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Department of Biology and University of California, San Diego, Cancer Center, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß
encompasses treatment for a variety of diseases; however, prolonged
exposure to IFN-/ß results in elevated levels of autoreactive Abs.
In this study, we investigated the potential of IFNs to modulate
apoptotic signals in B cells. We demonstrate that IFN- or IFN-ß
inhibit Ag receptor-mediated apoptosis in a dose-dependent manner.
Inhibition of phosphatidylinositol 3' (PI3)-kinase did not abolish the
effect of IFN, indicating that the antiapoptotic mechanism is
PI3-kinase- and protein kinase B/Akt-independent. Instead, IFN- and
IFN-ß, but not IFN-
, significantly increase the levels of the
survival protein Bcl-2, and to a lesser extent, Bcl-xL
expression. Thus, IFN-/ß-mediated inhibition of B cell Ag
receptor-triggered apoptosis may offer a model for the process that
leads to the escape of self-reactive B cells from negative selection
and consequently results in autoantibody
production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-ß, is associated with a
variety of undesirable side effects, one of the most common being the
development of autoreactive Abs (3, 4, 5, 6, 7, 8, 9). A similar autoimmune response
has been observed in instances of systemic infectious disease, a
condition that is also likely to result in a massive production of type
I IFNs. The rise of autoreactive Abs is ordinarily prevented through the elimination of B cells recognizing self Ag at various stages during their development in the bone marrow and in peripheral lymphoid organs (10, 11). B cell Ag receptor (BCR)3-mediated programmed cell death in germinal centers can be triggered by Ag binding in the absence of interaction with CD40 ligand-bearing CD4+ Th cells (11). These findings can be reproduced in vitro with immature B cell lines by initiating apoptosis via cross-linking of the surface Ig with anti-IgM µ-chain Abs, and by the prevention of apoptotic cell death through simultaneous activation of CD40 signaling (12, 13). The abrogation of the apoptotic program after CD40 ligation is likely mediated through the induction of the survival factors and protooncogenes Bcl-xL and Bcl-2 (13). Indeed, transgenic mice that express Bcl-2 in a disregulated manner display substantial production of autoreactive Abs, presumably due to impaired negative selection of self Ag-directed B cells (14, 15, 16). Based on these observations, we decided to investigate whether IFNs could promote B cell survival by way of a similar mechanism. Using the EBV-negative germinal center B cell line Ramos, we demonstrate that type I IFNs not only cause up-regulation of Bcl-2 and, to a much lesser extent, Bcl-xL expression, but are, in fact, also able to prevent BCR-mediated apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ramos cells were cultured in RPMI 1640 supplemented with 10%
FCS, L-glutamine, penicillin, and streptomycin (Irvine
Scientific, Santa Ana, CA). Goat F(ab')2 anti-human IgM
(µ-chain-specific) was obtained from Southern Biotechnology
Associates (Birmingham, AL). mAb to human Bcl-2 was purchased from
Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal Ab to human
Bcl-xL and murine mAb to human PTP1D were ordered from
Transduction Laboratories (Lexington, KY). Phospho-specific Akt and
STAT1 Abs were obtained from New England Biolabs (Beverly, MA).
Merocyanin 540 (MC540) and propidium iodine were purchased from Sigma
(St. Louis, MO), and genistein, wortmannin, and rapamycin were obtained
from Calbiochem (La Jolla, CA). IFN-, IFN-ß, and IFN- were
generous gifts from Hoffman LaRoche (Nutley, NJ), Chiron
(Emeryville, CA), and Genentech (South San Francisco, CA),
respectively.
Induction and detection of apoptosis
All experiments were performed in RPMI 1640 with 10% FCS,
L-glutamine, penicillin, and streptomycin. Ramos cells
(5 x 105 cells/ml) were cultured with anti-IgM
mAb (10 µg/ml) in the presence or absence of IFN-, IFN-ß, or
IFN- in 24-well culture plates for 24 h. After treatment, cells
were harvested and diluted in ice-cold PBS, pelleted, and resuspended
in FACS staining buffer (1x PBS, 1% FCS, 0.1%NaN3).
Cells were stained with MC540 and immediately analyzed by flow
cytometry (FACScalibur System; Becton Dickinson, Mountain View, CA;
excitation 488 nm, detection 560–600 nm).
