IL 1, IL4 and IL6 induce human B cell colony formation
Eur. J. Immunol. 1991. 21: 1271-1275
Kimberley McGinnes. and Christopher J. Paige Division of Cell and Molecular Biology, Ontario Cancer Institute, Toronto
Interleukins 1, 4 and 6 induce the colony formation of human bone marrow B lineage cells*
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An agar-based, B cell colony assay (McGinnes, K. et al., Blood 1990. 76: 896) has been used to study the influence of known cytokines on the growth of B lineage colonies initiated by cells from normal, human bone marrow samples. We demonstrate that a combination of interleukin (IL) 1, IL4 and IL6 act directly to promote the generation of plaque-forming colonies. IL6 was shown to act at a late stage of colony formation, which is consistent with its role in the induction of immunoglobulin secretion from mature B cells. In contrast, IL 1 and IL4 were required at earlier stages in the formation of colonies containing cells which secrete immunoglobulin.
1 Introduction B lymphopoiesis occurs in two prominent stages. The first takes place in the adult BM [l-31, is independent of antigenic stimulation, is characterized by the rearrangement of the Ig genes [4], and results in the generation of mature, sIg+ B cells. These mature lymphocytes are then transported to peripheral lymphoid tissues [5]where resting B cells are activated by antigen and as a result undergo terminal differentiation into Ig-secreting plasma cells [6-81. The differentiation of immature B lineage cells into sIg+ B cells is thought to be regulated by the stromal cells, extracellular matrices and secreted growth factors which constitute the BM microenvironment. In the mouse, BM stromal cells have been shown to support B lymphopoiesis, probably via secreted growth factors [9, 101. In addition to these observations, several independent cytokines have been demonstrated to stimulate the proliferation of B cell progenitors. These include IL7 [ l l , 121 and an as yet uncharacterized factor from a stromal cell line [13] in the mouse; and in humans, IL3 [14, 151, low- and highmolecular-weight B cell growth factors [16] and possibly IL 1 [17]. Another group of factors are proposed to induce the differentiation of these B cell progenitors. These include a cyclic neutropenia factor [MI, an unidentified S17 stromal cell line factor [19], a factor from stromal cell line TC-1 [20,21], and IL4 which probably acts to promote pre-B to B cell maturation [22,23]. The molecules regulating the terminal differentiation of mature, resting B cells into antibody-secreting plasma cells in humans has been extensively studied and reviewed [24-261. In summary, IL4 and B cell activation factor are
[I 91791
* This work was supported by the Medical Research Council of Canada, and the National Cancer Institute of Canada. Commonwealth Scholar. Correspondence: Christopher J. Paige, Division of Cell and
Molecular Biology, Ontario Cancer Institute, 500 Sherbourne St., Toronto, M4X 1K9,Canada Abbreviation:
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TCM:T cell-conditioned medium
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
activation factors. IL 2 and low-molecular-weight B cell growth factor stimulate the proliferation of activated B cells. While IL6 and IL2 can induce Ig secretion and terminal differentiation of plasma cells. Additional molecules such as IL 1, IL3, TNF, IFN-a and IFN-y act as cofactors in these scheme. The majority of studies investigating the influence of growth factors on human B lymphopoiesis have been performed using bulk liquid culture assays of B lineage cells enriched from peripheral blood, tonsils, fetal BM or fetal liver, EBV-transformed cell lines, or lymphoblastic leukemia cells. While such studies have provided valuable information regarding the regulation of B lymphopoiesis, in particular during terminal differentiation, in that they do not allow the estimation of the frequency of responding cells nor do they distinguish between the direct and indirect actions of factors under study.We have recently reported an agar assay which allows the clonal growth of B lineage cells from normal human BM samples [27]. These culture conditions allow both B cell precursors and mature B cells to grow and differentiate into colonies containing cells which secrete Ig [28]. In the present study we have analyzed a panel of recombinant, human growth factors for their ability to support the growth of such colonies in this assay system. We demonstrate that a combination of IL 1, IL4 and IL6 promotes the growth of B lineage colonies containing cells which secrete Ig from normal human BM samples.
2 Materials and methods 2.1 B cell colony assay Normal human BM samples were obtained from the femur of osteoarthritis patients undergoing hip replacement surgery. Heparinized peripheral blood, obtained from healthy volunteers, was used to prepare Tcell conditioned medium (TCM). These protocols were approved by the Wellesley Hospital, and the Ontario Cancer Institute, Toronto, Canada. After collection, BM samples were fractionated on a discontinuous Percoll (Pharmacia, Baie d’Urfe, Canada) gradient prior to culture in the top layer of the double agar B cell colony assay described elsewhere [27]. B lineage colony growth was supported by the addition of a source of 0014-2980/91/0505-1271$3S O + .25/0
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Eur. J. Immunol. 1991. 21: 1271-1275
K. McGinnes and C. J. Paige
growth factors, namely 10%-20% TCM, 5% SN from the 5637 cell line, or recombinant interleukins. After a 10-day incubation at 37 "C in 5% COz, each 35-mm culture dish was examined for the growth of B lineage colonies. The colony plaque assay [27] was employed as a means of enumerating those colonies which contained cells secreting Ig. Plaques associated with colonies (> 20 cells) identified these as belonging to the B lineage. Such colonies are referred to as plaque-forming colonies in this study.
with growth factors secreted by 5637 cells are able to support the growth of BM B linege cells. Candidate factors in the 5637 conditioned medium included IL1, IL6, IL3 and TNF-a [31-331.
