Abstract
The use of 129 strain-derived embryonic stem cell lines for targeted gene mutation in mice has led directly to an expanded use of this inbred strain worldwide. It has been noted, however, that the 129 genetic background can make a significant contribution to the severity of a mutant phenotype. In this study, we reveal a specific defect in the IL-5 and Rp105 responses of B lymphocytes from two widely used 129 mouse substrains. The response to stimulation through surface IgM is also diminished, although to a lesser degree, in these mice. The lesion appears to reduce significantly the expression of the α-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.
Occasionally in the long pedigree of an inbred mouse strain, a phenotypic variant arises such as the immunodeficient CBA/N and SCID mice (1, 2) and the LPS nonresponsive C3H/HeJ mouse (3). The characteristic phenotypes in these three cases are now known to be caused by mutations in genes for Bruton’s tyrosine kinase (Btk),3 the catalytic subunit of DNA-dependent protein kinase, and the Toll-like receptor, Tlr4, respectively (4, 5, 6, 7). The 129 inbred mouse strain, founded in 1928 (8), has become the strain of choice for gene-targeting experiments because of the facility with which 129-derived embryonic stem cell lines can colonize an embryo and contribute to the germline of the resultant chimeric animal. Chimeric founders are commonly backcrossed to the 129 strain to obtain a uniform genetic background upon which to assess the consequences of an engineered mutation. These characteristics and practices have led directly to an increased use of the 129 inbred strain in the recent past. However, there have been several examples of contributions to the penetrance or severity of a phenotype by the 129 genetic background itself (e.g., Refs. 9, 10, 11).
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.
Materials and Methods
Proliferation assays
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 β-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 β-actin product were used in PCR assays for either the IL-5Rα (5′ primer CAG TGG GAG AAA CCA CTT TCT GCC; 3′ primer GAG ATG CCA TTC TAC CAA GGA CTT A) or the βc-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 α-chain and βc 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-5Rα-chain, βc-chain, or β-actin.
Flow-cytometric analyses
Cell suspensions harvested from the peritoneal cavity, or prepared from the spleen, were stained as described previously (12).
Results
Anomalous behavior of 129/Sv B cells in response to IL-5
Mice bearing a null mutation in the βc-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 βc−/− 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. 1⇓A). 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 βc−/− component of the phenotype segregated from the 129/Sv effect, with βc+/+ littermates responding normally to all stimuli (Fig. 1⇓B and data not shown).
Hyporesponsiveness of 129/Sv splenocytes in vitro. A, Responses to LPS, IL-4, IL-5, and anti-CD38 Abs (either alone or in combination with individual ILs) by splenocytes from C57BL/6, 129/Sv, or a βc−/− mouse on a 129/Sv genetic background. Each stimulation index was calculated as described in Materials and Methods, using counts incorporated in unstimulated cultures (C57BL/6, 867 ± 171; 129/Sv, 1291 ± 352; βc−/−, 455 ± 114 cpm). B, Responses of splenocytes from C57BL/6, 129/Sv mice, and βc−/− or βc+/+ mice on a C57BL/6 background, to a subset of the stimuli used in A. Each bar represents a single mouse, and the results are representative of several experiments. Unstimulated cultures had incorporated 2785 ± 276, 5231 ± 429, 2986 ± 955 (the mean for three mice), and 3235 ± 1187 (the mean for three mice), respectively. Error bars indicate the SDs of triplicate determinations.
129/Sv eosinophil precursors respond normally to IL-5
The behavior of another IL-5-responsive cell type, the eosinophil precursor, was examined. Bone marrow cells from C57BL/6, 129/Sv, and βc−/− (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 βc−/− 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.
Frequency of progenitor cells per 50,000 bone marrow cells
Splenocytes from two 129 mouse substrains are hyporesponsive to stimuli that require Btk
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. 2⇓A), 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. 2⇓A). 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. 2⇓B). Furthermore, the 129/Ola substrain was, like 129/Sv, unresponsive to stimulation with anti-CD38 and IL-5 (Fig. 2⇓B). 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).
Splenocytes from two 129 mouse substrains are hyporesponsive to several stimuli. A, Proliferation of C57BL/6, 129/Sv, 129/Ola, and Xid/CBA/N splenocytes in response to LPS or to anti-μ stimulation. B, Proliferation of the same set of splenocytes as in A, in response to anti-CD38 stimulation alone, or in the presence of IL-5, and to stimulation by the anti-p105 Ab, RP/14. Each bar represents a single mouse, but the data are representative of several experiments (see text). Unstimulated cultures incorporated a mean of 1766 ± 295 (C57BL/6), 2555 ± 582 (129/Sv), 3490 ± 760 (129/Ola), and 659 (n = 2, Xid) cpm.
A closer examination of the proliferative responses of 129/Sv splenocytes to all three stimuli was undertaken to ensure that the differences were not merely kinetic differences. The 129/Sv response to anti-μ stimulation did not achieve wild-type levels when the concentration of Ab was doubled, nor did the response differ in kinetics from the C57BL/6 control (Fig. 3⇓). 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.
