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BLyS BINDS TO B CELLS WITH HIGH AFFINITY AND INDUCES ACTIVATION OF THE TRANSCRIPTION FACTORS NF-κB AND ELF-1
doi:10.1006/cyto.2000.0793, available online at http://www.idealibrary.com on
BLyS BINDS TO B CELLS WITH HIGH AFFINITY AND INDUCES ACTIVATION OF THE TRANSCRIPTION FACTORS NF-B AND ELF-1 Palanisamy Kanakaraj, Thi-Sau Migone, Bernardetta Nardelli, Stephen Ullrich, Yuling Li, Henrik S. Olsen, Theodora W. Salcedo, Thomas Kaufman, Erika Cochrane, Yuxiang Gan, David M. Hilbert, Judith Giri B lymphocyte stimulator (BLyS) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms. BLyS was shown to act specifically on B cells, inducing proliferation and immunoglobulin production both in vitro and in vivo. The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding. Similar to other TNF family members, BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis. BLyS binding to B cells is specific as other TNF family members tested did not compete for 125I-BLyS binding. Analysis of equilibrium binding of 125I-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding. Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant (KD) of about 0.1 nM. In addition we report that BLyS binding to B cells results in the activation of NF-B and the Ets family transcription factor, ELF-1, and in the induction of mRNA for Polo-like kinase (PLK). 2001 Academic Press
B lymphocyte stimulator (BLyS), also identified independently as TALL-1,1 BAFF,2 THANK,3 TNFS204 and zTNF4,5 is a new member of an ever growing TNF superfamily of proteins.6 Members of this family including TNF-, TNF-, FasL, CD40L, OX40L, lymphotoxin-, TRAIL/Apo-2L, CD27L, CD30L, 4-1BBL, TRANCE/RANKL, Light, TWEAK, TL-1 and APRIL, are expressed on a variety of cells and mediate pleiotropic cellular responses including proliferation, apoptosis and immune regulation.7–11 Like other TNF family proteins, with the exception of TNF-/LT-, BLyS is a type II From the Human Genome Sciences, Inc., Rockville, MD 20850, USA Correspondence to: Dr Palanisamy Kanakaraj, 9410 Key West Avenue, Human Genome Sciences, Inc., Rockville, MD 20850, USA. E-mail: [email protected] Received 27 July 2000; accepted for publication 28 July 2000 2001 Academic Press 1043–4666/01/010025+07 $35.00/0 KEY WORDS: B lymphocyte stimulator (BLyS)/ signaling/ binding/ transcription factors/ tumour necrosis factor (TNF) Abbreviations: TNF, tumour necrosis factor; TNFR, tumour necrosis factor receptor; TRAF, tumour necrosis factor associated factor; BLyS, B lymphocyte stimulator; DSS, disccinimidyl suberate; SDS, sodium dodecyl sulfate; PAGE, poly acrylamide gel electrophoresis; PLK, Polo-like kinase; ERK, extracellular signal regulated kinase CYTOKINE, Vol. 13, No. 1 (7 January), 2001: pp 25–31
transmembrane protein expressed as a membranebound form on monocytic cells that is also cleaved and released by proteases as a soluble protein.2,6 Recent studies indicate that BLyS binding is restricted to B cells.2,6 Both the membrane and soluble forms of BLyS are biologically active in promoting proliferation of B cells treated with anti-IgM.2,6 Animals injected with soluble recombinant BLyS exhibit disrupted splenic T and B cell zones with increased B cell counts and immunoglobulin levels.6 Mice transgenic for BLyS exhibit autoimmune lupus-like characteristics with high levels of rheumatoid factors, immune complexes, anti-DNA auto-antibodies and increased numbers of B and effector T cells.12,13 Taken together these results demonstrate that BLyS plays a major role in regulating B cell immune responses. The receptors for TNF family proteins also belong to a conserved family of proteins called the TNF receptor superfamily with homologous extracellular cysteine rich domains which form specific linear ligand binding structures (reviewed in ref. 14). The TNFR family proteins can be classified in to two types, based on the presence or absence of a conserved cytoplasmic domain responsible for apoptosis called death domain. Although receptors with death domains in some cases 25
RESULTS AND DISCUSSION
– 17 kDa
– 44 kDa
0.50
0.40
0.30 AU
have been shown to induce cell proliferation and/or survival signals, death domains play an essential role in mediating apoptosis via activation of caspases (15, review ref. 16). Receptors without death domains such as TNF receptor-2, HVEM/ATAR, RANK, CD27, CD30, CD40 and OX40 interact with TNF receptorassociated factors (TRAF 1–6) and mediate antiapoptotic survival or/and proliferative responses via activation of the transcription factor NF-B (review ref. 17). Three dimensional structural analysis of the TNF family proteins CD40 ligand,18 TNF-19,20 and TNF-21 show that these ligands form homotrimers and interact with their cognate receptors with a stoichiometry of one trimeric ligand to three receptors. With the exception of OX4022 and TNF23–25 detailed biochemical analysis of binding of the other TNF family ligands have not been reported. Here, using 125 I-labeled BLyS we demonstrate binding characteristics of BLyS to cell surface receptors on B cells. Our studies also show that activation of transcription factors NF-B and ELF-1 may play a role in BLySmediated B cell responses.
CYTOKINE, Vol. 13, No. 1 (7 January, 2001: 25–31)
– 200 kDa – 158 kDa
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0.20
0.10
0.00
2.00
4.00
6.00
8.00
10.00
Time (min) Figure 1.
BLyS forms a homotrimer.
Size exclusion HPLC analysis of BLyS. Purified BLyS protein is analyzed by SEC-HPLC using a Waters Protein-Pak HPLC column (7.8300 mm) equilibrated in 50 mM sodium acetate, 150 mM sodium chloride at pH 6.0. The column is calibrated with a gel filtration standards (Bio-Rad) which consists of thyroglobulin (MW 670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B-12 (1.35 kDa).
BLyS is present as a homotrimer in solution X-ray crystallographic structural studies of TNF- complexed with its receptor showed that TNF- is present as a homotrimer and that one molecule of trimeric TNF- interacts with three receptor molecules presumably resulting in multimerization of the TNF receptor on the cell surface.21 It was predicted by molecular modeling that all TNF proteins except NGF form trimeric complexes.27,28 We determined the molecular size of BLyS by gel filtration. The data (Fig. 1) showed that BLyS migrated as a single peak with an apparent molecular weight of 44–46 kDa. The molecular weight of monomeric soluble BLyS determined by SDS-PAGE was 17.4 kDa. The difference between the predicted apparent molecular weight of 52 kDa of trimeric BLyS and the molecular mass of the gel filtration peak may be due to folding of the native form of BLyS which migrates differently in gel filtration when compared to the denatured protein on SDS PAGE. The light scattering analysis of the samples from size exclusion chromatography revealed a major peak (96.5% area) corresponding to trimeric BLyS with molecular weight of 54 500 g/mole (data not shown). The trimeric form of BLyS was also confirmed by crosslinking studies using 125I-labeled BLyS. BLyS was iodinated by using iodobead method as described in Materials and Methods. The iodination procedure did not affect the biological activity of BLyS as determined by costimulatory activity of mock iodinated BLyS in a B cell proliferation assay (data not shown).
