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Identification of an IL-7-Associated Pre-Pro-B
Cell Growth-Stimulating Factor (PPBSF). II.
PPBSF Is a Covalently Linked Heterodimer of
IL-7 and a Mr 30,000 Cofactor
Laijun Lai, Fangqi Chen, Sean McKenna and Irving
Goldschneider
J Immunol 1998; 160:2280-2286; ;
http://www.jimmunol.org/content/160/5/2280
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References
Identification of an IL-7-Associated Pre-Pro-B Cell
Growth-Stimulating Factor (PPBSF). II. PPBSF Is a
Covalently Linked Heterodimer of IL-7 and a Mr 30,000
Cofactor1
Laijun Lai, Fangqi Chen,2 Sean McKenna,3 and Irving Goldschneider4
D
espite the demonstration over the past decade of the key
role of IL-7 in the development and differentiation of
murine pre-B cells and pro-B cells, the nature of its involvement, if any, at the pre-pro-B cell stage of lymphopoiesis
remains controversial (1–3). Inasmuch as our long-term xenogeneic (rat/mouse/human) bone marrow (BM)5 lymphoid culture
system selectively generates pre-pro-B cells and pro-B cells (4 –7),
we have used it as a model with which to identify the stromal
cell-derived growth factors that are responsible for regulating the
proliferation and/or differentiation of these primitive lymphoid
precursors.
Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030
Received for publication July 28, 1997. Accepted for publication November 4, 1997.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by Grant No. AI-32752 from the National Institutes
of Health.
2
Present address: Department of Medicine, University of Pennsylvania School of
Medicine, 909 Biomedical Research Building, 422 Curie Drive, Philadelphia, PA
19104.
3
Present address: Ares Advanced Technology, Inc., 280 Pond St., Randolph, MA
02368.
4
Address correspondence and reprint requests to Dr. I. Goldschneider, Department of
Pathology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030-3105. E-mail address: [email protected]
5
Abbreviations used in this paper: BM, bone marrow; CM, conditioned medium;
PPBSF, pre-pro-B cell growth-stimulating factor; TSLP, thymic stromal-derived lymphopoietin; SDF, stromal cell-derived factor; HSA, heat-stable Ag; BrdU, bromodeoxyuridine; TRITC, tetramethylrhodamine isothiocyanate; SCF, stem cell factor; IGF,
insulin-like growth factor; TdT, terminal deoxynucleotidyl transferase; ECM, extracellular matrix; sIgM, surface IgM; HRP, horseradish peroxidase.
Copyright © 1998 by The American Association of Immunologists
Results of earlier studies have demonstrated that serum-free BM
stromal cell conditioned medium (CM) from our culture system
selectively stimulates the proliferation of pre-pro-B cells from
freshly harvested rat BM and supports the accumulation, but not
the proliferation, of pro-B cells in vitro (8). Adsorption of CM with
anti-IL-7 mAb removes this activity, whereas rIL-7 restores this
activity to CM by BM stromal cells from IL-7 gene-deleted mice
(9). Furthermore, IL-7 with a nominal molecular mass of 25 kDa
coisolates with pre-pro-B cell growth-stimulating activity in the
apparent 50- to 100-kDa molecular mass fraction as determined by
ultrafiltration. Yet IL-7 itself does not induce proliferation of prepro-B cells, even in the presence of stem cell factor (SCF) or
insulin-like growth factor (IGF)-1, and anti-IL-7 mAb is unable to
neutralize the growth-stimulating activity in CM. These results, in
aggregate, suggested that the unique lymphopoietic properties of
our BM lymphoid culture system were due to the presence of a
molecular complex of IL-7 and a second stromal cell-derived factor. Although this molecular complex can also induce thymocyte
proliferation (9), we have not yet detected its presence in thymic
stromal cell CM (4, 8). Therefore, we have designated it pre-pro-B
cell growth-stimulating factor (PPBSF).
The present study directly confirms, by Western blot analysis, that
PPBSF is an ;55-kDa heterodimer that consists of one molecule of
IL-7 and one molecule of an as yet unidentified 30-kDa cofactor. The
results also demonstrate that the cofactor is constitutively produced by
IL-7(2/2) stromal cells under pro-B cell- but not pre-B cell-type
culture conditions and that it associates covalently with IL-7. Finally,
the results confirm our previous observation (9) that PPBSF “primes”
pre-pro-B cells and their immediate descendants to proliferate in response to IL-7 alone. The possible role of PPBSF in early B-lineage
development and the exclusion of several candidates for the PPBSF
cofactor are discussed below.
