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*
Faculty of Medicine, Department of Immunology, Technion, Haifa, Israel; and
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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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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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal C57/BL6 (B6), C57/BL6-Faslpr/Faslpr (B6/lpr), C57/BL6-µMT/µMT (µMT), and C57/BL6 gld/gld (gld mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice carrying both lpr and µMT homozygous mutations (µMT/lpr) were generated by crossing B6/lpr and µMT mice. µMT/gld mice were the progeny of the F2 generation of a µMT x gld cross. Littermates were typed for µMT, lpr, and gld homozygosity by PCR as previously described (4).
Analysis of serum Ig
Ab in serum was determined by sandwich ELISA. Sera from 4- to 6-mo-old mice was collected, and total IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM concentrations were measured using specific goat anti-mouse polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL). Purified Ig of each isotype provided the standard curve for calculation of Ab concentration, and results are expressed as micrograms per milliliter. Anti-chromatin IgG in serum was detected by ELISA as previously described (6). Affinity-purified mouse monoclonal IgG anti-dsDNA 3H9 (12) of known concentration served as a positive control and as a reference standard curve. Titers of anti-chromatin Abs for individual mice were calculated using the 3H9 Ab control standard curve and are expressed as micrograms per milliliter of 3H9 equivalent (amount of signal obtained in a tested sample equal to that obtained by a known concentration of the anti-dsDNA 3H9 mAb). In some experiments mice were immunized by OVA emulsified in CFA (100 µg/mouse, administered i.p.) or with dextran (Sigma, St. Louis, MO; 10 µg/mouse, administered i.v.). Mice were bled at various times after immunization, and Ab production was measured by specific ELISA. Dextran immunization and anti-dextran ELISA were performed as previously described (13).
RT-PCR assay
Total RNA was purified from RBC-depleted spleen cells derived
from the indicated mice. Samples were reverse transcribed to cDNA and
PCR amplified for V
-C to detect light chain expression and for
Gs
as a control as previously described
(14).
Ab and flow cytometry
Expression of surface molecules was detected using the following
Abs: CD43, S7 biotin (PharMingen); , goat anti-mouse light
chain biotin (Southern Biotechnology Associates); TCRß, H57 biotin
(PharMingen, San Diego, CA); CD19, 1D3 FITC (PharMingen), GL-7, GL-7
FITC (PharMingen); and B220, RA3-6B2 PE (Caltag, San Francisco, CA).
Biotinylated Abs were visualized with streptavidin-TriColor (Caltag)
for three-color analysis. Stained samples were analyzed by FACScan
(Becton Dickinson, Mountain View, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In µMT mice, B cell development in the bone marrow is blocked,
and cells die at the pro-B stage, resulting in complete deficiency of
mature B cells and agammaglobulinemia (Fig. 1
A) (11). This
occurs despite the fact that in µMT mice the genes encoding the
secreted form of IgM and all other Ig isotypes are intact. In striking
contrast to the µMT mice, introduction of the lpr mutation
rescues secretion of non-IgM serum Igs in µMT/lpr mice to
levels that are similar or even significantly elevated (IgG2a, IgA)
compared with those in their IgM-sufficient B6 and B6/lpr
counterparts (Fig. 1A). Careful analysis within the
µMT/lpr group revealed a skewed pattern of Ab production
in some individual mice. In some mice one Ab isotype was dominantly
found, whereas other mice produced high titers of all non-IgM serum
isotypes (not shown). Further light chain isotyping revealed that most
of the serum Abs were , but significant levels (up to 10-fold more
than in normal B6 or B6/lpr mice) were 
(data not shown).
Mice that showed a severe skewing of IgH isotypes had also a severely
skewed / ratio, which was dominated by either or light
chain (30–90% of the total light chain found in serum). The /
ratio in mice that produced all non-IgM serum Ig was also skewed, but
to a lesser extent, ranging from 2 to 25% (of the total light
chain found in serum).
