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Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Taejon, Republic of Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many different stimuli are known to induce B cell activation. LPS, viral hemagglutinins, CpG bacterial DNA, anti-Ig Ab, and PMA are well-known polyclonal B cell activators (2, 3, 4, 5). However, their roles in B cell activation are not same. For example, anti-Ig Ab stimulates B1 cells to induce DNA synthesis (6). Maturation of blast cells into Ab-secreting cells requires T cell-derived differentiation factors. CD40 ligand (CD40L)3 expressed by T cells delivers important signals in B cells to regulate cell proliferation, production of Ig, Ig class switching, rescuing cells from apoptosis, and germinal center formation (7). The roles of CD40L in the regulation of the B cell response have been confirmed in CD40L-deficient mice (8, 9). However, CD40L-CD40 interaction does not account for all contact-dependent T cell help for B cells. CD40L-deficient T cells have been shown to induce the proliferation and differentiation of B cells successfully (10). CD30L (11) and BAFF (12) expressed by T cells induce B cell activation in a CD40-independent manner. Recently, a B cell-specific transmembrane protein, RP-105, was also known to trigger B cell activation through a unique pathway that was different from IgM-mediated or CD40-mediated pathways (13).
Histamine-releasing factors (HRFs) are a group of factors that release histamine and other mediators from mast cells and basophils. It has been reported that HRF is involved in the pathogenesis of allergic diseases. There are two types of HRFs: one induces histamine release in the presence of IgE, and the other operates independently of IgE. IgE-dependent HRF (IgE-HRF) was first molecularly identified by MacDonald et al. (14). This HRF is a unique molecule with no homology to any known IL, chemokine, or Ag. Subsequent studies have been shown that IgE-HRF plays an important role in perpetuating late phase allergic reaction (15, 16). In addition, IgE-HRF stimulates the production of IL-4 and IL-13 from all basophils in the presence of IgE (17, 18). However, additional data suggest that IgE-HRF has a unique signaling pathway and binds to a specific receptor other than IgE (19). The molecular information for the IgE-HRF receptor is not known yet.
Recent results (11, 12, 13) of CD40- or T cell-independent B cell stimulatory factors impelled us to search for novel B cell stimulatory factors. In this regard we recently established a cell line, LK1 (20), derived from the spleen of a mouse that showed typical splenectasia and expressed several cytokines. In addition, the culture supernatants of LK1 cells stimulated B cell proliferation compared with those of similar lineage cell lines. As an effort to purify novel B cell stimulatory factors secreted from this cell line, we identified IgE-HRF as a B cell activation factor. IgE-HRF stimulated B cell growth and differentiation, which was different from the effects of LPS. It bound to B cells and induced cytokine production from B cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS, MTT, and polymyxin B sulfate (PMB) were purchased from Sigma (St. Louis, MO). Recombinant murine IL-2, IL-4, and IL-5 were purchased from R&D Systems (Minneapolis, MN). Anti-mouse CD40, anti-mouse IgM, and anti-mouse class II Ab were purchased from Immunotech (Miami, FL). Sephadex G-10 and Mono-Q column were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden). The mAb against Thy 1.2 was obtained from the ascites of HO-13-4 hybridoma. Rabbit complement was purchased from Serotec (Oxford, U.K.). FITC-conjugated anti-CD3 and anti-CD45R/B220 were purchased from PharMingen (San Diego, CA). For the production of anti-HRF polyclonal Ab, purified recombinant IgE-HRF (rHRF; 250 µg) was mixed with the same volume of IFA (Sigma) and injected i.m. three times into a male rabbit (New Zealand White) at 2-wk intervals. The activity and specificity of this Ab were assessed by ELISA and Western blotting. PV200 malaria Ag peptide was a gift from David C. Kaslow, National Institutes of Health (Bethesda, MD).
Cell culture and DNA transfection
LK1 cells were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FBS (Life Technologies), penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (0.3 mg/ml). No additional growth factors were used, and cells were fed twice weekly by partial replacement with fresh medium. For transient transfection assays, HRF cDNA was subcloned into the mammalian expression vector pFLAG-CMV, and 293 cells were transfected with the plasmids by the calcium phosphate-DNA coprecipitation method (21).
