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IMMUNOBIOLOGY
A splice variant of human ephrin-A4 encodes a soluble molecule that is
secreted by activated human B lymphocytes
Hans-Christian Aasheim, Else Munthe, Steinar Funderud, Erlend B. Smeland, Klaus Beiske, and Ton Logtenberg
Ephrin-A4 is a ligand for the erythropoietin-producing hepatocellular (Eph) receptor family of tyrosine kinases. We have
identified a secreted form of ephrin-A4,
denoted ephrin-A4 (s), which is encoded
by an alternatively spliced mRNA and is
produced by in vivo activated B cells in
tonsils. Blood B cells secrete ephrin-A4
(s) upon stimulation via the B-cell antigen
receptor. A subpopulation of tonsil cells
in the crypts with a dendritic cell phenotype was shown to express EphA2, an
Eph receptor tyrosine kinase that was
found to be capable of binding an eph-
rin-A4 immunoglobulin chimeric protein.
We conclude that ephrin-A4 (s) may play
a role in the interaction between activated
B lymphocytes and dendritic cells in
human tonsils. (Blood. 2000;95:221-230)
r 2000 by The American Society of Hematology
Introduction
The development of hematopoietic cells involves the commitment
and differentiation of self-renewing pluripotent stem cells into
mature cells of various lineages including B lymphocytes. In the
human bone marrow, discrete stages of B lymphopoiesis can be
discerned based on the ordered loss and acquisition of B–lineagespecific proteins and the state of rearrangement and expression of
immunoglobulin (Ig) genes. Mature B cells that express a membrane-bound IgM receptor leave the bone marrow and migrate to
peripheral lymphoid organs. Here, upon contact with an antigen, B
cells may enter a second round of clonal expansion and differentiation, resulting in the formation of antibody-secreting plasma cells
or memory B cells. These processes are controlled by interactions
between differentiating B lymphocytes, B cells, and soluble
molecules in the microenvironment. Although a number of key
membrane-bound and soluble molecules have been identified in
recent years, it has also become apparent that additional and as yet
unknown ligands and receptors play a role in early and late B-cell
differentiation processes.1-6
Receptor tyrosine kinases and their ligands play a critical role in
regulating cellular survival, proliferation, and differentiation.7 On
the basis of predicted structural homologies, sequence conservation, and similarity of ligands, receptor tyrosine kinases have been
assigned to several subclasses.8 One subclass, the erythropoietinproducing hepatocellular (Eph) carcinoma family of receptors,
constitutes the largest known family of receptor tyrosine kinases.
The Eph family of receptors comprises at least 14 distinct
members,9,10 from Xenopus to man,9,11-16 that are highly conserved.
Recently, a family of at least 8 membrane-bound ligands for Eph
receptors, termed ephrins, has been identified.17 Members of this
family share between 23% and 56% identity at the amino acid level
and display promiscuous binding to different Eph receptors.18
Efficient activation of Eph receptors by ephrins requires anchor-
ing the ligands to the cell membrane, either through a hydrophobic
transmembrane region or a glycosyl phosphatidylinositol (GPI)
group.19 Interestingly, membrane-bound ephrins may transduce
signals upon interaction with their cognate receptor.20 Signaling via
Eph receptors and their ligands has been implicated in axon
guidance and fasciculation, regulation of cell migration, and
definition of compartments in the developing embryo.17,21 In
addition, Eph receptors appear to play a role in angiogenesis,22 fetal
human B lymphopoiesis,23 and erythropoiesis.24
The recent notion that Eph receptors and their ligands may be
selectively expressed in subpopulations of hematopoietic cells
prompted us to search for expression of members of the ephrin
family of ligands in human B-lineage cells. Here we report the
identification of a splice variant of the ephrin-A4 gene, ephrin-A4
(s), which encodes a secreted form of this ligand. Soluble
ephrin-A4 is produced by mature B cells in the tonsil and by blood
B cells that are activated in vitro via their B-cell antigen receptor.
An ephrin-A4 binding tonsillar cell subpopulation with a dendritic
cell phenotype was shown to express the Eph-A2 receptor, 1 of 3
Eph receptors known to autophosphorylate upon ephrin-A4 binding.18 These results suggest that Eph receptors and their ligands
play a role in interactions between activated B lymphocytes and
other cell types in the microenvironment.
From the Departments of Immunology and Pathology, Institute for Cancer
Research, The Norwegian Radium Hospital, Oslo, Norway, and the Department of Immunology, University Hospital Utrecht, Utrecht, The Netherlands.
submitted to the European Bioinformatic Institute nucleotide sequence database under the accession numbers AJ006352 and AJ006353, respectively.
Submitted December 16, 1998; accepted August 28, 1999.
Materials and methods
Cell separation procedures
Tonsils, obtained from children undergoing routine tonsillectomy, were
minced, and mononuclear cells (MNC) were purified by Lymphoprep
(Nycomed Pharma, Oslo, Norway) density gradient centrifugation. In some
experiments, tonsillar MNC were depleted of T cells by 2 rounds of
Reprints: Hans-Christian Aasheim, PhD, Department of Immunology, Institute
for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway;
e-mail: [email protected].
H. C. A. is a post-doctoral fellow at the Norwegian Cancer Society, and E. M. is
a doctoral student supported by the Norwegian Counsel of Research.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
The nucleotide sequences of ephrin-A4 (m) and ephrin-A4 (s) have been
r 2000 by The American Society of Hematology
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
221
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222
AASHEIM et al
rosetting with 2-aminoethyl-isothiouronium-bromide-treated sheep red blood
cells.25 In other experiments, B cells and T cells were isolated with
anti-CD19–coated or CD4-coated beads26 (Dynabeads; Dynal, Oslo, Norway). Detachment of beads from the cells was performed (DETACHaBEAD,
Dynal) at ambient temperature for 45 minutes.27 The cells were washed
twice in RPMI 1640 with 1% FCS before immunofluorescent staining.
For isolation of adherent cells, tonsils were minced and washed before
adding a collagenase solution (Collagenase/Dispase/DnaseI; Boehringer
Mannheim, Mannheim, Germany) followed by incubation for 15 minutes at
37°C. The solution was discarded, and fresh solution was added for an
additional 2 hours at 37°C. The cells were washed twice in phosphatebuffered saline (PBS), resuspended in RPMI 1640 with 5% FCS, and
seeded in tissue culture flasks coated with bovine collagen (Vitrogen;
Collagen Biomaterials, Palo Alto, CA). The cells were incubated overnight
and detached from the flasks with PBS/1 mmol/L EDTA.
