interleukin 5 induces not only B-cell growth and differentiation, but also increased expression of interleukin 2 receptor on activated B-cells

Immunology Letters, 15 (1987)205-215

Elsevier IML 00899

T cell replacing factor/interleukin 5 induces not only B-cell growth and differentiation, but also increased expression of interleukin 2 receptor on activated B-cells N o b u y u k i H a r a d a ~, M i t s u h i r o M a t s u m o t o 1, N o b u o K o y a m a ~, A k i r a Shimizu 2, T a s u k u H o n j o 2, A k i r a T o m i n a g a ~ a n d Kiyoshi Takatsu 1 IDepartment of Biology, Institute for Medical Immunology, Kumamoto University Medical School, Honjo, Kumamoto, Japan; ZDepartment of Medical Chemistry, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan

(Received 26 March 1987) (Revision received9 April 1987) (Accepted 9 April 1987)

1. Summary A T-cell replacing factor (TRF)/interleukin-5 (11.,-5) is a B-cell growth and differentiation factor. In the present study, we examined the role of TRF/IL-5 in the increase in the levels of interleukin-2 (IL-2) receptor expression on activated B-cells. High pressure liquid chromatography (HPLC)-purified TRF/IL-5 (B151-TRF) from TRFproducing T-cell hybridoma, B151K12, as well as recombinant TRF/IL-5 (rec-TRF) were used for the analysis. Maximum anti-2,4-dinitrophenyl (DNP) IgG antibody response of DNP-primed B-cells or polyclonal IgM secretion o f B-cell tumor line BCL 1 was seen when HPLC-purified B151-TRF was added or when suboptimal doses of B151-TRF were added to the culture in the presence of 11.1-2. Normal resting B-cells gave maximum anti-SRBC IgM PFC responses when HPLC-purified B151-TRF and IL-2 were present. The purified B151-TRF as well as rec-TRF also induced on B-cells increased expression of IL-2 receptors that react with monoclonal anti-murine IL-2 receptor antibody, PC61, and 125I-labelled IL-2. The numbers Key words: B-cellgrowth and differentiationfactor; TRF/IL-5;

Activated B-cell; IL-2 receptor expression Correspondence to: Kiyoshi Takatsu, Department of Biology,

Institute for Medical Immunology,KumamotoUniversityMedical School, 2-2-1, Honjo, 860, Kumamoto, Japan.

of functional high affinity IL-2 receptors on activated B cells increased at least 20-fold by culturing them with purified B151-TRE Moreover, B151-TRF induced increase in the levels o f steady-state mRNA for IL-2 receptor by approximately 8-fold. These results suggest that activated B-cells as well as BCLl-cells may express functional IL-2 receptors or closely related molecules when stimulated with HPLC-purified B151-TRF as well as rec-TRF.

2. Introduction There is a body of evidence for showing that Tcell-dependent B-cell activation involves cellular interactions between helper T-cells, accessory cells and B cells [1]. Helper T-cells recognize antigens in the context of class II major histocompatibility complex (MHC) molecules on accessory cells or on resting B-cells and render B-cells susceptible to the subsequent action of B-cell stimulatory factors (BSF) or T-cell-replacing factor (TRF) which can induce B cell growth and differentiation [2-5]. However, precise role of each of these lymphokines in the various stages of the activation pathway is still uncertain, due in part to the use of crude or partially purified material in which the response could be influenced by contaminating lymphokines. Early studies with recombinant materials which are becoming increasingly available have

0165-2478 / 87 / $ 3.50 © 1987 ElsevierScience Publishers B.V. (BiomedicalDivision)

205

confirmed that one molecule may have multiple biological activities. For example, the molecular cloning of interleukin-4 (IL-4) confirmed that the activities described as BSF-1 and IgG1 induction factor (or B cell differentiation factor 3') could be attributed to the same molecule [6-9]. We described another B-cell active factor, TRF, originally derived from the B151K12 (B151) T-cell hybridoma. TRF is defined by two activities: induction of IgM secretion by the BCL l B-cell line and anti-IgM- and BSF-l-stimulated B-cells; and induction of hapten-specific IgG secretion in vitro by in vivo antigen-primed B-cells [10-14]. Hamaoka et al. reported that B151 produces two different kinds of TRFs, namely B151-TRF1 and B151-TRF2 [15]. B151-TRF described in this study is considered to be identical to B151-TRF1. More recently purified TRF from B151 has been shown to have B-cell growth factor II (BCGF II) activity [16]. BCGF II activity was first described by Swain and Dutton as the ability to induce proliferation in BCLl-cells [17]. Subsequent studies by them revealed that BCGF II molecule is also capable of promoting differentiation of normal B-cells and BCLl-cells to Igsecreting cells [18]. Recently it was shown that eosinophil differentiation factor (EDF), which also has BCGF II activity [191, induces both growth and differentiation of pre-activated normal mouse B-cells [20]. We have recently confirmed using recombinant TRF derived from complementary DNA encoding TRF that a single molecule is responsible for both TRF and BCGF II activity [21], and most likely EDF activity. Therefore, we proposed that TRF/BCGF II will be called interleukin-5 (IL-5) [21, 22]. The role of interleukin-2 (IL-2) in the B-cell differentiation has aroused the most controversy [23-30]. In our previous work, we found that IL-2 acted on antigen-primed B-cells synergistically with suboptimal doses of TRF-containing cell free supernatant of B151K12 to cause them to differentiate into Ig-secreting cells [31, 32], although IL-2 itself did not induce the same B-cell population for differentiation. In this study, we report on the stimulating activity of HPLC-purified B151-TRF and recombinant murine TRF (rec-TRF) in various stages of the Bcell activation. We demonstrate that HPLCpurified TRF as well as rec-TRF can synergize with 206