0022-1767/98/$02.00
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Evidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation
and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and “primes” them to proliferate
in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor
(PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a Mr of 30,000. Thus, when developed with anti-IL-7 mAb,
PPBSF migrates electrophoretically as a covalently bound ;55-kDa molecule under nonreducing conditions but dissociates under
reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 genedeleted (2/2) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared
in IL-7(2/2) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of
PPBSF. The PPBSF cofactor detected by this antiserum migrates as an ;30-kDa molecule and is able to maintain the viability,
but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(2/2) and IL-7(1/1)
bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely
as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures.
Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth
factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R. The Journal of Immunology, 1998,
160: 2280 –2286.
The Journal of Immunology
Materials and Methods
Animals
Male 4- to 6-wk-old IL-7 gene-deleted (IL-7(2/2)) and nondeleted (IL7(1/1)) mice (10) were bred from (129 3 B6)F2 stock that was generously
provided by Drs. Richard Murray and Ursula von Freeden-Jeffry (DNAX
Research Institute of Cellular and Molecular Biology, Palo Alto, CA). The
mice were used as donors of BM-adherent cells and stromal cell lines. Male
4- to 6-wk-old Lewis strain rats bred from stock originally obtained from
the National Cancer Institute, National Institutes of Health (Bethesda, MD)
were used as donors of BM lymphoid precursor cells.
Cytokines
rIL-7 was purchased from Genzyme Corporation (Cambridge, MA). Thymic stromal-derived lymphopoietin (TSLP) (11, 12) was generously provided by Dr. Philip J. Morrissey (Immunex Research and Development
Corporation, Seattle, WA); rflt-3 ligand (13) was generously provided by
Dr. Satish Menon (DNAX); and rSDF-1b (14) was purchased from R&D
Systems (Minneapolis, MN).
Antibodies
Immunofluorescence
Indirect immunofluorescence of cell surface Ags was performed by incubating 1 3 106 freshly harvested or culture-generated BM cells with mouse
or rat primary Abs (10 ml) and developing with appropriate FITC- or phycoerythrin-conjugated goat anti-IgG or anti-IgM Abs. To detect intranuclear TdT, cytocentrifuge-prepared cell smears were fixed in 4°C absolute
methanol, stained with rabbit Abs to TdT, and developed with FITC- or
TRITC-conjugated Abs to rabbit IgG (19). Double immunofluorescence for
Cm or Sm Ig heavy chains and TdT was performed on cell smears that were
fixed in cold absolute ethanol with 5% glacial acetic acid for 20 min at 4°C,
sequentially stained for TdT and HIS40, and developed with FITC goat
anti-mouse IgG and TRITC goat anti-rabbit IgG (17).
To detect the incorporation of BrdU, cultured cells were pulsed overnight with BrdU cell-proliferation labeling reagent (Amersham International) in a final concentration of 1:1000. Cytosmears prepared from these
cells were fixed in cold absolute ethanol with 5% glacial acetic acid,
stained with the anti-BrdU/nuclease reaction mixture for 60 min, and developed with FITC goat anti-mouse IgG. Double immunofluorescence for
BrdU and TdT was accomplished by staining for TdT at this step. Double
immunofluorescence for BrdU and cell-surface Ags was performed by
staining viable cells in suspension with the appropriate Abs and then staining cytocentrifuge smears of the same cells for BrdU.
Lymphoid culture systems
Pro-B-type cultures. Rat BM pre-pro-B cells and pro-B cells were generated in our culture system as previously described (4). Briefly, single cell
suspensions of mouse BM (8 3 106 cells) were added to 2 ml RPMI 1640
containing 20% lot-selected, defined FBS (HyClone, Logan, UT) in 35-mm
diameter culture plate wells and incubated at 37°C in 5% CO2. After 10
days, the confluent adherent cell layers were washed and seeded with 5 3
105 freshly harvested rat BM cells/ml. In some experiments, the rat BM
cells were seeded into microporous membrane culture inserts (0.4-mm pore
size; Transwell-3408, Costar, Cambridge, MA) that were placed over, but
not in contact with, the mouse BM-adherent cell layers. Total cells from the
culture inserts and nonadherent lymphoid cells from the standard cultures
were recovered in serum-free medium on day 10 for cytologic and phenotypic analysis (8).
Pre-B-type cultures. Rat BM pre-B cells were generated in long-term culture by a modification of the method of Whitlock and Witte (20). Briefly,
adherent cell layers were established by incubating 8 3 106 mouse BM
cells/well at 37°C (5% CO2) in 2 ml RPMI 1640 medium containing 5%
lot-selected, defined FBS (HyClone), 5 3 1025 M 2-ME, and 40 mg/L
gentamicin. The cultures were refed with 50% fresh medium twice weekly.
