The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia. Contributed by G. J. V. Nossal, ...
Proc. Nail. Acad. Sci. USA Vol. 84, pp. 1389-1393, March 1987 Immunology
Single cell studies on the role of B-cell stimulatory factor 1 in B-cell activation (cytokines/hapten-gelatin fractionation/T-cell independent antigens/B-cell clones)
MARK R. ALDERSON, BEVERLEY L. PIKE, AND G. J. V. NOSSAL The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia
Contributed by G. J. V. Nossal, November 4, 1986
The role of the T-cell-derived lymphokine ABSTRACT B-cell stimulatory factor 1 (BSF-1) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein-specific B lymphocytes isolated from normal mouse spleens by hapten-gelatin adherence has been studied in vitro. BSF-1 acting alone induced early B-cell activation, as assessed by a significant increase in cell diameter of single B cells cultured for 24 hr. A small but significant number of these B cells formed proliferating clones, some of which secreted antibody. When acting with the specific antigen fluorescein-polymerized flagellin, BSF-1 augmented early cell enlargement and markedly enhanced proliferation, but it did not increase the frequency of AFC clones stimulated by fluorescein-polymerized flagellin alone. The further addition of recombinant murine interleukin 1 (IL-1) marginally enhanced proliferation caused by antigen plus BSF-1. No synergy was observed between BSF-1 and IL-1 for antibody formation. In the presence of fibroblast filler cells, BSF-1 substantially inhibited AFC clone development achieved by antigen plus IL-1. BSF-1 was also found to be inhibitory to AFC clone development stimulated by specific antigen acting with either recombinant human interleukin 2 (IL-2) or with IL-2 plus IL-1, both in the presence or absence of filler cells. The results suggest that BSF-1 plays a complex role in the regulation of the B-cell activation pathway by enhancing early activation and antigen-specific proliferation as well as inhibiting the effects of other B-cell factors on antibody formation. BSF-1 is the only cytokine so far tested in the single B-cell system that acts with antigen to promote proliferation without concomitant antibody production. The T-cell-derived lymphokine B-cell stimulatory factor 1 (BSF-1) has recently been shown to act on a number of different cell types in a number of biological assays (1-11). BSF-1 (formerly termed B-cell growth factor 1 or BCGF-1) was originally described as acting with anti-IgM antibodies to induce proliferation of murine B lymphocytes (1). BSF-1 also activates resting B cells to increase their expression of class II major histocompatibility complex antigens (2), effects increases in cell volume (3, 4), and prepares them for the action of anti-IgM antibodies (4, 5). It has been shown that BSF-1 and B-cell differentiation factor y (BCDF-y) (or IgG1 induction factor) can be copurified (6). The recent molecular cloning of these molecules has shown them to be the same (7, 8). BSF-1 has also been shown to act on T cells, mast cells, and a pre-B-cell line (8-11). Our studies on the antigen-specific activation of B lymphocytes have shown a number of lymphokines and cytokines to possess both growth and differentiation-promoting activity (12-16). When acting with a specific T-independent antigen, recombinant murine interleukin 1 (IL-1), The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
recombinant human interleukin 2 (IL-2), and partially purified fractions of medium conditioned by concanavalin Astimulated EL4 cells, all promote both proliferation and antibody-forming cell (AFC) clone formation by single antigen-specific B cells. We have recently described an assay for assessing the effects of antigen and cytokines in the early activation of single B cells (17), allowing us to measure yet another distinct parameter of the activation pathway. The present studies describe the role of highly purified BSF-1 in early activation, proliferation, and antibody formation by single hapten-specific B cells, whether acting alone, with antigen, and/or in combination with IL-1 and/or IL-2.
MATERIALS AND METHODS Mice and Preparation of Fluorescein-Specific Splenic B Cells. Specific pathogen-free CBA/CaH/WEHI mice were used at 8-10 weeks of age. Hapten-speciflic B cells were prepared from spleen cell suspensions by fractionation on plastic dishes coated with fluorescein-gelatin as described (18-20). Adherent fluorescein-gelatin was removed from the recovered binding cells by collagenase. The binding population is 97% B cells, -70% fluorescein-binding, and -200-fold enriched for in vitro reactivity to fluorescein conjugates (19-21). Antigens. The hapten fluorescein was coupled onto polymerized flagellin and aminoethylcarbonylmethylated Ficoll (AECM53 Ficoll) as described (19, 20). Fluorescein1-polymerized flagellin was used at a final concentration of 50 ng/ml and fluorescein53-Ficoll was used at 0.1 ng/ml. BSF-1. Affinity-purified BSF-1, prepared as described (22), was the generous gift of J. Ohara and W. E. Paul. Unless stated otherwise, BSF-1 was used at 10 units/ml, with the BSF-1 activity being determined by the providers using the anti-IgM costimulation assay (22). IL-1. The IL-1 prepared as described (23) was kindly provided by Hoffmann-LaRoche (lot 11319-229-48). The IL-1 was used at 100 units/ml, with the IL-1 activity being determined by the providers using the thymocyte proliferation assay (24). IL-2. The IL-2 prepared as described (25) was kindly provided by Cetus Immune (Palo Alto, CA) (lot LP-222). The IL-2 was used at 100 units/ml, with the IL-2 activity being determined by the providers using a murine CTL-L line (25). EL4 Thymoma Cell-Derived B-Cell Growth and Differentiation Factors (BGDF) (EL-BGDF-pik). A 10x concentrate of medium conditioned by concanavalin A-stimulated EL4 thymoma cells prepared as described (13) at a final concentration of 5% (vol/vol) was used as a crude source of BGDF. This BGDF activity is termed EL-BGDF-pik (26). Abbreviations: BSF-1, B-cell stimulatory factor 1; AFC, antibodyforming cell; IL-1, recombinant murine interleukin 1; IL-2, recombinant human interleukin 2; BGDF, B-cell growth and differentiation factor(s); EL-BGDF-pik, medium conditioned by concanavalin Astimulated EL4 cells.