IL-2 to augment differentiation of pre-activated Bceils into Ig-secreting cells. Evidence to support the notion that HPLC-purified B151-TRF as well as recTRF can induce augmented expression of IL-2 receptors on activated B-cells will be provided.

3. Materials and Methods

3.1. Animals BALB/cCr nu/nu and BALB/cCr normal mice were obtained from the Shizuoka Laboratory Animal Center, Hamamatsu City, Japan. 3.2. Monoclonal antibody Monoclonal antibody (PC61) directed against mouse IL-2 receptor (IL-2R) was prepared according to the described method [30], and was a generous gift from Dr. Marcus Nabholz (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland). The cultured fluid of PC61 cells was subjected to a protein A-coupled Sepharose CL-4B beads column, and the eluate from the column with 3M potassium thiocyanate (pH 8.0) was used as the purified PC61 antibody. The purified PC61 antibody (50 ng/ml) can inhibit the IL-2-dependent proliferation of cloned T-cells (MTH) by about 50°70 when 5 × 103 MTH-cells were cultured for 24 h in 0.75 units/ml IL-2. Monoclonal anti-Thy 1.2 antibody, F7D6 was purchased from Olac. Ltd., (Serotec, Oxon, UK). 3.3. Preparation of HPLC-purified B151-TRF and recombinant TRF Cells of B151K12 T-cell hybridoma which produces TRF were cultured at 1× 105/ml for 48 h. B151-TRF was purified from a large batch of B151K12 supernatant by the procedures previously described [10, 13, 16]. B151KI2 supernatants were fractionated by ammonium sulfate precipitation in 50-80°7o saturation. The precipitate was dialyzed against 10 mM Tris-HC1 buffer (pH 8.5) and were applied on a DE52 (Whatman, Clifton, N J) column using a linear gradient of NaC1 (0- 0.25 M) in 10 mM Tris-buffer (pH 8.5). Eluate containing TRF activity from the DE52 column was dialyzed against 0.025 M bis-Tris-HC1 buffer (pH 6.3) and applied on a Mono P column (Pharmacia, Uppsala, Sweden). The samples were eluted with 10o70

Polybuffer at a pH range between 6.3 and 4.0. TRF active materials from the Mono P column were applied on a Superose 12 column (Pharmacia) for gel permeation, and were eluted with 10 mM H E P E S buffered saline (pH 7.0). Finally, TRF active fractions after the gel permeation were applied on a Protein C 4 column (Vydac, Hesperia, CA) for reverse H P L C and were eluted with a linear gradient of acetonitrile ( 0 - 80°70) containing 0.1°70 trifluoroacetic acid. Specific activity of the samples finally obtained were increased by an approximately 1,400,000-fold from the original supernatant, and contained no detectable BSF-1, I ~ 2 , and IFN3' activity. Recombinant murine TRF (rec-TRF) was prepared as previously described [21]. In brief, the cDNA for murine TRF (pSP6K-mTRF23) was cleaved with Sail to linearize plasmid DNA, and mRNAs were synthesized using SP6 RNA polymerase. The synthesized RNAs were injected into Xenopus oocytes. Incubation media were collected after 36 h. 3.4. IL-2 Purified recombinant human IL-2 (rec-IL-2) prepared from Escherichia coil that was induced with the hybrid plasmid containing human IL-2 cDNA cloned from Jurkat [33], was kindly provided by Dr. Junji Hamuro, Central Laboratories of Ajinomoto Chemical Co. Ltd., Yokohama, Japan. The absence of lipopolysaccharide (LPS) was ascertained by B-cell mitogenicity and the Limulus test. The specific activity of IL-2 was 5 × 10 7 U/mg protein in our assay. A unit of IL-2 was defined by IL-2 dependent proliferation of murine alloreactive cloned T-cells M T H as the reciprocal of the dilution yielding a cpm response that is 33°70 of the maximal response [13]. 3.5. IFN, y Murine IFN3, generated by recombinant DNA technology (rec-IFNy) was kindly provided by Dr. Kohsaburo Sato (Shionogi Central Laboratories, Osaka, Japan). IFN3, activity was assayed on mouse L929-cells by the 50°70 plaque reduction method as described [13]. 3.6. BSF-1 Incubation media of Xenopus oocytes which