After 20 days, the confluent adherent cell layers, containing only an occasional mouse lymphoid cell colony, were washed with RPMI 1640 and
seeded with 5 3 105 freshly harvested rat BM cells/ml as above. Culturegenerated lymphoid cells (.98% rat origin) were recovered on day 15 for
cytologic and phenotypic analysis.
Conditioned medium
Washed confluent mouse BM-adherent cell layers or stromal cell lines
therefrom were used to condition medium for 10 days (8). The CM for cell
stimulation was filtered to remove any cells, concentrated twofold by ultrafiltration in Centriprep-10 concentrator units (Amicon, Danvers, MA),
dialyzed for 16 h in serum-free normal medium at 4°C, and stored at
270°C. For cell stimulation, CM was diluted to twofold its original concentration with medium containing 20% FBS; for immunoadsorption or
Western immunoblotting, 103 concentrated CM in serum-free normal medium was used.
Immunoadsorption of CM with anti-IL-7 mAb
Anti-IL-7 mAb (mouse IgG2b) was conjugated to protein A-Sepharose by
incubating 15 ml of Ab with 80 ml of packed beads for 4 h. The beads were
extensively washed with PBS to remove unbound Ab. Immunoadsorption
was accomplished by incubating 103 concentrated CM with Ab-conjugated protein A-Sepharose beads (1 ml CM/80 ml packed beads) in a rotating mixer for 2 h at 4°C. The beads were pelleted in a microfuge (8000
rpm), and the supernatant was removed. This process was repeated three
times. Nonspecific binding was controlled by incubating CM with unconjugated protein A-Sepharose beads and beads conjugated with a mouse
IgG2b isotype control. The bound Ag was recovered from the beads by
elution with 0.1 M NaHCO3 buffer (pH 9.3) containing 0.5 M NaCl, and
the eluate was dialyzed for 16 h in PBS (pH 7.2) at 4°C.
Thymidine incorporation
To evaluate cell proliferation induced by CM, 1 3 105 freshly harvested rat
thymocytes or day 10 culture-generated rat BM lymphoid cells were pulsed
with 1 mCi/well of [3H]TdR (New England Nuclear, Boston, MA) 12 h
before harvesting. Incorporation of [3H]TdR was determined by liquid
scintillation spectroscopy.
Preparation of antisera to PPBSF
An anti-IL-7 immunoaffinity column was prepared by mixing 3 mg IL-7specific mAb in coupling buffer with 0.3 g cyanogen bromide-activated
Sepharose 4B according to the manufacturer’s instructions. One hundred
milliliters of 103 concentrated serum-free CM was loaded onto the column, which was then washed with 50 ml PBS. The bound Ag was eluted
with 10 ml of 0.1 M NaHCO3 buffer (pH 9.3) containing 0.5 M NaCl and
dialyzed for 16 h in PBS (pH 7.2) at 4°C. The activity of the eluate was
then tested by thymocyte proliferation analysis.
Equal volumes of eluate and Freund’s adjuvant were mixed, and IL7(2/2) mice were injected with a total of 2 ml eluate from IL-7(1/1)
pro-B CM, IL-7(1/1) pre-B CM, or 20 mg rIL-7 at multiple s.c. sites on
a biweekly basis. The first two biweekly immunizations were performed
using Ag emulsified with CFA. All subsequent immunizations were carried
out with the same Ag emulsified with IFA. After four successive biweekly
immunizations, sera were collected and pooled. To remove anti-IL-7 Abs,
some aliquots of antisera were adsorbed with rIL-7 coupled to cyanogen
bromide-activated Sepharose 4B (5 mmol rIL-7/ml gel).
Western immunoblotting of CM for PPBSF
For Western immunoblotting, 25 ml of eluate from anti-IL-7, anti-PPBSF,
or anti-IL-7 mAb-adsorbed anti-PPBSF Ab immunoaffinity columns was
mixed with 25 ml of 23 SDS sample buffer, with or without 0.1 M DTT,
and boiled for 5 min. The samples were loaded onto different slots of a 12%
SDS-PAGE gel and run overnight at 45 V. The proteins were then transferred onto Immobilon-P membrane (Millipore, Bedford, MA) using a
trans-Blot SD Semidry Transfer Cell (model 200/2.0, Bio-Rad, Hercules,
CA) at 300 mA for 1 h. After blocking with 5% blocking reagent in PBSTween 20, the membrane was incubated with appropriate dilutions of
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Neutralizing mAbs cross-reactive with both human and mouse IL-7 or
SDF-1b were purchased from Genzyme Corporation or R&D Systems,
respectively. Rabbit polyclonal Abs against TSLP or mAbs against flt-3
ligand were kindly provided by Immunex or DNAX, respectively. Mouse
IgG2b isotype control was obtained from Sigma (St. Louis, MO). Murine
mAbs to the HIS40 (anti-IgM) (15), HIS24 (anti-CD45RC-B220) (16, 17),
and HIS50 (anti-heat stable Ag (HSA)) (18) rat B-lineage-associated Ags
were generously provided by Dr. Davine Opstelten (Department of Pathology, University of Hong Kong, Hong Kong, China). Mouse antibromodeoxyuridine (anti-BrdU) mAb (with nuclease) was purchased from
Amersham International (Little Chalfont, U.K.). Affinity-purified FITCconjugated goat IgG F(ab9)2 anti-mouse IgM (heavy chain-specific) Ab
was obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD).