Purification of HRF
Culture supernatants were collected from 25 liters of LK1 cells (1 x 106 cells/ml) cultured in serum-free and protein-free hybridoma medium (Sigma) for 24 h. The culture supernatants were concentrated by ultrafiltration (YM-10 membrane, Amicon, Beverly, MA) to 10 ml and dialyzed for 24 h in 10 mM Tris-HCl (pH 7.6). The dialyzed sample was then applied at a flow rate of 60 ml/h to a Mono-Q HR5/5 column equilibrated in 10 mM Tris-HCl (pH 7.6), and the adsorbed proteins were eluted with a linear gradient of NaCl from 0 to 1 M. Aliquots of each fraction were assayed for B cell-stimulating activity. The partially purified proteins possessing biological activity were collected and further purified onto a Mono-Q column with a modification of a linear gradient of NaCl. The fractions showing maximal biological activity were concentrated by 10% TCA (Sigma) and applied to the SDS-PAGE. Then proteins were electrophoretically transferred from the gel to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The amino acid sequences of NH2-terminal region were determined by automated Edman degradation using a gas phase sequencer.
Expression and purification of rHRF in Escherichia coli
The HRF-encoding region was obtained from the human keratinocyte
cDNA library using PCR and cloned into the pMAL-c2 expression vector
(New England Biolabs, Beverly, MA). PCR primers were made to introduce
an EcoRI site at the initiation codon (5'-cggaattc ATG ATC
ATC TAC CGG GAC) and a SalI site downstream of the stop
codon (5'-cgcgtcgac TTA ACA TTT CTC CAT CTC TAA GCC). The thrombin
cleavage site (gaattc CTG GTT CCG CGT GGA TCC gaattc) was inserted into
the EcoRI site of pMAL-c2-HRF for the cleavage of
maltose-binding protein (MBP)-HRF fusion protein (pMALc2-Thr-HRF).
E. coli strain JM109, containing the pMALc2-Thr-HRF was
grown in Luria Bertonia broth supplemented with 0.2% glucose. When the
OD at 600 nm was 0.8,
isopropylthio-
-D-galactoside was added to a final
concentration of 1 mM and incubated for 24 h at 20°C. The cells
were centrifuged and disrupted by sonication in 20 mM Tris (pH 7.5).
The supernatants were filtered with a 0.45-µm pore size filter, and
the MBP-HRF fusion protein was isolated using the amylose column (New
England Biolabs). The fusion protein was cleaved by thrombin treatment
and then isolated into MBP and HRF using a DEAE-Sepharose column
(Pharmacia Biotech). Endotoxin was removed using a PMB-agarose column
(Sigma) according to the method suggested by the manufacturer.
Limulus amebocyte lysate (LAL) assay
Detection of endotoxin was determined by LAL assay. The assay kit was purchased from Hemachem (Ringmer, East Sussex, U.K.), and the amount of endotoxin in rHRF or MBP was measured using LAL reagent according to the manufacturer’s instruction. Control standard endotoxin was used as a positive control, and pyrogen-free 10 mM Tris (pH 8.0) as a negative control.
Isolation of splenic B cells
Splenic B cells were isolated from 6- to 8-wk-old BALB/c mice as
previously described (22). RBC were removed by treatment
of RBC lysis buffer (Sigma). Splenic T cells were depleted by treatment
of anti-Thy 1.2 and rabbit complement-mediated lysis (Serotec). The
cells were applied to a Sephadex G-10 column to remove cell lysates and
other cell populations except B cells (Pharmacia, Piscataway, NJ). The
purity of B cells (B220-positive population) was 
90%.