were injected with mRNAs of pSP6-IL4-374 synthesized with SP6 RNA polymerase [7] was used as a source of recombinant BSF-1 (rec-BSF-1). A unit of BSF-1 was defined by BSF-1 assay using antiIgM antibody [11] as the reciprocal of the dilution yielding a cpm response that is 50% of the maximal response. 3.7. Immunization of mice BALB/c mice were immunized i.p. 6 - 8 wk before use with 100 /zg of DNP-KLH included in aluminium hydroxide gel along with pertussis vaccine as previously described [34]. 3.8. BCL l-cells The BCL 1 tumor, originally donated by Dr. Ellen S. Vitetta (University of Texas Health Science Center at Dallas, TX), was maintained by in vivo passage in BALB/c mice. BCLl-cells were prepared from mice which had carried the BCL 1 tumor more than 4 wk, according to the method described [15]. 3.9. Preparation of purified B-cells Spleen cells (1 × 107/ml) from either DNP-KLHprimed or BCLl-bearing BALB/c mice which had received i.p. injection of 0.2 ml of rabbit antithymocyte serum two days before sacrifice were mixed with anti-Thy 1.2 antibody (1:2,000) 1:1 on volume basis and were incubated for 20 min at 4 °C and then at 37 °C for another 20 min. Excess antibody was removed by centrifugation of the ceils and 25 ~1 of well-selected rabbit complement to 1×107 cells in 1 ml tissue culture medium. Complement-dependent lysis of T-cells was allowed to proceed at 37 °C for 40 min. After washing the remaining cells the procedure was repeated once. The recovered cell populations were analyzed for surface IgM positivity by staining with fluorescence labeled goat anti-mouse IgM (Cedarlane, New York, NY) and fluorescence activated cell sorter analysis using EPICS V. Following the above fractionated process, cells were found to be 87°7o surface IgM-positive and less than 3°70 Thy 1.2-positive. The cells thus obtained responded in vitro to lipopolysaccharide (5 /~g/ml) but not to concanavalin A (5 /zg/ml) for [3H]thymidine uptake. After the culture the cells contained less than 1070 Thy 1.2 positive cells. 207

3.10. Cell culture Purified splenic B-cells or BCLl-cells, prepared as described, were cultured in serum-free RITC medium [35] (a kind gift from Dr. J. Hamuro) supplemented with 0.5% bovine serum albumin (BSA), 50/~M 2-mercaptoethanol and antibiotics. In some experiments, spleen cells from BALB/c nu/nu mice were activated in mass cultures by 50 /~g/ml LPS at 5 × 105 cells/ml for 2 days. Activated B-cell blasts or non-replicating, blast-like B-cells were separated by velocity sedimentation [36], thereafter washed extensively to free them of BSA used in the separation procedure, and were used as LPS-blasts. 3.11. Assessment of TRF activity Two assay systems for TRF activity were employed according to the described methods [10, 13, 16]. First, purified B-cells (5 × 105/0.2 ml culture) from DNP-KLH-primed mice were cultured with TRF-containing samples or other lymphokines, and stimulated with DNP-OA (12 ng/culture). After a 5-day culture period, the numbers of antiDNP IgG PFC were determined. Second, BCLrcells (1.5×105/0.2 ml culture) or LPSactivated B blasts (1 × 104/0.2 ml culture) were cultured with lymphokines for 2 days. After the culture the numbers o f IgM-secreting cells were enumerated as described [13]. A unit of TRF activity was defined by BCL1 assay as the reciprocal dilution yielding a 50% PFC response which is induced by a standard TRF preparation. 3.12. Assessment of anti-SRBC antibody response Purified splenic B-cells from 7 to 10-wk-old BALB/c mice were cultured at 1 × 106 ceils with final 0.05% SRBC and with or without lymphokines in 0.2 ml/well in 96 well flat-bottom tissue culture plate (Corning 25860, Corning Glass Works, Corning, NY). Culture medium consisted o f RPMI 1640 medium supplemented with 15% FCS, glutamine (2 raM), sodium pyruvate (1 mM), nonessential amino acid (Flow Laboratories), 50 /~M 2-mercaptoethanol and antibiotics. The cells were incubated for 5 days, and were assayed for direct PFC against SRBC by plaque assay.

