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*
Immunology Division and
Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of the
IL-5R, but may also influence events downstream of the IL-5R. This
phenotype displays a codominant inheritance pattern, and is accompanied
by a variable but significant depression of peritoneal B-1 cell numbers
in 50% of the mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 129/Sv mouse substrain routinely used in this institute, designated 129/SvEms-+Ter?, was originally obtained from The Jackson Laboratory (Bar Harbor, ME) in the early 1990s. A recent description of the 129 strain pedigree details the history and derivation of this substrain (10). While the phenotype described below was initially characterized in mice bred and housed locally, it has been confirmed in mice (now designated 129/SvEms-+Ter?/J; stock number 002065) purchased directly from The Jackson Laboratory, and in a second 129 substrain, 129/Ola. We noted that the B cells of these mice behaved aberrantly when compared with those from other inbred strains (e.g., C57BL/6 and CBA). We have characterized the lesion as one affecting responses to signals delivered through at least three receptors (surface Ig, Rp105, and the IL-5R), all of which depend upon the Btk kinase for signaling. We provide some evidence to suggest that the lesion affects IL-5R expression and signal transduction, but does not lie in the btk gene itself.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Single cell suspensions were prepared from spleens by physical disruption of the tissue, and cells were cultured and stimulated, as described previously (12). After 3 days, cells were pulsed with [3H]thymidine, as described (13). Murine rIL-5 (PharMingen, San Diego, CA) was used at 100 U/ml, LPS at 10 µg/ml, anti-CD38 (NimR-5) at 10 µg/ml, and RP/14 at 1 µg/ml. The stimulation index was calculated as cpm in a stimulated culture divided by cpm in a matched, but unstimulated culture, and the values are shown ± the SD for triplicate wells. The counts in unstimulated cultures are given in each figure legend. All mice used in this study ranged from 6–16 wk of age.
Colony assays
Bone marrow cells were harvested and washed, and 50,000 cells/ml were plated in semisolid medium, as described (14). Titrations of IL-5 were tested to determine the 50% endpoint, which was similar for each sample. Colonies were scored from stained preparations of the cultures after 7 days of incubation.
ELISA
Serum Ig titers in unimmunized male mice (6–7 wk old) were determined as described previously (13). OD were read on a Molecular Dynamics (Sunnyvale, CA) vmax Kinetic Microplate Reader.
RT-PCR
Sorted splenic B cells (B220+) were
cultured with stimuli noted in the text. Cytoplasmic RNA was prepared
(15), and first strand cDNA was synthesized according to
manufacturer’s instructions (Pharmacia Biotech, Uppsala, Sweden).
Template cDNAs were titrated using serial dilutions and a
ß-actin-specific PCR (5' primer GTG GGC CGC TCT AGG CAC CAA; 3'
primer CTC TTT GAT GTC ACG CAC GAT TTC), with 25 cycles of PCR
(94oC for 1 min, 55oC for 1
min, 72oC for 1 min). Dilutions giving equivalent
levels of ß-actin product were used in PCR assays for either the
IL-5R (5' primer CAG TGG GAG AAA CCA CTT TCT GCC; 3' primer GAG ATG
CCA TTC TAC CAA GGA CTT A) or the ßc-chain (5' primer GAA CCT TCA ATG
CTT CTT TGA TGG GAT; 3' primer GTG TAG ACA CTG GCC CCC G). The PCR
conditions used were similar to those described above, except that the
annealing temperature for the -chain and ßc reactions was
60oC, and 30 cycles were performed. Reaction
products were resolved on agarose gels, transferred to filters, and
probed with cDNAs corresponding to the IL-5R-chain, ßc-chain, or
ß-actin.
Flow-cytometric analyses
Cell suspensions harvested from the peritoneal cavity, or prepared from the spleen, were stained as described previously (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice bearing a null mutation in the ßc-chain of the IL-3, IL-5,
and GM-CSF receptors (16) had been backcrossed to the
129/Sv mice described above. Examination of mitogenic responses of B
cells from ßc-/- mice gave the expected
result: the cells did not proliferate in response to IL-5, either alone
or in combination with an Ab to CD38 (Fig. 1
A). IL-5 alone is weakly
mitogenic for B cells, but synergizes powerfully with anti-CD38 to
stimulate B cell proliferation (17). Unexpectedly,
however, cells from 129/Sv control mice were also nonresponsive to IL-5
stimulation, with or without anti-CD38 (Fig. 1A). The
129/Sv cells did respond to anti-CD38 when IL-4 was provided as the
costimulus, indicating a specific defect in the IL-5 component of the
response. 129/Sv cells also exhibited a vigorous response to LPS. When
backcrossed to C57BL/6 mice for five generations, the
ßc-/- component of the phenotype segregated
from the 129/Sv effect, with ßc+/+ littermates
responding normally to all stimuli (Fig. 1B and data not
shown).
