis. They differed in their state of maturation and in their capability to produce antibodies in vitro. These B-cell subpopulations were used to study features of a drug such as BAY R 1005. BAY R 1005 is a synthetic glycolipid analogue (GLA), which is supposed to modulate antibody synthesis. Mature, immunoglobulin- (Ig-) secreting B-lymphocytes secreted equal quantities of antibodies in the presence and in the absence of the GLA. BAY R 1005 was found to be without mitogenic activity on resting B-cells and did not induce them to produce antibodies. However, it supported the antibody production of preactivated B-lymphocytes. The in vitro preactivated B-cells were affected via monocytes. Only in vivo preactivated B-lymphocytes increased their antibody production under the direct influence of BAY R 1005.
Introduction Recently, a physical cell isolation procedure has been developed to separate resting, in vitro preactivated, and mature B-cell subpopulations of freshly drawn human peripheral blood on a preparative scale (Bauer et al., 1988). Mature B-lymphocytes secrete antibodies directly following isolation (Thomson & Harris, 1977; Fauci et al., 1980). Their antibody production capability could not be increased by any kind of stimulatory agent (Bauer et al., 1988). Resting B-lymphocytes can be stimulated in vitro by mitogens, antigens, and allogeneicleukocytesto secrete antibodies after an incubation period of 5-8 days. These B-cells require T-lymphocytes and monocytes for optimal growth (Waldman & Broder, 1982;Howard &Paul, 1983). The third group are the so-called in vivo preactivated B-lymphocytes. These cells have already received their activation signal in the body prior to isolation (Kikutani et al., 1986; Anderson et al., 1986). During further incubation in vitro they spontaneously produce antibodies after 3 days (Bauer et al., 1988). Their antibody production can be enhanced by B-cell growth and differentiation factors (Kehrl et al., 1985). In this study we have investigated the influence of BAY R 1005, a synthetic immunoenhancing agent (Hakomori, 1981; Lockhoff et al., 1985; Stunkel et al., 1988a,b,1989), on the antibody production capability of the B-lymphocyte subpopulations preparatively separated by our physical methods. BAY R 1005belongs to a new class of synthetic glycolipid analogues that show structural similarity to naturally occurring glycolipids found in mammalian cell membranes. Earlier studies of ita biological activity in vitro, using murine lymphocytes, have revealed that it augments the number of plaque-forming units in the Mishell-Dutton system without exerting mitogenic activity. On the basis of kinetic studies and B-cell proliferation studies it was concluded that BAY R 1005 acts subsequently to the antigenic stimulus as a second signal I This paper is dedicatedto Professor Karl Heinz Btichel,Member of the Board, Bayer AG, Leverkusen, FRG,on the occasion of his 60th birthday. * Correspondence should be addressed to this author. f Max-Planck-Institut fur Biochemie. t Institut fur Immunologie und Onkologie, Bayer AG.
87567936/91/3007-0391$02.50/0
(Stunkel et al., 198813,1989). Here we confirm that BAY R 1005affects human B-cells like murine B-cells,delivering a secondary signal during the first 3 days after primary stimulation in vitro. This secondarysignal requires monocytes. In addition we found that BAY R 1005 acta independently of monocytes also on in vivo preactivated human B-cells during a final period of maturation. The latter result seems to be important, because it points to further in vivo effects of the drug, which has the potential to be used as an immunosupporting agent in immunodeficient patients and for vaccination programs.