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B indicate that
µMT/FasL-deficient (µMT/gld) mice have a phenotype
similar to that of µMT/lpr mice. In these experiments
serum IgG was measured in
(µMT/µMT x
gld/gld)F2 mice at various times after
birth. As expected, in µMT, FasL-sufficient littermates, IgG levels
dropped rapidly after birth due to the loss of maternal Ig, but
µMT/gld offspring had high IgG levels that rose to levels
similar to those of wild-type littermates by 90 days after birth (Fig. 1B). These findings suggest that in mice deficient in the
Fas/FasL pathway, defective B cells can circumvent developmental check
points, such as the block caused by the µMT mutation, mature, and
differentiate to Ab-producing cells. Detection of mature Ig+ B cells in µMT/lpr mice
To quantify peripheral B cells in µMT/lpr mice,
spleen cells were stained for membrane expression of the pan B cell
marker CD19 and the light chain and were analyzed by flow cytometry
(Fig. 2). As previously shown,
CD19+/Ig- pro-B cells are
found in spleens of µMT mice (Fig. 2A, right,
and Fig. 2C) (11). In contrast, significant
numbers of Ig-bearing B cells, which in several mice reached 10–15%
of lymphocytes, were detected in spleens of µMT/lpr mice
(Fig. 2A, middle, and Fig. 2C). This
finding was confirmed independently by the detection of Ig- light
chain mRNA in µMT/lpr, but not µMT, spleen (Fig. 2B). In addition, a high frequency of µMT/lpr B
cells, which was not different from that found in control B6 and
B6/lpr mice, expressed the germinal center marker GL-7 (Fig. 2C summarizes absolute numbers and frequencies of
Ig+, Ig-, and
GL-7+ B cells in total spleen cells). It is thus
suggested that µMT/lpr B cells can undergo cellular
activation and are able to participate in an immune response. However,
as shown in Fig. 2C, numbers of Ig+ B
cells in the µMT/lpr spleens were still 10- to 20-fold
lower than those found in spleens derived from normal B6 or
B6/lpr mice.
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, or heavy chain constant
regions. FACS analysis shown in Fig. 2D reveal that B cells
expressing or heavy chain are found in large numbers in
spleens of µMT/lpr mice, but not in Fas-sufficient µMT
spleens. As expected, no surface IgM (sIgM) expression was found in
µMT or µMT/lpr mice. sIgD expression was not detected in
both µMT/lpr and µMT spleens (not shown). The
possibility that detection of surface Ig- or heavy chain on
µMT/lpr spleen B cells reflected coating of the
CD19+ cells with serum Ig was excluded by
prestaining of µMT spleen cells (containing
CD19+/Ig- pro B cells)
with Ig+ serum collected from
µMT/lpr mice (not shown). It therefore appears that in
mice deficient of functional Fas or FasL, B cells bearing the µMT
mutation can undergo isotype switching, mature, and differentiate to
plasma cells. This process, however, may not be efficient as only small
number of Ig+ B cells could be found in
µMT/lpr spleens relative to control. Analysis of B cell development in bone marrow
To determine whether the previously reported µMT-induced block
in the pro-B to pre-B transitional stage of development was breached in
µMT mice lacking functional Fas/FasL, bone marrow B cell
developmental compartments were analyzed by flow cytometry. In mouse
bone marrow, pro-B cells are distinguished from later developmental
stages by surface coexpression of CD43 along with low levels of B220
(CD45R; Fig. 3, upper boxes)
(15). Pre-B cells lack CD43 and express higher levels of
B220 (Fig. 3, lower boxes) (15). As shown in
Fig. 3, the transitional block in early B cell development evident in
µMT mice is not substantially rescued in µMT/lpr mice.
These findings suggest that only few µMT/lpr B cells can
circumvent the µMT-induced developmental block by undergoing isotype
switching, and that this process does not necessarily occur in the bone
marrow.
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Because B cells have been shown to be critical in the induction
and propagation of the lupus-like disease in mice lacking functional
Fas or FasL (16, 17, 18), we studied the onset of lupus in
µMT/lpr mice. At 8–10 wk of age, µMT/lpr
mice developed significant lymphadenopathy (a lupus-like symptom that
is a feature of lpr mice) that was more severe than in their
IgM-sufficient B6/lpr counterparts, which developed this
symptom only at later ages (Fig. 4A). Also,
µMT/lpr mice at this age had significantly exacerbated
proteinuria (not shown). Further surface staining for lymphoid markers
and FACS analysis revealed that the cell outgrowth in
µMT/lpr mice was dominated by
TCRß+/B220+ cells (Fig. 4B). These cells have been shown previously to accumulate in
lymph nodes of old and sick lpr mice causing
lymphadenopathy. The results therefore suggest that the partial B cell
lymphopenia in µMT/lpr mice exacerbates the lupus-like
disease in lpr mice, as has been proposed
(6).