Affinity-purified rabbit Ab to calf thymus terminal deoxynucleotidyl transferase (TdT) as well as FITC- and tetramethylrhodamine isothiocyanate
(TRITC)-conjugated goat anti-rabbit IgG were purchased from Supertechs
(Bethesda, MD). Phycoerythrin-conjugated goat anti-mouse IgG was obtained from Caltag Laboratories (San Francisco, CA). Horseradish peroxidase (HRP)-linked sheep anti-mouse IgG or anti-rabbit IgG were purchased from Amersham Life Sciences (Arlington Heights, IL).
2281
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PPBSF IS A HETERODIMER OF IL-7 AND A Mr 30,000 COFACTOR
FIGURE 2. Ability of BM-adherent cell CM generated under pro-Band pre-B-type culture conditions to support lymphopoiesis in vitro. Triplicate wells containing 5 3 105 freshly harvested rat BM cells/ml were
incubated in IL-7(1/1) CM generated under pro-B or pre-B culture conditions. Nonadherent lymphoid cells were harvested on day 10 and phenotyped as in Figure 1. Results represent the means of three separate
experiments.
Electrophoretic mobility and molecular mass of PPBSF
anti-IL-7 mAb, anti-TSLP polyclonal Ab, anti-flt-3 ligand mAb, antiSDF-1b mAb, or antiserum to PPBSF; washed; incubated with 1:2000
HRP-labeled anti-mouse IgG or HRP-labeled anti-rabbit IgG; washed
again; and developed with enhanced chemiluminescence Western blotting
analysis system (Amersham Life Sciences).
As shown in Figure 4, the difference in form of IL-7 in pro-B and
pre-B CM was confirmed by electrophoresis and Western immunoblotting. Under nonreducing conditions, the IL-7 in pro-B CM
Results
PPBSF activity is present in pro-B- but not pre-B-type cultures
As previously documented (6) and illustrated in Figure 1A, the
pre-pro-B cell and pro-B cell compartments in our culture system
progressively expand with time after inoculation with freshly harvested rat BM cells, whereas the pre-B cell compartment progressively contracts. In contrast (Fig. 1B), the pre-B cell compartment
progressively expands with time, after a brief lag, under WhitlockWitte (W-W)-type culture conditions (20), whereas the pre-pro-B
cell and pro-B cell compartments progressively contract, after a
brief period of expansion. Therefore, for convenience, these culture systems will be referred to as pro-B-type and pre-B-type cultures, respectively.
The differences in the generative potentials of the two culture
systems were further documented using inoculum of .95% pure
mixtures of pre-pro-B cells and pro-B cells obtained from day 10
pro-B-type cultures (6). Under pro-B-type culture conditions (Fig.
1C), the pre-pro-B cell/pro-B cell compartments progressively expanded and, as reported (8), there was a disproportional increase of
pre-pro-B cells with time. However, under pre-B-type culture con-
FIGURE 3. Ability of anti-IL-7 mAb to neutralize and/or adsorb the
growth-stimulating activity for rat thymocytes from IL-7(1/1) CM generated under pro-B- and pre-B-type culture conditions. Rat thymocytes
(1 3 105/well) were incubated for 3 days in IL-7(1/1) pro-B or pre-B CM
in the presence or absence of anti-IL-7 mAb (10 mg/ml) or after adsorption
with anti-IL-7 mAb. Incorporation of tritiated thymidine was determined
after a 12-h pulse. Results represent mean cpm 6 SD. *, p , 0.05 as
compared with respective values for normal culture medium.
