Autocrine B-CLL proliferation by BCGF-12KD
Eur. J. Immunol. 1992. 22: 1927-1930
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Short paper Sylvie FournierA+, James Jacksono, Ashok KumarO, Thomas Kingo, Surendra SharmaO, Guy Biron., Manuel RubioA, Guy DelespesseA+ and Marika SarfatiA+ University of MontrealA, Notre-Dame Hospital, Laboratory for Allergy Research, Montreal and Department of Pathologyo, Roger Williams Medical Center, Brown University, Providence
Low-molecular weight B cell growth factor (BCGF-12KD) as an autocrine growth factor in B cell chronic lymphocytic leukemia* The role of cytokines in the growth and spreading of human hematologic malignancies has been underlined in recent years. Here we report evidence that a human 12-kDa B cell growth factor (BCGF-12KD) may function as a growth stimulatory factor in B cell chronic lymphocytic leukemia (B-CLL). First, recombinant BCGF-12KD induced dose-dependent DNA synthesis in neoplastic Bcells of four B-CLL patients tested. Second, seven different B-CLL clones secreted a BCGF-12KD-like product that accounted for their proliferation. A neutralizing monoclonal antibody (mAb; Ac8) directed against a BCGF-12KD synthetic peptide inhibited the spontaneous growth of the leukemic B cells. The same mAb blocked DNA synthesis in normal tonsillar B lymphocytes induced by the culture supernatant of spontaneously proliferating B-CLL cells. Finally, BCGF-12KD mRNA was expressed in freshly isolated (two of three patients) as well as in vitro proliferating B-CLL cells (three of three patients). These findings strongly suggest that BCGF-12KD can modulate the growth of B-CLL cells in vivo as well as in vitro. They may offer significant insights into the biology of this frequent B lymphoproliferative disorder.
1 Introduction B cell chronic lymphocytic leukemia is a common lymphoproliferative disorder, accounting for 30% of all leukemia in the Western world. The disease is characterized by the proliferation and accumulation of B cells arrested at the intermediate stage of their differentiation [1]. We and others have shown that B-CLL cells can proliferate in response to several exogenous cytokines, including semipurified BCGF-12KD and that they exhibit a variable pattern in their responsiveness [2-31. BCGF-12KD is a human T cell-derived cytokine which promotes the proliferation of normal B cells preactivated with anti-Ig or other polyclonal activators [4]. Most importantly, BCGF-12KD induces S-phase entry in B cells from hairy cell leukemia [5], chronic lymphocytic leukemia [6], EBV-positive and EBV-negative Burkitt's lymphoma [7], and systemic lupus erythematosus [8]. In most of these studies, including human B-CLL [6], BCGF-12KD was used as a commercially available semi-purified material isolated from lectin-activated lymphocytes. A cDNA encoding BCGF-12KD has been recently charaterized and
expressed [9]; the recombinant product (rBCGF-12KD) is as effective as its natural counterpart in inducing DNA synthesis of preactivated normal B cells and of neoplastic Bcells [7, 101. We suggest here that BCGF-12KD is an autocrine growth factor for B-CLL cells by showing that neoplastic B cells: (a) proliferate in response to rBCGF-12KD; (b) express BCGF-12KD mRNA and (c) secrete a BCGF-12KD-like activity neutralized by an mAb (Ac8) directed against a synthetic BCGF-12KD peptide.
2 Patients and methods 2.1 Patients' details Seven B-CLL patients were studied on 17 separate occasions in various parts of the study.The patients were at Rai stage 0 to 111, they were not receiving therapy at the time of the study, their ages ranged between 39 and 75 years.Their peripheral blood contained 40 x lo3 to 90 x lo3 leukocytes/mm3 of which 65% to 90% were lymphocytes.
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M. Sarfati is an MRC scholar, G. Delespesse an MRC associate and S. Fournier an MRC student. This work was supported by a grant from the MRC of Canada and by NIH grant CA54763.