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Nuclear Ags are predominant self-Ags targeted by autoantibodies
that develop in lpr mice (6, 8, 17).
Anti-chromatin reactivity was measured in serum of young and old
µMT/lpr mice and compared with that of age-matched
B6/lpr, B6, and µMT mice (Fig. 5A). µMT/lpr mice
developed high titers of chromatin autoantibodies, and these titers
increased with age even more rapidly than in B6/lpr mice
(Fig. 5A). No direct correlation between levels of serum Ig
and anti-chromatin reactivity was found. Other autoantibodies
detected in µMT/lpr sera had anti-cardiolipin
specificity (40% of the mice; data not shown). In addition, we tested
the ability of µMT/lpr mice to produce Abs in response to
exogenous antigenic stimulation. Mice were immunized with OVA (a T
cell-dependent Ag) and with dextran (a T-independent Ag), and specific
serum Ab responses were measured over time. Results in Fig. 5B show that µMT/lpr mice failed to respond to
each of these Ags and were not different from the Fas-sufficient µMT
mice, which completely lack mature B cells and serum Abs. It is thus
possible that maturation of B cells in µMT/lpr mice is
specifically correlated with reactivity to self-Ags.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(19) or transgenic Ig-2b (20)
chains can support B cell development in the bone marrow. Thus,
defective B cells generated as a consequence of the µMT mutation
might be rescued by isotype switching, as we have shown in
µMT/lpr mice (Fig. 2), which is probably a limiting factor
in the survival of these cells. Unlike IgH- and IgH- expression
found in µMT/lpr spleen (Fig. 2D), we were not
able to detect significant expression of sIgD in spleen or bone marrow
of µMT/lpr mice (not shown). This, however, could be due
to the low frequency of IgD-expressing cells and the process of isotype
switching in these cells. While we cannot exclude the possibility that
isotype switching occurs in µMT B cells, our results clearly show
that in a Fas-deficient environment such B cells can mature and
differentiate to plasma cells and participate in an autoimmune
response.
Breakdown of self-tolerance in mice deficient of functional Fas
(MRL-lpr/lpr or lpr/lpr) results in lupus-like
disease, which is characterized by production of high titers of
autoantibodies such as anti-nuclear Ags (DNA, histone), rheumatoid
factors, and cardiolipin (6, 8, 17, 21). Early studies
used Ig-Tg/lpr mice to study the role of Fas in central B
cell tolerance and showed that Fas-deficient B cells bearing the Tg
specificity were efficiently eliminated in the bone marrow or the
periphery (6, 21). However, a very small population of
variant B cells with different specificities was still generated in
these mice, underwent activation and isotype switching, and was able to
produce high titers of anti-DNA Abs (6). Similarly, in
µMT/lpr (Fig. 2A) a relatively small
subpopulation of class-switched B cells, visible in spleen and lymph
nodes, gave rise to a severe autoimmune Ab response
(anti-chromatin, Fig. 5A and anti-cardiolipin; not
shown). In both experimental systems the ability of these cells to
develop exclusively depends on lack of Fas functionality. It was
previously suggested that these autoreactive B cells represent a
limited B cell repertoire with anti-nuclear specificities, which in
the absence of suppression mediated by nonautoreactive clones
exacerbates the disease (6, 22). Supporting this
hypothesis are recent findings showing that lack of functional Fas
promoted selection of anti-dsDNA B cells into the follicle and
production of autoantibodies in anti-dsDNA Ig/Tg MRL/lpr
mice (23). In addition, the absence of secreted IgM
accelerates IgG autoantibody production and lupus in lpr
mice (24). Consistent with all the above is our
observation that µMT/lpr mice have high titer of
anti-chromatin IgG, which develops more readily than in the
B6/lpr control mice (Fig. 5A), and their failure
to respond to external Ags upon immunization (Fig. 5B). In
addition, preliminary experiments of isoelectric focusing separation of
µMT/lpr serum IgG suggested the appearance of
oligo-monoclonal IgG in these mice (data not shown). Thus, as has been
proposed for MRL-lpr/lpr and lpr/lpr mice, a B
cell population with a limited repertoire of self-reactivity
(anti-histone and rheumatoid factor) may be expanded in the
periphery in a T cell-dependent (16, 25) or -independent
(6, 26) mechanism, thereby facilitating the development of
autoimmunity in µMT/lpr mice. It is also possible that
such autoreactive cells are generated in Fas-sufficient mice, but
undergo Fas-mediated apoptosis at the outer T cell zone, thus
preventing their migration into primary follicles, a paradigm that has
previously been suggested by Goodnow and colleagues
(10).