Venous blood was obtained from healthy volunteers, and MNC were
obtained by Ficoll-Paque density centrifugation. CD4⫹ T cells or CD19⫹
B cells were isolated by magnetic bead separation, as described for the
tonsil cells.
Cell culture
Blood B cells were stimulated in RPMI 1640 medium containing 10% FCS
with anti-µ antibodies (F[ab8]2 fragment) (Dako, Glostrup, Denmark) at a
final concentration of 37.5 µg/mL fixed Staphylococcus aureus bacteria
(SAC 1/20 000; Calbiochem-Behring, Cambridge, England), 5 ⫻ 10-8
mol/L of the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), or
5% T-cell supernatant. T-cell supernatant is collected from a 24-hour PHA
stimulation of MNC from 5 different donors.28 CD4⫹ blood T cells were
stimulated with TPA as described for the B cells. CD4⫹ and CD8⫹ T cells
were stimulated with anti-CD3 coated beads (Dynal) at a concentration of 2
beads per cell for the indicated times.
Cell lines
The following human cell lines were used in this study: pre-B cell line Reh
(ATCC CRL 8286) and Nalm 6;29 mature-B cell lines Bjab (Dr G.
Moldenhauer, University of Heidelberg, Heidelberg, Germany) and Daudi
(ATCC CCL 213); plasmacytoid cell lines U266 (ATCC TIB 196); T cell
lines JM, Jurkat (ATCC TIB 152), and HPB ALL; myeloid cell lines KG1-A
(ATCC CCL 246); HL-60 (ATCC CCL 240) and U937 (ATCC CRL-1596);
erythroid precursor cell line K562 (ATCC CCL-243); and cervical carcinoma cell line HeLa (ATCC CCL 2). All cell lines were grown in RPMI
1640 medium supplemented with 5% FCS at 37°C in a humidified
atmosphere with 5% CO2.
RNA isolation, Northern blot analysis, and first strand
cDNA synthesis
Total RNA was extracted from cells or tissues by standard methods, and 10
µg was size-fractionated on a 1% agarose formaldehyde denaturing gel,
transferred to nitrocellulose membranes, and cross-linked by baking for 2
hours at 80°C. We used a commercially available multiple tissue Northern
blot (Immune blot 1; Clontech, Palo Alto, CA). Prehybridization (1 hour)
and hybridization were performed in hybridization buffer (5 ⫻ SSPE, 10%
dextran sulfate, 0.1% SDS, 50% formamide, 100 µg/mL sheared salmon
sperm DNA) at 42°C. The membranes were hybridized overnight with
either a 32P-dCTP–labeled ephrin-A4 cDNA probe or a control ␤-actin
probe. After hybridization, the membranes were washed under high
stringency in 0.2 SSC/0.1% SDS at 65°C. PolyA⫹ mRNA was isolated
from cells or tissues using oligo-dT beads,30 and first-strand cDNA was
synthesized directly on mRNA bound to oligo-dT beads,30 as previously
described. Finally the first-strand cDNA beads were washed twice in 100 µL
TE buffer, solved in 25 µL TE, and stored at -20°C.
Isolation and nucleotide sequence analysis of
ephrin-A4 cDNAs
An inventory of ephrin sequences was performed on first-strand cDNA
generated from mRNA isolated from the pro-B cell line Reh. The primers
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
used were based on the conserved amino acid sequences LY(L/M)V (primer
ephlig58: CGG ATC CGT (C/T/A/G)TA TA(T/C) ATG GT) and
(D/Y)YYY(S/T) (primer ephlig38: CGA ATT C(A/G)(A/T) (G/T/A)AT
(G/A)TC (G/A)(A/T)A (A/T/C/G)T(A/C)) present in ephrin-A1, ephrinA3, ephrin-A4, and ephrin-B1 sequences.31,32 PCR amplification was
performed on oligo-dT–immobilized cDNA reverse transcribed from 500
ng of mRNA, using 0.75 µg of each primer and 40 cycles of 1 minute at
94°C, 2 minutes at 37°C, and 3 minutes at 63°C. Amplified products with
the expected size (approximately 180 base pair [bp]) were isolated from
agarose gels, ligated into T-vector (Promega, Madison, WI), and used for
dideoxy sequencing with the sequenase system (Stratagene, La Jolla, CA).
The ephrin-A4 PCR fragment, obtained after cloning and sequence
analysis, was labeled with 32P-dCTP in a PCR reaction using the degenerate
primers ephlig38 and ephlig58. Fifty ng of ephrin-A4 PCR fragment was
used as template in a 50-µL reaction containing 0.05 mmol/L dATP, dGTP,
and dTTP; 5 µL 32P-dCTP (300 Ci/mmol/L; Amersham Pharmacia Biotech,
Uppsala, Sweden); and 250 ng of each degenerate primer. The PCR
conditions were 12 cycles with 1 minute at 94°C, 1 minute at 45°C, and 1
minute at 72°C. The resulting probe was used to screen an Reh cell line
cDNA library constructed in the expression vector pCDM8.34 Four cDNA
clones were isolated. All clones were sequenced from both ends using the
T7 primer and a pCDM8 specific reverse primer. Homology to known
sequences was assessed (Blast program; National Center for Biotechnology
Information, Bethesda, MD).
Amplification of ephrin-A4 transcripts
To detect both ephrin-A4 transcripts, we employed a semiquantitative PCR
approach using a forward primer common to both variants and reverse
primers specific for each splice variant. Primers to amplify ephrin-A4 (s)
were forward primer 1A: 58-GTG GAG CTG GGC CTC AAC GAT TAC
C-38 (nucleotides 169-186) and reverse primer 2: 58-GGA GAG GAA CCT
TCC CTC-38 (nucleotides 489-506 in ephrin-A4 (s) sequence) yielding a
PCR product of 337 bp. Primers to amplify ephrin-A4 (m) were forward
primer 1A (same as for ephrin-A4(s)) and reverse primer 3: 58 GAG TCA
GGC CAT CCT GTT G (nucleotides 500-520 in ephrin-A4 (m) sequence),
yielding a PCR product of 351 bp. The PCR conditions were 2 minutes at
94°C followed by 40-second cycles at 94°C, 56°C, and 72°C. As previously
described, semiquantitative PCR and control ␤-actin PCR using primers23
were performed.