DSS crosslinking of 125I-labeled BLyS showed three bands of molecular weights corresponding to monomeric 17.4 kDa, dimeric 35 kDa and trimeric 52 kDa forms of BLyS by SDS-PAGE. No band beyond 52 kD was observed in the presence of crosslinking agent indicating that there was no multimerization of trimeric BLyS (data not shown). Monomeric and dimeric forms of BLyS in the presence of DSS crosslinking were due to partial crosslinking of BLyS molecules.
Number and affinity of BLyS binding sites on B lymphocytes Saturation binding analysis was performed on tonsillar B cells using varying concentrations of 125Ilabeled BLyS to determine affinity (KD) and number of binding sites (Fig. 2A). The BLyS binding to B cells was saturable and Scatchard plot analysis of the binding data revealed a single high affinity site with a KD of 0.1 nM. The estimated number of binding sites was approximately 2600 per cell. 125I-labeled BLyS binding experiments, were also performed using a variety of human and murine B cells. The binding affinity (KD) and number of binding sites were determined as shown in Table 1. Comparison of the BLyS binding affinity to mouse and human cells indicate that 125I-BLyS binds with similar high affinity to both human and murine cells.
BLyS binding and signaling in B cells / 27
30 000
TABLE 1. B cells
A
Binding of 125I-labeled BLyS to human and mouse
Cells (nM) Tonsillar B cells IM-9 Raji Mouse B cells A20 BCL-1
100 Bound/free
Counts (cpm)
20 000
10 000
75 50
2.5
1
5.0 Bound 2
7.5 3
4
B
Iodinated BLyS bound (%)
100
80
60
2600 3200 1702 179 778 4800
family proteins LIGHT, TNF- and FasL. As shown in Figure 2B, 125I-labeled BLyS binding to IM-9 cells was displaced only by unlabeled BLyS, but not by other TNF family proteins indicating that BLyS binds to a specific receptor on the B cell surface not shared by other TNF family proteins tested. The specific binding of 125I-labeled BLyS was time and temperature dependent. Binding occurred very fast following addition of 125 I-labeled BLyS; approximately 40% of maximal binding was observed after 5 min incubation at 24C and 4C. Moreover, binding reached a plateau level after 60 min incubation at both temperatures (data not shown).
40
Crosslinking of 125I-labeled BLyS to B cells and identification of BLyS binding protein(s)
20
0 0.01
0.1
1
10
100
Unlabeled cytokines (nM) Figure 2.
0.1 0.19 0.16 0.35 1.1 0.93
10.0
I125-labeled BLyS (nM) 120
Binding sites/cell
25 0
0
Dissociation constant KD
Characterization of
125
I-labeled BLyS binding to B cells.
A: Direct saturation binding of 125I-labeled BLyS. The binding was performed in 2106 tonsillar B cells with increasing concentrations of 125I-labeled BLyS alone or in the presence of 100-fold excess of unlabeled BLyS. The specific binding at each concentration of 125 I-labeled BLyS was determined. The Scatchard analysis of the binding data was shown in the inset. B: Specificity of BLyS binding. 1106 IM-9 cells in triplicates were incubated with 1 nM 125Ilabeled BLyS in absence or presence of 100-fold excess of different TNF family cytokines for 2 h at RT as indicated. The binding was expressed as percentage of total 125I-BLyS bound to the cells in the presence of unlabeled proteins. BLyS, – –; LIGHT, – –; TNF-, – –; FasL, – –.
Specificity of
125
I-labeled BLyS binding to B cells
BLyS is a strong proliferative factor for primary B cells, but it does not appear to affect functional activities of other peripheral blood leukocytes including resting T cells and monocytes. Accordingly, biotinylated BLyS was reported to bind specifically to B cells but not to other cells.6 We determined the specificity of 125I-labeled BLyS binding using a competitive binding assay (Fig. 2B) in absence or presence of 100-fold excess of unlabeled BLyS and other TNF
To characterize better the BLyS binding site on B cells, we performed DSS crosslinking studies of 125Ilabeled BLyS to B cells. Separation of the resulting crosslinked complexes by SDS-PAGE showed protein bands corresponding to 50, 35, and 17 kDa (Fig. 3A). These bands are mostly likely the crosslinked BLyS trimer, partially crosslinked dimer and non-linked monomeric forms of BLyS. The diffused higher molecular weight band of 140–200 kDa may be the result of crosslinking of dimeric and trimeric BLyS to one or more molecules of the BLyS receptor. There were no crosslinked proteins visible with DSS and 125 I-labeled BLyS in K562 or in the presence of excess of cold BLyS indicating that BLyS binds to a specific receptor on B cells. BLyS ligand-affinity column was used to identify protein(s) solubilized from Raji and normal human B cells that bind BLyS. Control (NKEF-C) and BLyS affinity columns were used to analyze the proteins present in Raji cell extracts that specifically bind BLyS and are likely to represent the receptor. The proteins bound to the control or BLyS affinity column were examined for their ability to bind BLyS by subjecting them to SDS-PAGE analysis under non-reducing conditions followed by blotting onto Problot membranes; the blots were incubated with biotinylated-BLyS and
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A
CYTOKINE, Vol. 13, No. 1 (7 January, 2001: 25–31)
K562
BLyS (100¥)
+
bound biotinylated-BLyS detected with alkalinephosphatase-streptavidin. Two major bands were visualized having molecular masses of 50 and 40 kDa (Fig. 3B, lane 4); there was only background binding to the control NKEF-C affinity column fractions (Fig. 3B, lane 2). The absence of binding observed from the mock-eluted BLyS affinity column fractions (Fig. 3B, lane 6) ruled out any binding of biotinylated BLyS to BLyS leaching from the affinity column via subunit exchange. A similar experiment was performed using tonsillar B cells pre-activated with SAC for 3 days to detect the BLyS binding proteins. The major molecular mass of the BLyS binding proteins was 50 and 40 kDa, similar to that observed in Raji cells (data not shown). Thus both Raji and human B cells contain BLyS binding proteins of 40 and 50 kDa which appear to represent the B cell BLyS receptor and/or proteins interacting with the BLyS-receptor complex.
IM-9
+
250 148
60
42
22 17
BLyS induced signaling in B cells B Mr kDa 200
98 64 50
36
30
21 1
2
3
4
5
6
125
Figure 3. DSS crosslinking of I-labeled BLyS and identification of BLyS binding proteins in B cells. A: IM-9 and K562 cells were incubated with 125I-labeled BLyS in the absence or presence of excess of unlabeled BLyS and crosslinked with DSS for 25 min at room temperature. The cell lysates were prepared and resolved on a 10% SDS-PAGE. The gel was dried and subjected to autoradiography. B: The Raji cell lysates were prepared and affinity purified through BLyS affinity column as described in Materials and Methods. Affinity column fractions were analyzed by 12% SDS-PAGE under non-reducing conditions, transferred and blots incubated with biotinlyated-BLyS: eluates from control (lane 2) and BLyS (lane 4 ) affinity columns incubated with Raji lysate or mock-eluted BLyS affinity column (lane 6). Lane 1, pre-stained molecular weight standards; lanes 3 and 5, biotinylated molecular weight standards.