208

3.13. Fluorescence-activated cell sorter (FACS)

analysis of IL-2 receptor binding The expression of IL-2 receptor on the DNPprimed B-cells and MTH-cells was determined as previously described [37], using EPICS V (Coulter Electronics, Hialeah, FL) equipped with an argon ion laser (164-05, Spectro Physics, Mountain View, CA) operating at 400 mW of power at 488 nm. Cells were stained with biotinylated anti-IL-2 receptor antibody (PC61) and fluorescein isothiocyanate (FITC)-coupled avidin. Controls included monoclonal biotinylated anti-Thy 1.2 and FITCavidin or FITC-avidin alone. 3.14. Binding assay of radiolabeled IL-2 The IL-2 binding assay was performed by the methods described by Lowenthal et al. [30] and Smith [38]. Briefly, to determine the level of binding, serial dilutions of 125I-labeled IL-2 were incubated with 106 cells in a total volume of 200 tzl of RPMI 1640 supplemented with 1% BSA in 1.5-ml Eppendorf micro-test tubes (Brinkman Instruments, Westbury, NY). After incubation for 20 min at 37 °C, 1 ml of ice-cold RPMI-BSA was added to each tube, and then the ceils were spun down at 9000 g for 10 s in an Eppendorf model 5414 centrifuge. The supernatant was removed and counted to determine the unbound radioactivity. The cell pellet was resuspended in 100 tzl RPMI 1640-BSA at 4 °C, and the cell suspension was layered on a mixture of 84% silicone oil (SH-550; Nakarai Chemicals, Ltd., Kyoto) and 16% paraffin oil (Nakarai Chemicals, Ltd.) in a soft 400-/zl polyethylene tube (Bio-Rad Laboratories). After centrifugation at 9000 g for 90 s, the tip containing the cell pellet was cut off and placed in tubes, and counted. The level of nonsaturable binding was determined in the presence of 100-fold molar excess of unlabelled IL-2, and specific binding was calculated by subtracting the amount of the nonsaturable binding. 3.15. Northern blot analysis Total RNA was prepared according to the described methods using guanidium thiocyanate [39]. The methods used for Northern blotting are essentially the same as those described by Thomas [40]. In brief, twenty micrograms of RNA isolated from cells stimulated with or without B151-TRF were glyoxylated, electrophoresed in a 1.1% agarose

and transferred to a nitrocellulose filter. The filter was hybridized with a 32p-labelled nick translated PstI-PstI (414 bp) fragment of pmlL-2R-1 as probe [41, 42].

4. Results

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A 1G

10

% o

m o.

_m

4.1. Role of HPLC-purified B151-TRF in the

differentiation of BCL r or preactivated Bcells into antibody-producing cells As we reported previously [11, 13], the B151K12 supernatants can trigger DNP-primed B-cells in the presence of DNP-coupled antigen into anti-DNP IgG PFC, and also induce differentiation of BCLl-cells into IgM-secreting cells in the absence of mitogenic or antigenic stimulation [13, 16]. It is conceivable that BCLl-cells are already activated out of a resting state and do not require antigen. It was also reported that both IL-2 and interferon--/ (IFN-/) may induce growth and differentiation of activated B-cells [ 2 3 - 30, 4 3 - 4 6 ] in different assay systems from ours. To compare B-cell differentiation-inducing activities of B151-TRF with other lymphokines in the different assay systems in activated B-cell populations, either purified B-cells from DNP-primed BALB/c mice, BCL 1 cells, or B-cell blasts induced by LPS were cultured with various amounts of HPLC-purified B151-TRF, recombinant h u m a n IL-2 (rec-IL-2), recombinant murine BSF-1 (recBSF-1), or recombinant murine IFN-/ (rec-IFN-/). The results of a representative one of three different experiments are presented in Fig. 1. It is evident that addition of HPLC-purified B151-TRF (50 U/ml) can induce m a x i m u m antiD N P IgG PFC responses in DNP-primed B-cells, and IgM PFC responses in BCLl-cells or LPSinduced B cell blasts, respectively. Addition of recIL-2 up to 50 U / m l was not effective under the same conditions. Rec-IFN-/ also had no effect on plaque formation of each cell population. The recIFN-/ was tested repeatedly in doses from 400 U/culture down to 1 U/culture on BCLl-cells. In no case has it any significant PFC formation over background.

go

25

50

( t.l/ml )

Fig. 1. Differentiationinducing activity of lymphokineson activated B-cells. DNP-primed B-cells (5×105/0.2 ml/well) (A), BCLvcells (1.5× 105/0.2 ml/well) (B), or LPS-stimulated B-cell blasts (1x 104/0.2 ml/well) (C) were cultured in the presence of various lymphokines. DNP-primed B-cells were stimulated with DNP-OA (12 ng/well) in the presence of lymphokines. Various concentrations (units/ml) of HPLC-purified B151-TRF( o ), human rec-IL-2 (•), murine rec-BSF-1 (A), or murine rec-IFN-r ( [] ) were added to the culture on day 0. After the culture the numbers of antibody secreting cells were enumerated according to the methods described in Materials and Methods. The results were expressedas geometric means of triplicate culture and standard errors.

4.2. Synergy of IL-2 with HPLC-purified TRF in

differentiation of activated B-cells To evaluate the role o f IL-2 in the B-cell differentiation, we examined synergistic effect of rec-IL-2 with HPLC-purified B151-TRE Synergistic effect of rec-IFN-/ and rec-BSF-1 with B151-TRF was also tested. One of the representative results of a series of four different experiments is presented in Table 1. Table 1 is illustrative of many experiments in which we have mixed lymphokines to examine synergistic effects on differentiation of DNP-primed B-ceils, BCLl-cells or LPS-induced B-cell blasts. As shown in Table 1, rec-IL-2, and rec-BSF-1 again had no effect on their own (Groups 4 and 5) and did not synergize each other (Group 7). Only HPLC-purified B151-TRF exhibited remarkable activities at all times in all experimental systems (Groups 2 and 3). Rec-IL-2 could synergize with limiting doses of B151-TRF (10 U/ml) (Group 6), but higher doses of the T R F (50 U/ml) alone led to an equivalent response (Group 3 vs Group 6). Addition of rec-BSF-1 in conjunction with rec-IL-2 plus limiting doses of B151-TRF did not alter the 209

Table 1 Synergistic effect of IL-2 with suboptimal doses of TRF/IL-5 on the differentiation of activated B-cells into Ig-secreting cell. Group No.