Materials and Methods Source of Blood. The peripheral blood used in this study was purchased from the Red Cross, Munich. Sixtyeight buffy coats obtained from blood conserves were provided for research after they had been tested and proved to be healthy. Physical Cell Isolation Procedure. Fractionation of Peripheral Blood. Mononuclear leukocytes (MNL) were isolated from the peripheral blood of healthy donors (Red Cross, Munich) by Ficoll-Hypaque (MSL 2000, Eurobio, Paris, France) density gradient sedimentation (Boyum, 1968). The MNL were further separated by countercurrent centrifugal elutriation (CCE) in a Beckman J-21B centrifuge (Beckman, Palo Alto, CA) at arotor (JE6) speed of 2430 rpm (Sanderson, 1982). For further purification, preparative free-flow electrophoresis was performed in an ACE 710 (Hirschmann Geratebau, Unterhaching, Germany). The cells were separated in a low ionic strength buffer as described earlier (Zeiller et al., 1970; Hannig, 1982; Bauer et al., 1988). Fractionation of Cells after in Vitro Incubation. Small numbers of cultured cells were separated according to their volume in a self-made elutriator that was adapted for use in a tabletop centrifuge (Bauer & Hannig, 1988). The system was equipped with the CS1 rotor containing a separation chamber with 0.5-mL volume. Typically, 5 X 107 cells harvested from culture flasks were injected into the rotor at a rotor speed of 2800 rpm (=600g). Then unstimulated cells were washed out at counterflow rates of 4 and 6 mL/min. Finally, the large stimulated cells were isolated at a counterflow rate of 6 mL/min and a rotor speed of 500 rpm.
@ 1991 American Chemical Society and American Institute of Chemlcal Engineere
Biotechnol. hog., 1991, Vol. 7, No. 5
382 Table 1: Characterization of the Purified B-Lymphocyte Fractions fraction
a
type of B-cells
A
resting
A*
resting
B
in vivo preactivated
C
plasmacytoid
cellular composition 70% B-cells" no T-cells no monocytes 8% B-cells 75% T-cells 12% monocytes 15% B-cella 10% T-cells 60% monocytes 1%B-cells 45% T-cells 50% monocytes
stimulation by
reactivity to BAY R 1005
two-way MLC
yes
two-way MLC
no
reactivity to supernate of monocytes + BAY R 1005
Yes no
The B-cells in fraction A required additional T-cells and monocytes for antibody production.
Synthetic Glycolipid Analogues (GLA). The backbone of the glycolipid analogues is composed of a carbohydrate residue with alkylamine and fatty acids of different chain length. This new class of synthetic glycolipids represents a glucosylamide consisting of a glucose molecule with stearylamine (18 C) and lauric acid (12 C). Compounds of N-glycolipid composition display strong structural similarities to the naturally occurring glycolipids, the glucosylsphingolipids and the glucosylglycerolipids. The glucosylamide BAY R 1005 is substituted at C-2 of the glucose residue with a L-leucinamide group (Lockhoff et al., 1987/1985; Stunkel et al., 1988a). BAY R 1005 is dissolved in ethanol or dimethyl sulfoxide (DMSO, 20 pL/ mg of substance),adjusted to 1mL/mg in culture medium (stock solution), sonicated for 1 min (Branson Sonifier B-12, Branson Sonic Power Co., Danbury, CT), and diluted with culture medium to the desired concentrations. Cellular Subpopulations. The B-cell separation procedure was performed as described earlier (Bauer et al., 1988). Small resting (Table I, fraction A), enlarged resting (Table I, fraction A*), in vivo preactivated (Table I, fraction B), and mature (Table I, fraction C) B-lymphocytes were preseparated by CCE at counterflow rates of 16, 20, 23, and 28 mL/min, respectively. The cells in the elutriator fractions were further purified by free-flow electrophoresis. The cellular composition of the finally purified fractions is shown in Table I. Small resting B-lymphocytes enriched in fraction A to 70% could not be stimulated by PWM or heterologous leukocytes unless they were mixed with autologous T-lymphocytes and monocytes at a ratio of 1:l:l prior to incubation. These T-lymphocytes and monocytes have been enriched in different fractions to 90% and 98%,respectively, by using CCE and free-flow electrophoresis. The enlarged resting B-lymphocytes of fraction A* were still contaminated with 75% T-lymphocytesand 