To examine the intracellular mechanisms triggered by BLyS receptor binding, tonsillar B cells treated with BLyS in the presence of SAC were subjected to real time PCR (Taqman analysis) to determine the induction of mRNA of a panel of proteins known to play an important role in the activation of lymphoid cells. Among the proteins analyzed, we consistently found that dramatic increase in induction of mRNA for Polo-like kinase (PLK) (Fig. 4A) and two-fold increase of Erk-1 mRNA expression (not shown) in B cells. PLKs belong to a sub family of serine/threonine kinases related to Saccharomyces cerevisiae cell cycle protein CDC5.29 The expression of PLK is induced during G2 and S phases of the cell cycle. PLK is reported to play a role in cell proliferation.30 The role of extracellular signal regulated kinases (ERK1/2) in cell survival and proliferative effects of growth factors and other agonists has been extensively studied. The induced expression of PLK and ERK-1 is consistent with the survival and proliferative effect of BLyS on B cells. In addition to ERK-1 and PLK, mRNA for CD25 (IL-2R) was found to be upregulated in some of the donors we examined. The CD25 promoter has been extensively studied (reviewed in ref. 31), and three positive regulatory regions (PRR) that regulate the transcriptional activation of CD25 have been described. PRRI has binding sites for NF-B and SRF, while PRRII has been shown to bind ELF-1 and HMGI/Y proteins. Both promoter regions play a crucial role in the induction of CD25 during T cell activation. Finally, PRRIII has binding sites for STAT5/ and ELF-1 and has been shown to be important for IL-2-induced upregulation of CD25.
BLyS binding and signaling in B cells / 29
B
60
C
IM-9 cells
B cells
A
Fold increase
50 PLK
40 30 20 10 0 Blys
+
Blys
+
ELF-1 Figure 4.
+
NFkB/SRF
+
ELF-1
+
NFkB/SRF
BLyS induced signaling in B cells.
A: Induction of PLK mRNA expression. Total RNA was prepared from tonsillar B cells unstimulated or stimulated with SAC or SAC plus BLyS for 12 h. PLK mRNA detected by a one step RT-PCR procedure using specific primers and probes. Expression levels of mRNA shown are relative to expression levels in unstimulated B cells. B: IM-9 cells or primary tonsillar B cells (C) were serum starved for 16 h and treated or not treated with 100 ng/ml soluble BLyS for 45 min at 37C. The nuclear extracts were prepared and incubated with 32P-labeled NF-B/SRF or ELF-1-specific double-stranded oligonucleotides. The protein–DNA complexes were separated on 6% polyacrylamide gel in 0.5% Tris-borateEDTA buffer.
We analyzed nuclear extracts from primary B cells and B cell lines by EMSA and found no significant induction using a PRRIII probe (data not shown). This is consistent with the fact that BLyS does not appear to induce signaling via tyrosine phosphorylation of the Jak/STAT pathway (data not shown). In contrast we found induction of DNA-binding activity to the probes corresponding to PRRI and II in IM-9 (Fig. 4B) and primary tonsillar B cells (Fig. 4C). NF-B has been shown to be induced by a number of members of the TNFR family proteins upon ligand binding and suggests that the receptor for BLyS may also belong to this family. ELF-1 is a transcription factor that is part of the ETS family of proteins and whose expression appears to be restricted to T and B cells. Binding sites for ELF-1 have been described in the promoters of a number of proteins that are important in the regulation of the immune response. In this paper we show that, as reported for other TNF family proteins, BLyS exists as a trimer and the trimeric form of BLyS binds to a single high-affinity site on B cells. The costimulatory activity of BLyS in inducing proliferation of B cells along with the result presented here in induction of PLK and ERK-1 expression and activation of transcription factors NF-B and
ELF-1 suggest that BLyS receptor may belong to a category of non-death domain containing TNF family receptors involved in survival, proliferation and other immunoregulatory effects. While this manuscript was under preparation, Gross et al.5 reported that TACI32 and BCMA33 are receptors for BLyS. The reported high affinity binding of BLyS to TACI and TACI induced activation of NF-B are in line with our observation.
MATERIALS AND METHODS Expression and purification of recombinant BLyS Recombinant BLyS was expressed in baculovirus and purified by chromatography using a combination of ion exchange (Poros HS-50, Poros PI-50; PE Biosystems, Framingham, MA, USA), size exclusion (Sephacryl S100 HR; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and hydrophobic interaction (Toyopearl Hexyl 650 C; Tosohaas, Montgomeryville, PA, USA) columns. The purity of BLyS was determined as described.6
Preparation of
125
I-labeled BLyS
Radio-iodination of BLyS was performed using the Iodobead method as described.26 Briefly, one Iodobead per
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CYTOKINE, Vol. 13, No. 1 (7 January, 2001: 25–31)
reaction was pre-washed with PBS and added to 1 mCi of 125 NaI in 80 l of PBS pH 6.5. The reaction was allowed to proceed for 5 min and then 10 g of BLyS (1 mg/ml stock) was added and incubated for 5 min at room temperature. The iodinated protein was separated from unbound radioactivity using a G-25 Sephadex quick spin column previously equilibrated with PBS containing 0.1% BSA. Protein concentration and specific radioactivity of 125I-labeled BLyS were determined by TCA precipitation of pre-column and post-column samples. The specific activity of 125I-BLyS used in the experiment was 900–1600 Ci/mmol (17.5–34.6 Ci/g).
Preparation of tonsillar B cells Primary B cells were purified from tonsils by depleting CD3 + cells using magnetic bead (MACS) column. B cell preparations were >95% pure as determined by flow cytometric analyses of CD19 and CD20 expression.
Binding analysis Direct binding of radiolabeled BLyS was performed in triplicates in a 96-well plate using 2106 cells in 100 l of binding buffer (Ham’s F containing 0.5% BSA and 0.1% sodium azide) with indicated concentrations of 125I-labeled BLyS. The non-specific binding of BLyS to cells was determined in the presence of 100-fold excess of unlabeled BLyS in the binding mixture. The competitive binding was carried out using 0.3 nM of 125I-labeled BLyS in the absence or presence of varying concentrations of unlabeled BLyS. The binding reaction was performed at room temperature for 2 h on a shaker platform. We have previously determined that the incubation of 125I-labeled BLyS for 2 h at 25C was sufficient to reach equilibrium binding. After the incubation, the cell-bound BLyS was separated from unbound free 125Ilabeled BLyS by centrifugation through 200 l of 1.5 dibutylphthlate/1.0 bis (2-ethyl-hexyl) phthalate oil mixture in a polyethylene microfuge tubes (Bio-Rad, Hercules, CA, USA) for 20 s at 11 750g. The microfuge tubes were then frozen quickly in liquid nitrogen and the bottom tip of the tubes was cut off using a tube cutter. Radioactivity in the bottom containing the cell pellet (bound fraction) and the top (unbound fraction) of the tubes were counted by using a gamma counter. The binding was analyzed by Prizm (GraphPad Software, San Diego, CA, USA) to determine the dissociation constant (KD) and number of binding sites.