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Proc. Natl. Acad. Sci. USA 84
Immunology: Alderson et al.
B-Cell Cloning Systems. Fluorescein-specific B cells were cultured in 60-well Terasaki trays at an average of 0.4 to 12 cells per well in 10 ,il of RPMI 1640 medium supplemented with 5% (vol/vol) fetal calf serum and 100 ,uM 2-mercaptoethanol. The B cells were dispensed in 5 ,ul of medium, and then a further S 1.l of medium with or without antigen/factors at 2x the final concentration was added. For filler cellcontaining cultures, 500 3T3 cells were dispensed in 5 1.l of medium, and B cells in 5 /.l. The antigen/factors were added in a further 2 1.l at 6x the final optimal concentration. Trays were held at 370C in a humidified atmosphere of 10% C02/90% air. Assessment of Cell Enlargement at 24 hr. Approximately 2 fluorescein-specific B cells were dispensed per well as described above. The trays were centrifuged at 500 x g for 5 min to ensure that all cells were at the bottom of the well. A further 5 1.l of medium antigen/factors was then carefully added. After 2 hr at 370C, wells containing from one to four geographically separated cells were selected, with each cell being individually mapped in its well. The diameter of each individual cell was measured using an inverted phase contrast microscope at 10Ox magnification with a graduated scale in the eyepiece. After 24 hr, the diameter of each individual cell was remeasured. A cell was scored as having enlarged significantly if an increase in cell diameter of at least 0.9 gm was noted at 24 hr as described (17). From 30 to 40 individual B cells were measured per experimental group. Assessment of Clonal Proliferation. Before assay for antibody formation, culture wells were examined for the presence or absence of a proliferating B-cell clone using an inverted phase microscope as described (13-16). Assessment of Antibody Formation. Antibody formation was assessed using a sensitive enzyme-linked immunosorbent assay (ELISA) as described (14, 16, 27). Flexible U-bottomed polyvinyl 96-well ELISA trays (Dynatech, Alexandria, VA) were coated with an affinity-purified sheep anti-murine immunoglobulin antibody (SAM) (Silenus Laboratories, Dandenong, Australia). The supernatant fluid of each culture well was individually transferred into 50 ,ul of 0.3% skim milk powder and 0.05% Tween 20 in phosphatebuffered saline (pH 7.3) (PBS; 0.15M NaCl/0.02 M sodium phosphate). After 4 hr, the plates were washed and horseradish peroxidase-coupled SAM was added and left for a further 4 hr. After washing, the substrate 2,2'-azinobis(3ethylbenzthiazoline) sulfonic acid (Sigma) was added, and the absorbance of the wells was read 1 hr later using a Titertek Multiscan ML (Flow Laboratories) at dual wavelengths (414 nm and 492 nm). A well was considered positive if its absorbance exceeded the mean 3 SEM of the background as described (14, 16). The frequency of AFC clone precursors was determined by Poisson analysis as described (15, 16, 21). RESULTS ±
BSF-1 Acts with Antigen to Promote Proliferation but Not Differentiation of Single Hapten-Specific B Cells. The ability of
(1987)
B cells per well 8 10 6
36.7
20
10
FIG. 1. Limiting dilution analysis of proliferation and AFC clone development among fluorescein-specific B cells stimulated with either BSF-1 (10 units/ml) or EL-BGDF-pik (5%, vol/vol) in the presence of fluorescein-polymerized flagellin. The frequency values for proliferating clones were 10.9% ± 2.3% for fluorescein-polymerized flagellin and BSF-1 (&), and 13.0%6 ± 2.6% for fluoresceinpolymerized flagellin and EL-BGDF-pik (0). Values for AFC clones were 1.89% ± 0.73% for fluorescein-polymerized flagellin and BSF-1 (A&) and 5.46% ± 1.37% for fluorescein-polymerized flagellin and EL-BGDF-pik (e). Not shown are the values for fluoresceinpolymerized flagellin alone and BSF-1 alone, which for proliferation were 3.10% ± 0.97% and 4.04% ± 0.73%, respectively, and for AFC clone formation were 1.82% ± 0.71% and 1.19% ± 0.57%, respectively.
BSF-1 to promote clonal growth and/or differentiation amongst single B cells was assessed. Limiting numbers of
fluorescein-specific
B cells
were
cultured in the absence of
any other cell type with 10 units of BSF-1 per
ml, both in the
T-independent antigen fluorescein-polymerized flagellin. Clonal proliferation and AFC clone formation were assessed 5 days later. presence and absence of the
Table 1. Effect of BSF-1 on clone development amongst single fluorescein-specific B cells Without fluorescein-polymerized flagellin With fluorescein-polymerized flagellin % AFC % proliferating % AFC % proliferating P P P P clones clones clones clones Stimulus 0.5 ± 0.2 2.4 ± 0.6 Nil 1.0 ± 0.3 1.9 ± 0.4 7.2 ± 1.9 NS