Cell proliferation assay
Isolated splenic B cells (2 x 105/well) were plated on 96-well flat-bottom microtiter plates (Costar, Cambridge, MA) in 100 µl of RPMI 1640 supplemented with 10% FBS, and 100 µl of test samples were added. To destroy the biological activity of HRF, splenic B cell culture supernatants were treated with 1 mg/ml trypsin for 1 h at 25°C or boiled for 10 min. For the [3H]thymidine uptake assay, cells were incubated for 72 h in a humidified 5% CO2 incubator at 37°C. The cells were pulsed with 0.5 µCi of [3H]thymidine (sp. act., 84.8 Ci/mmol; New England Nuclear, Boston, MA) for the last 6 h of incubation and were harvested onto a glass-fiber filter using an automated cell harvester (Inotech, Zurich, Switzerland). The amount of radioactivity incorporated into the DNA was determined using a liquid scintillation counter (LS 6000A; Beckman, Palo Alto, CA). For the MTT assay, 100 µg of MTT was added to the each well, and the MTT assay was performed as previously described (23).
Ig production from splenic B cells
Splenic B cells were incubated with 1 µg/ml of LPS or 500 ng/ml of HRF in the presence or the absence of PMB (1 µg/ml) for 3 days. After incubation, cell culture supernatants were collected, and Ig production was measured with an mAb-based mouse Ig isotyping kit (PharMingen) according to the manufacturer’s instruction.
Flow cytometric analysis
Splenic B cells treated as indicated were washed with staining buffer (PBS containing 3% FBS and 0.1% NaN3) and stained with FITC-conjugated anti-CD3, anti-B220, or anti-MHC class II Ab for 30 min on ice. After incubation, the cells were washed and analyzed using a flow cytometer (Becton Dickinson, Mountain View, CA).
HRF binding assay
Whole splenocytes or splenic B cells (1 x 106 cells) were incubated with 1 µg/ml of LPS for 24 h at 37°C. After incubation, the cells were washed and incubated with biotin-conjugated rHRF for 30 min on ice. For competition experiments, a 10-fold excess of unconjugated rHRF or 2 µg/ml of anti-mouse IgE Ab was added to the cells together with biotin-labeled rHRF. Cells were washed with staining buffer, and streptavidin-conjugated PE (SA/PE) was added to each sample, followed by incubation for 30 min on ice. The cells were washed and incubated with FITC-labeled anti-B220 or anti-CD3 Ab for 30 min on ice. Binding of HRF was analyzed by flow cytometry.
Western blot analysis
Western blotting was conducted as previously described (24). 293 cells transiently transfected with pCMV-flag-human HRF were lysed in lysis buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 1 mM Na3VO4, 1 mM PMSF, 100 µg/ml aprotinin, and 1 µg/ml leupeptin). The protein concentrations were determined using Bradford reagent (Bio-Rad). Cell lysates containing equal amounts of protein were resolved by 10% PAGE and transferred to an immunoblot PVDF membrane (Bio-Rad). The blot was treated with anti-mouse HRF Ab followed by incubation with peroxidase-conjugated secondary Ab. The Ag-Ab complexes were detected using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). Following electroblotting, the blot was stained with Coomassie blue to normalize the protein concentrations of each lane.