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FIGURE 1. Kinetics of generation of rat BM lymphoid cells in pro-Band pre-B-type cultures. Confluent layers of IL-7(1/1) mouse BM-adherent cells established under pro-B- or pre-B-type culture conditions were
inoculated with either 5 3 105 freshly harvested rat BM cells/ml or 5 3 104
rat BM lymphoid cells/ml from day 10 pro-B-type cultures (see Materials
and Methods). The numbers and phenotypes of B-lineage cells/well in the
nonadherent compartment were determined at the indicated times during 3
wk of culture as follows: total (B2201); pre-pro-B (B2201 HSA2 cm2);
pro-B (B2201 HSA1 cm2); pre-B (B2201 HSA1 cm1); and B (B2201
HSA1 sIgM1). Results represent the means of triplicate wells of a representative experiment (one of three). The numbers of pre-pro-B cells and
pro-B cells are combined for ease of presentation. The numbers of sIgM1
B cells are not indicated, as these were seen only in the inoculum of freshly
harvested rat BM.
ditions (Fig. 1D), pre-pro-B cells decreased and pro-B cells increased during the first 2 wk of culture, after which pro-B cells
decreased and pre-B cells increased.
The results in Figure 2 show that CM from pro-B- and pre-Btype cultures supported the same patterns of lymphopoiesis, albeit
at lower efficiency, as cultures containing BM-adherent cells (cf
Figs. 1A and 1B; day 11). Furthermore, as shown in Figure 3, the
growth-stimulating activity for thymocytes in pre-B CM, unlike
that in pro-B CM, was both neutralized and adsorbed by anti-IL-7
mAb. These results suggested that the IL-7 in pre-B-type cultures,
unlike that in pro-B-type cultures, is not complexed with the cofactor previously detected in IL-7(2/2) pro-B CM (9). To verify
this, we evaluated the ability of rIL-7 to restore PPBSF activity to
IL-7(2/2) CM generated under pre-B-type culture conditions. No
activity for pre-pro-B cells was detected (data not shown). The
results below show that this was due to the absence of the PPBSF
cofactor from pre-B CM.
The Journal of Immunology
migrated with a molecular mass of 55 kDa (lane 2, arrow),
whereas that in pre-B CM migrated at 25 kDa (lane 1). However,
under reducing conditions, the IL-7 in both pro-B and pre-B CM
migrated as 25-kDa molecules. Furthermore, the IL-7-associated
molecule in pro-B CM did not dissociate after treatment with 8 M
urea, 1 M acetic acid, or 0.1 M NaOH (Fig. 5), suggesting that it
exists as a covalently bound (presumably disulfide-linked) molecular complex.
PPBSF is a heterodimer of IL-7 and an Mr 30,000 cofactor
To confirm that PPBSF is a self-associating heterodimer, rIL-7 was
added to IL-7(1/1) and IL-7(2/2) CM generated under pro-B-
FIGURE 5. Resistance of the IL-7-associated molecular complex to
noncovalent dissociation. The IL-7-associated molecular complex in pro-B
CM was affinity purified with anti-IL-7 mAb and treated with either 0.1 M
PBS, 8 M urea, 1 M acetic acid, or 0.1 M NaOH at 100°C for 10 min. The
samples were electrophoresed on 10% SDS-PAGE under nonreducing conditions and developed with anti-IL-7 mAb. All lanes continue to display the
55-kDa IL-7-associated molecular complex.
FIGURE 6. rIL-7 forms a heterodimer with a soluble cofactor in IL7(2/2) pro-B, but not pre-B, CM. Two hundred fifty ng rIL-7 (;14.5
kDa) were added to IL-7(1/1) or IL-7(2/2) CM generated under pro-B
(left lanes) or pre-B (right lanes)-type culture conditions. One hour later,
total IL-7 in these CM was affinity purified, electrophoresed under nonreducing conditions, and developed with anti-IL-7 mAb. Arrows (lane 5)
indicate newly formed (;45 kDa) rIL-7-associated molecular complex in
IL-7(2/2) pro-B, but not pre-B, CM.
and pre-B-type culture conditions. One hour later, the total amount
of IL-7 in these CM was affinity purified, electrophoresed under
nonreducing conditions, and subjected to Western blot analysis.
The results in Figure 6 show that all detectable rIL-7 added to
IL-7(2/2) pro-B CM (lane 5, arrow) migrated as part of a 45-kDa
molecule, whereas the rIL-7 added to IL-7(1/1) pro-B CM (lane
3) migrated at 14.5 kDa. This suggested that the rIL-7 formed a
heterodimer with an ;30-kDa molecule in IL-7(2/2) CM. Again,
the endogenous IL-7 in pro-B CM migrated as part of a 55-kDa
molecule (lanes 2 and 3). Conversely, all detectable rIL-7 added to
either IL-7(2/2) pre-B CM (lane 5, arrow) or IL-7(1/1) pre-B
CM (lane 3) migrated at 14.5 kDa, and the endogenous IL-7 migrated at 25 kDa (lanes 2 and 3).