12 5% monocytes. Hence the total fraction could be used for antibody production assays without adding further cells. The in vivo preactivated B-cells in fraction B produced spontaneously antibodies. This antibody secretion could be increased by IL-2 but not by heterologous leukocytes. The mature B-cells in fraction C started antibody production at day 0 of incubation. Their antibody production could not be manipulated by PWM, heterologous leukocytes, or IL-2. Cell Characterization. The purity of the lymphocyte and monocyte subpopulations was judged by light microscopy, volume analysis, and antibody binding assays. The morphology and the esterase activity of the different subpopulations were determined as described earlier (Bauer & Hannig, 1984). The volume analyses were performed in a Metricell flow cytometer (Kachel, 1976). The numbers of T-cells (anti-T3 from Coulter Electronics, Miami, FL) and of B-cells (anti-IgM and anti-IgG from
Behring, Marburg, Germany) were determined by direct immunofluorescence techniques (Bauer et al., 1988;Bauer & Kachel, 1988). Cell viability was checked by trypan blue exclusion. Cell Culturing. The isolated cells were adjusted to a concentration of 1.2 X 106/mL of RPMI 1640 (GIBCOEurope, Karlsruhe, Germany) supplemented with 107% (v/v) heat-inactivated fetal calf serum (FCS) and with 100 units/mL penicillin and 100 pg/mL streptomycin (GIBCO). Then they were seeded into 96-well microtiter plates (0.2 mL/well) or into 50-mL culture flasks (20mL/ flask). After 7 days of incubation at 37 "C, the cells were harvested by centrifugation. The supernatants of the microtiter plates were screened for human antibodies. The cells from the culture flasks were separated in a specially constructed tabletop elutriator according to Bauer and Hannig (1988). Primary i n Vitro Activation of B-Cells. In order to investigate the mitogenic activity of PWM or BAY R 1005, the B-cells isolated from one blood donor were cultured in the presence of 5 pg/mL PWM or various concentrations (between 1 and 50 pg/mL) of BAY R 1005. In order to perform B-cell activation by heterologous leukocytes, the MNLs of three unrelated donors were mixed (Munker et al., 1983). Subsequently, the cell mixture was fractionated by CCE. The B-cells leaving the elutriator at counterflow rates of 16 and 20 mL/min were further purified by freeflow electrophoresis and cultured for 7 days as described above. Quantification of Antibodies Released into the Culture Supernatants. Human antibodies produced by B-lymphocytes during incubation in vitro were absorbed by protein A coated sheep red blood cells (SRBC)(Gronowicz et al., 1976;Bauer et al., 1988). Protein A coated SRBC (5 X lo6 cells) were incubated in 0.4 mL of serially diluted culture supernatants for 1 h at 37 "C. Then the SRBC were washed in veronal-buffered saline and resuspended in 0.125 mL of this buffer containing 2 7% (v/v) guinea pig serum and either 1%(v/v) rabbit anti-human IgG or 1% (v/v) rabbit anti-human IgM antiserum (Behringwerke, Marburg, Germany). After an additional 1h of incubation at 37 OC, the hemoglobin released into the supernatant was determined spectrophotometricallyat 412 nm. In this system the protein A coated SRBC were completely lysed if 30 ng of IgG or IgM was present in the assay. Ultrafiltration of Media and Supernatants. Culture supernatants and culture medium containing BAY R 1005 were concentrated by ultrafiltration using the Amicon ultrafiltration system Centriflo CF50A (Amicon, Lexington, MA). Either culture supernatant or culture medium was centrifuged at 3000 rpm and 0 OC until the volume was reduced from 8 to 0.5 mL. This concentrate was mixed
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1 Co
a
b
c
d
e
Figure 1. Quantities of antibodies produced spontaneously by mature (a), resting (b), and in vivo preactivated B-cells (c) and by small resting B-cells stimulated either with PWM (d) or with heterologous leukocytes (e). The bars indicate the antibody concentrations found in the culture supernatants, when B-cells were incubated for 7 days in the presence (hatched bars) and in the absence (dotted bars) of BAY R 1005.
with 0.75 mL of medium and added to microtiter plates after being diluted 1:6.