Correspondence: Marika Sarfati, University of Montreal, NotreDame Hospital, Laboratory for Allergy Research, 1560 Sherbrooke Street East Montreal. Quebec H2L 4M1, Canada Abbreviations: B-CLL: B cell chronic lymphocytic leukemia BCGF-MKD: Low-molecular weight B cell growth factor
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
2.2 Lymphocyte preparations and culture conditions Peripheral blood mononuclear cells from CLL patients (>90% CD5+ and CD20+) or normal tonsillar lymphocytes were isolated with Lympho Prep (Cedarlane, Ontario, Canada); highly purified B cells (no detectable CD3+ or CD14+ cells) were negatively selected by a combination of rosetting (twice) with aminoethylisothiouronium bromidetreated sheep red blood cells, adherence to plastic and treatment with L-leucine methyl ester. 0014-2980/92/0707-1927$3.50+ .25/0
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S. Fournier, J. Jackson, A. Kumar et al.
For primary cultures, fresly isolated highly purified B cells (B-CLL cells) were cultured at 2 x 106/ml (96-well flatbottom plate; 200 pl/well) in HBlOl serum-free medium (Irvine Scientific, Santa Ana, CA) for 6 days. Secondary cultures were performed to obtain enhanced spontaneous DNA synsthesis [ 2 ] ;B-CLL cells were first plated in 6-well Costar plates (15 x 106/10ml) for 6 days in HBlOl serum-free medium, the cells were then washed and recultured for 2 days. The use of serum-free medium in all the above cultures avoided the disadvantages of serum factors that frequently increase the background proliferation and may mask any effects of highly purified regulatory molecules under study [ 111. Cells were pulsed with 0.5 pCi = 18.5 kBq [3H]thymidine (Amersham Corp.) during the last 16 h of the culture. Cells were then harvested and counted in a standard liquid scintillation counter.
Eur. J. Immunol. 1992. 22: 1927-1930
2.5 Production of rBCGF-12KD
rBCGF was prepared as described [7, 101. Briefly, E. coli containing BCGF expression plasmid pARJ43 was grown to an optical density of 0.3 at 600 nm, and induced for 2 h with 0.5 mM of isopropyl-P-D-thiogalactopyranoside. Bacteria were harvested by centrifugation, washed with lysis buffer (50 nM Tris acetate, pH 8.2, 1mM EDTA, 0.5 mM phenylmethysulfonyl fluoride, 1 pg/ml aprotinin), and lysed in buffer containing 50 pg/ml freshly prepared lysozyme (Sigma, St. Louis, MO) for 40 min at 4°C. The resulting lysates were centrifuged at 100000 x g for 30 min and supernatants collected. This material was adjusted to pH 5.9 with 0.1 N HCl and applied to 5 ml S-Sepharose Fast Flow column (Pharmacia, Piscataway, NJ). After washing, the bound material (containing < 1% of the total protein loaded) was eluted with 20 mM Tris acetate, pH 8.2. This material was further purified by passage through an affinity column conjugated with the Ac8 mAb. After extensive 2.3 Polymerase chain reaction (PCR) technique washing, the BCGF activity was eluted with 100mM sodium citrate, pH 3.6.The eluted material was neutralized Total RNA was isolated from cryoconserved B-CLL cells by with 1M Tris-HC1 (pH 8.0), dialyzed, and concentrated the cesium chloride-guanidinium isothiocyanate method with a Centricon filter (Amicon Corp., Danvers, MA). and poly(A)+ RNA was prepared by using an oligo(dT)BSA (1 mg/ml final) was added to stabilize BCGF biologic coated matrix (Dynabeads). Poly(A)+ RNA (0.2 pg) was activity.This affinity-purified material yielded a 14-16-kDa converted into cDNA with a commercial kit using the band on a SDS-PAGE gel and in a Western blot assay using oligo(dT) primer and the reverse transcriptase according to Ac8 mAb. This recombinant product (rBCGF-12KD) the manufacturer's (Cetus, Emeryville, CA) directions. showed biologic activity comparable to natural product DNA amplification was carried out exactly as previously BCGF [ 7 ] ; 2.5% rBCGF preparation is equivalent to described [ 101 using oligonucleotide primers derived from 1 U/ml BCGF activity (defined as 50% of the growththe BCGF-12KD coding region. The primer sequences promoting activity of natural product BCGF on an EBV+ included both sense (5'GGGGAACAAGGAAACCTCT- cell line, BI9). TGGG-3') and anti-sense (5'AAAGCCTCTCTTTTTTAATATAAGTGGTAG-3') combination. DNA was subjected to 45 cycles of PCR amplification using Taq polymerase (Bethesda Research Laboratories Life Technologies 3 Results and discussion Inc., Bethesda, MD) in a thermal cycler (Ericomp Inc., San Diego, CA). The PCR products were electrophoresed It has been postulated that autocrine loops are required for on a 1.2% agarose gel and stained with ethidium bromide the growth and progression of several hematological maland further processed from Southern analysis using 32P- ignancies [12]. B-CLL cells have been shown to both labeled Alu-free BCGF-12KD probe, when required. produce and respond to a variety of cytokines [ 131including IL-1[14],TNF-a [15] and IL-6 [16]. So far, all the published data describing the effect of BCGF-12KD on B-CLL cells have been obtained using commercially available BCGF2.4 Mouse mAb to BCGF-12KD synthetic peptides 12KD (which is known to contain small amounts of contaminating IFN-y, CSF, lymphotoxin) [2, 61. In the A neutralizing mAb to a BCGF-12KD synthetic peptide has recently been raised and the details will be provided present study, we first examined the ability of recombinant elsewhere". In brief, we selected two peptide regions from BCGF-12KD to stimulate DNA synsthesis by B-CLL cells. the BCGF-12KD coding region (based on hydrophobicity) Fig. 1 shows that rBCGF-12KD treatment increases and two putative anti-BCGF mAb have been obtained [3H]thymidine incorporation by neoplastic B cells from all using standard methods. One of these, Ac8, is an IgG four CLL patients tested, and that the effect is dose antibody of the IgGl isotype which shows specific binding dependent. We recently reported that B cells isolated from to native BCGF-12KD and rBCGF-12KD in Western blot untreated CLL patients with progressive disease displayed assays". Importantly, Ac8 is a neutralizing antibody which enhanced spontaneous DNA synthesis and cellular prolifeffectively blocks the bioactivity of rBCGF-12KD assayed eration following a two-step culture [2].We show here that a on anti-p-activated B cells and the undifferentiated lym- mouse mAb ( A d ) , directed against a BCGF-12KD pepphoma MC116 cell line [7]. Ac8 mAb was used as an tide [lo], markedly suppresses the spontaneous DNA ammonium sulfate cut preparation at concentrations rang- synthesis in B cells from seven different B-CLL patients ing from 10 to 200 pg/ml. Anti-IL-4 mAb (IgGlx; produced (Fig. 2). Although the precise mechanisms leading to the in our laboratory) was used as control mAb at the same spontaneous proliferation of B-CLL cells are not yet understood, these data suggest that the malignant proliferconcentrations. ating B cells may produce and respond to an endogenous BCGF-12KD-like molecule in vitro. This claim is further supported by the observation that the culture supernatants '. Sharma et al.. manuscript in preparation. of these spontaneously proliferating B-CLL cells induce a
Autocrine B-CLL proliferation by BCGF-12KD
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Figure 2. Effect of neutralizing anti-BCGF mAb on B-CLL proliferation. B-CLL cells from seven different patients (secondary culture) were cultured in the absence or presence of increasing concentrations of Ac8 (anti-BCGF neutralizing mAb) or isotypematched anti-IL-4 mAb (control mAb). Results are expressed as percent inhibition relative to parallel control cultures grown in the absence of mAb (mean ? 1 SEM of [3H]thymidineincorporation for control cultures of 11 experiments: 18445 f 6439 cpm).
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Figure 1. Effect of rBCGF-12KD on spontaneous DNA synthesis by B-CLL. (A) B-CLL cells from patients No. 1 , 2 and 3 (primary culture) or from patient No. 4 (secondary culture) cultured in the absence or presence of 10% rBCGF-12KD. (B) B-CLL cells from patient No. 1 (primary culture) incubated with increasing concentrations of rBCGF-12KD (one representative patient out of three). Results are expressed as the mean f 1 SD of quadriplicate cultures.