The 55-kDa molecular mass of PPBSF suggests that it consists
of one molecule of IL-7 and one molecule of cofactor. Nonetheless, to exclude the possibility that IL-7 and the cofactor can associate in multiple proportions, graded amounts of rIL-7 were
added to constant volumes of IL-7(2/2) pro-B CM. Only a single
species of PPBSF (;45 kDa) was detected by Western blot analysis, even when rIL-7 was added in amounts that ranged between
10-fold above and below that required for maximum complex formation (data not shown).
Similarly, results in Figure 7 show that native IL-7 in pre-B CM
complexes with an ;30-kDa molecule when added to IL-7(2/2)
pro-B CM (lane 3, arrow). However, when mixed with IL-7(1/1)
pro-B CM (lane 1), native IL-7 continues to migrate as a 25-kDa
molecule. The failure of IL-7 to exist as a heterodimer in pre-B
CM does not appear to be due to an inhibitory effect of 2-ME on
complex formation. This was shown by the addition of 2-ME to
pro-B CM. Under these conditions, 2-ME neither caused the
PPBSF in IL-7(1/1) CM to dissociate nor prevented rIL-7 from
forming PPBSF when added to IL-7(2/2) CM (data not shown).
Therefore, as confirmed below, the PPBSF cofactor appears to be
absent from pre-B CM.
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FIGURE 4. Western immunoblot detection of an IL-7-associated molecular complex in pro-B but not pre-B CM. Affinity-purified IL-7 from
103 concentrated pre-B or pro-B CM was subjected to 12% SDS-PAGE
under reducing or nonreducing conditions, and immunoblot analysis was
accomplished using anti-IL-7 mAb. The arrow indicates a 55-kDa IL-7associated molecular complex.
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PPBSF IS A HETERODIMER OF IL-7 AND A Mr 30,000 COFACTOR
Antiserum to PPBSF has separable specificities for IL-7 and the
PPBSF cofactor
Antisera were raised in IL-7(2/2) mice against Ags isolated from
IL-7(1/1) pro-B and pre-B CM by affinity purification with antiIL-7 mAb. The ability of these antisera to neutralize PPBSF activity in pro-B CM was then tested against freshly harvested rat
BM cells. The results in Figure 8 show that antiserum to the IL7-associated Ag(s) in pro-B CM completely neutralized PPBSF
activity. Therefore, we have termed this anti-PPBSF antiserum.
However, the antisera to the IL-7-associated Ag in pre-B CM and
to rIL-7 itself did not neutralize PPBSF activity, although both
neutralized rIL-7 activity (data not shown).
Western blot analysis with the anti-PPBSF antiserum (Fig. 9A)
FIGURE 8. Ability of antisera prepared in IL-7(2/2) mice against IL7-associated Ags from pro-B CM to neutralize PPBSF activity. Triplicate
wells containing 5 3 105 freshly harvested rat BM cells/ml were incubated
in IL-7(1/1) pro-B CM to which antisera to rIL-7 and IL-7-associated Ags
from pre-B CM or pro-B CM were added. Nonadherent lymphoid cells
were harvested on day 10 and phenotyped. Results represent the means 6
SD of three separate experiments. *, p , 0.05 as compared with results for
respective normal serum controls.
FIGURE 9. Western immunoblot detection of both components of the
PPBSF heterodimer by antiserum to PPBSF. IL-7(1/1) and IL-7(2/2)
pro-B and pre-B CM were affinity-purified with antiserum to PPBSF from
pro-B CM (see Fig. 8). The eluates were electrophoresed under reducing or
nonreducing conditions, and immunoblot analysis was conducted with (A)
unadsorbed or (B) rIL-7-adsorbed antiserum to PPBSF. Arrows indicate
;30-kDa PPBSF cofactor either alone (lane 4) or dissociated from 55-kDa
PPBSF (lane 2). Asterisks indicate ;25-kDa IL-7 either alone (lane 1) or
dissociated from PPBSF (lane 2). The ;25-kDa bands (asterisks) observed
in A are not detected in B, whereas both the ;30-kDa (arrows) and 55-kDa
(lane 2, left) bands remain.
detected a single band of ;55 kDa in IL-7(1/1) pro-B CM under
nonreducing conditions (lane 2), and two bands of ;25 kDa (asterisk) and 30 kDa (arrow) under reducing conditions. Only the
30-kDa band was observed in IL-7(2/2) pro-B CM (lane 4, arrows), and only the 25-kDa band was observed in IL-7(1/1)
pre-B CM (lane 1, asterisks). Neither band was detected in IL7(2/2) pre-B CM (lane 3). Hence, the 30-kDa molecule was selectively produced under pro-B cell culture conditions.