The samples were separated on a 1.5% agarose gel, blotted to
nitrocellulose filters, and probed with 32P-dCTP labeled full-length ephrin-A4 cDNA probe or ␤-actin. Both ephrin-A4 (s) and ephrin-A4 (m)
amplified fragments were cut out of the gel, cloned into T-vector (Promega),
and subsequently sequenced to confirm the identity of the sequences.
Nucleotide sequence analysis
Two full-length cDNA clones (ephrin-A4 [m] and ephrin-A4 [s]) and a
genomic ephrin-A4 clone were sequenced in both directions after restriction fragment subcloning (pBluescript SK, Stratagene) with either T3, T7,
or ephrin-A4 specific primers. Double-stranded sequencing was performed
on plasmid DNA using T7 DNA polymerase (Amersham Pharmacia
Biotech), dideoxy nucleotides (USB, Cleveland, OH), and 35S-dATP
(NEN, Boston, MA). Database searches were performed using a network
service (NCBI).
In situ hybridization
A dioxygenin-(Dig)-11-dUTP labeled probe for in situ hybridization was
synthesized by PCR34 using the forward primer 1B 58-GGG CGA TGC
GGC TGC TGC, nucleotides 23-40 and reverse primer 3 (described above)
and the cloned ephrin-A4 (m) cDNA as a template. A negative control probe
was prepared by amplification of a fragment of the Echerichia coli
neomycin-resistant gene using specific primers and Dig-11-dUTP.
Frozen tissue sections of 5-6 µm were fixed in 4% formaldehyde,
washed, dehydrated, and blocked for endogenous peroxidase with 1.5%
H2O2 in methanol. mRNA was made accessible for the probes by treatment
with proteinase K (1 µg/mL) and Triton- ⫻ 100 (0.005% in PBS). Sections
were preincubated for 10 minutes at 42°C with a 25-µL hybridization
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BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
mixture (30% formamide, Tris/EDTA buffer, 4 ⫻ standard saline citrate, 1
µg/µL yeast tRNA, 1 µg/µL herring sperm DNA) (Boehringer Mannheim)
before the denatured probe was added for 18 hours at 37°C. After
hybridization, the slides were washed, and the probe was detected with a
monoclonal anti-Dig antibody (1:50 in PBS/1% BSA; Boehringer
Mannheim). The signal was detected with horseradish-peroxidaseconjugated swine antirabbit antibody (1:100 in PBS/1% BSA; DAKO,
Glostrup, Denmark) and visualized with diaminobenzidine with nickel
intensification.
Isolation of genomic ephrin-A4 clones
We screened 106 plaque-forming units from a human lymphocyte genomic
library in ␭-DASH (Stratagene) with 32P-dCTP labeled ephrin-A4 cDNA
insert. Hybridizing clones were plaque purified in subsequent screening
rounds. BamHI fragments were subcloned from selected phages (pBluescript SK, Stratagene) for nucleotide sequence analysis.
Preparation of ephrin-A4 specific antibodies
Antisera were made against a synthetic peptide corresponding to peptide
sequence SHPKEPESSQDPLEE (amino acid number 160-175), in a
specific part of ephrin-A4 (s) (Figure 1A), thus only recognizing
ephrin-A4 (s). Rabbits were initially immunized in the scruff with 300 µg
KLH-coupled peptide mixed with Freunds complete adjuvant. Animals
were boosted once a month with the antigen in Freunds incomplete
adjuvant. Serum was collected before each new booster round and screened
for specific antibodies in an enzyme-linked immunosorbent assay (ELISA).
Affinity-purified antibodies were obtained by running high-titer rabbit
antiserum through a column of ephrin-A4 specific peptide coupled to 4
NHS-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden).
Specific antibodies were eluted with 0.1 mol/L glycin/HCl pH 2.5 and
dialyzed against PBS.
Construction of chimeric proteins and binding to cells
An expression vector with the mouse IgG2b heavy chain constant region
was constructed. The IgG2b sequence, encompassing the hinge and CH2
and CH3 regions, was amplified by PCR using a genomic IgG2b fragment
(accession number v00 763.em_ro) as the template. BamHI and XhoI
restriction sites were included in the forward and the reverse primer
respectively (2bfor: 58 CCG GGA TCC GAG CCC AGC GGG CCC ATT
Figure 1. Ephrin-A4 cDNA clones. Amino acid sequences of ephrin-A4 (m) and ephrin-A4 (s) and ephrin-A4 gene structure. (A) Comparison of the amino acid
sequences of ephrin-A4 (m) and ephrin-A4 (s). Amino
acid numbering is depicted on the right. (B) Schematic
presentation of the exon-intron organization of the ephrin-A4 gene. Exons are boxed. The size of the exons in
bp are indicated by numbers in the boxes. Shaded
sub-box in exon IV denotes the part of the mRNA that is
spliced out in the ephrin-A4 (s) variant. *Denotes translation stop codon in the ephrin-A4 (m) sequence. **Denotes translation stop codon in the ephrin-A4 (s) sequence. (C) Sequences of exon-intron junction and the
size of the introns. Capital letters denote the exons, and
small letters denote the introns.
A SPLICE VARIANT OF HUMAN EPHRIN-A4
223
TC and 2brev: 58 GGC TCT AGA TGC AGG CAG AAA CCT CAT TC).
PCR products were digested with BamHI and XhoI and ligated into the
pCDNA1 vector (InVitrogen, Carlsbad, CA). Two chimeric proteins were
generated with this vector, ephrin-A4-Fc and CD19short-Fc. To generate
ephrin-A4-Fc, the common part of the 2 ephrin-A4 variants was amplified
(excluding the GPI-signal sequence or 38 end of ephrin-A4 (s)), using a
vector-specific primer (T7) and a reverse ephrin-A4 primer (Ephrinrev: 58
CCG GGA TCC AAC AGG GAT GGG CTG ACT) including a BamHI site.
The resulting product was cleaved with HindIII and BamHI and ligated in
pCDNA1. A construct for production of a control Fc-chimeric protein was
generated with the signal sequence and the first 30 amino acids of the CD19
molecule. This fragment of CD19 was amplified from CD19 cDNA in ␲iH3
vector35 using a vector-specific primer and a reverse CD19 primer
(CD19rev: 58 CCG CGG ATC CGG TCA GCT GCT GAG TGG G)
including a BamHI site. The resulting product was cleaved with HindIII and
BamHI and ligated into pCDNA1␥ 2b.
Plasmids encoding ephrin-A4-Fc or CD19short-Fc were transfected
into COS cells,33 and the chimeric proteins were purified from culture
supernatant by affinity chromatography on a protein-G column (Pharmacia). Integrity of the fusion proteins was confirmed by labeling transfected
COS cells overnight with 35S-methionine, purifying the fusion proteins
from the culture medium, and separating purified products on a 12% acryl
amide gel (data not shown).