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Figure 3. Effect of B-CLL culture supernatant on normal B cell proliferation: neutralization by anti-BCGF-12KD mAb. Highly purified tonsillar B cells (1 X 106/ml)were preactivated with soluble goat F(ab‘)z anti-IgM (Cappel; 5 pg/ml) in HBlOl serum-free medium. After 3 days, cells were pulsed with 0.5 pCi [3H]thymidine for 16 h. (A) Anti-p-preactivated B cells were cultured with various concentrations of CSN (= pool of two unconcentrated culture supernatant of B-CLL secondary cultures from two different CLL patients). (B) Anti-p-preactivated B cells were cultured with 20% CSN in the presence of Ac8 mAb or control mAb (200 PgW.
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Figurc. 4. PCR amplification of BCGF-12KD mRNA in B-CLL. SS cDNA prepared by using poly(A)+ RNA isolated from an EBV- cell line MC 116 cells (lane 2); PHA-activated unfractionated lymphocytes (lane 3); freshly isolated unfractionated normal Iymphocytcs (lanc 4); freshly isolated B-CLL cells (patients 1,2,3; lane 5-7); in vitro spontaneous proliferating B-CLL cells (secondary culture) in the absence of rBCGF-12KD (patients 2,3.4; lane 8-10), Actin PCR products were visualized in each sample (not shown).
dose-dependent [3H]thymidine incorporation by anti-IgMstimulated normal B cells (Fig. 3 a); this BCGF activity is significantly inhibited by neutralizing anti-BCGF-12KD mAb (Ac8; Fig. 3 b). Of note, the same concentrations of anti-BCGF-12KD mAb did not affect DNA synthesis by EBV+ B cell lines or U937 monocytic cell line, ruling out a nonspecific blockade by the Ac8 mAb [lo] and data not shown. Furthermore, it is important to point out that high concentrations of Ac8 are needed to neutralize the BCGF-12KD activity as an ammonium sulfate preparation was used in these experiments. To explore the possible in vivo relevance of the above observations, we next examined the presence of BCGF-12KD mRNA in freshly isolated B-CLL cells. PCR analysis of the BCGF-12KD gene transcription indicates that the majority of freshly isolated B-CLL cells (two out of three patients) as well as all in vitro proliferating B-CLL cells (three out of three patients) express the BCGF-12KD gene at significant levels; whereas, this gene is silent in normal resting lymphocytes (Fig. 4).These data suggest that in vivo activated B-CLL may preferentially use BCGF-12KD as an autocrine growth factor, whereas, B-CLL consistently activate the BCGF-12KD gene when they are cultured (Figs. 2 and 4). This is the first report demonstrating that B-CLL cells express, produce and respond to BCGF-12KD. The ability of exogenous rBCGF-12KD to enhance the spontaneous proliferation of B-CLL cells (Fig. 1, patient No. 4) suggests, as it was shown for other cytokines (i.e. TNF-a), that this factor may increase its own production [14].These data clearly imply that B-CLL cells express a receptor for BCGF-12KD. Although several molecules have been proposed to serve this role including CD23 [17], B8.7 [18] and a 35-kDa molecule, appearing early in B cell activation [19], the receptor for BCGF-12KD remains to be identified [20]. The current studies strongly suggest that a deregulated production of BCGF-12KD may complement the autocrine growth requirements of neoplastic B cells including B-CLL
cells. In this context, it is important to point out that EBVbut not EBV+ transformed B cells express BCGF-12KD mRNA and utilize this cytokine as an autocrine factor [7, 101 and Sharma et al., personal observations). Given the fact that B-CLL is not clinically associated with EBV, the presence of BCGF-12KD mRNA in freshly isolated and in vitro cultured B-CLL cells further suggests a pathogenetic role of this cytokine in B-CLL disease. We wish to thank Norma Del Bosco for her excellent secretarial assistance.
Received December 30, 1991; in revised form March 16, 1992.
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