RT-PCR analysis
Total cellular RNA was extracted by using RNAzol B
(Tel-Test, Friendswood, TX) according to the manufacturer’s
instruction. Aliquots (3 µg) of total RNA were transcribed into cDNA
at 37°C for 1 h in a total volume of 20 µl with 2.5 U of
Moloney murine leukemia virus reverse transcriptase (Roche, Mannheim,
Germany). Reverse transcribed cDNA samples were added to a
PCR mixture consisting of Takara 10x PCR buffer, 0.2 mM dNTP, 0.5 U of
Taq DNA polymerase, and 10 pmol of primers of each cytokines. The
primer sequences are as follows: IL-1, 5'-GAAGGGCTGCTTCCAAACCTTTGACC-3'
and 5'-TGTGGATTGAGGTGGATTCTTGC-3'; IL-2, 5'-AACAGCGCACCCACTTCAA-3'
and 5'-TTGAGATGATGCTTTGACA-3'; IL-4, 5'-GTCTGCTGTGGCATATTCTG-3' and
5'-GGCATTTCTCATTCAGATTC-3'; IL-6, 5'-ATGAAGTTCCTCTCTGCAAGA-3' and
5'-GGTTTGCCGAGTACATCTCAA-3'; IL-10,
5'-TCCTTAATGCAGGACTTTAAGGGTTACTTG-3' and
5'-GACACCTTGGTCTTGGAGCTTATTAAAATC-3'; TNF-
,
5'-GGCAGGTCTACTTGGAGTCATTGC-3' and 5'-ACATTCGAGGCTCCAGTGAATTCGG-3';
TGF1, 5'-TGGACCGCAACAACGCCATCTATGAGAAAACC-3' and
5'-TGGAGCTGAAGCAATAGTTGGTATCCAGGGCT-3'; and -actin,
5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCC TTAATGTCACGCACGATTTC-3'. All
PCR mixtures were heated to 95°C for 1 min and cycled 30 times at
95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, followed by
an additional extension step at 72°C for 10 min. PCR products were
electrophoresed and visualized by ethidium bromide staining.
EMSA
Splenic B cells (1 x 107) were
stimulated with 1 µg/ml of LPS or 500 ng/ml of rHRF in the presence
or the absence of anti-CD40 Ab (250 ng/ml) for 6 h. The
nuclear extracts were prepared according to the procedure previously
described (25). DNA mobility shift assays were performed
using double-stranded oligonucleotides comprising the consensus
sequences for NF-
B (5'-GGGAGTTGAGGGGACTTTCCCAGGC-3').
Oligonucleotides were terminal-labeled with
[-32P]dCTP using a Klenow fragment of DNA
polymerase I. Aliquots of nuclear extracts (5 µg) were incubated at
room temperature for 30 min with labeled oligonucleotides in a total
volume of 20 µl under following conditions: 4% glycerol, 1 mM
MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM
Tris-HCl (pH 7.5), and 2 µg of poly(dI:dC). DNA-protein complexes
were electrophoresed on a 6% polyacrylamide gel, and the gel was dried
and autoradiographed.
In vivo treatment of HRF protein or plasmid DNA
To investigate in vivo effects of rHRF and the cDNA for HRF on Ig production, splenocyte proliferation, and cytokine expression, BALB/c female mice (three mice per group) between 6 and 8 wk of age were injected i.m. with 20 µg of rHRF or with 50 µg of pcDNA-HRF in combination with 20 µg of PV200 peptide Ag on days 21, 31, and 41. The mice were sacrificed on day 10 after the last injection, and antisera were collected for determination of Ig production. Anti-PV200 Ab production was determined by ELISA. Splenocytes derived from these mice were removed and then stimulated with 500 ng/ml of rHRF plus 250 ng/ml of anti-CD40 Ab for 3 days. The cells were pulsed with 0.5 µCi of [3H]thymidine for the last 6 h of incubation, and cell proliferation was determined by the amount of radioactivity incorporated into the DNA.
Statistical analysis
For statistical analysis of data, p values were analyzed using the paired Student’s t test program (StatView 5.1; Abacus Concepts, Berkeley, CA). Results were considered statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously we established a murine erythroleukemia cell
line, LK1 cells (20). Based on RT-PCR, LK1 cells produce
cytokines such as IL-5, IFN-
, and TNF- (20). When
the culture supernatants of LK1 cells were incubated with whole spleen
cells for 4 days, B cells multiplied, but the T cell population
decreased, suggesting that LK1 cells secrete B cell stimulatory factors
that decrease the proportional T cell population or that there are some
inhibitory factors for T cell proliferation in their culture
supernatants (Fig. 1
A). They
also increased the proliferation of purified B cells dose-dependently
(Fig. 1B). Combined treatment of rIL-2 and rIL-4 increased B
cell proliferation in a dose-dependent manner also. The B
cell-stimulating activity was abolished by boiling and trypsin
treatment of the culture supernatants (Fig. 1C). The culture
supernatant or other established murine erythroleukemia cell lines,
such as MEL and DS19, showed much less B cell proliferation activity
(Fig. 1C). As an effort to isolate potential B
cell-stimulating factors, 25 l of these supernatants were
concentrated, and the proteins contained therein were fractionated by
Sephadex G-75 gel filtration and Mono-Q anion exchange chromatography
(Fig. 2). Fractions 16–35 (Fig. 2A) were pooled and then rechromatographed. Fraction 16
(Fig. 2B) possessing biological activity was further
purified. Finally, fraction 20 (Fig. 2C) showing B
cell-proliferating activity was subjected to SDS-PAGE and blotted onto
a PVDF membrane for peptide sequencing. The
NH2-terminal amino acids
(NH2-MIIYRDLISHD-COOH) revealed 100% homology to
those of murine IgE-HRF. IgE-HRF is known to be involved in allergic
reaction and to stimulate the production of IL-4 and IL-13 from
basophils, but nothing has been known about its effects on B cell
function.