Cells in solution (PBS/0.1% BSA/0.1% Na-azide) were preincubated
for 10 minutes with human aggregated IgG (Pharmacia) and then incubated
with 25 µg/mL fusion protein for 1 hour at 4°C. The cells were washed and
stained with a PE-labeled anti-mouse Ig polyclonal antibody (Ig-RPE;
Southern Biotechnology Associates, Birmingham, AL) directed to the Ig
tail of the fusion proteins. Double staining of the cells was performed with
the following FITC-labeled antibodies: anti-CD4, anti-CD11c, anti-CD13,
anti-CD21, anti-CD45, and anti HLA-DR (all from DAKO); anti-CD19
(Becton Dickinson, San Jose, CA); anti-CD31 and anti-CD86 (Pharmingen,
San Diego, CA); anti-CD38 and anti-CD34 (Coulter Immunotech, Pittsburgh, PA); and anti-CD40 (Caltag, Burlingham, CA). We also used
biotin-labeled anti-CD123 (Pharmingen).
Western blotting
Whole cell lysates were made from different fractions of tonsil adherent
cells and tonsil B and T cells. Ephrin-A4 binding cells in the tonsil adherent
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224
AASHEIM et al
fraction were isolated by first binding ephrin-A4-Fc fusion protein to rabbit
anti-mouse IgG2b-coated beads (Dynabeads, Dynal) followed by incubation of the tonsil adherent cells with these beads for 1 hour at 4°C before
washing the beads twice in PBS. CD34⫹ endothelial cells were isolated
from the ephrin-A4 depleted cells using anti-CD34 coated beads (Dynal) as
described for the ephrin-A4 binding cells. Cells depleted for ephrin-A4
binding cells and CD34⫹ cells are denoted rest adherent.
Ten µg of cell lysate were separated by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to nitrocellulose membranes. The membranes were hybridized with a polyclonal
anti–EphA-2 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) and
visualized (ECL system; Amersham).
COS cells were either transfected with plasmids encoding ephrin-A4
(m) or ephrin-A4(s) or mock-transfected.33 Supernatants and cells were
collected 4 days after transfection. The cells were lysed (PBS/1% NP-40),
and 10 µg of total cellular protein or 10 µL of culture supernatants was
separated (SDS-PAGE). Tonsil B and T cells were lysed, and 10 µg of
protein was separated (SDS-PAGE). All samples were blotted to nitrocellulose and hybridized with anti–ephrin-A4(s) antiserum and visualized
(Amersham).
B cells (1 ⫻ 105 cells in 200 µL RPMI 1640 with 10% FCS per well of a
96-well plate) were stimulated with anti-µ, SAC, and T-cell supernatants in
different combinations.
After 6 days of stimulation, culture supernatants were harvested, and 10
µL of supernatant were separated (SDS-PAGE) and blotted to nitrocellulose
filter. The filters were hybridized with biotinylated anti–ephrin-A4 (s)
antiserum and developed as described above.
Immunohistochemistry
Frozen sections of tonsil were fixed with methanol and pretreated with
H2O2 to block endogenous peroxidase activity. The sections were incubated
with anti-Eck (Eph-A2) antiserum (1/50, Santa Cruz Biotechnology) or
normal rabbit antiserum with the same concentration. After washing, the
cells were incubated with biotinylated goat anti-rabbit Ig (Vector Laboratories, Burlingham, CA). The signal was developed with avidin-peroxidase
(Vector Laboratories) and diaminobenzidine. The tissues were counterstained with hematoxylin.
Results
Isolation and characterization of ephrin-A4 cDNA clones
The recent notion that Eph receptors may be selectively expressed
in subpopulations of human B lymphocytes prompted us to search
for expression of members of the ephrin family of ligands for Eph
receptors in human B-lineage cells. RNA extracted from the human
pro-B cell line Reh was analyzed for the presence of ephrin-A
transcripts in PCR using degenerate primers hybridizing to conserved stretches of nucleotides in the ephrin-A1, ephrin-A3,
ephrin-A4, and ephrin-B1 sequences.31,32 Nucleotide sequence
analysis of 12 cloned PCR fragments unveiled the presence of the
ephrin-A4 sequence.
A 32P-labeled ephrin-A4 PCR fragment was prepared and used
to probe an Reh pro-B cell plasmid cDNA library. The cDNA insert
of 2 hybridizing clones, 5.1 and 2.1, was sequenced from the 58 and
38 end, and the partial sequences were found to represent the
published ephrin-A4 sequence. In contrast to the published ephrin-A4 sequence,32 both clones contained the 38 untranslated
region, including a poly-A tail. In agarose gel electrophoresis, a
slight difference in size was observed between clones 5.1 and 2.1.
The complete nucleotide sequences, which are identical to the
published ephrin-A4 sequence, showed that clone 5.1 is 1182
nucleotides long and encodes a protein of 201 amino acid residues.
Clone 2.1 is 1036 nucleotides long and lacks a 146 bp stretch
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
(position 498-643) at the 38 end of the open reading frame. Clone
2.1 encodes a protein of 193 amino acid residues. As a result of the
frame shift incurred by the missing 146 bp, clone 2.1 differs by 37
amino acids from clone 5.1 at the carboxy terminus (Figure 1A).
This altered carboxy terminus does not contain a typical transmembrane region nor does it harbor the GPI-signal sequence present in
membrane-bound ephrin-A4.32 This suggests that clone 2.1 may
encode a secreted molecule. The mRNA corresponding to cDNA
clone 5.1 was named ephrin-A4 (m), and the mRNA corresponding
to cDNA clone 2.1 was named ephrin-A4 (s).
The ephrin-A4 (s) cDNA results from an alternative splice in
exon IV
To determine the molecular basis for the differences in ephrin-A4
(m) and ephrin-A4(s), a human genomic library in ␭-DASH was
screened with a 32P-labeled ephrin-A4 probe. A hybridizing clone
covering the entire ephrin-A4 gene of approximately 7.5 kilobases
was subjected to restriction mapping, subcloning, and partial
nucleotide sequence analysis. Exon sequences and exon-intron
boundaries were determined using vector and exon-specific primers. The ephrin-A4 gene consists of 4 exons; the translation start
codon is in the first exon, and the GPI-linkage signal sequence, the
stop codons, and the 38 untranslated sequence are in the fourth exon
(Figure 1B). The 146 bp stretch missing in the ephrin-A4 (s)
sequence was spliced out of exon IV using the internal consensus
splice donor site AG (Figure 1C).