Experiments, such as those reported in Fig. 1, demonstrated that a combination of IL 1, IL4 and IL6 promoted the growth of plaque-forming, B lineage colonies. This support was similar to that afforded by TCM, and in nine experiments IL 1plus IL 4 plus IL 6 supported the growth of an average of 19% more plaque-forming colonies than 2.2 Media TCM.These findings are consistent with the suggestion that IL 1and IL 6 were the active factors in the 5637 conditioned The culture medium used in the preparation of conditioned medium. IL1, IL4 and IL6 were titered into the double media and for the growth of fresh BM B lineage cells was agar B cell colony assay to determine the optimal concenOpti-MEM (Gibco/BRL, Burlington, Canada) supple- trations required for plaque-forming colony growth. In mented with 5% FBS (Gibco), 50 U/ml penicillin, 50 Fg/ml these experiments the concentrations of two factors was streptomycin (Gibco), and 5 x M 2-ME (Gibco). In kept constant and the third varied. It was determined that the culture of fresh BM cells, 10 Fg/ml Staphylococcus the optimal concentrations were 1 U/ml for IL 1, 150 U/ml aureus (Cowan strain) protein A (Pharmacia) andor for IL4 and 24 U/ml for IL6. 20 pg/ml dextran sulfate (Sigma, St. Louis, MO) was included in the culture medium. To determine whether IL 1 plus IL4 plus IL6 provide the necessary requirements for the growth of plaque-forming TCM was prepared by incubating normal human peripheral colonies, four titration experiments were performed in blood T cells (E-rosette positive; [29], at 1 x 106/ml, in which 2.5 X lo4 to 2 X 105 Percoll-enriched BM cells were culture medium containing 1% PHA (Wellcome, Hartfort, plated and the resultant plaque-forming colonies enumerGB). SN were collected after 3 days, filtered, stored at 4°C ated. The least squares regression line fitted to these data and used in the B cell colony assay within 1 month of after logarithmic transformation had a slope of approxipreparation. Conditioned medium was similarly prepared mately 1(range 0.93 to 1.20), as shown in Fig. 2.This result from 5637, a human bladder carcinoma cell line [30], and suggests that the growth of plaque-forming, B lineage used as a source of growth factors. colonies under these conditions was limiting only for the cell which initiated the colony growth, and further implies that under these culture conditions IL 1, IL 4 and IL 6 acted 2.3 Cytokines directly on B lineage cells. To determine the influence of known cytokines on the generation of plaque-forming colony growth, human recombinant interleukins were added to the bottom layer of the B cell colony assay in the absence of TCM. The following optimal concentrations of interleukins were used; IL 1 (1 U/ml; Genzyme, Boston, MA), IL2 (200 U/ml; Cetus Corporation, Emeryville, CA), IL4 (150 U/ml; gift from Dr. M. Schreier, Sandoz Pharma, Basel, Switzerland), IL5 (2 U/ml; Amgen Biologicals, Thousand Oaks, CA), IL6 (24 U/ml; Genetics Institute, Cambridge, MA), IL7 (250 U/ml; gift from Dr. S. Gillis, Immunex, Seattle, WA) .
GROWTH FACTORS
3 Results and discussion We have previously demonstrated that TCM promotes the clonal growth and differentiation of human, BM B lineage cells. The clonogenic cells included CD10+, Ig L chain uncommitted precursors and more mature CD20+ B cells, but not plasma cells [l,281. In order to further investigate the capacity of this conditioned medium to support the growth of plaque-forming B lineage colonies, TCM was replaced by combinations of 5637 conditioned medium and recombinant growth factors in the double agar B cell colony assay. It was observed that the combination of IL4 and 5% 5637 conditioned medium supported the growth of plaqueforming colonies at a similar level to that supported by TCM (105 k 16% of the TCM level; n = 4 experiments; Fig. 1). These results suggested that IL4 in combination
0
10
20
30
40
50
60
70
PLAQUE-FORMINGCOLONY NUMBER
Figure 1. The effect of growth factors on the generation of plaque-forming colonies. Combinations of TCM, 5637 conditioned medium and recombinant human growth factors were added to the bottom layer of a double agar culture to give a final concentration of 15% TCM, 5% 5637 conditioned medium, 1 U/ml, 200 U/ml IL2, 150 U/ml I L 4 , 2 U/ml IL5,24 U/ml IL6 and 250 U/ml IL7.
Cultures not containing exogenous growth factor were established in parallel. Percoll-enriched BM cells (1 X los) were plated in the top layer of the B cell colony assay and after a 10-dayincubation the resultant plaque-forming colonies were enumerated. The results (mean k SD of three replicate cultures) from one experiment are shown here.
IL 1, IL4 and IL6 induce human B cell colony formation
Eur. J. Immunol. 1991. 21: 1271-1275
In growth factor checkerboard experiments (Fig. 1) where IL1, IL2, IL4, IL5, IL6 and IL7 were tested in the B cell colony assay, no single factor supported the growth of plaque-forming colonies. Two-factor combinations of IL 1 plus IL4, IL 1 plus IL6, and IL4 plus I L 6 promoted some plaque-forming colony growth, but at levels which were at least fivefold less than that supported by a combination of these 3 factors (IL 1plus IL4 plus IL6; n = 3 experiments). IL 1plus IL 7, and IL 6 plus IL 7 also supported some colony growth (< 10% that of IL 1plus IL4plus IL6). In addition, the combination of IL 1 plus IL4 plus IL7 permitted the growth of 34% of the number of plaque-forming colonies
PLAQUE-FORMING COLONY NUMBER
'OoO
1
1 1000
r,
rT-
7 7 7
1 1,
T-I
10000 100000 NUMBER OF INPUT CELLS
1000000
Figure 2. Relationship between input cell numbers plated in the presence of IL 1, IL4 and IL 6 and resultant plaque-formingcolony numbers. Percoll-enrichedBM cells (2.5 X 1@,5 X 1@,1 X 1@and 2 x 105) were plated in a double agar culture which included optimal concentrations of IL1, IL4 and IL6. After lodays, plaque-formingcolonies were enumerated. Each data point represents the mean(* SD) of three replicate cultures. The linear regression line generated from the logarithm of the arithmetic means has a slope of 1.14.