An interesting question raised by the present data is how the lack of a functional Fas/FasL pathway facilitates the development of B cells in µMT/lpr mice. A possible explanation is that Fas has a direct role in the elimination of defective and self-reactive B cell precursors. Fas is known to be expressed by developing B cells in the bone marrow (27, 28). The in vivo source of FasL that may regulate the µMT B cells is not clear. Possible sources include circulating T lymphocytes (29) and activated B cells (30). Our bone marrow analysis revealed that the µMT-induced block in B cell development is not substantially circumvented in µMT/lpr mice. Along this line of explanation, our results suggest that in the absence of Fas-FasL signaling the developmental block imposed by the lack of µ-chain, although substantially intact, is rendered somewhat "leaky," allowing slow accumulation of rare variant B cells in the periphery. Similar accumulation of variant B cells in peripheral organs was described in different B lymphopenic mice (31, 32, 33). However, unlike in µMT mice, the lack of Fas functionality in µMT/lpr mice may allow the specific expansion and survival of autoreactive B cells, perhaps due to the availability of dysregulated, Fas-deficient T cells.
Alternatively, as class switching may rescue µMT B cells, it is also
possible that B cell Ag receptor expression and B cell maturation may
first occur in the peripheral lymphoid organs of µMT/lpr
mice, for example, during the germinal center reaction. It has been
suggested that B cells at early stages of development can be recruited
to the germinal centers (34, 35) and may undergo Ig gene
rearrangements during a germinal center response (reviewed in Ref.
36). Furthermore, ongoing gene rearrangements in germinal
center sIg+ cells may generate
sIg+ B cells (34, 35), which
phenotypically are similar to pre-B cells or µMT B cells found in
peripheral organs (Fig. 2A) that normally fail to
participate in an immune response.
Interestingly, a recent study showed that in lpr mice, a
sIg- B cell generated by a stop mutation in its
expressed Ig- gene was able to subsequently participate in the
germinal center response, acquire self-reactivity through new gene
rearrangement, and contribute to the autoimmune response
(25). The results described here suggest that
Fas-deficient mice may be particularly permissive for participation of
Ig- B cells in Ab responses. Such permissiveness
may be accommodated by the lpr T cells, which had been shown
to be important in the induction of murine lupus and isotype switching
in lpr B cells (37). It is possible, though,
that these lpr T cells may be actively involved in the
recruitment and activation processes of self-reactive B cells in
µMT/lpr mice. Our results showing high frequency of
germinal center B cells in µMT/lpr, but not in µMT, mice
(GL-7+; Fig. 2C), are in agreement
with this hypothesis. Taken together, our results show that lack of
µH expression can be circumvented in mice deficient of functional
Fas/FasL by isotype switching, which can take place in the bone marrow
or in the periphery, a process that can be regulated by different
mechanisms as discussed above. However, the small number of mature
cells found in the µMT/lpr mouse suggest that this process
is inefficient and may be limited by the availability of T cell
activation and the selection process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Doron Melamed, Faculty of Medicine, Department of Immunology, Technion, Bat Galim, Haifa 31096, Israel.