Results Different B-Cell Fractions Used for Characterization of Activation Signals Delivered by BAY R 1005. Resting (fractions A and A*), in vivo preactivated (fraction B), and mature (fraction C) B-lymphocyteswere separated and purified by the three step physical isolation procedure described above (Table I). These B-cell populations were used to answer the following questions concerning the pharmacological activity of BAY R 1005: (i) Does BAY R 1005 deliver a primary B-cell activation signal as the mitogen PWM or antigenic heterologous leukocytes do? (ii) Does it have secondary effects like B-cell growth factors? (iii) Does it support the final process of antibody secretion? In order to answer the third question, mature B-lymphocytes (Table I, fraction C) were cultured in the presence and in the absence of BAY R 1005. They produced equal quantities of antibodies in both assays (Figure la). This indicated that the final process of antibody secretion of mature B-lymphocytes is not affected by BAY R 1005. Investigating whether BAY R 1005 delivers a primary signal of B-cell activation, resting B-cells (Table I, fraction A) were cultured in the presence and in the absence of BAY R 1005. These antibody production assays were performed under the same conditions as the PWM stimulation assays. The concentrations of BAY R 1005 varied from 1 to 50 pg/mL. Nevertheless, the resting B-cells did not produce detectable quantities of antibodies under the influence of BAY R 1005 (Figure lb). Hence the experiments suggested that BAY R 1005 does not deliver a primary mitogenic or antigenic signal. Concerning the second question, we carried out two different series of experiments: First, the in vivo preactivated B-lymphocytes (Table I, fraction B) were incubated with and without BAY R 1005. Without the drug, the in vivo preactivated B-cells produced spontaneouslybetween 300 and 750 ng of Ig/mL of culture supernatant depending on the blood donor. If the culture medium contained between 12.5 and 50 pg/mL BAY R 1005, the Ig production was increased up to 3000 ng/mL (Figure IC). Concentrations below and above this range had less or no effect
3.1
6.2
126
26
60
100
w/ml
Figure 2. Quantities of antibodies (y-axis) produced during 7 days, when in vivo preactivated B-cells were incubated in the presence of varying concentrations of BAY R 1005 (x-axis).
(Figure2). The results suggested that BAY R 1005delivers a secondary signal to, in vivo preactivated B-cells. Second, small resting B-lymphocytes (Table I, fraction A) were stimulated by PWM or heterologous leukocytes as described above. In both stimulation experiments, parallel assays were performed either with 12.5 pg/mL BAY R 1005or without. PWM triggered the resting B-cells to produce 3000 ng/mL Ig whether or not BAY R 1005 was present in the assay (Figure Id). Then resting B-cells (Table I, fraction A) of three different donors were cultured together as described above (two-way MLC). After 7 days an average of 300 ng of Ig/mL of supernatant was found. If the MLC was performed in the presence of BAY R 1005, 3000 ng of Ig or even more was secreted into 1 mL of culture supernatant (Figure le). These results show that BAY R 1005enhances Ig production of small resting B-cells costimulated with heterologous leukocytes. This suggests that BAY R 1005 may deliver a secondary signal also to in vitro activated B-cells. Different Secondary Signals on in Vivoandin Vitro Preactivated B-Cells Period ofthe Responsive Stage. It is known that in vivo preactivated B-cells have obtained their primary signal of activation at least 3 days before they can be isolated from the peripheral blood (Bauer et al., 1988). Then they are responsive to BAY R 1005. Thus we investigated whether BAY R 1005 acts on in vitro activated B-cells within the first 3 days of an MLC or later. For this purpose small resting B-cells of three different blood donors were incubated in 50-mL culture flasks as described above (scaled-up MLC). The incubation was interrupted after 3 days, the cells were harvested and fractionated according to their volume by the tabletop elutriator. In this way six fractions were obtained containing cells of different volumes (Figure 3). The cells of these fractions were incubated at a concentration of 1.2 X lo6 cells/mL for another 4 days. The subsequent antibody quantification revealed that only one of the fractions (6 mL/min counterflow and 500 rpm, Figure 3, 0 ) contained B-cells that produced around 1500 ng/mL Ig. This fraction consisted of 70 % monocytes, 20 % T-cells, and 10% B-cells. When BAY R 1005 was added to this fraction during the final 4 days of maturation, no effect of the drug was seen. In control experiments, BAY R 1005 (12.5 pg/mL) was added to the culture flasks during the first 3 days of the MLC. Then the cells were separated by the tabletop elutriator. The cells in the different fractions were cultured again for another 4 days. Now the cells of the fraction collected at 6 mL/min counterflow rate and 500 rpm rotor speed produced up to 9000 ng/mL Ig. The results show that BAY R 1005 delivers the secondary signal to in vitro
.