Western blot analysis
Following treatment, cells were washed with PBS and lysed with lysis buffer (0.5 ml) containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 10 mM ß-glycerophosphate, 1 mM sodium-vanadate, and 1 mM PMSF. Lysates were centrifuged at 15,000 x g for 15 min at 4°C, and protein concentration was determined by Bradford (Bio-Rad, Richmond, CA). Equivalent concentrations of protein were boiled in SDS sample buffer containing 2-ME and resolved by 7.5% SDS-PAGE (Bio-Rad) and electrotransferred onto Immobilon-P membranes (Millipore, Bedford, MA). After transfer onto poly(vinylidene difluoride) membrane, proteins were detected with anti-Bcl-2, anti-Bcl-XL, anti-PTP1D, anti-phospho-Akt, and anti-phospho-STAT1. Blots were developed with HRP-conjugated secondary Abs and enhanced chemiluminescence (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, upper
panel) or with (lower panel)
anti-IgM Ab. Several other studies resorted to incubations for up
to 48 h; however, we observed only minor differences in the rate
of apoptosis between 24 and 48 h (data not shown). Therefore, we
restricted our analysis to 24 h intervals to avoid interference
with the antiproliferative effects of IFN. The rate of apoptosis was
determined by flow cytometry after staining cells with merocyanin, a
fluorescent dye that detects changes in the organization of the
membrane lipid bilayer associated with early apoptotic events (18). In
agreement with earlier reports, anti-IgM-mediated cross-linking
triggered programmed cell death in 
25% of the stimulated cells (12, 17). Similar results were obtained when propidium iodine staining was
employed to determine the extent of apoptosis (data not shown). To
establish a basic correlation between the level of cross-linking and
the magnitude of apoptosis, cells were treated with increasing amounts
of anti-IgM Ab and assayed for merocyanin binding. The
dose-dependent induction of apoptosis after a 24-h stimulation
reached a peak at a concentration of 10 µg/ml anti-IgM Ab (Fig. 1B).
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(10,000 U/ml). As
shown in Fig. 2A, addition of
IFN- during IgM cross-linking resulted in a 50% reduction of the
rate of anti-IgM-induced apoptosis (compare top right
and bottom panels). The observed inhibition of Ag
receptor-mediated programmed cell death correlated directly with the
concentration of IFN- (Fig. 2B, left). Next,
we wanted to test whether the ability to prevent apoptosis was
restricted to IFN-, or if IFN-ß and IFN- were also able to
interfere with the progression of BCR-induced cell death. Ramos cells
were triggered to undergo apoptosis via BCR cross-linking in the
presence of increasing concentrations of IFN-ß or IFN-, and the
rate of apoptosis was determined after 24 h. IFN-ß, which
utilizes the same cell surface receptor as IFN-, was able to prevent
apoptosis to a similar extent as IFN- (Fig. 2B,
center). In contrast, IFN- affected the apoptotic process
only marginally (Fig. 2B, right).
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. Interestingly, inhibition
of PI3-kinase activity by wortmannin or rapamycin (Fig. 2
C)
had no obvious effect on IFN-’s ability to prevent BCR-mediated
apoptosis. The complete inhibition of Akt activation by epidermal
growth factor in primary human foreskin fibroblasts demonstrates that
wortmannin at the applied concentration is highly effective as a
PI3-kinase inhibitor (Fig. 2E, lanes 2 vs
3).
The inhibitory effect of IFN- was not altered when it was added
1 h before or 1 h after stimulation with anti-IgM Ab
(data not shown). This indicated that IFN- acts by a mechanism
distinct from interference with early apoptotic signaling events and
suggested hindrance of the apoptotic program based on gene induction.
It has been extensively documented that IFN-mediated gene transcription
requires the activation of members of the STAT family of transcription
factors, which in turn depends on their tyrosine phosphorylation and
dimerization (24, 25, 26, 27, 28). Therefore, we pretreated cells with the tyrosine
kinase-specific inhibitor genistein before addition of IFN- and
initiation of apoptosis by anti-IgM Abs. As shown in Fig. 2C, genistein was indeed able to prevent IFN- from
modulating the apoptotic response, thereby strengthening the argument
for IFN acting on a transcriptional level. Effectiveness of the
genistein pretreatment is also evident from the complete inhibition of
STAT1 tyrosine phosphorylation in response to IFN- (Fig. 2E, lanes 5 vs 6). Neither inhibitors
alone displayed an inhibitory effect on the anti-IgM-induced
apoptosis per se (Fig. 2D).
In vivo, germinal center B cells are prevented from undergoing
apoptosis after Ag recognition by receiving additional signals through
CD40 via interaction with CD40 ligand-bearing Th cells (12, 13).
