Proc. Nati. Acad. Sci. USA Vol. 82, pp. 8153-8157, December 1985 Immunology Interleukin 1 can act as a B-cell growth and differentiation factor (cytokines/hapten-gelatin fractionation/T-cel-independent antigens/antibody-forming cell dones/enzyme-linked immunosorbent assay) 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, July 23, 1985 ABSTRACT Splenic B lymphocytes specifically reactive to B cell, the bioactivities exhibited by IL-1 were identical to the hapten fluorescein (FLU) were prepared from nonimmune those in the filler cell-free system. adult mice by affinity fractionation on hapten-gelatin. These FLU-specific B cells were cultured as single cells or in small numbers in 10-ul wells either in the absence of any feeder, MATERIALS AND METHODS filler, or accessory cell or in the presence of 3T3 fibroblasts Mice and Preparation of Fluorescein (FLU)-Specific Splenic acting as filer cells. A selected batch ofa "T-cell-independent" B Cells. Specific-pathogen-free CBA/CaH/Wehi mice were antigen, FLU-Ficoll, which induces growth and differentiation used as spleen donors at 8-10 weeks of age. Hapten-specific only in the presence of lymphokines or cytokines acting as B-cell populations were prepared from spleen cell suspen- B-cell growth and differentiation factors (BGDF), was used as sions by fractionation on FLU-gelatin as described (15-17). the antigenic stimulus. It was found that murine interleukin 1 Adherent FLU-gelatin was removed from the recovered prepared by recombinant DNA technology was an effective, binding cells by collagenase. The binding population is 97% although weak, BGDF when acting with antigen on B cells B cells, -z70% FLU-binding, and -200-fold enriched for in cultured either under filler cell-free conditions or in the vitro reactivity to FLU conjugates (13, 16, 17). presence of3T3 cells. When the murine interleukin 1 was used Antigen. The hapten FLU was coupled onto aminoethyl- in combination with recombinant human interleukin 2, itself a carbonylmethylated Ficoll (AECM53-Ficoll), as described weak but effective BGDF in the system, an additive effect was (16, 17), and the FLU53-Ficoll was used as a final concen- observed. The results challenge the notion that interleukin 1 is tration of 0.1 ng/ml. exclusively or even primarily an activating cytokine. This EL4 Thymoma Cell-Derived BGDF. A lOx concentrate of system, in which pure factors are able to act with specific medium conditioned by concanavalin A-stimulated EL4 antigen on single hapten-specific B cells, will prove helpful for thymoma cells prepared as described (12) was used as a the further dissection of the respective roles of the various source ofT-cell-derived BGDF at a final concentration of5% factors that can act on B cells. (vol/vol). According to the recently proposed convention (18) this is termed EL-BGDF-pik. The important macrophage-derived growth-regulatory mol- Recombinant Murine IL-1 (r-mu-IL-1). The r-mu-IL-1 was ecule interleukin 1 (IL-1) (1) has recently been produced in prepared as described (2) and kindly provided by Hoff- pure form through recombinant DNA technology (2). Ever mann-La Roche (lot 11319-229-48). The concentration of since the description of IL-1 as a lymphocyte-activating r-mu-IL-1 is expressed as units/ml of IL-1 activity as deter- factor (3, 4), its bioactivity has been assumed to relate mined by the providers, using the thymocyte proliferation primarily if not exclusively to early events in the activation assay (19). of a resting, Go, cell. For example, IL-1 is seen as a cofactor Recombinant Human IL-2 (r-hu-IL-2). The r-hu-IL-2 was required for concanavalin A to exert its activating effects on prepared as described (20) and kindly provided by Cetus T lymphocytes (5) with concomitant expression of IL-2 Immune (Palo Alto, CA) (lot LP-222). Activity is expressed receptors, after which IL-2 is seen as the only growth factor as units/ml of r-hu-IL-2 as determined by using a murine required for further proliferation ofthe T-cell clone. IL-1 has CTL-L line as described (21). also been implicated in the activation of B lymphocytes B-Cell Cloning Systems. FLU-specific B cells were cultured (6-10), though in this instance, some work has suggested that in 60-well Terasaki trays at a mean of0.4-12 cells per well in it acts later than another postulated activating factor, B-cell- 10 ,ud of RPMI 1640 medium supplemented with 5% (vol/vol) stimulating factor 1 (BSF-1) (6). fetal calf serum and 100 AM 2-mercaptoethanol, either in the We have reported (11-14) a system in which normal murine absence of any added filler cells or in the presence of 300 B lymphocytes, preselected