Biotechnoi. Rog., 1991, Vol. 7, No. 5
394
lJbl/ml 8
6o ielatim cell number
0
20
40
GO
80
relat Ive v d urm Figure 3. Volume distribution curves of MNL's separated by the tabletop elutriator after 3 days of incubation in an MLC. The fractions were collected at 2800 rpm and 2.5 mL/min counterflow (e), at 2800 rpm and 4 mL/min counterflow (+, *), at 2800 rpm and 6 mL/min counterflow (0, X), and a t 500 rpm and 6 mL/min counterflow ( 0 ) .
preactivated B-cells only during the first 3 days of incubation but not during the final period of maturation. This indicates that BAY R 1005 acts on in vitro and in vivo activated B-cells at different time points after stimulation. Direct and Indirect Effects. In further experiments we were interested whether BAY R 1005 acts directly on the B-cells or via other cells that are present in the assays. Hence the isolated monocytes were cultured for 3 days in the presence of BAY R 1005. Then the supernatant was aspirated, concentrated, and added to the antibody production assays. In vivo preactivated B-cells (Table I, fraction B), which responded to BAY R 1005 (Figure IC), produced around 550 ng/mL Ig in the presence and in the absence of the monocyte supernatant (Figure 4a). Then small resting B-cells (Table I, fraction A) were incubated in an MLC with and without the monocyte supernatant. In these assays the monocyte supernatant increased the antibody production of the B-cells from 300 to 3000 ng/ mL (Figure 4b), similarly to BAY R 1005 (Figure le). The results show that the supernatant of monocytes cultured in the presence of BAY R 1005 had an effect on in vitro but not on in vivo preactivated B-cells. Since BAY R 1005had a direct effect on in vivo preactivated B-cells we concluded that after 3 days of incubation the monocytes had eliminated the drug. The fact that the monocyte supernatant supported the MLC-induced antibody production may be explained by a monokine. During an MLC, BAY R 1005 and the monocyte supernatant increased the antibody production of small resting B-cells (Figures l e and 4b). So it was not certain whether the effect of BAY R 1005on in vitro preactivated B-cells was due only to stimulation of monocytes or also to a direct interaction with the B-cells. Thus we looked for an activation assay, which could be performed without or with a very small number of monocytes. We found a subgroup of enlarged, resting B-cells, fraction A* (Table I). They left the elutriator at a counterflow rate of 20 mL/min and after final purification in the free-flow electrophoresis the fraction contained 8% B-cells together with 75% T-cells and 12% monocytes. The B-cells were in a resting state, because they did not produce antibodies unless stimulated by PWM or in an MLC. During an MLC, the B-cells of fraction A* produced between 300 and 750 ng/mL antibodies depending on the blood donors. Neither BAY R 1005nor medium that contained the drug
a
b
C
Figure 4. Quantities of antibodies found in the supernatant of B-cells incubated in medium (dotted bars) and in medium containing the supernatant of BAY R 1005 treated monocytes (hatched bars). The antibodies were produced either spontaneously by in vivo preactivated B-cells (a) or by MLC-activated B-cells leaving the elutriator at a counterflow rate of 16 mL/min (b) or 20 mL/min (c).
nor supernatant of unstimulated monocytes, both concentrated by the Centriflo technique described above, increased the Ig production capability of these B-cells in the MLC. If, however, aconcentrated supernatant of BAY R 1005 treated monocytes was added to the assays, the cells produced up to 7000 ng/mL Ig (Figure 4c). The results show that the monocyte supernatant but not BAY R 1005 itself affects in vitro preactivated B-cells. If a large number of monocytes is required for a stimulation assay, BAY R 1005 may act via the monocytes. This suggests that BAY R 1005 has a direct effect only on in vivo and an indirect effect on in vitro preactivated B-cells.