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The behavior of another IL-5-responsive cell type, the eosinophil
precursor, was examined. Bone marrow cells from C57BL/6, 129/Sv, and
ßc-/- (here on a C57BL/6 genetic background)
were cultured in semisolid medium in the presence of IL-3, GM-CSF, or
IL-5 (Table I). Although the
ßc-/- cells were unable to form colonies in
response to GM-CSF or IL-5, 129/Sv bone marrow yielded colonies in
response to all stimuli, with numbers of IL-5-responsive eosinophil
precursors comparable with C57BL/6 bone marrow. The eosinophil colonies
formed after stimulation by IL-5 were of normal shape and size, with
colony cells exhibiting normal eosinophil maturation. Therefore, the
inability to respond to IL-5 is confined to the B cell lineage in these
129/Sv mice.
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In light of the abnormal response of 129/Sv B cells to IL-5, we looked for other characteristics that might begin to explain the phenotype in molecular terms. One protein that is known to be required for transduction of IL-5 signals in B cells, and to be dispensable for IL-5 signaling in eosinophils, is Btk (18, 19, 20). The behavior of B cells from 129/Sv mice and those from a second commonly used 129 substrain, 129/Ola, was compared with those from Xid mice (the CBA/N strain). CBA/N mice carry a debilitating point mutation in the btk gene (4, 5).
Xid B cells are known to be refractory to stimulation through the B
cell receptor, as the Btk kinase is essential for propagation of the
mitogenic signal (5). Stimulation of B cells through
surface IgM cross-linking with an anti-µ Ab invoked a strong
proliferative response from C57BL/6 splenocytes (Fig. 2A), but, as expected, no
significant response from cultures of Xid splenocytes. Cells from
129/Sv and 129/Ola mice proliferated, but both were significantly
hyporesponsive in this assay (Fig. 2A). Another mitogenic
response that requires Btk is that initiated through cross-linking of
the Rp105 surface protein, using the mAb RP/14 (21). Even
though Rp105 is expressed at the same level on 129/Sv and C57BL/6 B
cells (data not shown), 129/Sv and 129/Ola splenocytes were refractory
to stimulation by this potent mitogen (Fig. 2B).
Furthermore, the 129/Ola substrain was, like 129/Sv, unresponsive to
stimulation with anti-CD38 and IL-5 (Fig. 2B). There is,
therefore, some concordance between the phenotypes of Xid B cells and
those from these two 129 mouse substrains, in that proliferative
responses to IL-5, to anti-IgM, and to anti-Rp105 were absent
or diminished. (This was uniformly true for all 129/Sv
(n = 23) and 129/Ola mice (n = 3)
examined.) Sequencing of the btk gene of the 129/Sv strain
did not, however, reveal a Xid-like mutation or any other abnormality
(data not shown), and a normal copy of the btk gene did not
complement the 129/Sv defect (see below).
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). The same was true for the
anti-Rp105 response (data not shown). Similarly, raising either the
anti-CD38 or the IL-5 concentration in the cultures had no effect
on the 129/Sv response, and the cells did not acquire responsiveness
later in the assay period (Fig. 3). These data indicate that 129/Sv B
cells are simply less sensitive to the proliferative signals initiated
by anti-Rp105 and IL-5 in particular (and by anti-µ to a
lesser extent) than C57BL/6 B cells.