Ephrin-A4 gene expression in tissues and hematopoietic cells
It has previously been reported that the ephrin-A4 gene is
expressed in the adult human spleen, prostate, ovary, small
intestine, and colon and in the fetal heart, lung, and kidney.32 We
confirmed and extended these findings by showing, using Northern
blot analysis, that the ephrin-A4 gene is abundantly expressed in
adult spleen and lymph node and in fetal liver. It is weakly
expressed in adult peripheral blood leukocytes, thymus, and bone
marrow (Figure 2A). Northern blot analysis of purified hematopoietic cells showed high levels of ephrin-A4 expression in tonsil B
cells, intermediate levels of expression in tonsil T cells, and low
levels of expression in blood B cells and CD4⫹ and CD8⫹ T cells
(Figure 2B). In contrast to blood, human tonsils contain many
activated B cells, raising the possibility that ephrin-A4 expression
is induced during B-cell activation. Indeed, ephrin-A4 mRNA
levels were upregulated by stimulation of blood B cells through
their B cell receptor with anti-µ antibodies to a level comparable to
that observed in freshly isolated tonsil B cells. In addition,
ephrin-A4 expression is upregulated in anti-CD3 stimulated CD4⫹
and CD8⫹ blood T cells. Stimulation with the phorbol ester TPA
did not induce the expression of ephrin-A4 in either blood B cells
or T cells (Figure 2B).
High levels of ephrin-A4 expression were also detected in
hematopoietic cell lines representing various lineages and differentiation stages (Figure 2C). The early B cell lines (Tom-1, BV173,
Reh, Nalm-6), the mature B cell lines (Daudi, Bjab, U698) the
plasmacytoid B cell line (U266), and the T cell lines (JM and
Jurkat) all showed strong expression of ephrin-A4 mRNA, while a
weaker expression was observed in the promyeloid cell line KG1-A
and the erythroid cell line K562. No expression was observed in the
T cell line HPB-ALL and JY and in the myeloid cell lines HL-60
and U937.
The Northern blot analysis did not discriminate between cells
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BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
A SPLICE VARIANT OF HUMAN EPHRIN-A4
225
Ephrin-A4–expressing cells are detectable in situ in tonsil
germinal centers and extrafollicular areas
In a current model of peripheral B-cell development in secondary
lymphoid organs, newly formed bone marrow–derived B cells first
migrate into the extrafollicular T-cell zones. Here, the tripartite
interaction between antigen-specific B and T lymphocytes and
interdigitating cells leads to B-cell activation; B cells may differentiate into plasma cells or enter primary or secondary follicles to
initiate or sustain a germinal center reaction.5 In-situ hybridization
of sections of human tonsil with a dUTP-Dig–labeled ephrin-A4
probe unveiled strongly ephrin-A4 expressing cells in germinal
centers and weakly expressing cells in the follicular mantle zone
(Figures 4A and 4C). In the extrafollicular areas, scattered individual cells with a lymphoid appearance also displayed strong
staining (Figures 4A and 4E). No staining was observed with a
dUTP-Dig–labeled control probe (Figures 4B, 4D, 4F).
Ephrin-A4 (s) protein is produced by freshly isolated tonsil B
cells and by blood B cells after in vitro activation via the B-cell
antigen receptor
Figure 2. Expression of ephrin-A4 mRNA in different human tissues and cell
types. Upper panels show ephrin-A4 hybridization; lower panels, ␤-actin hybridization. (A) Expression in different hematopoietic tissues: (1) spleen, (2) lymph node, (3)
thymus, (4) peripheral blood leukocytes, (5) bone marrow, and (6) fetal liver. (B)
Expression in freshly isolated, cultured B and T lymphocytes: (1) peripheral blood B
cells, (2) 24-hour TPA-stimulated blood B cells, (3) 24-hour anti-µ–stimulated blood B
cells, (4) peripheral blood CD4⫹ T cells, (5) 24-hour TPA-stimulated blood CD4⫹ T
cells, (6) 24-hour anti-CD3–stimulated blood CD4⫹ T cells, (7) peripheral blood
CD8⫹ T cells, (8) 24-hour anti-CD3–stimulated blood CD8⫹ T cells, (9) tonsil B cells,
and (10) tonsil T cells. (C) Expression in hematopoietic cell lines: (1) Tom-1, (2)
BV173, (3) Reh, (4) Nalm-6, (5) Daudi, (6) Bjab, (7) U266, (8) U698, (9) JM, (10)
Jurkat, (11) HPB ALL, (12) JY, (13) KG1-A, 914) HL60, (15) U937, and (16) K562.
expressing the ephrin-A4 (m) or ephrin-A4 (s) variant. In subsequent experiments, a semiquantitative RT-PCR approach was
employed with primer sets that discriminate between ephrin-A4 (s)
and ephrin-A4 (m). In all PCR experiments, the quality and amount
of cDNA were assessed by PCR with primers specific for ␤-actin.
In all populations analyzed, high levels of expression of the
ephrin-A4 (m) form were detectable. In the Reh cell line, freshly
isolated tonsil B cells and 24-hour anti-µ–stimulated blood B cells,
both high levels of expression of the ephrin-A4 (s) form, were
detectable (Figure 3). In contrast, very low levels of expression of
ephrin-A4 (s) were detectable in tonsil T cells and in anti-CD3–
activated CD4⫹ or CD8⫹ T cells (Figure 3).
Figure 3. Semiquantitative PCR analysis of the expression of ephrin-A4 (m) and
ephrin-A4 (s) mRNA in freshly isolated and stimulated B and T lymphocytes. (1)
water control, (2) Reh pro-B cells, (3) tonsil T lymphocytes, (4) 24-hour anti-CD3–
stimulated CD8⫹ blood T lymphocytes, (5) 24-hour anti-CD3–stimulated CD4⫹
blood T lymphocytes, (6) tonsil B lymphocytes, and (7) 24-hour anti-µ–stimulated
blood B cells. The upper panel shows ephrin-A4 (s) specific PCR (28 cycles); the
middle panel, ephrin-A4 (m) specific PCR (30 cycles); and the lower panel, ␤-actin
specific PCR (24 cycles).