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supported by IL 1 plus IL4 plus I L 6 (n = 3 experiments). No effect on plaque-forming colony numbers was observed with the addition of either IL2 or IL5 to IL 1plus IL4 plus I L 6 culture conditions. The addition of IL7, at concentrations of 100 U/ml, 250 U/ml and 500 U/ml, to the combination of IL 1plus IL 4 plus IL6 led to a significant reduction (36%; p ~ 0 . 0 5 in ) the number of resultant plaque-forming colonies (n = 3 experiments). It is interesting that IL7 was not particularly active in the culture system employed in this study, and was in fact inhibitory to the generation of plaque-forming colony growth induced by IL 1, IL4 and IL6. There are several possible explanations for these observations. Firstly, additional growth factors may be required for IL7 to induce the proliferation of human pre-B cells in our assay system. Alternatively, the B cell colony assay may not be a suitable system for studying the effects of IL7 as the read-out assay used detects Ig-secreting colony cells. The inhibitory effect of IL7 on the induction of plaque-forming colony growth by IL 1plus IL 4 plus IL 6 may be a reflection that in vivo maturation of pre-B cells occurs in the BM whereas the generation of Ig-secreting plasma cells usually occurs in distant peripheral lymphoid organs. It should also be noted that there are few reports describing the proliferative effects of human IL7 on human B cell precursors. Goodwin et al. [34] enriched normal BM populations of B lineage (CD19+) cells and Touw et al. [35] studied pre-B ALL cells. In both reports an increased incorporation of [3H]thymidinewas observed following short-term culture in the presence of IL7, however the level of DNA synthesis measured was several fold lower than that induced in the murine system.
To assess the stage-specific requirements for IL 1, IL 4 and IL 6, experiments (n = 3) were performed in which the addition of one of these factors into the B cell colony assay was delayed. In each case, two factors were added to the cultures on day 0 while the third was added into the top agar
Table 1. Effect of delayed addition of IL1, IL4 and IL6 on colony growtha)
Presence of growth factor Day 0 3 6 9 10 IL~+L~: +ILl +ILl +IL1 +IL1 ILl+L6:
+IL4 +L4 +IL4 +n 4
ILl+IL4:
+IL6 +IL6 +IL6 +L6
J 1
Colony number lbtal PFCh)
1 I
20.2 f 2.9 42.2 f 4.1 41.6f 2.2 32.3 f 1.1 16.0 f 2.0
11.5 f 2.5 28.3 f 1.9 29.8 k 1.3 15.5 f 1.5 9.7 f 2.1
1
12.2 f 2.1 42.2 f 4.1 34.2 f 2.3 14.0 f 1.2 10.1 f 1.3
10.0 k 2.8 28.3 f 1.9 30.3 f 3.1 8.0 f 2.0 11.0 f 1.0
1
8.6 f 4.0 42.2 f 4.1 40.1f4.1 38.2 f 2.6 32.3 f 3.3
4.5 f 1.5 28.3 +. 1.9 28.0f 1.4 25.3 f 2.1 12.3 f 3.3
I
4
1 1 1 1
1 1 1 1
1
Percoll-enrichedBMcells (1 X 105) were plated in the B cell colony assay under the conditions shown and the resultant colonies enumerated after 10days. A combination of two growth factors (IL1 plus IL4, IL 1 plus IL6 or IL4 plus IL6) was added to the bottom layer at optimal concentrations, while the third factor, IL 6, IL4 or IL 1, respectively),was added to the top layer of agar cultures on day 0 , 3 , 6or 9, or was omitted completely. The results of a typical experiment (mean k SD of three replicate cultures) are shown in this table. The data were analyzed statistically using a Student's t-test (p< 0.05). PFC: plaque-forming colonies.
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K. McGinnes and C. J. Paige
layer on day 0 , 3 , 6 or 9 of a 10-day culture, as indicated in Table 1. It was observed that IL6 acted late in colony formation, in that a significant reduction in plaque-forming colony number only occurred when this factor was added at day 9. No reduction was observed when IL6 was included in the culture from daysO, 3 or 6. This decrease in plaque-forming colony number was not accompanied by a concomitant decrease in the total number of colonies (plaque-forming colonies others). These observations suggest that IL6 may act on cells in colonies of mature B cells, causing these cells to secrete Ig, and hence form plaques. This suggestion would be consistent with the current concept that IL6 is a terminal differentiation factor which induces the secretion of Ig from activated, mature, human B cells [36].
+
IL1 and IL4, on the other hand, appear to act at earlier stages of B lineage colony formation, in that addition to the cultures is required prior to day 6 to generate plaqueforming colonies. In the case of IL 1, addition at day 6 led t o a significant reduction in the number of plaque-forming colonies generated, although the total number of colonies observed during the culture period was unaffected. This result suggests that IL 1 may act at the level of Ig secretion, in addition to influencing the proliferation of B lineage cells. In the literature, IL1 has been shown to cause the proliferation of human leukemic pre-B cells [ 181, suggesting a role for this factor in B lineage differentiation in the BM. In addition, IL 1 reportedly acts as a cofactor for IL4 [37] in the proliferation of mature B cells and for I L 6 [38, 391 in plasma cell generation [26, 401. If IL 1is acting as a cofactor for IL4 and IL6 in the B cell colony assay this may explain the relatively low concentration required. A significant decrease in the number of plaque-forming colonies, as well as total colonies, was observed when IL4 was omitted from cultures for the first 6 days of incubation. This result suggests that IL4 probably acts at earlier stages of B lineage cell proliferation and differentiation, as has been implied in the literature. Hofman et al. [22] demonstrated that IL4 caused an increase in the number of cytoplasmic p+ (cp+), CD20+ and sIg+ cells in human fetal BM cultures grown in the presence of adherent BM cells. While King et al. [23] found that the secretion of IL4 from murine stromal cells caused the maturation of pre-B cells into sIg+ B cells. In addition, IL4 is reported to be a cofactor in B cell proliferation, as well as an activation factor causing mature B cells to enter the cell cycle [4 1-45].