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To prove its biological activity further, the cDNA for murine HRF
was subcloned into the pMAL-c2 plasmid, expressed as a fusion protein
with MBP, and isolated from MBP by thrombin treatment and column
chromatography (Fig. 3A). To
remove the endotoxin in the rHRF preparation, PMB-agarose column
chromatography was performed, and the endotoxin level was measured by
LAL assay. Endotoxin activity in the sample was removed to a level
below the limit of detection (<1 pg/µg). Recombinant HRF stimulated
mouse splenic B cell proliferation dose-dependently, with maximal
activity at 100 µg/ml (Fig. 3B). Control MBP had minimal
effects at high concentrations. This activity was completely abolished
by boiling and anti-HRF Ab treatment (Fig. 3C). HRF was
also transiently expressed in a mammalian cell line, 293 transformed
embryonal kidney cells (Fig. 3D). Culture supernatants of
HRF-transfected cells showed B cell stimulatory effects in a
dose-dependent manner, but normal medium or culture supernatants of
vector-transfectants had minimal effects (Fig. 3E). However,
murine HRF had no stimulatory effect on human B lymphocytes (data not
shown). IL-2, IL-4, IL-5, anti-CD40 Ab, and anti-IgM Ab are
well-known stimulators of murine B cells. Next, the combination effects
of rHRF on B cell proliferation were assayed in the presence of these B
cell stimulators. In the presence of anti-CD40 Ab (250 ng/ml), rHRF
increased B cell proliferation synergistically, and its activity peaked
at 500 ng/ml (Fig. 4A).
Thereafter, rHRF at a concentration of 500 ng/ml was used in the
cotreatment experiments with anti-CD40 Ab and other stimulators.
Recombinant HRF showed mild synergistic effects with IL-2, IL-4, and
IL-5 (Fig. 4B). Maximal activity was observed in the
combination of cytokines, rHRF, and anti-CD40 Ab. Homotypic
aggregation of B cells was examined by microscopic observation (Fig. 4C). Weak aggregation was observed when B cells were
cultured with the culture supernatant of LK1 cells (Fig. 4C,
b) or MBP-HRF (Fig. 4C, d). The maximal homotypic
aggregation of B cells was observed when B cells were cultured in the
presence of MBP-HRF, anti-CD40, and anti-IgM Ab (Fig. 4C,
f), even though MBP plus anti-CD40 and anti-IgM Ab had
minimal effects (Fig. 4C, e). MHC class II expression is
another marker for B cell activation (13). When B cells
were treated with LPS or rHRF, MHC class II expression was elevated
(Fig. 5A). The effects of LPS
on MHC class II expression were abolished by PMB (Fig. 5AI),
a cyclic cationic peptide antibiotic that neutralizes the biological
activity of LPS (26, 27), but those of rHRF were not
affected by PMB (Fig. 5AII), suggesting that the effects of
rHRF are different from those of LPS. Recombinant HRF also moderately
increased the expression of other B cell surface molecules, such as
CD22 (Fig. 5B) and CD54 (Fig. 5C), which are
related to cell adhesion. Interestingly, rHRF significantly induced
CD69 expression, which is the earliest leukocyte activation Ag and is
expressed on activated B cells (28) (Fig. 5D),
but it had little effect on CD23 and CD44 expression (data not shown).