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A), exemplified by the
down-modulation of the heat-stable Ag (25), and the
characteristic
IgMlow/IgDhigh profile of
the majority of cells. The 129/Sv mice also had normal serum IgM and
IgG3 titers. Interestingly, they consistently had higher IgG1 titers
than C57BL/6 and Xid mice. In these respects, the 129 mice do not
resemble Xid mice. However, 129/Sv mice frequently had reduced numbers
of peritoneal B-1 cells compared with control (C57BL/6) mice (Fig. 5). This observation was true for about
one-half of the 129/Sv and 129/Ola mice examined. There was variation
in the degree of reduction, and it did not seem to correlate strongly
with age. The low B-1 cell numbers, when they occur, might reflect the
inability of 129/Sv and 129/Ola B cells to respond to IL-5, a growth
factor for B-1 cells (26, 27, 28). When cells with a B-1-like
phenotype were present in the 129 mice (see Fig. 5, right
panel), they reproducibly displayed a slightly different labeling
pattern to that seen for C57BL/6 B-1 cells.
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In eosinophils and in B cells, IL-5 signaling is initiated at a
receptor that is comprised of an - and a ß-chain (ßc). Resting B
cells express very little of the -chain of the IL-5R; this chain is
induced upon cellular activation (29). Transcription of
the genes for both chains of the IL-5R was measured during activation
of 129/Sv B cells by CD38 and IL-5, a regimen known to induce
expression of the IL-5R (17). B220+
cells were purified from spleen by sorting, and the cells were
activated in vitro in the presence of anti-CD38 mAb and IL-5. After
24 h of culture, semiquantitative RT-PCR was performed on cDNA
generated from the cells, using primers for the IL-5R-chain, the
ßc-chain, and ß-actin (Fig. 6). A
small difference was seen in the expression of the ßc-chain of the
IL-5R in 129/Sv compared with C57BL/6 B cells: resting levels were low,
but these increased upon stimulation by 
2-fold and 6-fold,
respectively (values were normalizd to the ß-actin signal). In 129/Sv
B cells, both the resting and induced levels (normalized) of the
IL-5R-chain were 20-fold lower than in C57BL/6 B cells. Indeed,
there was little evidence for induction of the -chain in 129/Sv B
cells under these circumstances. This suggests that the defect in IL-5
signaling in the B cells of this mouse substrain is due largely to
their unusually low level of constitutive expression of the
IL-5R-chain and an inability to up-regulate its transcription upon
activation.
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129/Sv female mice were crossed to wild-type C57BL/6 males to
establish the mode of inheritance of the 129/Sv phenotype (with respect
to the proliferative responses assessed above). Splenocytes from all
F1 progeny responded normally to LPS, but poorly
to RP/14 or to IL-5 plus anti-CD38 (Fig. 7). The F1 level
was at best intermediate between the parental responses, indicating
that the phenotype was inherited in a codominant fashion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anomalous behavior of 129 B cells observed in this study was very specific, and seemed to partially overlap with responses that depend upon the Btk kinase. These included a diminished proliferative response to anti-µ signaling, and virtually no response to IL-5 (either alone or in combination with anti-CD38) or to cross-linking of surface Rp105, despite the fact that this molecule is present at normal levels on the surface of 129/Sv B cells. Rp105 shows homology to the Toll receptor family through characteristic leucine-rich repeats (31), and although its physiological ligand is not known, Rp105 may act to recognize molecular patterns specific to microorganisms (32). The signal emanating from Rp105 on B cells is not yet well defined, but a component of the pathway is clearly nonfunctional in the mice described in this study.
It is becoming clear that the IL-5 signaling pathway follows a
different course in mouse eosinophils and from that taken in B
lymphocytes. In B cells, the Btk, Lyn, and Fyn kinases all play
important roles, while in eosinophils the functions of these kinases
are dispensable for the IL-5 response (33). In the 129/Sv
mice described in this work, the B cell pathway has been specifically
impinged upon (Table I
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CD38 cross-linking up-regulates IL-5R expression, and this is thought
to be the means by which anti-CD38 synergizes with IL-5 to enhance
proliferation (29). Btk is required for the up-regulation
of the IL-5R-chain under these circumstances (29), and
the src family kinase Lyn has also been implicated
(34). In this study, anti-CD38 treatment failed to
induce expression of the IL-5R-chain in 129/Sv B cells (Fig. 6).