Crosslinking of
125
I-labeled BLyS to B cells
IM-9 or K562 cells (20106/200 l of binding buffer) were incubated with 125I-labeled BLyS (1 nM) for 2 h at room temperature in the absence or presence of excess unlabeled BLyS. After incubation, the cells were washed twice with ice-cold PBS and resuspended in 1 ml of PBS. DSS (1 mM) was added and incubated at room temperature for 30 min. The crosslinking reaction was terminated by addition of 5 mM ammonium acetate. Cells were then lysed in 200 l of 1% NP40 lysis solution (10 mM HEPES pH 7.5, 0.15 M NaCl, 10% glycerol, 10 g/ml aprotinin, 10 g/ml leupeptin and 1 mM PMSF). The proteins were separated by 10% SDS-PAGE and results were developed by autoradiography.
Ligand blot analysis of BLyS binding protein(s) from Raji cells The proteins, NKEF-C and BLyS, were dialyzed against 25 mM HEPES buffer, pH 7.2; the affinity columns were then prepared by mixing the dialyzed proteins with Affi-gel-15 (Bio-Rad) at room temperature for 3 h (0.8 mg BLyS/0.5 ml resin). Columns were extensively washed according to manufacturer’s directions. Raji cells (17109) were washed three times in PBS and then solubilized in 30 mM HEPES buffer, pH 7.5, 0.15 M NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM Pefabloc, 1 g/ml leupeptin, 10 g/ml aprotinin, 1.5 M pepstatin, 5 g/ml E64, and 1 mM EDTA. After 15 min on ice the lysate was centrifuged at 3000g for 20 min. The supernantant was centrifuged at 10 000g for 20 min and loaded first over control NKEF-C affinity column. The flow through was then loaded over the BLyS affinity column. The wash columns were washed with with 30 mM HEPES buffer, pH 7.5, 0.15 M NaCl. The bound material was eluted from the columns with 10 mM HCl collected in 0.5 ml fractions and neutralized immediately with TRIS buffer. The eluates were further concentrated using an Ultra-free MC 10 000 MW cut-off filtration unit to a volume of 50–100 l. The affinity purified proteins were analyzed under non-reducing conditions on 12% SDS-PAGE and blotted onto Problot membrane (ABI).26 The blots were blocked by incubation with blocking buffer [0.5 mg/ml of BSA in PBS containing 0.05% (V/V) Tween 20] for 30 min. The BLyS binding proteins were detected by incubating the blots in the presence of 1 g/ml biotinylated-BLyS for 1 h in blocking buffer. After washing with blocking buffer the blots were incubated for 45 min with 0.5 g/ml of streptavidin– alkaline phosphatase conjugate (KPL, Gaithersburg, MD, USA) diluted in blocking buffer. The blot was washed three times with 10 ml PBS with 0.05% Tween-20. Bound BLyS was detected by development with BCIP/NBT substrate (KPL, Gaithersburg, MD, USA).
Electrophoretic mobility shift assay (EMSA) Primary tonsillar B cells or IM-9 cells were serum starved for 16 h and treated or not treated with 100 ng/ml soluble BLyS for 45 min at 37C. The nuclear extracts were prepared in high salt buffer (20 mM Hepes pH 7.9, 1 mM EDTA, 420 mM NaCl, 15% glycerol and protease and phosphatase inhibitors). Extracts were incubated on ice in the presence of phosphatase and protease inhibitors and a 32Plabeled probe corresponding to the positive regulatory regions present in the IL-2R alpha promoter. Reaction mixtures were separated on 6% acrylamide gels in Trisbuffered EDTA, dried, and autoradiographed.
Quantitative RT-PCR analysis Total RNA was prepared from unstimulated or SACand SAC+BLyS-stimulated tonsillar B cells. Messenger RNA levels of PLK and ERK-1 B cells were determined by real time quantitative PCR using a ABI 7700 Taqman Sequence Detector. Amplification primers and probe were designed to span the region from nucleotides 252 to 332 of the human PLK sequence and nucleotides 373 to 446 of the human ERK-1 mRNA (Genbank accession no. X75932 and
BLyS binding and signaling in B cells / 31
no. X60188). For quantitation of mRNA levels the comparative Delta Ct method was used (Perkin-Elmer user bulletin no. 4, 1997) using a 18S ribosomal RNA probe as endogenous reference. Expression levels are shown relative to observed levels in unstimulated B cells.
REFERENCES 1. Shu HB, Hu WH, Johnson H (1999) TALL-1 is a novel member of the TNF family that is downregulated by mitogens. J Leukoc Biol 65:680–683. 2. Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, Ambrose C, Lawton P, Bixler S, Acha-Orbea H, Valmori D, Romero P, Werner-Favre C, Zubler RH, Browning JL, Tschopp J (1999) BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med 189:1747–1756. 3. Mukhopadhyay A, Ni J, Zhai Y, Yu G, Aggarwal B (1999) Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J Biol Chem 274:15978–15981. 4. Tribouley C, Wallroth M, Chan V, Paliard X, Fang, Lamson G, Pot, Escobedo J, Williams LT (1999) Characterization of a new member of the TNF family expressed on antigen presenting cells. Biol Chem 380:1443–1447. 5. Gross J, Johnston J, Mudri S, Enselman R, Dillon S, Madden KXW, Parrish-Novak J, Foster D, Lofton-Day C, Moore M, Littau A, Grossman A, Haugen H, Foly K, Blumberg H, Harrison K, Kindsvogel W, Clegg C (2000) TACI and BCMA are receptors for a TNF homologue implicated in B-cell immune autoimmune disease. Nature 404:995–999. 6. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, Soppet D, Charters M, Gentz R, Parmelle D, Li Y, Galperina O, Giri J, Roschke V, Nardelli B, Carrell J, Sosnovtseva S, Greenfield W, Ruben SM, Olsen HS, Fikes J, Hilbert DM (1999) BLyS: a member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260–263. 7. Marsters SA, Sheridan JP, Piiti RM, Brush J, Goddard A, Ashkenazi A (1998) Identification of a ligand for the death-domaincontaining receptor Apo3. Curr Biol 8:525–528. 8. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Davis-Smith T, Rauch C, Smith CA, Goodwin RG (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3:673–682. 9. Mauri DN, Ebner R, Montgomery RI, Kochel KD, Cheung TC, Yu GL, Ruben TC, Murphy M, Eisenberg RJ, Cohen GH, Spear PG, Ware CF (1998) LIGHT, a new member of the TNF superfamily, and lymphotoxin alpha are ligands for herpesvirus entry mediator. Immunity 8:21–30. 10. Wong BR, Josien R, Lee SY, Sauter B, Li HL, Steinman RM, Choi Y (1997) TRANCE (tumor necrosis factor (TNF)-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 186:2075–2080. 11. Yasuda H, Shiman N, Nakagawa N, Yamaguchi K, Kinisaki M, Mochizuki SI, Tomoyasu A, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahasi N, Suda T (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602. 12. Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, Tschoop J, Browning JL (1999) Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med 190:1697–1710. 13. Khare SD, Sarosi I, Xia XZ, McCabe S, Miner K, Solovyev I, Hawkin N, Kelley M, Chang D, Van G, Ross L, Delaney J, Wang