Next, Ig production from B cells was monitored by isotype-specific
ELISA. Recombinant HRF induced Ig production from B cells (Table 1). In comparison with LPS, rHRF induced
less IgG, or IgA, but it induced more IgM in the absence or the
presence of anti-CD40 Ab, suggesting that rHRF induces a different
stage of B cell differentiation compared with LPS. PMB abolished the
effects of LPS on Ig production dramatically, except for IgM, but it
had no effect on those of rHRF. HRF enhanced IL-4 and IL-13 secretion
by human basophils (18), and it was observed that HRF
induced IL-6 and IL-10 gene expression in B cells (see below). Its
effects on Ig production may be due to the induced cytokines. When
anti-IL-6 Ab or anti-IL-10 Ab was added to the culture,
rHRF-induced IgM production was reduced partially (19.5 ± 5.3%
reduction by anti-IL-6 Ab and 14.4 ± 2.7% reduction by
anti-IL-10 Ab, respectively).
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Based on its effects on B cell activation, it is likely that the
HRF-binding molecule would be expressed on B cells. Binding of HRF was
monitored using SA/PE-conjugated rHRF (HRF-SA/PE) and anti-B220 Ab.
HRF-SA/PE bound B cells as expected compared with IgG plus SA/PE or
unlabeled HRF control (Fig. 6A). Its binding was competed
with unlabeled cold rHRF (Fig. 6B). Previously, HRF was
thought to bind and stimulate target cell by interacting with IgE
molecules on the surface of these cells. However, additional studies
showed that HRF stimulates cells by binding to an unknown receptor,
which is distinct from IgE. When cells were incubated with anti-IgE
Ab, there was no difference in HRF binding to B cells, further
suggesting that HRF binds to cells through its own receptor, which is
not related to IgE.
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Murine B cells produce several cytokines in response to mitogens.
Recombinant HRF induced IL-1, IL-6, and IL-10 expression from B cells
in the absence or the presence of anti-CD40 Ab, but it had no
effect on IL-2, TNF-, and TGF-1 expression (Fig. 7A). NF-B is known to be
one of important transcription factors regulated during B cell
activation. NF-B was activated slightly by rHRF alone, but it was
dramatically activated by rHRF in combination with anti-CD40 Ab
(Fig. 7B).
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To analyze the in vivo effects of HRF on Ig production and
cytokine expression, BALB/c mice were injected with pcDNA-HRF or rHRF
in combination with PV200 peptide Ag. Antisera from these mice were
collected for determination of Ig production. As shown in Table 2, total Ig production was increased by
rHRF treatment. Consistent with in vitro results, IgM production was
dominant. When pcDNA-HRF was injected, a similar pattern was observed
compared with pcDNA or pcDNA-IL-18 injection. Ag-specific Ig production
was monitored by PV200 ELISA (Fig. 8A). As expected, rHRF
increased the PV200-specific Ig production. However, infusion of IgE
with rHRF had no effect on Ig production in vivo (data not shown).
Next, splenocytes from immunized mice were restimulated with
anti-CD40 Ab in vitro. As shown in Fig. 8B, splenocyte
proliferation was remarkably increased in the case of cells obtained
from mice injected with rHRF or rHRF plus PV200 Ag compared with cells
obtained from mice that did not receive rHRF. In addition, the secreted
Ig was measured (Table 3). As shown for
cell proliferation, Ig production was elevated after in vivo injection
of rHRF of HRF cDNA compared with the control level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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produced from NK cells enhances IgG3 production, and TGF- causes IgA
secretion from B cells (10). A Toll-like receptor protein,
RP-105, can trigger B cell activation without any costimulators
(13). IgE-HRF was originally known as a complete secretagogue for histamine and IL-4 from basophils expressing IgE+. However, HRF could increase histamine and IL-4 from IgE- basophils when preincubated with HRF and challenged with anti-IgE Ab (18). Also, HRF did not bind to IgE. Rottlerin, a nonstaurosporine-derived kinase inhibitor isolated from Mallotus philippinensi, discriminated between HRF and other IgE-dependent (anti-IgE and Ag) histamine release (19). It had no effect on histamine release induced by anti-IgE, but it enhanced rHRF-mediated histamine release. These results suggest that HRF activates cells by interacting with a specific receptor on the cell surface.