The results presented in this report clearly suggest that IL 1, IL4 and IL6 play a role in human B lymphopoiesis. The importance of these cytokines for BM B lineage differentiation is verified by the observation that these three factors are produced by BM stromal cells, some of which have been shown to support B lineage cell proliferation and differentiation. For example, it has been demonstrated that IL4 is produced by murine stromal cells [21,23]; I L 6 production has been reported from both murine [46,47] and human [48,49] BM stromal cells; while IL 1 is secreted in human long-term BM cultures (LTBMC; [49]). In addition to these reports, we have previously demonstrated that human BM stromal cells from spicule-derived LTBMC were able to support the growth and differentiation of B lineage cells into colonies which contain cells secreting Ig. Concentrated
Eur. J. Immunol. 1991. 21: 1271-1275
SN from these LTBMC were shown to contain IL 1and JL 6 but not IL4 [50].The reason why IL4 was not detected may be because this cytokine was bound to the extracellular matrix surrounding stromal cells, as has been reported for granulocyte-macrophage CSF and IL3 [51, 521, and therefore was not present in the LTBMC SN.
4 Concluding remarks In conclusion, this report demonstrates that three growth factors, IL 1, IL 4 and IL 6, are important in the growth and differentiation of human B lineage cells of BM origin. Using a newly developed B cell colony assay we have shown that these three interleukins, in combination, induce the formation of colonies containing Ig-secreting cells. It will be of further interest to determine precisely at which stage in the differentiation process these factors act. The authors are grateful to Drs. Earl Bogoch and David Hastings, Wellesley Hospital, Toronto, for supplying bone marrow samples; and also thank Dr. Max Schreier and Dr. H. l? Kocher, Sandoz Pharma, Basel, for providing IL 4 and Dr. Steve Gillis, Immunex. Seattle, for supplying I L 7. Received January 2, 1991.
5 References 1 Osmond, D. G., J. Reticuloendothel. Soc. 1975. 17: 97. 2 Rosse, C., Int. Rev. Cytol. 1976. 45: 155. 3 Gathings, W. E., Lawton, A. L. and Cooper, M. D., Eur. J. Immunol. 1977. 7: 804. 4 Yancopoulos, G. D. and Alt, F. W., Annu. Rev. Immunol. 1986. 4: 339. 5 Osmond, D. G., Monogr. Allergy 1980. 16: 157. 6 Calvert, J. E., Maruyama, S., Tedder, T. F., Webb, C. F. and Cooper, M. D., Semin. Hematol. 1984. 21: 26. 7 Zola, H., Pathology 1985. 17: 365. 8 Melchers, F. and Anderson, J., Annu. Rev. Immunol. 1986.4: 13. 9 Dorshkind, K., Annu. Rev. Immunol. 19W. 8: 111. 10 Kincade, P.W., Lee, G., Pietrangeli, C. E.. Hayashi, S.4. and Gimble, J. M.,Annu. Rev. Immunol. 1989. 7: 111. 11 Namen, A. E., Scheimer, A. E., March, C. J., Overell, R. W., 12 13 14 15 16 17 18 19
Park, L. S., Urdal, D. L. and Mochizuki, D. Y., J. Exp. Med. 1988. 167: 988. Lee, G . , Namen, A. E., Gillis, S., Ellingworth, L. R. and Kincade, P.W., Immunology 1989. 142: 3875. Rennick, D., Jackson, J., Moulds, C., Lee, F. and Yang. G., Nature 1989. 142: 161. Uckun, F. M., Gesner,T. G., Song, C.W., Myers, D. E. and Mufson, A., Blood 1989. 73: 533. Wormann, B., Gesner,T. G., Mufson, R. A. and LeBien,T.W., Leukemia 1989. 3: 399. Uckun, F. M., Fauci, A. S., Heerema, N. A., Song, C. W., Mehta, S. R., Gajl-Pezalska, K., Chandan, M. and Ambrus, J. L., Blood 1987. 70: 1020. Uckun,F. M., Myers, D. E.,Fauci, A. S., Chandan-Langlie, M. and Ambrus, J. L., Blood 1989. 74: 761. Landreth, K. S., Engelhard, D., Beare, M. H., Kincade, P.W.. Kapoor, N. and Good, R., J. Immunol. 1985. 134: 2305. Landreth, K. S. and Dorshkind, K., J. Imrnunol. 1988. 140:
845. 20 Song, Z. X., Shadduck, R. K., Innes, D. J., Jr..Waheed. A. and Quesenberry, P. J., Blood 1985. 66: 273.