L, Lacey D, Boyle WJ, Hsu H (2000) Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad Sci USA 97:3370–3376. 14. Armitage RJ (1994) Tumor necrosis factor receptor superfamily and their ligands. Curr Opin Immunol 6:407–413. 15. Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308. 16. Gravestein LA, Borst J (1998) Tumor necrosis factor superfamily members in the immune system. Seminar Immunol 10:423– 434. 17. Wajant H, Grell M, Scheurich P (1999) TNF receptor associated factors in cytokine signaling. Cytokine & Growth Factor Rev 10:15–26. 18. Karpusas M, Hsu YM, Wang JH, Thomson J, Lederman S, Chess L, Thomas D (1995) 2A crystal structure of an extracellular fragment of human CD40 ligand. Structure 3:1031–1039. 19. Eck M, Sprang SR (1989) The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding. J Biol Chem 264:17595–17605. 20. Jones EY, Stuart DI, Walker N (1989) Structure of tumor necrosis factor. Nature 338:225–228. 21. Banner D, D’Arcy A, Janes W, Gentz R, Schoenfeld HJ, Broger C, Loetscher H, Lesslaur W (1993) Crystal structure of the soluble 55kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 73:431–445. 22. Al-Shamkhani A, Mallett S, Brown MH, James W, Barclay AN (1997) Affinity and kinetics of the interaction between solublew trimeric OX40 ligand, a member of the tumor necrosis factor superfamily, and its receptor OX40 on activated T cells. J Biol Chem 272:5275–5282. 23. Yoshie O, Tada K, Ishida N (1986) Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors. J Biochem 100:531–541. 24. Tsujimoto M, Feinman R, Kohase M, Vilcek J (1986) Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells. Archives of Biochemistry and Biophysics 249:563–568. 25. Pennica D, Lam VT, Mize NK, Weber RF, Lewis M, Fendly BM, Lipari MT, Goeddel DV (1992) Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization and receptor phosphorylation. J Biol Chem 267:21172–21178. 26. Bennett GL, Horuk R (1997) Iodination of chemokines for use in receptor binding analysis. Methods Enzymol 288:34–148. 27. Loetscher H, Schlaeger EJ, Lahm HW, Pan YC, Lesslauer W, Brockhau M (1990) J Biol Chem 265:20131–20138. 28. Peitsch MC, Tschopp J (1995) Comparative molecular modelling of the Fas-ligand and other memebers of the TNF family. Mol Immunol 32:761–772. 29. Clay FJ, McEwen SJ, Bertoncello I, Wilks AF, Dunn AR (1993) Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila. Proc Natl Acad Sci USA 90:4882–4886. 30. Lee K, Grenfell TZ, Yarm FR, Erikson RL (1998) Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk. Proc Natl Acad Sci USA 95:9301–9306. 31. Lin JX, Leonard WJ (1997) Signaling from the IL-2 receptor to the nucleus. Cytokine and Growth Factor Reviews 8:313–332. 32. von Bulow GU, Bram RJ (1997) NF-AT activation by a CAML-interacting member of the tumor necrosis superfamily. Science 278; 138–141. 33. Madry C, Laabi Y, Callebaut I, Roussel J, Hatzoglou A, Le Coniat M, Mornon JP, Berger R, Tsapis A (1998) The characterization of murine BCMA gene defines it is anew memebr of the tumor necrosis factor receptor superfamily. Int Immunol 10:1693–1702.
BLyS BINDS TO B CELLS WITH HIGH AFFINITY AND INDUCES ACTIVATION OF THE TRANSCRIPTION FACTORS NF-B AND ELF-1 Palanisamy Kanakaraj, Thi-Sau Migone, Bernardetta Nardelli, Stephen Ullrich, Yuling Li, Henrik S. Olsen, Theodora W. Salcedo, Thomas Kaufman, Erika Cochrane, Yuxiang Gan, David M. Hilbert, Judith Giri B lymphocyte stimulator (BLyS) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms. BLyS was shown to act specifically on B cells, inducing proliferation and immunoglobulin production both in vitro and in vivo. The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding. Similar to other TNF family members, BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis. BLyS binding to B cells is specific as other TNF family members tested did not compete for 125I-BLyS binding. Analysis of equilibrium binding of 125I-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding. Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant (KD) of about 0.1 nM. In addition we report that BLyS binding to B cells results in the activation of NF-B and the Ets family transcription factor, ELF-1, and in the induction of mRNA for Polo-like kinase (PLK). 2001 Academic Press
B lymphocyte stimulator (BLyS), also identified independently as TALL-1,1 BAFF,2 THANK,3 TNFS204 and zTNF4,5 is a new member of an ever growing TNF superfamily of proteins.6 Members of this family including TNF-, TNF-, FasL, CD40L, OX40L, lymphotoxin-, TRAIL/Apo-2L, CD27L, CD30L, 4-1BBL, TRANCE/RANKL, Light, TWEAK, TL-1 and APRIL, are expressed on a variety of cells and mediate pleiotropic cellular responses including proliferation, apoptosis and immune regulation.7–11 Like other TNF family proteins, with the exception of TNF-/LT-, BLyS is a type II From the Human Genome Sciences, Inc., Rockville, MD 20850, USA Correspondence to: Dr Palanisamy Kanakaraj, 9410 Key West Avenue, Human Genome Sciences, Inc., Rockville, MD 20850, USA. E-mail: [email protected] Received 27 July 2000; accepted for publication 28 July 2000 2001 Academic Press 1043–4666/01/010025+07 $35.00/0 KEY WORDS: B lymphocyte stimulator (BLyS)/ signaling/ binding/ transcription factors/ tumour necrosis factor (TNF) Abbreviations: TNF, tumour necrosis factor; TNFR, tumour necrosis factor receptor; TRAF, tumour necrosis factor associated factor; BLyS, B lymphocyte stimulator; DSS, disccinimidyl suberate; SDS, sodium dodecyl sulfate; PAGE, poly acrylamide gel electrophoresis; PLK, Polo-like kinase; ERK, extracellular signal regulated kinase CYTOKINE, Vol. 13, No. 1 (7 January), 2001: pp 25–31
transmembrane protein expressed as a membranebound form on monocytic cells that is also cleaved and released by proteases as a soluble protein.2,6 Recent studies indicate that BLyS binding is restricted to B cells.2,6 Both the membrane and soluble forms of BLyS are biologically active in promoting proliferation of B cells treated with anti-IgM.2,6 Animals injected with soluble recombinant BLyS exhibit disrupted splenic T and B cell zones with increased B cell counts and immunoglobulin levels.6 Mice transgenic for BLyS exhibit autoimmune lupus-like characteristics with high levels of rheumatoid factors, immune complexes, anti-DNA auto-antibodies and increased numbers of B and effector T cells.12,13 Taken together these results demonstrate that BLyS plays a major role in regulating B cell immune responses. The receptors for TNF family proteins also belong to a conserved family of proteins called the TNF receptor superfamily with homologous extracellular cysteine rich domains which form specific linear ligand binding structures (reviewed in ref. 14). The TNFR family proteins can be classified in to two types, based on the presence or absence of a conserved cytoplasmic domain responsible for apoptosis called death domain. Although receptors with death domains in some cases 25
RESULTS AND DISCUSSION
– 17 kDa
– 44 kDa
0.50
0.40
0.30 AU
have been shown to induce cell proliferation and/or survival signals, death domains play an essential role in mediating apoptosis via activation of caspases (15, review ref. 16). Receptors without death domains such as TNF receptor-2, HVEM/ATAR, RANK, CD27, CD30, CD40 and OX40 interact with TNF receptorassociated factors (TRAF 1–6) and mediate antiapoptotic survival or/and proliferative responses via activation of the transcription factor NF-B (review ref. 17). Three dimensional structural analysis of the TNF family proteins CD40 ligand,18 TNF-19,20 and TNF-21 show that these ligands form homotrimers and interact with their cognate receptors with a stoichiometry of one trimeric ligand to three receptors. With the exception of OX4022 and TNF23–25 detailed biochemical analysis of binding of the other TNF family ligands have not been reported. Here, using 125 I-labeled BLyS we demonstrate binding characteristics of BLyS to cell surface receptors on B cells. Our studies also show that activation of transcription factors NF-B and ELF-1 may play a role in BLySmediated B cell responses.