A rabbit antiserum was raised against a 15-residue peptide corresponding to a region in the carboxy terminus that is specific for the
ephrin-A4 (s) protein, and it will not recognize the ephrin-A4 (m)
protein. The antiserum was affinity-purified on a column with
peptide-coupled sepharose beads. The specificity of the affinitypurified rabbit anti–ephrin-A4 (s) polyclonal antibody was first
analyzed in Western blots of crude cell lysates and culture
supernatants from COS cells transfected with ephrin-A4 (m) or
ephrin-A4 (s). A band of the expected size was present in lanes
containing cell lysates or culture supernatant from COS cells
transfected with the ephrin-A4 (s) cDNA but not from COS cells
transfected with the ephrin-A4 (m) cDNA or mock-transfected
cells (Figure 5A). Note the presence of a second band in the cell
lysate of ephrin-A4 transfected cells, which may represent an
unprocessed product. In Western blots of lysates from purified
tonsil B and T lymphocytes, the anti–ephrin-A4 (s) antiserum
detected a single band of 28 kDa molecular weight in lysates from
tonsil B cells but not T cells (Figure 5B). In lysates of freshly
isolated blood B cells, ephrin-A4 (s) proteins were not detectable
(data not shown).
In the supernatant of blood B cells stimulated for 6 days with
anti-µ antibodies, SAC or TPA, secreted ephrin-A4 (s) protein
could be detected in the culture supernatant after stimulation with
anti-µ or SAC but not after stimulation with TPA (Figure 5C).
Addition of supernatant from PHA-stimulated T cells (Tsup)
containing cytokines that progress anti-µ stimulated B cells through
the cell cycle36 had no or slightly inhibitory effect, while the
combination of Tsup and SAC stimulation of B cells showed a
more pronounced inhibitory effect on the expression of ephrinA4(s) protein when compared with SAC alone or SAC and anti-µ.
These protein data confirm the predicted amino acid sequence of
the carboxy terminus of the ephrin-A4 (s) cDNA and show that
ephrin-A4 (s) is secreted by in vitro activated B cells. Repeated
attempts to utilize the anti–ephrin-A4 (s) antiserum in immunohistochemical analysis of tonsil sections or transfected COS cells were
unsuccessful.
The high expression of ephrin-A4 mRNA in anti-CD3 stimulated T cells led us to perform the same experiment with these cells.
T cells (both CD4⫹ and CD8⫹) were stimulated for 6 days with
anti-CD3–coated beads (Dynabeads) or TPA. Ephrin-A4 (s) protein
could not be detected in the supernatants or cell lysates of these
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AASHEIM et al
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
Figure 4. In situ (mRNA) hybridization of tonsil sections with ephrin-A4 probe and control probe. (A, B)
Overview of a germinal center (GC) and extrafollicular
area; objective ⫻25. (C, D) Close-up of a germinal center
with follicular mantle (FM) zone; objective ⫻40. (E, F)
Close-up of an extrafollicular (EF) area; objective ⫻40.
Panels A, C, and E show the ephrin-A4 Dig-labeled
probe, and panels B, D, and F, the control Dig-labeled
probe. Brown staining shows ephrin-A4 mRNA hybridization in the GC and EF areas.
cells (data not shown). These observations correspond to the
semiquantitative PCR data showing no detection of ephrin-A4 (s)
message in anti-CD3–stimulated T cells.
Staining of cells with an ephrin-A4-Fc chimeric protein
To detect cells capable of binding ephrin-A4, a fusion protein,
which contained the shared sequence between the membrane and
soluble forms of the molecule and the constant region of mouse
IgG2b, was generated (ephrin-A4-Fc). As a negative control, we
generated a fusion protein comprising the amino terminal 30
residues of human CD19 and the murine IgG2b constant region
Figure 5. Western blot analysis with ephrin-A4 (s)
specific antiserum. (A) COS cells were transfected with
ephrin-A4 (m) cDNA or ephrin-A4 (s) cDNA, and cells
and culture supernatant were harvested 3 days posttransfection. Cells were lysed. Either 10 µg protein was
applied in each lane (left panel), or 10 µL culture
supernatant was applied in each lane (right panel). (B)
Tonsil T cells or tonsil B cells were isolated and lysed, and
10 µg of protein was applied in each lane. (C) 105 blood B
cells were stimulated for 6 days, as indicated in the figure
text. Control is no stimulation. The culture supernatant
was harvested, and 10 µL was applied in each lane. All
blots were stained with ephrin-A4 (s) specific polyclonal
antiserum. The arrowhead denotes the ephrin-A4 (s)
protein band. Molecular weight in kDa is indicated to the
left of each panel. Tsup: supernatant of PHA-stimulated
pooled T cells.
(CD19short-Fc). The fusion proteins were first analyzed for
reactivity with different cell lines of the B-cell, T-cell, and myeloid
lineage. The B lymphoma cell line Bjab specifically bound the
ephrin-A4-Fc fusion protein, while the T-cell line Jurkat did not.
The negative control protein, CD19short-Fc, did not bind to either
of the cell lines (Figure 6).
In tonsil, purified B and T lymphocytes did not bind the
ephrin-A4-Fc fusion protein (results not shown). Within the
population of tonsil cells that adhered to collagen-coated tissue
flasks, approximately 5% of cells stained with the ephrin-A4-Fc
protein. Double immunofluorescent staining with different markers
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BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
A SPLICE VARIANT OF HUMAN EPHRIN-A4
227
Figure 6. Ephrin-A4-Fc binding to cell lines. The
Jurkat and the Bjab cell line were stained with either the
control CD19short-Fc (dotted line) or the ephrin-A4-Fc
(bold line) fusion protein.
for adherent cell populations in the tonsil showed that the
ephrin-A4⫹ cells expressed low to intermediate levels of CD40,
high or intermediate levels of HLA-DR, and barely detectable
levels of CD14. Staining was not observed with anti-CD11c,
anti-CD19, anti-CD4, anti-CD2, anti-CD13, anti-CD31, antiCD34, anti-CD86, anti-CD21, anti-CD45, anti-CD38, anti-CD71,
and anti-CD123 antibodies (Figure 7). Immunohistochemical staining of frozen tonsil sections with ephrin-A4 fusion protein was not
successful. Ephrin-A4-Fc did not stain cytospin preparations of the
Bjab cell line using different fixation protocols, indicating that the
receptor-ligand interaction is abrogated under these conditions.