Therefore, 129/Sv B cells may be defective in some component of the
signal that allows CD38 to influence the IL-5R promoter. Failure of
129/Sv and 129/Ola B cells to respond to IL-5 may simply be due to the
inability to express sufficient IL-5R-chain mRNA to provide high
affinity IL-5R at the cell surface. Indeed, the kinetics of the
proliferative response, with abnormally low initial levels that did not
increase with time (Fig. 3B), correlate with the
transcription of the IL-5R-chain. This started low and did not rise
in response to a known inducer (Fig. 6). In vitro, we used
anti-CD38 as the inducer, but a number of stimuli have been shown
to up-regulate IL-5R-chain expression, including LPS and IL-4
(31). Proliferative responses to both of these agents were
shown to be normal in 129/Sv mice (Fig. 1A), so it is
possible that IL-5R expression may be induced normally and
physiologically by a number of means in vivo, other than through
CD38.
Despite some similarity to the Xid phenotype, other characteristics
were not shared between 129/Sv and Xid mice. 129/Sv B cells do respond,
albeit somewhat poorly, to surface Ig cross-linking (Fig. 2A), while Btk is absolutely essential for this
proliferative response (1). 129/Sv B cells can proliferate
in response to anti-CD38 when IL-4, rather than IL-5, is the
costimulus (Fig. 1A), while Xid B cells are refractory to
both CD38 and IL-5 signaling (20, 21). Xid B cells exhibit
a maturation block in the periphery (22, 23), which is not
apparent in 129/Sv B cells (Fig. 4). And finally, the normal levels of
serum IgM, IgG1, and IgG3 (Fig. 4) in 129/Sv mice attest to a less
severe restriction on B cell maturation and function than the loss of
Btk activity imposes. Indeed, sequencing of cDNA encoding the Btk
kinase from C57BL/6 and from 129/Sv mice failed to reveal any mutations
in the structural gene of the latter strain, nor gross differences in
the levels of Btk expression in B cells between the two strains (data
not shown).
The genetic basis for the phenotype of 129/Sv B cells may reflect a
polymorphism in a gene whose function is shared between the Rp105 (and
to a lesser degree the B cell receptor) signal for proliferation, and
for the correct induction of transcription of the IL-5R-chain gene
in response to a signal through CD38. Although we have shown that a
wild-type C57BL/6 btk allele was unable to correct the
nonresponsive phenotype in F1 mice (129/Sv
x C57BL/6; Fig. 7), we have some preliminary evidence for a functional
interaction between Btk and the 129/Sv mutation. In a cross between a
129/Sv female and a Xid (CBA/N) male, the F1
females (which would bear one normal and one mutant allele of the
X-linked btk gene) showed a consistently lower level of
proliferation to RP/14 and to anti-CD38 plus IL-5 than their
brothers (who would carry only the wild-type btk allele;
data not shown). The differences were small, but were consistent with
the mutant Btk protein enhancing the 129/Sv effect. No such difference
was noted when 129/Sv x CBA/J F1 mice were
examined (data not shown).
B-1 cells in 129 mice were often diminished in number (Fig. 5), but
this was variable and may be transient. In this regard, the 129 mice
resemble mice bearing targeted mutations in genes for IL-5
(35) or the IL-5R-chain (36). These mutant
mice display a transient deficit in B-1 cells that is resolved by
adulthood, presumably as a result of other signals promoting B-1 cell
growth apart from IL-5. Xid mice are unable to maintain a normal B-1
cell population (24), most likely because both IL-5 and Ag
receptor signaling are defective, so cells cannot receive the necessary
self-renewal signals.
This 129/Sv substrain comprises a production colony of The Jackson Laboratory, and mice are actively being sold to a number of different institutions (8 and personal communications). 129/Ola is also commonly used, and as the E14 embryonic stem cell line was originally derived from the Ola branch of the 129 strain (10), mutant mice derived from this cell line may display some of the characteristics described in this study. We have shown that several facets of B cell biology, including responses to certain mitogens and cytokines, and the dynamics of the B-1 lymphocyte population, are anomalous in this mouse substrain, and that the phenotype persists through at least one generation of outcrossing, through a codominant pattern of inheritance. Other branches of the 129 pedigree should be tested for the characteristics described in this work, and investigators should be aware of the influence of this genetic background on immune cell behavior, in the absence of any additional genetic manipulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lynn M. Corcoran, Immunology Division, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria 3050, Australia. E-mail address:
3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; ßc, common ß.
Received for publication May 24, 1999. Accepted for publication September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain: synergistic effects with interleukin 5. Proc. Natl. Acad. Sci. USA 92:11814.-deficient mice. Immunity 4:483.[Medline]
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