Lymphokines I HPLC-TRF

B-cell source 2 IL-2

BSF-1

1 2 3 4 5 6 7 8

DNP-primed B-cells Anti-DNP IgG P F C

42 (1.09) 10 50 50 10 10

20 50 50 50

20 20

512 1373 53 43 1353 57 1291

(1.03) (1.21) (1.02) (1.04) (1.21) (1.25) (1.07)

BCL 1 IgM PFC

50 (1.03) 1150 2870 70 65 2353 73 2681

(1.20) (1.17) (1.25) (1.30) (1.09) (1.08) (1.21)

LPS-blasts IgM PFC

20 (1.02) 246 591 33 53 580 28 603

(1.17) (1.04) (1.23) (1.26) (1.07) (1.06) (1.13)

i Units/ml. 2 Responding cells were cultured with various lymphokines in the presence or absence of other lymphokines indicated according to the procedures described in Materials and Methods. After the culture, numbers of P F C were enumerated according to the methods described in Fig. 1. Results were expressed as geometric means of triplicate culture and their standard errors.

responses induced by rec-IL-2 plus B151-TRF (Group 8 vs Group 6). 4.3. Synergy of rec-IL-2 with HPLC-purified B151-TRF in primary anti-SRBC antibody responses As originally demonstrated by Dutton et al. [47], Schimpl and Wecker [48] followed by many investigators including us [10], TRF induces SRBC specific primary IgM PFC responses when it is added on day 2 to the 5-day culture of SRBC-stimulated resting B-cells. It is also demonstrated by Leibson et al. [43] and Sidman et al. [44] that IL-2 and IFNy in conjunction with other B-cell stimulatory factors are essentially required to induce primary antiSRBC IgM PFC responses. To evaluate whether we could reconcile the observations in these different experimental models, we tested the same lymphokine preparations used in the previous Table 1 for their activities in the responses to SRBC. To obtain reproducible results the cultures are set up with the use of purified B-cells at a higher cell density (1 x 106/well/0.2 ml). A representative result of three different series of experiments is displayed in Table 2. HPLC-purified B151-TRF induced a significant anti-SRBC IgM PFC response, although more than 50 U/ml of B151TRF was required and the responses induced by 100 U/ml of B151-TRF were not maximum (Group 1 vs Groups 2 and 3). Similar results were obtained using 210

rec-TRF in place of HPLC-purified B151-TRF (Groups 4 and 5). Interestingly, rec-IL-2 (50 U/ml) synergized with limiting doses of HPLC-purified B151-TRF as well as rec-TRF (50 U/ml) to induce remarkable anti-SRBC IgM PFC responses (Groups 8 and 9), although rec-IL-2 itself induced a substantial response (Group 6). Addition of recIL-2 alone beyond 150 U/ml gave a high number of anti-SRBC IgM PFC response (data not shown). Recombinant IFNy was not active on its own, and did not synergize with rec-IL-2 (Groups 7 and 10). 4.4. Effects of monoclonal anti-IL-2 receptor antibody on synergy between B151-TRF and IL-2 on pre-activated B-cells As shown in Table 1, IL-2 can augment responses of activated B-cells in the presence of suboptimal doses of HPLC-purified B151-TRF, suggesting that IL-2 may act on B-cells through the same receptor for B151-TRF or that B151-TRF may induce and/or augment expression of functional IL-2-receptor on activated B-cells. To evaluate either possibility, we tested the blocking effect of monoclonal anti-IL-2 receptor antibody on antibody forming cell responses of DNP-primed B-cells or BCLl-cells. As it can be seen in Fig. 2A, a significant antiDNP IgG PFC response was again observed in DNP-primed B-cells by rec-TRF, and addition of monoclonal anti-IL-2 receptor antibody PC61 (final 2/zg/ml) to the culture at the commencement

Table 2 Synergistic effect of IL-2 with TRF/IL-5 on the generation of primary anti-SRBC IgM PFC responses. Group No.

Anti-SRBC IgM PFC/culture

Lymphokines1 HPLC-TRF

Rec-TRF

IL-2

1

2 3 4 5 6 7 8 9 10

50 100

50 100 50

50 -

IFN3"

50 50 50

-

Expt. 2

-

5 (1.26)

-

21 (1.40) 48 (1.21) 30 (1.05) 55 (1.18) 18 (1.26) 6 (1.02) 138 (1.26) 198 (1.12)

29 (1.09) 51 (1.32) 24 (1.12) 62 (1.07) 28 (1.08) 4 (1.01) 219 (1.02) 238 (1.13)

9 (1.08)

11 ( 1 . 2 6 )

50

Expt. 1

150 150

8 (1.21)

1 Units/ml. 2 Purified resting B-cells (1 x 106/well) were stimulated with 0.05% SRBC for 5 days in the presence or absence of various lymphokines. After the culture, anti-SRBC IgM PFC assay was conducted. Results were expressed as the geometric means of triplicate culture and their standard errors.