Kinetics of the proliferation response to anti-μ and anti-CD38 plus IL-5. A, Proliferation of splenocytes from the indicated mouse strains to two concentrations of anti-μ Ab, or to the standard (see Materials and Methods) concentrations of anti-CD38 plus IL-5, and to cultures in which each stimulant was doubled in turn. “CD38 hi” indicates the Ab concentration was doubled, but IL-5 was unchanged, and “IL-5 hi” indicates the converse. Each bar represents a single mouse. B, Proliferation in response to standard concentrations of anti-μ Abs or to anti-CD38 plus IL-5 over the course of 4 days of culture. Each line represents a single mouse.
As the proliferation assays indicated some overlap between the phenotypes of the 129 and Xid mice, further comparisons were undertaken. In Xid mice, as a result of a failure of signaling through the B cell Ag receptor, B cell maturation is blocked at an immature stage, and this is manifest in an immature phenotype of B cells in the periphery (22, 23). Low titers of certain serum Ig classes and a lack of peritoneal B-1 lymphocytes have also been documented (24). By comparison, 129/Sv B cells showed a normal maturation profile in the spleen (Fig. 4⇓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.
Maturation of peripheral B cells and serum Ig titers is normal in129/Sv mice. A, The phenotypes of splenic B cells from three different strains of mice, stained to differentiate immature from mature cells. In the HSA histograms, the vertical lines indicate the position of the mature populations, while in the IgM vs IgD dot plot, mature cells are IgDhigh and IgMlow. B, Titres of serum Ig in naive animals.
Reduction in peritoneal B-1 cells in 129/Sv mice. Peritoneal cells from three 129/Sv mice and a control C57BL/6 mouse were stained for the indicated surface markers, chosen to highlight B-1 lymphocytes (the expected position for B-1 cells is boxed in each panel). The B220/CD5 staining pattern was consistent in each case, with the results shown using other markers for B-1 cells (data not shown).
Activated 129/Sv B cells express abnormally low levels of IL-5R genes
In eosinophils and in B cells, IL-5 signaling is initiated at a receptor that is comprised of an α- and a β-chain (βc). Resting B cells express very little of the α-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-5Rα-chain, the βc-chain, and β-actin (Fig. 6⇓). A small difference was seen in the expression of the βc-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 β-actin signal). In 129/Sv B cells, both the resting and induced levels (normalized) of the IL-5Rα-chain were ∼20-fold lower than in C57BL/6 B cells. Indeed, there was little evidence for induction of the α-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-5Rα-chain and an inability to up-regulate its transcription upon activation.
129/Sv B cells transcribe both chains of the IL-5R upon activation. Sorted B cells were cultured without stimulation (Unst), or were stimulated with anti-CD38 and IL-5. RT, reverse transcriptase; C, no template control. RT-PCR products corresponding to each of the three genes are labeled.
Inheritance of the hyporesponsive phenotype
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.
Inheritance of the 129/Sv phenotype. Proliferative responses of spleen cell suspensions to stimulation with LPS, RP/14, or anti-CD38 in combination with IL-5. IL-5 or anti-CD38 alone gave stimulation indices of less than 5 for all cell samples (data not shown). Counts in unstimulated cultures were C57BL/6, 1766 ± 295 cpm; 129/Sv, 2555 ± 582 cpm; F1, 2317 ± 381 cpm.
Discussion
The work described in this study highlights the unusual behavior of B cells from two substrains of 129 mice, when compared with C57BL/6 control mice. C57BL/6 mice were chosen as controls because of their wide use and paradigmatic status in immunological studies, but more importantly because they are one of the most closely related inbred strains to 129, as defined genealogically (30). Two 129 substrains (Sv and Ola) were shown to behave similarly, but anomalously, in a number of in vitro assays.
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⇑).
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-5Rα-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-5Rα-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-5Rα 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-5Rα-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. 3⇑B), correlate with the transcription of the IL-5Rα-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-5Rα-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. 1⇑A), so it is possible that IL-5Rα 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. 2⇑A), 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. 1⇑A), 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-5Rα-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 × 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 × 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-5Rα-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.
Acknowledgments
We thank Prof. N. Nicola for IL-5, Dr. A. Strasser for NimR-5 (anti-CD38), Dr. K. Miyake for RP/14 (anti-Rp105), and D. Grail and Dr. A. DeFranco for mice. L. Barlow, L. Dirago, and S. Mifsud provided expert technical assistance.
Footnotes
-
↵1 This work was supported by the National Health and Medical Research Council.
-
↵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: corcoran{at}wehi.edu.au
-
↵3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; βc, common β.
- Received May 24, 1999.
- Accepted September 14, 1999.
- Copyright © 1999 by The American Association of Immunologists
References
In this issue