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IL 1. IL4 and IL6 induce human B cell colony formation
21 Woodward, T. A., McNiece, I. K., Witte, P. L., Bender, P., Crittenden, R.,Temeles, D. S., Robinson, B. E., Baber, G . B., Deacon, D. H., Isakson, P. C. and Quesenberry, F! J., Blood 1990. 7.5: 2130. 22 Hofman, F. M., Brock, M., Taylor, C. R. and Lyons, B., J. Immunol. 1988. 141: 1185. 23 King, A. G.,Weirda, D. andLandreth, K. S., J. Immunol. 1988. 141: 2016. 24 Kishimoto, T., Prog. Allergy 1988. 42: 280. 25 Lipsky, F! E., Hirohata, S., Jelinek, D. F., McAnally, L. and Splawski, J. B., J. Rheumatol. Suppl. 1988. 76: 229. 26 Steel, C. M. and Hutchings, D., Biochim. Biophys. Acta 1989. 989: 133. 27 McGinnes, K., Keystone, E., Bogoch, E., Hastings, D., Messner, H. A., Jamal, N. and Paige, C. J., Blood 1990. 76: 896. 28 McGinnes, K., Letarte, M. and Paige, C. J., Blood 1991, in press. 29 Kaplan, M. E., Woodson, M. and Clark, C., in Bloom, B. R. and David, J. R. (Eds.), In Vitro Methods in Cell Mediated and Tumor Immunity, Academic Press, San Diego 1976, p. 83. 30 Fogh, J., J. Natl. Cancer Inst. Monogr. 1978. 49: 5 . 31 Jublinsky, F!T. and Stanley, E. R., Proc. Natl. Acad. Sci. USA 1985. 82: 2764. 32 Gabrilove. J. E.,Welte, K., Hams, I?, Platzer, E., Lu, I., Levi, E., Mertelsmann, R. and Moore, M. A. S., Proc. Natl. Acad. Sci. USA 1986. 83: 2478. 33 Rawle, E C., Shields, J., Smith, S. H., Iliescu, V., Merkenschlager, M., Beverley, I? C. L. and Callard, R. E., Eur. J. Immunol. 1986. 16: 1017. 34 Goodwin, R. G., Lupton, S., Schmierer, A., Hjemld, K. J., Jerzy, R., Clevenger,W., Gillis,S., Cosman, D. and Namen, A., Proc. Natl. Acad. Sci. USA 1988. 86: 302. 35 Touw, I., Powels, K. ,Van Agthoven, T. ,Van Gurp, R., Budel, L., Hoogerbrugge, H., Delkwel, R., Goodwin, R., Namen, A. and Lowenberg, B., Blood 1990. 75: 2097.
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36 Muraguchi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y., Nakajima, K. and Kishimoto, T., J. Exp. Med. 1988. 167: 332. 37 Hivroz, C., Valle, A., Brouet, J. C.. Banchereau, J. and Grillot-Courvalin, C., Eur. J. Immunol. 1989. 19: 1025. 38 Emilie, D., Crevon, M.-C., Auffredou, M.T. and Galanaud, I?, Eur. J. lmmunol. 1988. 18: 2043. 39 Jandl, R. C., Flanagan, R. G. and Schur, I? H., Clin. Immunof. Immunopathol. 1988. 46: 115. 40 Gordon,J. and Guy, G. R., Immunol. Today 1987. 8: 339. 41 Defrance,T.,Vanbervleit,B., Aubry, J.-I?,Takebe,Y., Arai,N., Miyajima, A.,Yokota,T., Lee, F., Arai, K . 4 . ,Vries, J. E. and Banchereau, J., J. Immunol. 1987. 139: 1135. 42 Gordon, J., Cairns, J. A., Millsum, M. J., Gillis, S. and Guy, G. R., Eur. . I . Immunol. 1988. 18: 1561. 43 Kishimoto, T. and Hirano, T., Annu. Rev. Immunol. 1988. 6: 485. 44 Splawski, J. B., Jelinek, D. and Lipsky, P. E., J. Immunol. 1989. 142: 1569. 45 Carlsson, M., Sundstrom, C., Gengtsson, M.,Totterman,T. H., Rosen, A. and Nilsson, K.,Eur. J. Immunol. 1989. 19: 913. 46 Chui, C. P., Moulds, C. Coffman, R. L., Rennick, D. and Lee. F., Proc. Natl. Acad. Sci. USA 1988. 8.5: 7099. 47 Gimble, J. M., Pietrangeli, C., Henley, A., Dorheim, M. A., Silver, J., Namen, A.,Takeichi, M., Goridis, C. and Kincade, I? W., Blood 1989. 74: 303. 48 Nemunaitis, J., Andrews, D. F., Mochizuki, D.Y., Lilly, M. B. and Singer, J. W., Blood 1989. 74: 1929. 49 Slack, J. L., Nemunaitis, J., Andrews, D. F., I11 and Singer, J. W., Blood 1990. 75: 2319. 50 McGinnes, K., Quesniaux,V., Hitzler, J., Paige, C. J., Exp. Hematol. 1991, in press. 51 Gordon, M.Y., Riley, G. P., Watt, S. M. and Greaves, M. F., Nature 1987. 326: 403. 52 Roberts, R., Gallagher, J., Spooncer, E., Allen,T. D., Bloomfield, F. and Dexter, T. W., Nature 1988. 332: 376.
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Kimberley McGinnes. and Christopher J. Paige Division of Cell and Molecular Biology, Ontario Cancer Institute, Toronto
Interleukins 1, 4 and 6 induce the colony formation of human bone marrow B lineage cells*
-
An agar-based, B cell colony assay (McGinnes, K. et al., Blood 1990. 76: 896) has been used to study the influence of known cytokines on the growth of B lineage colonies initiated by cells from normal, human bone marrow samples. We demonstrate that a combination of interleukin (IL) 1, IL4 and IL6 act directly to promote the generation of plaque-forming colonies. IL6 was shown to act at a late stage of colony formation, which is consistent with its role in the induction of immunoglobulin secretion from mature B cells. In contrast, IL 1 and IL4 were required at earlier stages in the formation of colonies containing cells which secrete immunoglobulin.