In this paper we identified IgE-HRF as a B cell stimulatory factor from
the culture supernatants of LK1 cells that also expressed IL-5 and
IFN- (20). The effects of rHRF on B cell functions were
demonstrated in several aspects. First, rHRF directly enhanced B cell
proliferation at a concentration of less than 1µg/ml. It had strong
synergies with the known B cell stimulatory cytokines and anti-CD40
Ab. In the presence of anti-CD40 Ab, rHRF could activate B cell
growth at a concentration of <10 ng/ml. It induced B cell blast
formation maximally in the presence of anti-CD40 Ab and
anti-IgM Ab. Second, rHRF induced MHC class II expression, a marker
for B cell activation (13). Third, it activated NF-B
activity of B cells especially in the presence of anti-CD40 Ab.
Finally, it could stimulate in vitro and in vivo Ig production from B
cells. In addition to functional aspects, a binding study of HRF to B
cells demonstrated that HRF binds B cells via its own receptor, which
is different from IgE as suggested previously (19). These
data from studies of B cell proliferation and differentiation clearly
demonstrated that rHRF is another B cell stimulatory factor that has
synergies with other known B cell activation factors.
Immature B cells, characterized by the expression of mIgM, migrate to
the periphery where they transverse a transitional stage to become
naive mature B cells (29). CD69 has been known as an
earliest leukocyte activation maker. A recent study reported that
CD69-deficient mice showed normal functions of T cells, platelets,
neutrophils, and eosinophils (28). However, CD69 knockout
mice showed a significant increase in the number of pre-B and immature
B cells compared with wild-type mice. These results indicate that CD69
plays a critical role in B cell development and early activation.
Recombinant HRF increased CD69 expression significantly (Fig. 5
), and
it affected IgM production the most, especially in the presence of
anti-CD40 Ab (Tables 1 and 3). Other differentiation Ags, such as
CD23 and CD44 (30, 31), were not affected by rHRF. In
addition, its effect on Ig production was partially inhibited by Abs
against IL-6 or IL-10, which were induced by rHRF. Based on these
results, the effects of rHRF on B cell functions suggest that rHRF
induces B cell proliferation more than differentiation into
Ig-secreting plasma cells compared with polyclonal B cell
activation.
Removal of bacterial endotoxin (LPS) contamination was one of the major
concerns for the purification of rHRF. It was also monitored in
different ways. First, boiling or Ab treatment completely abolished the
HRF effects on B cell growth. Second, rHRF from 293 mammalian cells
showed the similar B cell growth activity. Third, Ig production induced
by LPS or rHRF showed different patterns: rHRF induced higher levels of
IgM than LPS did, but to a lesser extent in the cases of IgG and IgA.
Fourth, PMB which neutralizes the biological activity of LPS (26, 27), reduced LPS effects on Ig production (Table 1) and MHC
class II expression (Fig. 5), but did not have any effect on rHRF
activity. Furthermore, LPS and rHRF exhibited the different effects on
the induction of IL-6 gene expression and NF-B activation in the
presence of anti-CD40 Ab (Fig. 7). Collectively, the effects of
rHRF on B cell functions such as Ig secretion, MHC class II, and gene
expression were distinguished from those of LPS.
Histamine itself affects B cell proliferation and differentiation. Histamine induces B cell proliferation in the presence of anti-IgM Ab only when the serum was dialyzed and c-Kit-positive cells were removed (32). It enhances IgE and IgG4, but has no effect on IgG, IgM, or IgA production in the presence of IL-4 or IL-13 (33). Based on the culture conditions and Ig production patterns, it is unlikely that the effects of rHRF on B cell activities in this study are due to the indirect effects of histamine production.