Discussion In this study cell populations of the human mononuclear leukocytes were highly enriched by a physical cell isolation procedure, which includes countercurrent centrifugal elutriation and free-flow electrophoresis. This method provides a source of B-cells in their native state of defined differentiationand activation (Bauer et al., 1988; Bauer, 1987). They proved suitable to investigate biologicaleffects of a pharmacologicalcompound such as BAY R 1005, because their in vivo status is maintained for in vitro experiments. BAY R 1005 has the potential to be used in men as an adjuvant for vaccination programs and/or as an immunotherapeutic agent in immunodeficient patients. At present it is necessary to get information about the ability of BAY R 1005to support a humoral immune response by enhancingthe antibody production of preactivated B-cells in humans. Neither resting nor mature B-cells responded to BAY R 1005 (Figure lb,a). Thus we concluded that the drug does not have mitogenic activity and does not affect the final process of antibody secretion. However, it delivers secondary signals on B-cells, preactivated by a first antigenic signal. At optimal concentrations BAY R 1005 increased the antibody production capability 10-fold, if control and drug-added experiments, performed with B-cellsof one blood donor, were compared. Differentblood donors had B-cells with different basic spontaneous antibody production capability (Bauer et al., 1988). This was the main reason for variations indicated in Figures 1, 2, and 4.
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Sofar the findings are in accordance with in vitro studies on murine B-cells (Stiinkel et al., 1988a,b). However, the concentration of BAY R 1005required for an optimal effect seems to be lower in the human system than in the mouse system, where at least 30 rg/mL is necessary in order to achieve a 6-fold increase of antibody production (Stiinkel et al., 1988a,b). In addition we found differences between in vivo and in vitro preactivated B-cells. Both kinds of activated B-cells are affected a t different time points after stimulation and by different ways. It is still unknown why in vivoand in vitro preactivated B-cellsshow different functional behavior at the third day after the primary activation. Now monoclonal antibodies are used for characterization of the differentiation stages of both kinds of B-cells at several time points after stimulation. When BAY R 1005 increases the antibody production of in vivo preactivated B-cellsit seems to mimic the activity of a natural growth factor such as IL-2, IL-5, or IL-6 (Kehrl et al., 1985; Vazquez et al., 1987; Wong & Clark, 1988; Takatsu et al., 1988). The antibody production of in vitro preactivated B-cells is affected by a supernatant of BAY R 1005treated monocytes. During an MLC of small resting B-cells (Table I, fraction A), not only the supernatant but also BAY R 1005 were active, though in these assays a large number of monocytes (33%)had to be present in order to achieve antibody production. Thus, it is not fully clear whether in addition to the monocyte supernatant BAY R 1005 is acting directly on small resting B-cells (Table I, fraction A) or via the monocytes present in the assays. At the moment we do not know the active factor in the monocyte supernatant. It may be identical with a monokine supporting the activation of human B-cells as described by others (Falkhoff et al., 1983; Tosato et al., 1988; Navarro et al., 1989). The in vivo preactivated B-cells are affected by BAY R 1005 during a late period of maturation. Earlier kinetic studies have shown that in vivo preactivated B-cells have obtained their first activation signal at least 3 days prior to isolation (Bauer et al., 1988). In contrast, human in vitro preactivated B-cells are affected by BAY R 1005 a t an early time point after the primary activation, similarly as it was observed for murine in vitro preactivated B-cells (Stiinkel et al., 1988a,b). After three days of incubation they could be enriched in one fraction by the tabletop elutriator (500 rpm rotor speed and 6 mL/min counterflow rate). At this time they were able to produce antibodies without further mitogenic stimulation (Bauer & Hannig, 1988,1986) like the in vivo preactivated B-cells. However, their ability to respond to BAY R 1005 has disappeared. So we concluded that it is important to use in vivo preactivated B-cells for pharmacological studies, because they may provide more information about the effects of a drug in a human body. The cell populations highly purified by our physical cell separation method (Bauer, 1987) were helpful to discover several steps of the mode of action of a pharmacological compound. Since they have maintained their in vivo status during the isolation procedure, the results of the experiments appear to be relevant for the in vivo situation.
CCE EM GLA HBS Ig IgG
Notation countercurrent centrifugal elutriation electrophoretic mobility glycolipid analogue Hanks' balanced salt medium immunoglobulin immunoglobulin G
IgM MLC MNL
PWM SRBC
immunoglobulin M mixed leukocyte culture mononuclear leukocytes pokeweed mitogen sheep red blood cells
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