1 Introduction B lymphopoiesis occurs in two prominent stages. The first takes place in the adult BM [l-31, is independent of antigenic stimulation, is characterized by the rearrangement of the Ig genes [4], and results in the generation of mature, sIg+ B cells. These mature lymphocytes are then transported to peripheral lymphoid tissues [5]where resting B cells are activated by antigen and as a result undergo terminal differentiation into Ig-secreting plasma cells [6-81. The differentiation of immature B lineage cells into sIg+ B cells is thought to be regulated by the stromal cells, extracellular matrices and secreted growth factors which constitute the BM microenvironment. In the mouse, BM stromal cells have been shown to support B lymphopoiesis, probably via secreted growth factors [9, 101. In addition to these observations, several independent cytokines have been demonstrated to stimulate the proliferation of B cell progenitors. These include IL7 [ l l , 121 and an as yet uncharacterized factor from a stromal cell line [13] in the mouse; and in humans, IL3 [14, 151, low- and highmolecular-weight B cell growth factors [16] and possibly IL 1 [17]. Another group of factors are proposed to induce the differentiation of these B cell progenitors. These include a cyclic neutropenia factor [MI, an unidentified S17 stromal cell line factor [19], a factor from stromal cell line TC-1 [20,21], and IL4 which probably acts to promote pre-B to B cell maturation [22,23]. The molecules regulating the terminal differentiation of mature, resting B cells into antibody-secreting plasma cells in humans has been extensively studied and reviewed [24-261. In summary, IL4 and B cell activation factor are
[I 91791
* This work was supported by the Medical Research Council of Canada, and the National Cancer Institute of Canada. Commonwealth Scholar. Correspondence: Christopher J. Paige, Division of Cell and
Molecular Biology, Ontario Cancer Institute, 500 Sherbourne St., Toronto, M4X 1K9,Canada Abbreviation:
1271
TCM:T cell-conditioned medium
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
activation factors. IL 2 and low-molecular-weight B cell growth factor stimulate the proliferation of activated B cells. While IL6 and IL2 can induce Ig secretion and terminal differentiation of plasma cells. Additional molecules such as IL 1, IL3, TNF, IFN-a and IFN-y act as cofactors in these scheme. The majority of studies investigating the influence of growth factors on human B lymphopoiesis have been performed using bulk liquid culture assays of B lineage cells enriched from peripheral blood, tonsils, fetal BM or fetal liver, EBV-transformed cell lines, or lymphoblastic leukemia cells. While such studies have provided valuable information regarding the regulation of B lymphopoiesis, in particular during terminal differentiation, in that they do not allow the estimation of the frequency of responding cells nor do they distinguish between the direct and indirect actions of factors under study.We have recently reported an agar assay which allows the clonal growth of B lineage cells from normal human BM samples [27]. These culture conditions allow both B cell precursors and mature B cells to grow and differentiate into colonies containing cells which secrete Ig [28]. In the present study we have analyzed a panel of recombinant, human growth factors for their ability to support the growth of such colonies in this assay system. We demonstrate that a combination of IL 1, IL4 and IL6 promotes the growth of B lineage colonies containing cells which secrete Ig from normal human BM samples.
2 Materials and methods 2.1 B cell colony assay Normal human BM samples were obtained from the femur of osteoarthritis patients undergoing hip replacement surgery. Heparinized peripheral blood, obtained from healthy volunteers, was used to prepare Tcell conditioned medium (TCM). These protocols were approved by the Wellesley Hospital, and the Ontario Cancer Institute, Toronto, Canada. After collection, BM samples were fractionated on a discontinuous Percoll (Pharmacia, Baie d’Urfe, Canada) gradient prior to culture in the top layer of the double agar B cell colony assay described elsewhere [27]. B lineage colony growth was supported by the addition of a source of 0014-2980/91/0505-1271$3S O + .25/0
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K. McGinnes and C. J. Paige
growth factors, namely 10%-20% TCM, 5% SN from the 5637 cell line, or recombinant interleukins. After a 10-day incubation at 37 "C in 5% COz, each 35-mm culture dish was examined for the growth of B lineage colonies. The colony plaque assay [27] was employed as a means of enumerating those colonies which contained cells secreting Ig. Plaques associated with colonies (> 20 cells) identified these as belonging to the B lineage. Such colonies are referred to as plaque-forming colonies in this study.
with growth factors secreted by 5637 cells are able to support the growth of BM B linege cells. Candidate factors in the 5637 conditioned medium included IL1, IL6, IL3 and TNF-a [31-331.
Experiments, such as those reported in Fig. 1, demonstrated that a combination of IL 1, IL4 and IL6 promoted the growth of plaque-forming, B lineage colonies. This support was similar to that afforded by TCM, and in nine experiments IL 1plus IL 4 plus IL 6 supported the growth of an average of 19% more plaque-forming colonies than 2.2 Media TCM.These findings are consistent with the suggestion that IL 1and IL 6 were the active factors in the 5637 conditioned The culture medium used in the preparation of conditioned medium. IL1, IL4 and IL6 were titered into the double media and for the growth of fresh BM B lineage cells was agar B cell colony assay to determine the optimal concenOpti-MEM (Gibco/BRL, Burlington, Canada) supple- trations required for plaque-forming colony growth. In mented with 5% FBS (Gibco), 50 U/ml penicillin, 50 Fg/ml these experiments the concentrations of two factors was streptomycin (Gibco), and 5 x M 2-ME (Gibco). In kept constant and the third varied. It was determined that the culture of fresh BM cells, 10 Fg/ml Staphylococcus the optimal concentrations were 1 U/ml for IL 1, 150 U/ml aureus (Cowan strain) protein A (Pharmacia) andor for IL4 and 24 U/ml for IL6. 20 pg/ml dextran sulfate (Sigma, St. Louis, MO) was included in the culture medium. To determine whether IL 1 plus IL4 plus IL6 provide the necessary requirements for the growth of plaque-forming TCM was prepared by incubating normal human peripheral colonies, four titration experiments were performed in blood T cells (E-rosette positive; [29], at 1 x 106/ml, in which 2.5 X lo4 to 2 X 105 Percoll-enriched BM cells were culture medium containing 1% PHA (Wellcome, Hartfort, plated and the resultant plaque-forming colonies enumerGB). SN were collected after 3 days, filtered, stored at 4°C ated. The least squares regression line fitted to these data and used in the B cell colony assay within 1 month of after logarithmic transformation had a slope of approxipreparation. Conditioned medium was similarly prepared mately 1(range 0.93 to 1.20), as shown in Fig. 2.This result from 5637, a human bladder carcinoma cell line [30], and suggests that the growth of plaque-forming, B lineage used as a source of growth factors. colonies under these conditions was limiting only for the cell which initiated the colony growth, and further implies that under these culture conditions IL 1, IL 4 and IL 6 acted 2.3 Cytokines directly on B lineage cells. To determine the influence of known cytokines on the generation of plaque-forming colony growth, human recombinant interleukins were added to the bottom layer of the B cell colony assay in the absence of TCM. The following optimal concentrations of interleukins were used; IL 1 (1 U/ml; Genzyme, Boston, MA), IL2 (200 U/ml; Cetus Corporation, Emeryville, CA), IL4 (150 U/ml; gift from Dr. M. Schreier, Sandoz Pharma, Basel, Switzerland), IL5 (2 U/ml; Amgen Biologicals, Thousand Oaks, CA), IL6 (24 U/ml; Genetics Institute, Cambridge, MA), IL7 (250 U/ml; gift from Dr. S. Gillis, Immunex, Seattle, WA) .