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A

~

is

O ,r-

@12

identical results were o b t a i n e d using H P L C purified B151-TRF. These results suggest that the culture o f pre-activated B-cells with B151-TRF m a y induce increased expression o f f u n c t i o n a l IL-2 receptors o n B-cell surface.

a

~ 10 0 M. a- 6 n. Z 0 -.I 3

0

4.5. lncrease in the level of lL-2 receptor expression

on activated B-cells by B151-TRF 6

12 18 24. IL-2 ( U/ml )

.-

(5

e.

=

12 18 IL-2 ( U / m l )

-7

24

Fig. 2. Effect of anti-IL-2 receptor antibody on PFC responses induced by TRWIL-5 plus IL-2. Either DNP-primed B-cells (5×105/0.2 ml/well) (A) or BCLl-cells (1.5x105/0.2 ml/well) (B) were cultured with either HPLC-TRF (o, • ) or rec-TRF (zx, • ) (1 U/well) in the presence of various concentrations (units/ml) IL-2. In some cultures, the purified PC61 anti-IL-2 receptor antibodies (final 2 #g/ml) was added on day 0 ( • , • ). (©) and ( • ) denote background PFC in the absence of recTRF and PFC responses induced by IL-2 alone, respectively.

did n o t affect at all o n a n t i - D N P IgG P F C responses. I n contrast, the same a n t i b o d y strikingly i n h i b i t e d that a n t i - D N P P F C responses i n d u c e d by rec-IL-2 plus s u b o p t i m a l doses o f rec-TRF to the level o f those i n d u c e d by rec-TRF alone. Similar results were o b t a i n e d with the use of B C L 1 cells in place o f D N P - p r i m e d B-cells as r e s p o n d i n g cells (Fig. 2B). I n b o t h experimental systems, essentially

We examined expression o f IL-2 receptors o n activated B-ceils in two ways. First, we tested the capacity o f b i n d i n g o f m o n o c l o n a l anti-IL-2 receptor a n t i b o d y a n d 125I-labeled 11_,-2 to D N P - p r i m e d Bceils, respectively. Secondly, we examined m R N A expression for IL-2 receptors in D N P - p r i m e d Bcells. D N P - p r i m e d B-cells were cultured with or without H P L C - p u r i f i e d B151-TRF for 3 days. Cells harvested at that time were more t h a n 87% surface Igpositive a n d were less t h a n 1070 Thy 1-positive cells (Fig. 3A). T h e ceils were stained with b i o t i n y l a t e d anti-IL-2 receptor a n t i b o d y (PC61) in c o n j u n c t i o n with F I T C - a v i d i n . As a positive control for IL-2 receptor expression, cloned alloreactive T-cells ( M T H ) were stained. The cells stained were analyzed with a fluorescence activated cell sorter. As expected, the m a j o r i t y (88070) o f M T H - c e l l s were IL-2 receptor-positive (data n o t shown). As can be seen in Fig. 3C, D N P - p r i m e d B-cells cultured with H P L C - p u r i f i e d B151-TRF for 72 h were also IL-2 211

A

B

4

t

A

No

B

u~ ;)

Relative fluorescence

intensity

Fig. 3. Effect of TRF/IL-5 on the expression of IL-2 receptors on B-cells. DNP-primed B-cells (5 x 105/0.2 ml) were cultured in 96-well microplate in the presence (C) or in the absence of 15 U/ml of HPLC-TRF (B) for 72 h. After the culture cells were collected and washed with PBS. The cells thus obtained (1 × 106) were incubated with 2 ~1 of biotinylated rat monoclonal antiIL-2 receptor antibody (PC61, 2.1 mg/ml) for 20 rain at 4°C ( ). After the washing they were stained with 5/~1 of FITClabeled avidin (1 mg/ml) for another 20 rain at 4 °C. The portion of DNP-primed B-cells which had been cultured with HPLC-TRF were incubated with biotinylated anti-Thy 1.2 antibody in place of anti-IL-2 receptor antibody (A). After the extensive washing the positively stained cells were analyzed with FACS using EPICS V operating at 400 mW of power at 488 nm. Fluorescence histograms were obtained 10,000 viable cells. (----) denotes the pattern observed by staining the cells with FITCavidin alone.