Adsorption of the anti-PPBSF antiserum with rIL-7 (Fig. 9B)
eliminated reaction with the 25-kDa band but not with the 30-kDa
band (lanes 2 and 4, arrows). However, adsorption with rIL-7 did
not alter the ability of this antiserum to neutralize PPBSF activity
in IL-7(1/1) pro-B CM (data not shown). These results suggested
that the 30-kDa molecule was the PPBSF cofactor. Evidence confirming this possibility was obtained by adsorbing IL-7(1/1)
pro-B CM with anti-IL-7 mAb before Western blot analysis and
demonstrating the absence of both the 30-kDa (PPBSF cofactor)
and 55-kDa (PPBSF) bands as well as the 25-kDa (IL-7) band
(data not shown).
The PPBSF cofactor is not TSLP, flt3 ligand, or SDF-1
We have previously demonstrated that neither rSCF nor rIGF-1
can substitute for the PPBSF cofactor in enabling IL-7 to induce
proliferation of pre-pro-B cells in vitro (9). Here, we determined
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FIGURE 7. Formation of a heterodimer of native IL-7 with a soluble
cofactor in IL-7(2/2) pro-B CM. Equal volumes of IL-7(1/1) or IL7(2/2) pre-B CM were mixed with IL-7(1/1) or IL-7(2/2) pro-B CM as
indicated. One hour later, the total amount of IL-7 in these CM was affinity
purified, electrophoresed under nonreducing conditions, and developed
with anti-IL-7 mAb. The arrow (lane 3) indicates a newly formed ;55kDa IL-7-associated molecular complex that uses pre-B CM as a source of
native IL-7 (lane 1; 25-kDa band).
The Journal of Immunology
FIGURE 10. The PPBSF cofactor does not cross-react serologically
with TSLP, flt3, SDF-1, or IL-7. Equivalent amounts (500 ng) of affinitypurified PPBSF cofactor (see Fig. 9B) and rIL-7, TSLP, flt3, and SDF/1b
were electrophoresed under reducing conditions and developed with a
panel of Abs prepared against each of these factors. No cross-reactions
were observed when Ab concentrations were used that were ;10-fold
greater than those required to detect homologous reactions.
Discussion
When combined with the observations in a companion paper (9),
the present results appear to permit the following conclusions regarding PPBSF. Structurally, 1) PPBSF is a covalently linked heterodimer consisting of IL-7 and a cofactor with an Mr of ;30,000;
2) PPBSF can form in solution by the spontaneous association of
its two components; 3) the production of the two components of
PPBSF by BM stromal cells is independently regulated; but 4) the
formation and/or release of PPBSF under pro-B culture conditions
is coordinated such that little if any of either component normally
appears in monomeric form in the supernatant; and 5) the PPBSF
cofactor, and hence, PPBSF, is neither formed nor released under
pre-B cell culture conditions, thereby leaving only monomeric
IL-7 in the supernatant. Although the nature of the covalent binding reaction is not known, dissociation of the PPBSF complex by
DTT suggests that it occurs through a disulfide/sulfhydryl exchange mechanism similar to that observed in solution between
platelet-derived growth factor and a2-macroglobulin (21). Functionally, 1) PPBSF stimulates the proliferation of pre-pro-B cells
and some thymocytes but not pro-B cells or pre-B cells; 2) PPBSF
primes pre-pro-B cells and their immediate descendants to proliferate in the presence of IL-7 alone; and 3) both IL-7 and the
PPBSF cofactor maintain the viability of “unprimed” pre-pro-B
cells, but neither induces their proliferation.
Having previously demonstrated that CM from pro-B-type cultures selectively stimulates the proliferation of pre-pro-B cells in
vitro (8), the combined results of the present studies suggest that
PPBSF, rather than IL-7 alone or in combination with SCF and/or
IGF-1 (9), is the responsible agent. We also postulate that PPBSF
normally induces the observed differentiation of pre-pro-B cells to
pro-B cells (5, 6, 8, 22, 23). However, other cytokines, including
monomeric IL-7 itself (9) and some as yet undefined SDFs, appear
to amplify this process by inducing proliferation and/or differen-
tiation of pro-B cells (2, 3, 11–14, 24 –28). Although none of these
defined factors efficiently induces the development of pre-B cells
in our culture system (our unpublished observations), such differentiation is rapidly induced in vitro by incubation of pro-B cells in
IL-7(1/1) pre-B CM (29). Furthermore, surface IgM (sIgM)1 B
cells appear in vivo within 2 wk of adoptive transfer of culturegenerated pre-pro-B/pro-B cells to irradiated recipients (8; and our
unpublished observations). Hence, additional factors not effectively represented in our culture system appear to be required for
the differentiation of pro-B cells to pre-B cells (3, 14, 30, 31).