GROWTH FACTORS
3 Results and discussion We have previously demonstrated that TCM promotes the clonal growth and differentiation of human, BM B lineage cells. The clonogenic cells included CD10+, Ig L chain uncommitted precursors and more mature CD20+ B cells, but not plasma cells [l,281. In order to further investigate the capacity of this conditioned medium to support the growth of plaque-forming B lineage colonies, TCM was replaced by combinations of 5637 conditioned medium and recombinant growth factors in the double agar B cell colony assay. It was observed that the combination of IL4 and 5% 5637 conditioned medium supported the growth of plaqueforming colonies at a similar level to that supported by TCM (105 k 16% of the TCM level; n = 4 experiments; Fig. 1). These results suggested that IL4 in combination
0
10
20
30
40
50
60
70
PLAQUE-FORMINGCOLONY NUMBER
Figure 1. The effect of growth factors on the generation of plaque-forming colonies. Combinations of TCM, 5637 conditioned medium and recombinant human growth factors were added to the bottom layer of a double agar culture to give a final concentration of 15% TCM, 5% 5637 conditioned medium, 1 U/ml, 200 U/ml IL2, 150 U/ml I L 4 , 2 U/ml IL5,24 U/ml IL6 and 250 U/ml IL7.
Cultures not containing exogenous growth factor were established in parallel. Percoll-enriched BM cells (1 X los) were plated in the top layer of the B cell colony assay and after a 10-dayincubation the resultant plaque-forming colonies were enumerated. The results (mean k SD of three replicate cultures) from one experiment are shown here.
IL 1, IL4 and IL6 induce human B cell colony formation
Eur. J. Immunol. 1991. 21: 1271-1275
In growth factor checkerboard experiments (Fig. 1) where IL1, IL2, IL4, IL5, IL6 and IL7 were tested in the B cell colony assay, no single factor supported the growth of plaque-forming colonies. Two-factor combinations of IL 1 plus IL4, IL 1 plus IL6, and IL4 plus I L 6 promoted some plaque-forming colony growth, but at levels which were at least fivefold less than that supported by a combination of these 3 factors (IL 1plus IL4 plus IL6; n = 3 experiments). IL 1plus IL 7, and IL 6 plus IL 7 also supported some colony growth (< 10% that of IL 1plus IL4plus IL6). In addition, the combination of IL 1 plus IL4 plus IL7 permitted the growth of 34% of the number of plaque-forming colonies
PLAQUE-FORMING COLONY NUMBER
'OoO
1
1 1000
r,
rT-
7 7 7
1 1,
T-I
10000 100000 NUMBER OF INPUT CELLS
1000000
Figure 2. Relationship between input cell numbers plated in the presence of IL 1, IL4 and IL 6 and resultant plaque-formingcolony numbers. Percoll-enrichedBM cells (2.5 X 1@,5 X 1@,1 X 1@and 2 x 105) were plated in a double agar culture which included optimal concentrations of IL1, IL4 and IL6. After lodays, plaque-formingcolonies were enumerated. Each data point represents the mean(* SD) of three replicate cultures. The linear regression line generated from the logarithm of the arithmetic means has a slope of 1.14.
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supported by IL 1 plus IL4 plus I L 6 (n = 3 experiments). No effect on plaque-forming colony numbers was observed with the addition of either IL2 or IL5 to IL 1plus IL4 plus I L 6 culture conditions. The addition of IL7, at concentrations of 100 U/ml, 250 U/ml and 500 U/ml, to the combination of IL 1plus IL 4 plus IL6 led to a significant reduction (36%; p ~ 0 . 0 5 in ) the number of resultant plaque-forming colonies (n = 3 experiments). It is interesting that IL7 was not particularly active in the culture system employed in this study, and was in fact inhibitory to the generation of plaque-forming colony growth induced by IL 1, IL4 and IL6. There are several possible explanations for these observations. Firstly, additional growth factors may be required for IL7 to induce the proliferation of human pre-B cells in our assay system. Alternatively, the B cell colony assay may not be a suitable system for studying the effects of IL7 as the read-out assay used detects Ig-secreting colony cells. The inhibitory effect of IL7 on the induction of plaque-forming colony growth by IL 1plus IL 4 plus IL 6 may be a reflection that in vivo maturation of pre-B cells occurs in the BM whereas the generation of Ig-secreting plasma cells usually occurs in distant peripheral lymphoid organs. It should also be noted that there are few reports describing the proliferative effects of human IL7 on human B cell precursors. Goodwin et al. [34] enriched normal BM populations of B lineage (CD19+) cells and Touw et al. [35] studied pre-B ALL cells. In both reports an increased incorporation of [3H]thymidinewas observed following short-term culture in the presence of IL7, however the level of DNA synthesis measured was several fold lower than that induced in the murine system.