3 Abbreviations used in this paper: FasL, Fas ligand; sIgM, surface IgM.
Received for publication February 23, 2000. Accepted for publication July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2b transgenic mice as a model for the rule of immunoglobulins in B cell development. Immunol. Res. 13:291.[Medline]
/ß" T cells. J. Exp. Med. 183:2271.5 protein in B cell development. Cell 69:823.[Medline]
ß T cells in the pathogenesis of murine lupus. J. Immunol. 162:7241.This article has been cited by other articles:
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  E. Edry, H. Azulay-Debby, and D. Melamed TOLL-like receptor ligands stimulate aberrant class switch recombination in early B cell precursors Int. Immunol., December 1, 2008; 20(12): 1575 - 1585. [Abstract] [Full Text] [PDF]
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 G. Perona-Wright, K. Mohrs, J. Taylor, C. Zaph, D. Artis, E. J. Pearce, and M. Mohrs Cutting Edge: Helminth Infection Induces IgE in the Absence of {micro}- or {delta}-Chain Expression J. Immunol., November 15, 2008; 181(10): 6697 - 6701. [Abstract] [Full Text] [PDF]
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E. Edry, S. B. Koralov, K. Rajewsky, and D. Melamed Spontaneous Class Switch Recombination in B Cell Lymphopoiesis Generates Aberrant Switch Junctions and Is Increased after VDJ Rearrangement J. Immunol., November 15, 2007; 179(10): 6555 - 6560. [Abstract] [Full Text] [PDF]
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A. Choudhury, P. L. Cohen, and R. A. Eisenberg Mature B Cells Preferentially Lose Tolerance in the Chronic Graft-versus-Host Disease Model of Systemic Lupus Erythematosus J. Immunol., October 15, 2007; 179(8): 5564 - 5570. [Abstract] [Full Text] [PDF]
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C. Jiang, J. Foley, N. Clayton, G. Kissling, M. Jokinen, R. Herbert, and M. Diaz Abrogation of Lupus Nephritis in Activation-Induced Deaminase-Deficient MRL/lpr Mice J. Immunol., June 1, 2007; 178(11): 7422 - 7431. [Abstract] [Full Text] [PDF]
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Y. Ueda, D. Liao, K. Yang, A. Patel, and G. Kelsoe T-Independent Activation-Induced Cytidine Deaminase Expression, Class-Switch Recombination, and Antibody Production by Immature/Transitional 1 B Cells J. Immunol., March 15, 2007; 178(6): 3593 - 3601. [Abstract] [Full Text] [PDF]
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J. E. Butler and N. Wertz Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG3 J. Immunol., October 15, 2006; 177(8): 5480 - 5489. [Abstract] [Full Text] [PDF]
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T. M Strutt, J. Uzonna, K. K McKinstry, and P. A Bretscher Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC Int. Immunol., May 1, 2006; 18(5): 719 - 728. [Abstract] [Full Text] [PDF]
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E. Edry and D. Melamed Receptor Editing in Positive and Negative Selection of B Lymphopoiesis J. Immunol., October 1, 2004; 173(7): 4265 - 4271. [Abstract] [Full Text] [PDF]
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P. Nanni, L. Landuzzi, G. Nicoletti, C. De Giovanni, I. Rossi, S. Croci, A. Astolfi, M. Iezzi, E. Di Carlo, P. Musiani, et al. Immunoprevention of Mammary Carcinoma in HER-2/neu Transgenic Mice Is IFN-{gamma} and B Cell Dependent J. Immunol., August 15, 2004; 173(4): 2288 - 2296. [Abstract] [Full Text] [PDF]
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J. Seagal, E. Edry, H. Naftali, and D. Melamed Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Ig{micro}-deficient/lpr mice Int. Immunol., July 1, 2004; 16(7): 905 - 913. [Abstract] [Full Text] [PDF]
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R. Marquina, M. A. Diez, M. Lopez-Hoyos, L. Buelta, A. Kuroki, S. Kikuchi, J. Villegas, M. Pihlgren, C.-A. Siegrist, M. Arias, et al. Inhibition of B Cell Death Causes the Development of an IgA Nephropathy in (New Zealand White x C57BL/6)F1-bcl-2 Transgenic Mice J. Immunol., June 1, 2004; 172(11): 7177 - 7185. [Abstract] [Full Text] [PDF]
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 J. Seagal, E. Edry, Z. Keren, N. Leider, O. Benny, M. Machluf, and D. Melamed A Fail-safe Mechanism for Negative Selection of Isotype-switched B Cell Precursors Is Regulated by the Fas/FasL Pathway J. Exp. Med., November 17, 2003; 198(10): 1609 - 1619. [Abstract] [Full Text] [PDF]
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J. Seagal, N. Leider, G. Wildbaum, N. Karin, and D. Melamed Increased plasma cell frequency and accumulation of abnormal syndecan-1plus T-cells in Ig{micro}-deficient/lpr mice Int. Immunol., September 1, 2003; 15(9): 1045 - 1052. [Abstract] [Full Text] [PDF]
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S. Shivtiel, N. Leider, O. Sadeh, Z. Kraiem, and D. Melamed Impaired Light Chain Allelic Exclusion and Lack of Positive Selection in Immature B Cells Expressing Incompetent Receptor Deficient of CD19 J. Immunol., June 1, 2002; 168(11): 5596 - 5604. [Abstract] [Full Text] [PDF]
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