receptor-positive (24%), whereas the cells cultured with medium alone were less than 4% IL-2 receptor-positive (Fig. 3B). To determine the number and affinity of IL-2 receptors on DNP-primed B-cells cultured with B151-TRF, a binding assay using 125I~IL-2 was conducted, and compared with those expressed on cloned T-cells (MTH). It was demonstrated that the average number of high- and low-affinity of IL-2 binding sites per B151-TRF-activated DNP-primed B-cell was 400 and 6500, respectively (Fig. 4A), and was less that that of M T H (6400 for highaffinity and 124,000 for low-affinity, respectively) (Fig. 4B). DNP-primed B-cells cultured with medium alone expressed less than 20 low-affinity IL-2 receptors, if any (data not shown), It was also demonstrated by Scatchard plot analysis that DNPprimed B-cells cultured with B151-TRF expressed two kinds of receptors, high- and low-affinity. Dissociation constants obtained were 57.1 pM for high-affinity and 8.13 nM for low-affinity, and that for M T H were 33.3 pM for high-affinity and 12.5 nM for low-affinity. The expression of mRNA for 1L-2 receptor was 212

m O0

25

5

5

Bound IL-2(rnoiecuies/cell,

xlo -3 )

10 -4 )

Bound IL-2(molecules/cell, xlO

Fig. 4. Scatchard plot analysis of equilibrium binding analysis of 1251-IL-2 to DNP-primed B-cells (A) or MTH-cells (B). DNPprimed B-cells cultured with rec-TRF (15 U/ml) for 3 days in the presence of DNP-OA (12 ng/well) or MTH ceils were incubated at 37 °C in the presence of various concentrations of I251-IL-2 (20 p M - 2 0 nM) for 1 h. Specific equilibrium binding was determined after subtraction of nonspecific binding, and the data were re-expressed as a Scatchard plot.

examined by Northern blot analysis with the use of cDNA (pmIL-2R-1) for IL-2 receptor. As can be seen in Fig. 5, DNP-primed B-cells cultured with HPLC-purified B151-TRF showed increased expression of 3.5 Kb, 2.2 Kb and 1.5 Kb mRNA for IL-2

A

B TRF

(-)'

3 . S K b -~ 2 . 2 K b -> 1.SKb

TRF

(+)

(-)

(+)

e O

Fig. 5. Northern blot analysis (A) and dot-blot hybridization analysis (B), of RNA from DNP-primed B-cells. Total RNA (20 ~tg) of DNP-primed B-cells that had been cultured for 4 days in the presence of DNP-OA with (+) or without ( - ) rec-TRF (15 U/ml) were obtained. (A) RNA were glyoxylated, electrophoresed in a 1.1% agarose and transferred to a nitrocellulose filter. (B) RNA were serially diluted with 20xSSC and dot hybridization assay was performed. In the case, the filter was hybridized with a 32p-labeled nick-translated PstI-PstI (414 bp) fragment of pmIL-2R-1 as probe. Autoradiographs were made by exposing X-ray film to the filter. Exposures were 16 h in duration.

receptor (panel A). Essentially similar results were obtained using BCL1 cells (data not shown). To confirm above results, dot-blot analysis was also performed. As shown in Fig. 5B, DNP-primed Bcells stimulated with HPLC-purified B151-TRF induced at least 8-fold increase in the level of IL-2 receptor mRNA expression. 5. Discussion The experiments presented in this study explore the effects of B151-TRF and other factors on the development of Ig-secreting cells and the induction of increase in the level of IL-2 receptor expression in activated B-cells. The major findings in this study can be summarized as follows. In all experimental systems employed, HPLC-purified B151-TRF as well as rec-TRF (devoid of IL-1, IL-2, IL-3, IFN'r, and BSF-1 activities) can induce differentiation of activated B-cells into Ig-secreting cells. This effect is seen in the anti-DNP IgG PFC responses of DNP-primed B-cells and anti-SRBC primary IgM PFC responses, and in the polyclonal responses of BCL 1 cells and LPS-induced B-cell blasts. Rec-IL-2 itself was inactive in the BCL 1 as well as DNP-primed B-cell assay under the conditions employed (Fig. 1 and Table 1). However, a moderate concentration of rec-IL-2 ( 5 - 2 0 0 U/ml) enhanced the development of Ig-secreting cells from DNPprimed B-cells or BCLl-cells in the presence of suboptimal doses of B151-TRF (Table 1 and Fig. 2). In the case of primary SRBC-specific response of normal resting B-cells, the addition of B151-TRF and IL-2 was obligatory to induce maximum responses (Table 2). These data are essentially compatible with the data published by Swain et al. [25]. Rec-IFN3, alone was inactive in assays employed here and gave no variable and marginal synergy with any other factors. Rec-BSF-1 showed no effects on its own, nor did it synergize with IFNT, B151-TRF or IL-2 in any of the assay systems. A number of reports have concerned IL-2 as a necessary component of antigen-specific primary PFC responses of non-primed B-cells [23-25]. In contrast, other reports including ours with the use of neoplastic B-cells showed no effect of IL-2 itself on PFC formation (Fig. 1). In this paper, we describe these apparently contradictory results by showing that different kinds of PFC responses