During the process of B cell activation and differentiation, phenotypic
and functional changes occur, such as Ig rearrangement and expression
of cell surface molecules. Ú\/These cellular events are integrated by
transcription factors, which execute a program of activation and
differentiation by regulating gene expression. Among them, PU.1 has
been known to regulate various B cell-specific genes, including mb-1,
Ig J chain, and Ig -chain, by cooperating with other transcription
factors, such as Pip and c-Fos (34). NF-B is also
involved in regulating the Ig light chain gene and B cell activation.
In vivo, NF-B-null mice have a defect in B cell activation and Ig
secretion. Recombinant HRF activated NF-B activity and
NF-B-related cytokine gene expression, especially in the presence of
anti-CD40 Ab. Identification of an HRF receptor and
receptor-mediated signaling will elucidate the molecular actions of HRF
in B cells.
CD40L cooperates with various cytokines to induce B cell activation,
proliferation, and Ig isotype switching. CD40L, BAFF, and CD30L are
expressed by T cells and are important for the functions of B cells at
multiple steps of the T cell-dependent immune response. CD40L
counteracts apoptotic signals in B cells after B cell receptor
engagement, but BAFF is not able to rescue B cells from
anti-µ-mediated apoptosis (12). HRF is expressed in
various types of cells, including T cells, monocytes, fibroblasts, and
some tumors (14). It is also released from macrophages,
which are stimulated with macrophage CSF (35). HRF binding
patterns (Fig. 6) indicated that B cells have unidentified HRF-binding
molecules that were functionally active in basophils, mast cells, and
eosinophils also, suggesting that it may be involved in a broad
spectrum of immune responses. Functionally, HRF has been well known as
a critical factor in late phase allergic reaction (15, 16). In addition, our report and other studies (15, 19) have shown that HRF stimulates target cells in the absence
of IgE. Taken together, these results indicate that HRF itself
functions as an initiator for multi-immune regulators, including B cell
activation, mediating the possible interaction between allergic
response and Ab production. More dissected studies, including
identification of its receptor and possible roles of HRF in B cell
apoptosis, are required to understand the HRF-mediated molecular events
leading to humoral immune responses.
In this paper we identified the IgE-HRF as a B cell activation factor. IgE-HRF showed synergy with anti-CD40 Ab in B cell proliferation and activation. However, its effects were different from the effects of LPS on Ig production and inhibitor sensitivity.
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2 Address correspondence and reprint requests to Dr. Inpyo Choi, Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Eoun-Dong 52, Yusong, Taejon 305-333, Republic of Korea. E-mail address: ipchoi{at}kribb4680.kribb.re.kr
3 Abbreviations used in this paper: CD40L, CD40 ligand; HRF, histamine-releasing factor; SA/PE, streptavidin-conjugated PE; IgE-HRF, IgE-dependent HRF; PMB, polymyxin B sulfate; PVDF, polyvinylidene fluoride; MBP, maltose-binding protein; LAL, Limulus amebocyte lysate; rHRF, recombinant IgE-HRF.
Received for publication September 20, 2000. Accepted for publication March 21, 2001.
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,
p < 0.05 vs no rHRF group; **,
p < 0.01 vs all other groups. B,
The cells were stimulated with various cytokines (100 U/ml) or rHRF
(500 ng/ml) in the presence or the absence of anti-CD40 Ab (250
ng/ml) for 3 days. Cell proliferation was determined as described.
**, p < 0.01 vs all other groups.
C, After 3 days of incubation, homotypic aggregation of
splenic B cells was observed by light microscopy (x100).
a, Medium; b, LK1 cell culture
supernatants (10%); c, MBP (100 µg/ml);
d, MBP-HRF (100 µg/ml); e, MBP (100
µg/ml), anti-CD40 (250 ng/ml), and anti-IgM (100 ng/ml);
f, MBP-HRF (100 µg/ml), anti-CD40 (250 ng/ml), and
anti-IgM (100 ng/ml).