EphA2 is a candidate ephrin-A4 receptor in human tonsils
Ephrin-A4 has been shown to bind to 6 different members of the
family of Eph receptor tyrosine kinases. Binding of ephrin-A4 to 3
of these, EphA2, EphA5, and EphA6, reportedly results in autophosphorylation of the receptor and has been interpreted to reflect
high-affinity, functional receptor-ligand interaction.18 In humans,
Figure 7. Immunofluorescent analysis of ephrin-A4 binding tonsil adherent cells. Tonsil adherent cells were stained with ephrin-A4-Fc (A) or ephrin-CD19short-Fc (D)
chimeric proteins in combination with a panel of fluorochrome-labeled monoclonal antibodies. (B, C, E, F) Histograms represent the staining patterns of ephrin-A4 binding cells
gated in the upper left panel (A): shaded histograms represent ephrin-A4 binding cells costained with relevant FITC control, and open histograms represent costaining of
ephrin-A4 binding cells with FITC-labeled antibodies directed to indicated surface markers.
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228
AASHEIM et al
expression of EphA5 has been detected in the brain and placenta.9
While expression data have been reported for the EphA6 gene in
humans, high levels of expression in rats and mice are noted in the
brain.37,38 EphA2 mRNA has been found in different rat tissues,
including the spleen; in human cell lines of epithelial origin; and in
human umbilical vein endothelial cells.22,39 In Northern blots, we
detected high levels of EphA2 mRNA in adult bone marrow,
spleen, and lymph node, and in fetal liver, whereas no message was
found in adult thymus and peripheral blood leukocytes (Figure 8A).
The expression pattern of EphA2 in lymphoid tissues was similar to
the expression pattern observed for ephrin-A4.
Based on the presence of EphA2 message in these lymphoid
organs, we further investigated EphA2 as a putative candidate
receptor for the ephrin-A4 ligand in human tonsil. First, we
confirmed that COS cells transiently transfected with the ephrin-A4
(m) cDNA are capable of binding an EphA2-Fc fusion protein,
confirming results previously published by Gale et al18 (results not
shown). In immunohistochemical staining of frozen tonsil sections,
an anti-EphA2 antiserum showed reactivity with cells in the crypts
of the tonsils (Figure 9), whereas, tonsil B and T lymphocytes
completely lacked reactivity with this antibody.
The immunohistochemical data were further confirmed by
isolating ephrin-A4 binding cells from the fraction of adherent
tonsil cells by immunomagnetic separation using beads (Dynal)
coupled to the ephrin-A4-Fc chimeric protein. Lysates of ephrinA4-Fc binding cells strongly reacted with the anti–EphA-2 antibody in Western blot analysis, whereas isolated CD34⫹ endothelial cells, or the tonsil adherent cell fraction depleted of both
ephrin-A4-Fc binding cells and CD34⫹ cells, did not react or only
weakly reacted with this antibody (Figure 8B).
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
Figure 9. Immunohistochemical staining of a tonsil section with anti-EphA2
antibodies. Frozen tonsil sections were stained with EphA2 specific antiserum (A) or
normal rabbit serum Ig (B) (objective ⫻40. Red EphA2⫹ cells are present in the tonsil
crypt area only.
Discussion
We have identified a mRNA splice variant of the human ephrin-A4
gene, denoted ephrin-A4 (s), a member of a family consisting of at
Figure 8. EphA2 mRNA expression in hematopoietic tissues and EphA2 protein
production by purified populations of cells. (A) EphA2 mRNA expression in
different hematopoietic tissues. The upper panel shows EphA2 hybridization, and the
lower panel, ␤-actin hybridization to the same blot. Molecular weight in kb is indicated
to the right. (B) Western blot analysis of lysates of purified tonsil cell subpopulations
using the EphA2-specific anti-serum. No specific staining was observed with lysates
from purified T or B lymphocytes, CD34⫹ (endothelial) cell, and collagen-adherent
cells depleted of CD34⫹ cells and ephrin-A4 binding cells (‘‘rest adherent’). The
arrow indicates the EphA2 protein specifically detected in the fraction of ephrin-A4
binding adherent cells. Molecular weight in kDa is indicated to the right.
least 8 predominantly membrane-bound ligands for Eph receptor
tyrosine kinases. Characterization of the genomic structure of the
ephrin-A4 gene unveiled that this splice variant lacks 146 nucleotides at the 38 end of the open reading frame in the first part of exon
IV when compared to the published ephrin-A4 sequence, here denoted
ephrinA-4 (m).32 This results in an altered carboxy terminus of 37
amino acids compared to the ephrin-A4 (m) protein and the
absence of the GPI-signal sequence present in ephrin-A4 (m). The
prediction that this mRNA encodes a soluble molecule was substantiated by the finding that COS cells transfected with ephrin-A4(s)
cDNA produced soluble ephrin-A4 protein, and more importantly,
in vitro activated human blood B-lineage cells were found to
secrete the ephrin-A4 (s) protein into the culture supernatant.
Moreover, cell lysates of freshly isolated human tonsil B lymphocytes containing a large fraction of in vivo activated B cells were
shown by Western blotting to contain the ephrin-A4 (s) protein.
The significance of these findings is two-fold. First, this is only
the second member of the ephrin gene family that can reportedly
exist in a membrane-bound and soluble form, and it is the first
soluble ephrin ligand that originates from an alternatively spliced
mRNA. Ephrin-A1, a cytokine-inducible endothelial cell product,
appears to be shed from the cell membrane.22 Second, in the
peripheral B-cell compartment, ephrin A4 (s) expression is associated with B-cell activation. So far, ephrins and their ligands have
almost exclusively been studied in the developing embryo, where
they act as instructive molecules that guide the topographic
movement of cells and growth cones and play a role in defining
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BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
compartments. The current data add to the more recent notion that
Eph receptors and ephrins play a role outside the developing
nervous system, ie, in hematopoiesis and angiogenesis.22-24
The analysis of ephrin-A4 (m) and ephrin-A4 (s) expression at
the mRNA and protein level in peripheral B-cell populations
yielded important clues as to the possible functional role of these
molecules. Very weak ephrin-A4 mRNA expression was observed
in blood B cells, and ephrin-A4 (s) protein could not be detected in
lysates of freshly isolated blood B cells. In contrast, tonsil B cells
appeared to express mRNA for soluble ephrin-A4, and ephrin-A4
(s) protein could be readily detected in lysates of freshly purified
tonsil B cells. Tonsil and blood T cells expressed barely detectable
levels of ephrin-A4 mRNA in Northern blot analysis and did not
produce detectable levels of ephrin-A4 (s) protein. Anti-CD3
stimulated T cells expressed high levels of ephrin-A4 mRNA, but
neither ephrin-A4 (s) mRNA nor protein could be detected from
these cells. Because of the lack of ephrin-A4 (m) specific antibodies, we were not able to assess whether activated T cells produce
membrane-bound ephrin-A4 protein.