on the basis of their antigen BALB/c 3T3 fibroblast cells as described (13, 14, 17). For specificity, are able to be stimulated to proliferate and some purposes-e.g., the delineation of factor concentra- differentiate to immunoglobulin secretion status when cul- tion-response characteristics-an oligoclonal approach using tured singly in the presence of antigen and a source of B-cell a mean of 10 B cells per well was used as described (14). The growth and differentiation factors (BGDF). Under these additional presence of filler cells markedly improves both conditions, the B cell itself is the only possible target for the cloning efficiency and clone size (13, 14, 17). Routinely, 300 action of antigen and factors. In the belief that this system 3T3 cells were dispensed into the culture wells in 5 Al of was ideal for testing various purified factors for bioactivity on medium prior to the addition of the B cells to allow time for B cells, we have used it to assess the bioactivity of murine adherence. To avoid intertray variance, B cells are dispensed IL-1, prepared by recombinant DNA technology (r-mu-IL-1). into the wells of all trays in 5 1.d of medium containing twice The results show IL-1 to have a combination ofbioactivities, the required concentration of antigen. For filler cell-free including the promotion ofboth growth and differentiation of B cells. Moreover, when filler cells were added to the single Abbreviations: FLU, fluorescein; AFC, antibody-forming cell; BSF- 1, B-cell-stimulating factor 1; BGDF, B-cell growth and differenti- ation factor(s); BCGF, B-cell growth factor; IL-1 and IL-2, inter- The publication costs ofthis article were defrayed in part by page charge leukin 1 and 2; r-mu-IL-1, recombinant murine IL-1; r-hu-IL-2, payment. This article must therefore be hereby marked "advertisement" recombinant human IL-2; SAM, sheep anti-murine immunoglobulin in accordance with 18 U.S.C. §1734 solely to indicate this fact. antibody. 8153 Downloaded by guest on September 27, 2021 8154 Immunology: Pike and Nossal Proc. Natl. Acad Sci. USA 82 (1985) conditions, a further 5 ul of medium alone was added. Number of B cells per well Factors were added in a further 1 /.l of medium at lix the 0.4 0.6 0.8 1.0 1.2 1 required final concentration. Assessment of Clonal Proliferation. Before assay for anti- body formation, culture wells were examined, by using an inverted phase microscope as described (12, 13, 17), for the presence or absence of a proliferating B-cell clone. In the presence of 3T3 cells, the proliferating B cells could usually be distinguished, due to the adherence to plastic, the different morphology, and the substantially larger size ofthe 3T3 cells. Assessment of Antibody Formation. Antibody formation was assessed by using a sensitive enzyme-linked immuno- sorbent assay (ELISA) procedure as described (14). As the In target B cells were selected for specificity for FLU by a) prefractionation on hapten-gelatin, a sheep anti-murine 0) immunoglobulin antibody (SAM) was used as the polyvalent GJ- antigen in the ELISA assay rather than to FLU coupled cmC bovine serum albumin. This change was to measure antibody a) production per se, regardless of its affinity, as a result of particular stimuli. Briefly, the supernatant fluid of each culture well was individually transferred into the wells of a 96-well flexible U-bottomed polyvinyl plate (Dynatech, Alexandria, VA) precoated with affinity-purified SAM (Silenus Laboratories, Dandenong, Australia) at 3 ,ug/ml and containing 50 /.l of0.3% skim milk powder and 0.05% Tween 20 in phosphate-buffered saline. After a period of >2 hr at room temperature, the plates were washed in PBS-tween 20 and horseradish peroxidase-coupled SAM was added for a period of >4 hr. After washing, the substrate 2,2'-azinobis(3- ethylbenzthiazolinesulfonic acid) (ABTS) (Sigma) was added and the absorbance ofthe fluid in the wells was read 1 hr later with a Titertek Multiscan ML (Flow Laboratories) using dual wavelengths (414 and 492 nm). A well was considered positive if its absorbance exceeded the mean ± 3 SEM of the FIG. 1. Limiting dilution analysis ofthe response ofFLU-specific background as calculated on the basis of a large number of B cells as assessed by AFC clone formation by ELISA. (A) Response replicates of supernatants from wells lacking B-cell input. to FLU-Ficoll and EL-BGDF-pik (x-x) (frequency value, 46.3% ± Control wells lacking antibody consistently gave an absorb- 12.5%). (B) Response to FLU-Ficoll alone (x-x) (frequency value, 2.76% ± 0.6%) or FLU-Ficoll plus r-mu-IL-1 at 100 units/ml (a-0) ance value of <0.010 unit.