show a differential dependence on IL-2. The SRBC-driven response of normal B-cells required II~2 and was optimal only when IL-2 plus TRF were added (Table 2). It is too early to fully understand these differences in B-cell responses to IL-2, but three possibilities can be suggested. First, IL-2 can work indirectly in systems with contaminating cells of the T-cell lineages. To exclude this possibility, we used highly purified B-cells. The contamination of Thy 1.2-positive cells in the B-cell population used in this study was less than 3% and 1% before and after the culture, respectively. Addition of relatively high concentrations of rec-IL-2 (25 - 100 U/ml) was ineffective in this study. It is conceivable, therefore, that this possibility is unlikely. Secondly, different subsets of B-cells, perhaps reflecting different states of differentiation, have distinct lymphokine requirements. Differences between polyclonal and antigen-specific B-cell responses can perhaps be understood if the B-cells engaged in polyclonal maturation are distinct from those B-cells that require specific antigen to respond. Thirdly, B151-TRF or some antigen like SRBC may induce in vitro increase in the levels of IL-2 receptor expression on activated B-cells as well as BCL 1. Although other possibilities are not fully excluded at this moment, we are in favour of the last possibility based on the following reasons. First, anti-II_~2 receptor antibody suppressed the enhanced antibody-forming cell response by IL-2, while the same antibody showed little suppressive effect on antibody formation induced by B151-TRF alone (Fig. 2). Secondly, surface expression of IL-2 receptor was enhanced by stimulation of activated B-cells with B151-TRE This was assessed by FACS analysis (Fig. 3), and IL-2 binding assay using I25Ilabeled IL-2 (Fig. 4). The results of binding assay revealed that numbers of IL-2 receptors on DNPprimed B-cells were increased only when DNPprimed B-cells were cultured in the presence of B151-TRF (6500 sites/cell, in the presence of B151-TRF and less than 20 sites/cell, in the absence of B151-TRF, respectively). Thirdly, increase in the expression o f steady-state mRNA expression for IL-2 was observed by Northern blot analysis (Fig. 5). Sizes of mRNA detected for IL-2 receptor by our hand are similar to those detected in activated T-cells [42]. 213

The expression o f cell surface receptors for IL-2 on activated T-cells has long been established [49], and under certain conditions B-cells can also express IL-2 receptors and respond to IL-2 [29]. For example, B-cell leukemias express IL-2 receptors [50] and the existence o f low numbers o f IL-2 receptors on n o r m a l B-cells has been reported [51]. It is also reported that some forms o f activation o f n o r m a l B-cells can induce high numbers o f IL-2 receptors. Zubler et al. [27] has demonstrated that splenic B-cells cultured in vitro in the presence o f LPS, anti-Ig antibody-coated beads and EL-4 thym o m a cell conditioned medium, expressed IL-2 receptors in similar numbers and o f similar affinity to those present on concanavalin A-activated T-cells [30]. In addition, Nakanishi et al. also demonstrated that B-cells stimulated with anti-IgM and BSF-1 expressed IL-2 receptors, and that addition o f B151 conditioned m e d i u m to the culture m e d i u m led to enhancement o f this expression [28]. This was a clear indication that activity present within the B151 culture supernatant may play some role in the induction o f IL-2 receptor expression. W h e t h e r murine B-cells express IL-2 receptors u p o n activation in vivo is still u n k n o w n . In some experiments, the B-cells cultured with FCS-containing m e d i u m alone also expressed a significant level o f IL-2 receptor, while freshly prepared B-cells did rarely express IL-2 receptor on their surface (data not shown). In this regard, studies delineating the role o f IL-2 in the B-cell differentiation should be o f considerable interest. In summary, the l y m p h o k i n e requirements seen in various assays o f the ability o f murine B-cells to m o u n t P F C responses suggest that there m a y exist distinct pathways o f differentiation to Ig-secretion. In addition, these assays show that T R F / I L - 5 can play a key role in the responses o f BCL~ and normal B-cells, that differentiate into polyclonal I g M responses, and in the antigen-specific IgG P F C responses o f antigen-specific B-cells. In this study, we clearly show the data to be interpreted that T R F / I L - 5 is able to induce in vitro increase in the expression o f functional IL-2 receptors on activated B-cells. This is the first evidence that there is a Tcell derived molecule which can induce IL-2 receptors on B-cell surface. This m a y suggest that T R F / I L - 5 is able to induce IL-2 receptor expression on the cell lineage other than B-cells and m a y have 214

another role in the regulation o f i m m u n e responses t h r o u g h IL-2 receptor expression.

Acknowledgements We wish to thank Drs. Junji H a m u r o and Kohsaburo Sato for their generous gift o f recombinant h u m a n IL-2 and recombinant murine IFN-y, respectively, Dr. Marcus Nabholz for providing anti-IL-2 receptor antibody, Drs. Yoshimi Sano and Toshiyuki H a m a o k a for their criticism and valuable discussions t h r o u g h o u t this study. The secretarial assistance o f Ms. Sayuri Tachimoto is very m u c h appreciated. This work was supported in part by a Grant-in-Aid for Scientific Research and Cancer Research from the Ministry o f Education, Culture and Science, and a Grant for Research on Intractable Diseases from the Ministry o f Health and Welfare, Japan.

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