The observation that pro-B cells develop in IL-7 gene-deleted
mice (10) would appear to challenge the postulated role of PPBSF
in early B-lineage development. A more cautious interpretation,
which we favor, is that PPBSF is the preferred ligand under physiologic conditions but that compensatory mechanisms for stimulating the proliferation and, at the very least, the phenotypic differentiation of pre-pro-B cells exist under nonphysiologic
circumstances. Furthermore, such compensatory mechanisms may
be incomplete, given that phenotypic differentiation to pro-B cells
may occur in the absence of IgH gene rearrangements (32), and
that pro-B cell proliferation is markedly reduced in IL-7(2/2)
mice (C. Wei and I. Goldschneider, unpublished observations). It
is important to emphasize, therefore, that the in vivo administration of anti-IL-7 mAb prevents the development of pro-B cells in
normal mice (33), and that pro-B cells apparently fail to develop in
IL-7R a-chain (2/2) mice (34). Inasmuch as the absence of IL-7
itself does not prevent pro-B cell formation (10), the former results
suggest that anti-IL-7 mAb causes the coordinate elimination of
IL-7 and an associated cofactor, thereby providing indirect evidence for the existence of PPBSF in vivo (also see Ref. 35). In
addition, the latter results suggest that the compensatory factor in
IL-7(2/2) mice transduces a signal via the IL-7R. An intriguing
candidate is a TSLP-like molecule (11, 12), whose function might
be negatively affected in IL-7R a-chain (2/2) mice. Whatever its
identity, the present results suggest that this compensatory factor is
absent from IL-7(2/2) stromal cell CM.
Regarding the nature of the PPBSF cofactor, the most obvious
candidate is the soluble form of the IL-7R (36), especially because
other soluble ligand-receptor complexes in the hemopoietin family
have been found to have enhanced functional activity over the
ligand alone (37). Although not formally excluded, this possibility
seems unlikely for several reasons. First, the form of the soluble
IL-7R with the lowest molecular mass thus far described (38) is
still significantly greater than that of the PPBSF cofactor. Second,
the PPBSF cofactor appears to bind IL-7 covalently. Third, adsorption of CM with anti-IL-7R mAb does not remove the PPBSF
cofactor (our unpublished observations). Nevertheless, it will be
important to demonstrate that the PPBSF cofactor is produced by
BM stromal cells from IL-7R a-chain (2/2) mice.
Inasmuch as IL-7 is avidly bound by heparin (39), it is possible
that the PPBSF cofactor is a component of the stromal cell-associated extracellular matrix in our culture system (5, 6). Despite our
inability to detect PPBSF activity in extracellular matrices (ECMs)
extracted from BM-adherent cell layers with hypertonic saline (8),
continued efforts are warranted based upon reports of the regulation of growth-factor signaling by ECM proteins (40, 41) and especially the description by Oritani and Kincade (42) of a series of
ECM glycoproteins that selectively increase the IL-7-dependent
proliferation of pre-B cells.
A number of stromal cell-derived cytokines/chemokines that act
synergistically with IL-7 have been described, and, based on molecular mass and certain functional attributes, at least some might
theoretically be candidates for the PPBSF cofactor. Therefore,
Western blot analysis was conducted to determine whether any of
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whether any of three other cloned cytokines/chemokines that synergize with IL-7 in regulating early B-lineage development were
identical with the PPBSF cofactor. The results in Figure 10 show
that neither TSLP (11, 12), flt3 ligand (13), nor pre-B cell stimulation factor/SDF-1b (14) is detected by Western blot analysis using antiserum to the PPBSF cofactor, and that the PPBSF cofactor
is not detected by mAbs to TSLP, flt3 ligand, or SDF-1b. Furthermore, none of these cytokines/chemokines formed a heterodimer
when mixed with rIL-7 (data not shown).
2285
2286
PPBSF IS A HETERODIMER OF IL-7 AND A Mr 30,000 COFACTOR
the candidates bind to IL-7 and/or cross-react serologically with
the PPBSF cofactor. The negative results obtained with TSLP (11,
12), flt3 ligand (13), and pre-B cell stimulation factor/SDF-1 (14)
appear to exclude these factors, and previous mixing experiments
with rIL-7 have similarly excluded SCF and IGF-1 (9). Other IL7-synergizing factors are similarly being analyzed. However, studies of the primary amino acid sequence of affinity-purified PPBSF
cofactor are most likely to reveal its identity.
Acknowledgments
We thank Drs. Richard Murray and Ursula von Freeden-Jeffry (DNAX
Research Institute of Cellular and Molecular Biology) for providing the
breeding stock of the IL-7 gene-deleted mice and Dr. Paul Kincade (Oklahoma Medical Research Foundation) for his insightful review of the manuscript. We also thank Mrs. Leigh Maher for expert technical assistance and
Ms. Ruth Faasen and Ms. Cathy Mitchell for excellent secretarial
assistance.
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