To further elucidate the nature of the ephrin-A4 producing cells,
we examined B-cell populations in human tonsils, a lymphoid
organ that represents a working model for in vivo peripheral B-cell
activation and differentiation. With a probe detecting both ephrin-A4 mRNA species, strongly ephrin-A4⫹ cells, with a round,
lymphoid appearance, were found in germinal centers and in
extrafollicular areas in tonsil by in situ hybridization. Weakly
ephrin-A4 mRNA expressing cells were observed in the follicular
mantle zone. Of note, the extrafollicular ephrin-A4⫹ cells may
represent naive B-cell blasts that have been activated by T cells and
interdigitating cells.5
The suggestion that ephrin-A4 (s) secretion is associated with B
lymphocytes in an activated state was further supported by the
observation that resting blood B cells could be induced to secrete
the soluble ligand through agents that cross-link the B-cell antigen
receptor and induce B-cell activation. In that respect it is noteworthy that the phorbol ester TPA, a potent B-cell antigen receptorindependent stimulator of B-cell proliferation, did not induce
secretion of ephrin-A4 (s) protein. Thus, B-cell antigen receptor
cross-linking appears to be a prerequisite for the induction of
ephrin-A4 secretion, presumably mediated by B-cell receptorassociated Src-family kinases such as Lyn, Fyn, and Blk.
Eph receptors have been shown to bind multiple ligands, and
ephrin ligands are capable of binding multiple Eph receptors.
Binding of ephrin-A4 to EphA2, EphA5, and EphA6 has been
shown to result in autophosphorylation of the receptor and has been
interpreted to reflect high-affinity, physiologically relevant ligandreceptor interaction. The overlap in expression pattern of EphA2
(lymphoid organs but not in thymus or blood) and ephrin-A4
prompted us to investigate whether EphA2 could be a candidate
receptor for ephrin-A4 in human tonsils. In immunohistochemical
analysis, EphA2⫹ cells were detectable in the crypts of tonsils with
a polyclonal antibody specific for the intracellular portion of the
EphA2 receptor. An ephrin-A4 IgG fusion protein did not perform
in immunohistochemistry, but ephrin-A4 binding cells could be
found by immunofluorescence analysis in the adherent fraction of
isolated tonsil MNC. In lysates of purified ephrin-A4 binding cells,
EphA2 protein was detectable in Western blotting, whereas in
lysates of the remaining purified CD34⫹ tonsillar endothelial cells,
EphA2 receptor protein was not detectable.
A SPLICE VARIANT OF HUMAN EPHRIN-A4
229
In double-immunofluorescent staining analysis, ephrin-A4 binding
cells expressed intermediate to high levels of HLA-DR, intermediate to
low levels of CD40, and barely detectable CD14, but lacked other
markers for myeloid or endothelial cells or markers specific for B- and
T-lineage cells. The localization of the EphA2 receptor to cells in the
crypts and the HLA-DR⫹⫹ or HLA-DR⫹/CD40⫹/CD14low/lineage
phenotype suggests that these cells may be dendritic cells. The phenotype of the EphA2⫹ adherent cells partially overlaps with the phenotype
of interdigitating dendritic cells, including the characteristic presence of
HLA-DR⫹⫹ and HLA-DR⫹ cells, but lacks other characteristics of
interdigitating dendritic cells such as expression of low levels of CD4
and CD86.40 Thus, within the tonsil micro environment, EphA2⫹ cells
with a dendritic cell phenotype may communicate with activated B cells
through the soluble ephrin-A4 protein.
It is noteworthy that B-ephrins, anchored to the cell membrane
via a transmembrane region, can transduce signals initiated by
cell–cell contact.20,41 Although not proven, it has been suggested
that A-type ephrins, such as ephrin-A4, may exert a similar activity
through association with transmembrane proteins.42 In B and T
cells from blood and tonsils, the membrane-bound form of
ephrin-A4 was detectable, indicating that putative signaling events
may be mediated via this ligand.
The functional consequence of the interaction between soluble
ephrin-A4 and the Eph2⫹ cells in tonsil with a dendritic cell
phenotype remains to be elucidated. The ephrin-A1 soluble ligand,
the only other reported naturally occurring soluble ephrin, has been
shown to act as an angiogenic factor in vivo,22 as a growth factor
for melanoma cell lines,43 and as a neurotrophic factor in cultures
of rat spinal cord neurons.44 Along the same line, it may be envisaged
that in tonsil, ephrin-A4 (s) acts as a stimulator or chemoattractant of
dendritic cells. Indeed, dendritic cells in tonsil have recently been shown
to be capable of directly interacting with B cells and inducing
immunoglobulin production.45 Alternatively, ephrin-A4 (s) may act as
an antagonist of the EphA2 receptor, similar to what has been proposed
for (nonnatural) recombinant soluble ephrin ligands, such as ephrin-B1
and ephrin-A3,19,46 produced in vitro. The observation that EphA2-Fc
fusion protein does not appear to bind to any cell population in tonsil
lends support to a model of interaction between soluble ephrin-A4 and
EphA2⫹ cells, without cell–cell interactions involving EphA2⫹ and
ephrin-A4 (m)⫹ cells. The notion that the only reported naturally
occurring soluble ephrins, ephrin-A1 and ephrin-A4, both appear to act
through the EphA2 receptor expressed on different cell types supports
the view that the spatially and temporarily regulated expression of Eph
receptors and their ligands is a key determinant in the specificity of their
interactions. Whatever the outcome of this interaction, the data reported
here add to the growing notion that Eph receptors and their ligands may
play an important role outside the developing nervous system, and they
appear to have a role in the hematopoietic system.
Acknowledgments
We thank Dick van Wichen for expert help with in situ hybridizations; Kari Hildrum for help with producing the ephrin-A4 (s)
antiserum; and Goril Olsen, Toril Holien, and Ruth Solien for
expert technical assistance. We also thank Espen Bekkevold at
LIIPAT, The Norwegian National Hospital, for invaluable help with
the isolation of adherent cells from tonsils.
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230
BLOOD, 1 JANUARY 2000 • VOLUME 95, NUMBER 1
AASHEIM et al
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2000 95: 221-230
A splice variant of human ephrin-A4 encodes a soluble molecule that is
secreted by activated human B lymphocytes
Hans-Christian Aasheim, Else Munthe, Steinar Funderud, Erlend B. Smeland, Klaus Beiske and Ton
Logtenberg
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