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– 200 kDa – 158 kDa
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0.20
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0.00
2.00
4.00
6.00
8.00
10.00
Time (min) Figure 1.
BLyS forms a homotrimer.
Size exclusion HPLC analysis of BLyS. Purified BLyS protein is analyzed by SEC-HPLC using a Waters Protein-Pak HPLC column (7.8300 mm) equilibrated in 50 mM sodium acetate, 150 mM sodium chloride at pH 6.0. The column is calibrated with a gel filtration standards (Bio-Rad) which consists of thyroglobulin (MW 670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa) and vitamin B-12 (1.35 kDa).
BLyS is present as a homotrimer in solution X-ray crystallographic structural studies of TNF- complexed with its receptor showed that TNF- is present as a homotrimer and that one molecule of trimeric TNF- interacts with three receptor molecules presumably resulting in multimerization of the TNF receptor on the cell surface.21 It was predicted by molecular modeling that all TNF proteins except NGF form trimeric complexes.27,28 We determined the molecular size of BLyS by gel filtration. The data (Fig. 1) showed that BLyS migrated as a single peak with an apparent molecular weight of 44–46 kDa. The molecular weight of monomeric soluble BLyS determined by SDS-PAGE was 17.4 kDa. The difference between the predicted apparent molecular weight of 52 kDa of trimeric BLyS and the molecular mass of the gel filtration peak may be due to folding of the native form of BLyS which migrates differently in gel filtration when compared to the denatured protein on SDS PAGE. The light scattering analysis of the samples from size exclusion chromatography revealed a major peak (96.5% area) corresponding to trimeric BLyS with molecular weight of 54 500 g/mole (data not shown). The trimeric form of BLyS was also confirmed by crosslinking studies using 125I-labeled BLyS. BLyS was iodinated by using iodobead method as described in Materials and Methods. The iodination procedure did not affect the biological activity of BLyS as determined by costimulatory activity of mock iodinated BLyS in a B cell proliferation assay (data not shown).
DSS crosslinking of 125I-labeled BLyS showed three bands of molecular weights corresponding to monomeric 17.4 kDa, dimeric 35 kDa and trimeric 52 kDa forms of BLyS by SDS-PAGE. No band beyond 52 kD was observed in the presence of crosslinking agent indicating that there was no multimerization of trimeric BLyS (data not shown). Monomeric and dimeric forms of BLyS in the presence of DSS crosslinking were due to partial crosslinking of BLyS molecules.
Number and affinity of BLyS binding sites on B lymphocytes Saturation binding analysis was performed on tonsillar B cells using varying concentrations of 125Ilabeled BLyS to determine affinity (KD) and number of binding sites (Fig. 2A). The BLyS binding to B cells was saturable and Scatchard plot analysis of the binding data revealed a single high affinity site with a KD of 0.1 nM. The estimated number of binding sites was approximately 2600 per cell. 125I-labeled BLyS binding experiments, were also performed using a variety of human and murine B cells. The binding affinity (KD) and number of binding sites were determined as shown in Table 1. Comparison of the BLyS binding affinity to mouse and human cells indicate that 125I-BLyS binds with similar high affinity to both human and murine cells.
BLyS binding and signaling in B cells / 27
30 000
TABLE 1. B cells
A
Binding of 125I-labeled BLyS to human and mouse
Cells (nM) Tonsillar B cells IM-9 Raji Mouse B cells A20 BCL-1
100 Bound/free
Counts (cpm)
20 000
10 000
75 50
2.5
1
5.0 Bound 2
7.5 3
4
B
Iodinated BLyS bound (%)
100
80
60
2600 3200 1702 179 778 4800
family proteins LIGHT, TNF- and FasL. As shown in Figure 2B, 125I-labeled BLyS binding to IM-9 cells was displaced only by unlabeled BLyS, but not by other TNF family proteins indicating that BLyS binds to a specific receptor on the B cell surface not shared by other TNF family proteins tested. The specific binding of 125I-labeled BLyS was time and temperature dependent. Binding occurred very fast following addition of 125 I-labeled BLyS; approximately 40% of maximal binding was observed after 5 min incubation at 24C and 4C. Moreover, binding reached a plateau level after 60 min incubation at both temperatures (data not shown).
40
Crosslinking of 125I-labeled BLyS to B cells and identification of BLyS binding protein(s)
20
0 0.01
0.1
1
10
100
Unlabeled cytokines (nM) Figure 2.
0.1 0.19 0.16 0.35 1.1 0.93
10.0
I125-labeled BLyS (nM) 120
Binding sites/cell
25 0
0
Dissociation constant KD
Characterization of
125
I-labeled BLyS binding to B cells.
A: Direct saturation binding of 125I-labeled BLyS. The binding was performed in 2106 tonsillar B cells with increasing concentrations of 125I-labeled BLyS alone or in the presence of 100-fold excess of unlabeled BLyS. The specific binding at each concentration of 125 I-labeled BLyS was determined. The Scatchard analysis of the binding data was shown in the inset. B: Specificity of BLyS binding. 1106 IM-9 cells in triplicates were incubated with 1 nM 125Ilabeled BLyS in absence or presence of 100-fold excess of different TNF family cytokines for 2 h at RT as indicated. The binding was expressed as percentage of total 125I-BLyS bound to the cells in the presence of unlabeled proteins. BLyS, – –; LIGHT, – –; TNF-, – –; FasL, – –.