Activation of CD40 has been shown to result in the transcriptional
up-regulation of the antiapoptotic protooncogenes Bcl-2 and
Bcl-xL (29). Furthermore, both Bcl proteins have been
recognized for their ability to prevent anti-IgM-induced cell death
(17). Therefore, we decided to analyze whether stimulation of Ramos
cells with IFN-/ß or IFN- would increase the expression levels
of either Bcl protein. Exposure of cells to IFN- or IFN-ß for
various times resulted in substantially increased amounts of Bcl-2,
whereas IFN- treatment had only a marginal effect on the expression
levels of this survival factor (Fig. 3A). Although the regulation
of Bcl-xL expression by IFNs seems to follow the pattern
set by Bcl-2, the observed changes in protein levels were only marginal
(Fig. 3B). The levels of STAT1 tyrosine phosphorylation
induced by the different IFNs correlates directly with the extent of
up-regulation of the Bcl proteins (Fig. 3C). To ensure that
the demonstrated up-regulation of Bcl-2 and Bcl-xL was
specific for those proteins, and to verify equality of loading of the
samples, the blot shown in Fig. 3A was stripped and reprobed
for the unrelated tyrosine phosphatase SHP-2, revealing identical
amounts of the protein in each sample (Fig. 3D). Addition of
anti-IgM Ab to Ramos cells during stimulation with IFN- (Fig. 3E, lane 3) or IFN-ß (data not shown) did not
alter IFN-mediated up-regulation of Bcl-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A critical outcome of B cell development is the selection of cells expressing functional Ag receptors lacking autoreactivity. Maturing B cells undergo positive selection in the bone marrow to ensure the presence of a functional B cell receptor. At later stages of lymphocyte development, autoreactive B cells are then eliminated from the mature B cell population by means of Ag receptor-mediated programmed cell death. Interference with this negative selection process, i.e., through the ectopic expression of the protooncogene Bcl-2, results in the abrogation of the apoptosis protocol and the escape of autoreactive B cells into the periphery with the subsequent production of self-directed Abs (14, 15, 16).
Recent studies have resulted in an increased appreciation of immunomodulatory responses elicited by IFNs that are distinct from their ability to interfere with cell cycle progression and viral replication (30). These biological functions of IFNs include their capability to manipulate the events that mediate programmed cell death. Interestingly, most of the investigations demonstrate the ability of IFNs to initiate apoptosis in a variety of cell types (31), presumably through the transcriptional up-regulation of components of the Fas-initiated apoptotic signaling cascade (32). However, a few studies also illustrate the potential of IFNs to act as negative regulators of programmed cell death, even though the underlying mechanism remained unidentified (33, 34).
Based on this information, we decided to investigate whether IFN treatment could lead to autoantibody production through interference with the apoptosis-based negative selection process of self-directed B cells. The Burkitt lymphoma line Ramos, which in its developmental stage resembles germinal center B cells, yields a robust apoptotic response after the cross-linking of cell surface IgM. As such, these cells provided an ideal model to investigate the putative modulatory actions of IFNs.
In the present study, we show that type I IFNs are indeed able to
drastically reduce the level of apoptosis initiated through Ag receptor
cross-linking. The type I IFN receptor has been shown to associate with
the lipid kinase PI3K (19), whose phosphorylation product is a known
activator of the antiapoptotic serine/threonine kinase Akt (PKB) (22, 23, 35). Consequently, it seemed likely that the prevention of
IgM-mediated apoptosis by IFN-/ß involves the activation of Akt.
However, our results clearly show that the prevention of apoptosis by
IFN-/ß occurs independently of PI3K and subsequent Akt activity,
since inhibition of the lipid kinase did not abrogate the antiapoptotic
properties of IFN-/ß. Furthermore, we were unable to observe an
IFN-mediated increase of Akt kinase activity (unpublished
observations). Additional experiments elucidating the kinetic
requirements for IFN-/ß to prevent apoptosis suggested that the
underlying mechanism depended on IFN-induced gene transcription.
Abrogation of the antiapoptotic properties of IFN-/ß in response
to the tyrosine kinase inhibitor genistein further supported the notion
of survival signal originating from a transcriptional response mediated
through the Jak/STAT signaling cascade.
Previous studies had shown that CD40 ligation is able to prevent
programmed cell death in Ramos B cells (13), and that increased
expression levels of the protooncogene Bcl-2 are the basis for this
event. The results presented here demonstrate that the survival factor
Bcl-2 is substantially up-regulated in response to type I IFNs, whereas
the related factor Bcl-xL is only slightly affected.
Interestingly, IFN-, which only marginally influenced BCR-mediated
apoptosis, also failed to induce a significant change in Bcl-2
expression levels. These observations were found to further correlate
directly with the level of STAT1 tyrosine phosphorylation induced in
response to the individual IFNs.
In summary, our results demonstrate that type I IFNs, in contrast to
IFN-, are able to prevent Ag receptor-mediated programmed cell
death. This abrogation of apoptosis is PI3-kinase-independent, but
displays a direct correlation with the IFN-/ß-induced
up-regulation of the antiapoptotic survival factors Bcl-2 and, to a
lesser extent, Bcl-xL. As inappropriate expression of
Bcl-2/Bcl-xL is a recognized cause for the development of
self-directed Abs by limiting apoptotic deletion of autoreactive B
cells, our results provide an attractive hypothesis by which the
evolvement of autoimmune responses in patients undergoing IFN therapy
could be explained.
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Acknowledgments |
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, IFN-ß, and IFN- were
generously provided by Hoffman LaRoche, Chiron, and Genentech,
respectively. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael David, University of California, San Diego, Department of Biology, Bonner Hall 3138, 9500 Gilman Drive, La Jolla, CA 92093-0322. E-mail address:
3 Abbreviations used in this paper: BCR, B cell Ag receptor; MC540, merocyanin 540; PI3, phosphatidylinositol 3'.
Received for publication January 19, 1999. Accepted for publication March 1, 1999.
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