To assess the stage-specific requirements for IL 1, IL 4 and IL 6, experiments (n = 3) were performed in which the addition of one of these factors into the B cell colony assay was delayed. In each case, two factors were added to the cultures on day 0 while the third was added into the top agar
Table 1. Effect of delayed addition of IL1, IL4 and IL6 on colony growtha)
Presence of growth factor Day 0 3 6 9 10 IL~+L~: +ILl +ILl +IL1 +IL1 ILl+L6:
+IL4 +L4 +IL4 +n 4
ILl+IL4:
+IL6 +IL6 +IL6 +L6
J 1
Colony number lbtal PFCh)
1 I
20.2 f 2.9 42.2 f 4.1 41.6f 2.2 32.3 f 1.1 16.0 f 2.0
11.5 f 2.5 28.3 f 1.9 29.8 k 1.3 15.5 f 1.5 9.7 f 2.1
1
12.2 f 2.1 42.2 f 4.1 34.2 f 2.3 14.0 f 1.2 10.1 f 1.3
10.0 k 2.8 28.3 f 1.9 30.3 f 3.1 8.0 f 2.0 11.0 f 1.0
1
8.6 f 4.0 42.2 f 4.1 40.1f4.1 38.2 f 2.6 32.3 f 3.3
4.5 f 1.5 28.3 +. 1.9 28.0f 1.4 25.3 f 2.1 12.3 f 3.3
I
4
1 1 1 1
1 1 1 1
1
Percoll-enrichedBMcells (1 X 105) were plated in the B cell colony assay under the conditions shown and the resultant colonies enumerated after 10days. A combination of two growth factors (IL1 plus IL4, IL 1 plus IL6 or IL4 plus IL6) was added to the bottom layer at optimal concentrations, while the third factor, IL 6, IL4 or IL 1, respectively),was added to the top layer of agar cultures on day 0 , 3 , 6or 9, or was omitted completely. The results of a typical experiment (mean k SD of three replicate cultures) are shown in this table. The data were analyzed statistically using a Student's t-test (p< 0.05). PFC: plaque-forming colonies.
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K. McGinnes and C. J. Paige
layer on day 0 , 3 , 6 or 9 of a 10-day culture, as indicated in Table 1. It was observed that IL6 acted late in colony formation, in that a significant reduction in plaque-forming colony number only occurred when this factor was added at day 9. No reduction was observed when IL6 was included in the culture from daysO, 3 or 6. This decrease in plaque-forming colony number was not accompanied by a concomitant decrease in the total number of colonies (plaque-forming colonies others). These observations suggest that IL6 may act on cells in colonies of mature B cells, causing these cells to secrete Ig, and hence form plaques. This suggestion would be consistent with the current concept that IL6 is a terminal differentiation factor which induces the secretion of Ig from activated, mature, human B cells [36].
+
IL1 and IL4, on the other hand, appear to act at earlier stages of B lineage colony formation, in that addition to the cultures is required prior to day 6 to generate plaqueforming colonies. In the case of IL 1, addition at day 6 led t o a significant reduction in the number of plaque-forming colonies generated, although the total number of colonies observed during the culture period was unaffected. This result suggests that IL 1 may act at the level of Ig secretion, in addition to influencing the proliferation of B lineage cells. In the literature, IL1 has been shown to cause the proliferation of human leukemic pre-B cells [ 181, suggesting a role for this factor in B lineage differentiation in the BM. In addition, IL 1 reportedly acts as a cofactor for IL4 [37] in the proliferation of mature B cells and for I L 6 [38, 391 in plasma cell generation [26, 401. If IL 1is acting as a cofactor for IL4 and IL6 in the B cell colony assay this may explain the relatively low concentration required. A significant decrease in the number of plaque-forming colonies, as well as total colonies, was observed when IL4 was omitted from cultures for the first 6 days of incubation. This result suggests that IL4 probably acts at earlier stages of B lineage cell proliferation and differentiation, as has been implied in the literature. Hofman et al. [22] demonstrated that IL4 caused an increase in the number of cytoplasmic p+ (cp+), CD20+ and sIg+ cells in human fetal BM cultures grown in the presence of adherent BM cells. While King et al. [23] found that the secretion of IL4 from murine stromal cells caused the maturation of pre-B cells into sIg+ B cells. In addition, IL4 is reported to be a cofactor in B cell proliferation, as well as an activation factor causing mature B cells to enter the cell cycle [4 1-45].
The results presented in this report clearly suggest that IL 1, IL4 and IL6 play a role in human B lymphopoiesis. The importance of these cytokines for BM B lineage differentiation is verified by the observation that these three factors are produced by BM stromal cells, some of which have been shown to support B lineage cell proliferation and differentiation. For example, it has been demonstrated that IL4 is produced by murine stromal cells [21,23]; I L 6 production has been reported from both murine [46,47] and human [48,49] BM stromal cells; while IL 1 is secreted in human long-term BM cultures (LTBMC; [49]). In addition to these reports, we have previously demonstrated that human BM stromal cells from spicule-derived LTBMC were able to support the growth and differentiation of B lineage cells into colonies which contain cells secreting Ig. Concentrated
Eur. J. Immunol. 1991. 21: 1271-1275
SN from these LTBMC were shown to contain IL 1and JL 6 but not IL4 [50].The reason why IL4 was not detected may be because this cytokine was bound to the extracellular matrix surrounding stromal cells, as has been reported for granulocyte-macrophage CSF and IL3 [51, 521, and therefore was not present in the LTBMC SN.
4 Concluding remarks In conclusion, this report demonstrates that three growth factors, IL 1, IL 4 and IL 6, are important in the growth and differentiation of human B lineage cells of BM origin. Using a newly developed B cell colony assay we have shown that these three interleukins, in combination, induce the formation of colonies containing Ig-secreting cells. It will be of further interest to determine precisely at which stage in the differentiation process these factors act. The authors are grateful to Drs. Earl Bogoch and David Hastings, Wellesley Hospital, Toronto, for supplying bone marrow samples; and also thank Dr. Max Schreier and Dr. H. l? Kocher, Sandoz Pharma, Basel, for providing IL 4 and Dr. Steve Gillis, Immunex. Seattle, for supplying I L 7. Received January 2, 1991.
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