Specificity of
125
I-labeled BLyS binding to B cells
BLyS is a strong proliferative factor for primary B cells, but it does not appear to affect functional activities of other peripheral blood leukocytes including resting T cells and monocytes. Accordingly, biotinylated BLyS was reported to bind specifically to B cells but not to other cells.6 We determined the specificity of 125I-labeled BLyS binding using a competitive binding assay (Fig. 2B) in absence or presence of 100-fold excess of unlabeled BLyS and other TNF
To characterize better the BLyS binding site on B cells, we performed DSS crosslinking studies of 125Ilabeled BLyS to B cells. Separation of the resulting crosslinked complexes by SDS-PAGE showed protein bands corresponding to 50, 35, and 17 kDa (Fig. 3A). These bands are mostly likely the crosslinked BLyS trimer, partially crosslinked dimer and non-linked monomeric forms of BLyS. The diffused higher molecular weight band of 140–200 kDa may be the result of crosslinking of dimeric and trimeric BLyS to one or more molecules of the BLyS receptor. There were no crosslinked proteins visible with DSS and 125 I-labeled BLyS in K562 or in the presence of excess of cold BLyS indicating that BLyS binds to a specific receptor on B cells. BLyS ligand-affinity column was used to identify protein(s) solubilized from Raji and normal human B cells that bind BLyS. Control (NKEF-C) and BLyS affinity columns were used to analyze the proteins present in Raji cell extracts that specifically bind BLyS and are likely to represent the receptor. The proteins bound to the control or BLyS affinity column were examined for their ability to bind BLyS by subjecting them to SDS-PAGE analysis under non-reducing conditions followed by blotting onto Problot membranes; the blots were incubated with biotinylated-BLyS and
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A
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K562
BLyS (100¥)
+
bound biotinylated-BLyS detected with alkalinephosphatase-streptavidin. Two major bands were visualized having molecular masses of 50 and 40 kDa (Fig. 3B, lane 4); there was only background binding to the control NKEF-C affinity column fractions (Fig. 3B, lane 2). The absence of binding observed from the mock-eluted BLyS affinity column fractions (Fig. 3B, lane 6) ruled out any binding of biotinylated BLyS to BLyS leaching from the affinity column via subunit exchange. A similar experiment was performed using tonsillar B cells pre-activated with SAC for 3 days to detect the BLyS binding proteins. The major molecular mass of the BLyS binding proteins was 50 and 40 kDa, similar to that observed in Raji cells (data not shown). Thus both Raji and human B cells contain BLyS binding proteins of 40 and 50 kDa which appear to represent the B cell BLyS receptor and/or proteins interacting with the BLyS-receptor complex.
IM-9
+
250 148
60
42
22 17
BLyS induced signaling in B cells B Mr kDa 200
98 64 50
36
30
21 1
2
3
4
5
6
125
Figure 3. DSS crosslinking of I-labeled BLyS and identification of BLyS binding proteins in B cells. A: IM-9 and K562 cells were incubated with 125I-labeled BLyS in the absence or presence of excess of unlabeled BLyS and crosslinked with DSS for 25 min at room temperature. The cell lysates were prepared and resolved on a 10% SDS-PAGE. The gel was dried and subjected to autoradiography. B: The Raji cell lysates were prepared and affinity purified through BLyS affinity column as described in Materials and Methods. Affinity column fractions were analyzed by 12% SDS-PAGE under non-reducing conditions, transferred and blots incubated with biotinlyated-BLyS: eluates from control (lane 2) and BLyS (lane 4 ) affinity columns incubated with Raji lysate or mock-eluted BLyS affinity column (lane 6). Lane 1, pre-stained molecular weight standards; lanes 3 and 5, biotinylated molecular weight standards.
To examine the intracellular mechanisms triggered by BLyS receptor binding, tonsillar B cells treated with BLyS in the presence of SAC were subjected to real time PCR (Taqman analysis) to determine the induction of mRNA of a panel of proteins known to play an important role in the activation of lymphoid cells. Among the proteins analyzed, we consistently found that dramatic increase in induction of mRNA for Polo-like kinase (PLK) (Fig. 4A) and two-fold increase of Erk-1 mRNA expression (not shown) in B cells. PLKs belong to a sub family of serine/threonine kinases related to Saccharomyces cerevisiae cell cycle protein CDC5.29 The expression of PLK is induced during G2 and S phases of the cell cycle. PLK is reported to play a role in cell proliferation.30 The role of extracellular signal regulated kinases (ERK1/2) in cell survival and proliferative effects of growth factors and other agonists has been extensively studied. The induced expression of PLK and ERK-1 is consistent with the survival and proliferative effect of BLyS on B cells. In addition to ERK-1 and PLK, mRNA for CD25 (IL-2R) was found to be upregulated in some of the donors we examined. The CD25 promoter has been extensively studied (reviewed in ref. 31), and three positive regulatory regions (PRR) that regulate the transcriptional activation of CD25 have been described. PRRI has binding sites for NF-B and SRF, while PRRII has been shown to bind ELF-1 and HMGI/Y proteins. Both promoter regions play a crucial role in the induction of CD25 during T cell activation. Finally, PRRIII has binding sites for STAT5/ and ELF-1 and has been shown to be important for IL-2-induced upregulation of CD25.
BLyS binding and signaling in B cells / 29
B
60
C
IM-9 cells
B cells
A
Fold increase
50 PLK
40 30 20 10 0 Blys
+
Blys
+
ELF-1 Figure 4.
+
NFkB/SRF
+
ELF-1
+
NFkB/SRF
BLyS induced signaling in B cells.
A: Induction of PLK mRNA expression. Total RNA was prepared from tonsillar B cells unstimulated or stimulated with SAC or SAC plus BLyS for 12 h. PLK mRNA detected by a one step RT-PCR procedure using specific primers and probes. Expression levels of mRNA shown are relative to expression levels in unstimulated B cells. B: IM-9 cells or primary tonsillar B cells (C) were serum starved for 16 h and treated or not treated with 100 ng/ml soluble BLyS for 45 min at 37C. The nuclear extracts were prepared and incubated with 32P-labeled NF-B/SRF or ELF-1-specific double-stranded oligonucleotides. The protein–DNA complexes were separated on 6% polyacrylamide gel in 0.5% Tris-borateEDTA buffer.
We analyzed nuclear extracts from primary B cells and B cell lines by EMSA and found no significant induction using a PRRIII probe (data not shown). This is consistent with the fact that BLyS does not appear to induce signaling via tyrosine phosphorylation of the Jak/STAT pathway (data not shown). In contrast we found induction of DNA-binding activity to the probes corresponding to PRRI and II in IM-9 (Fig. 4B) and primary tonsillar B cells (Fig. 4C). NF-B has been shown to be induced by a number of members of the TNFR family proteins upon ligand binding and suggests that the receptor for BLyS may also belong to this family. ELF-1 is a transcription factor that is part of the ETS family of proteins and whose expression appears to be restricted to T and B cells. Binding sites for ELF-1 have been described in the promoters of a number of proteins that are important in the regulation of the immune response. In this paper we show that, as reported for other TNF family proteins, BLyS exists as a trimer and the trimeric form of BLyS binds to a single high-affinity site on B cells. The costimulatory activity of BLyS in inducing proliferation of B cells along with the result presented here in induction of PLK and ERK-1 expression and activation of transcription factors NF-B and
ELF-1 suggest that BLyS receptor may belong to a category of non-death domain containing TNF family receptors involved in survival, proliferation and other immunoregulatory effects. While this manuscript was under preparation, Gross et al.5 reported that TACI32 and BCMA33 are receptors for BLyS. The reported high affinity binding of BLyS to TACI and TACI induced activation of NF-B are in line with our observation.
MATERIALS AND METHODS Expression and purification of recombinant BLyS Recombinant BLyS was expressed in baculovirus and purified by chromatography using a combination of ion exchange (Poros HS-50, Poros PI-50; PE Biosystems, Framingham, MA, USA), size exclusion (Sephacryl S100 HR; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and hydrophobic interaction (Toyopearl Hexyl 650 C; Tosohaas, Montgomeryville, PA, USA) columns. The purity of BLyS was determined as described.6
Preparation of
125
I-labeled BLyS
Radio-iodination of BLyS was performed using the Iodobead method as described.26 Briefly, one Iodobead per
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reaction was pre-washed with PBS and added to 1 mCi of 125 NaI in 80 l of PBS pH 6.5. The reaction was allowed to proceed for 5 min and then 10 g of BLyS (1 mg/ml stock) was added and incubated for 5 min at room temperature. The iodinated protein was separated from unbound radioactivity using a G-25 Sephadex quick spin column previously equilibrated with PBS containing 0.1% BSA. Protein concentration and specific radioactivity of 125I-labeled BLyS were determined by TCA precipitation of pre-column and post-column samples. The specific activity of 125I-BLyS used in the experiment was 900–1600 Ci/mmol (17.5–34.6 Ci/g).
Preparation of tonsillar B cells Primary B cells were purified from tonsils by depleting CD3 + cells using magnetic bead (MACS) column. B cell preparations were >95% pure as determined by flow cytometric analyses of CD19 and CD20 expression.
Binding analysis Direct binding of radiolabeled BLyS was performed in triplicates in a 96-well plate using 2106 cells in 100 l of binding buffer (Ham’s F containing 0.5% BSA and 0.1% sodium azide) with indicated concentrations of 125I-labeled BLyS. The non-specific binding of BLyS to cells was determined in the presence of 100-fold excess of unlabeled BLyS in the binding mixture. The competitive binding was carried out using 0.3 nM of 125I-labeled BLyS in the absence or presence of varying concentrations of unlabeled BLyS. The binding reaction was performed at room temperature for 2 h on a shaker platform. We have previously determined that the incubation of 125I-labeled BLyS for 2 h at 25C was sufficient to reach equilibrium binding. After the incubation, the cell-bound BLyS was separated from unbound free 125Ilabeled BLyS by centrifugation through 200 l of 1.5 dibutylphthlate/1.0 bis (2-ethyl-hexyl) phthalate oil mixture in a polyethylene microfuge tubes (Bio-Rad, Hercules, CA, USA) for 20 s at 11 750g. The microfuge tubes were then frozen quickly in liquid nitrogen and the bottom tip of the tubes was cut off using a tube cutter. Radioactivity in the bottom containing the cell pellet (bound fraction) and the top (unbound fraction) of the tubes were counted by using a gamma counter. The binding was analyzed by Prizm (GraphPad Software, San Diego, CA, USA) to determine the dissociation constant (KD) and number of binding sites.
Crosslinking of
125
I-labeled BLyS to B cells
IM-9 or K562 cells (20106/200 l of binding buffer) were incubated with 125I-labeled BLyS (1 nM) for 2 h at room temperature in the absence or presence of excess unlabeled BLyS. After incubation, the cells were washed twice with ice-cold PBS and resuspended in 1 ml of PBS. DSS (1 mM) was added and incubated at room temperature for 30 min. The crosslinking reaction was terminated by addition of 5 mM ammonium acetate. Cells were then lysed in 200 l of 1% NP40 lysis solution (10 mM HEPES pH 7.5, 0.15 M NaCl, 10% glycerol, 10 g/ml aprotinin, 10 g/ml leupeptin and 1 mM PMSF). The proteins were separated by 10% SDS-PAGE and results were developed by autoradiography.
Ligand blot analysis of BLyS binding protein(s) from Raji cells The proteins, NKEF-C and BLyS, were dialyzed against 25 mM HEPES buffer, pH 7.2; the affinity columns were then prepared by mixing the dialyzed proteins with Affi-gel-15 (Bio-Rad) at room temperature for 3 h (0.8 mg BLyS/0.5 ml resin). Columns were extensively washed according to manufacturer’s directions. Raji cells (17109) were washed three times in PBS and then solubilized in 30 mM HEPES buffer, pH 7.5, 0.15 M NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM Pefabloc, 1 g/ml leupeptin, 10 g/ml aprotinin, 1.5 M pepstatin, 5 g/ml E64, and 1 mM EDTA. After 15 min on ice the lysate was centrifuged at 3000g for 20 min. The supernantant was centrifuged at 10 000g for 20 min and loaded first over control NKEF-C affinity column. The flow through was then loaded over the BLyS affinity column. The wash columns were washed with with 30 mM HEPES buffer, pH 7.5, 0.15 M NaCl. The bound material was eluted from the columns with 10 mM HCl collected in 0.5 ml fractions and neutralized immediately with TRIS buffer. The eluates were further concentrated using an Ultra-free MC 10 000 MW cut-off filtration unit to a volume of 50–100 l. The affinity purified proteins were analyzed under non-reducing conditions on 12% SDS-PAGE and blotted onto Problot membrane (ABI).26 The blots were blocked by incubation with blocking buffer [0.5 mg/ml of BSA in PBS containing 0.05% (V/V) Tween 20] for 30 min. The BLyS binding proteins were detected by incubating the blots in the presence of 1 g/ml biotinylated-BLyS for 1 h in blocking buffer. After washing with blocking buffer the blots were incubated for 45 min with 0.5 g/ml of streptavidin– alkaline phosphatase conjugate (KPL, Gaithersburg, MD, USA) diluted in blocking buffer. The blot was washed three times with 10 ml PBS with 0.05% Tween-20. Bound BLyS was detected by development with BCIP/NBT substrate (KPL, Gaithersburg, MD, USA).
Electrophoretic mobility shift assay (EMSA) Primary tonsillar B cells or IM-9 cells were serum starved for 16 h and treated or not treated with 100 ng/ml soluble BLyS for 45 min at 37C. The nuclear extracts were prepared in high salt buffer (20 mM Hepes pH 7.9, 1 mM EDTA, 420 mM NaCl, 15% glycerol and protease and phosphatase inhibitors). Extracts were incubated on ice in the presence of phosphatase and protease inhibitors and a 32Plabeled probe corresponding to the positive regulatory regions present in the IL-2R alpha promoter. Reaction mixtures were separated on 6% acrylamide gels in Trisbuffered EDTA, dried, and autoradiographed.
Quantitative RT-PCR analysis Total RNA was prepared from unstimulated or SACand SAC+BLyS-stimulated tonsillar B cells. Messenger RNA levels of PLK and ERK-1 B cells were determined by real time quantitative PCR using a ABI 7700 Taqman Sequence Detector. Amplification primers and probe were designed to span the region from nucleotides 252 to 332 of the human PLK sequence and nucleotides 373 to 446 of the human ERK-1 mRNA (Genbank accession no. X75932 and
BLyS binding and signaling in B cells / 31
no. X60188). For quantitation of mRNA levels the comparative Delta Ct method was used (Perkin-Elmer user bulletin no. 4, 1997) using a 18S ribosomal RNA probe as endogenous reference. Expression levels are shown relative to observed levels in unstimulated B cells.
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