MAPK p38 Is Dispensable for Lymphocyte Development and Proliferation
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免疫学杂志 2005年第1期
Abstract
Signals mediated by the p38 MAPK have been implicated in many processes required for the development and effector functions of innate and adaptive immune responses. As mice deficient in p38 exhibit embryonic lethality, most analyses of p38 function in lymphocytes have relied on the use of pharmacologic inhibitors and dominant-negative or constitutively active transgenes. In this study, we have generated a panel of low passage p38+/+, p38+/–, and p38–/– embryonic stem (ES) cells through the intercrossing of p38+/– mice. These ES cells were used to generate chimeric mice by RAG-deficient blastocyst complementation, with the lymphocytes in these mice being derived entirely from the ES cells. Surprisingly, B and T cell development were indistinguishable when comparing chimeric mice generated with p38+/+, p38+/–, and p38–/– ES cell lines. Moreover, proliferation of p38–/– B and T cells in response to Ag receptor and non-Ag receptor stimuli was intact. Thus, p38 is not an essential component of signaling pathways required for robust B and T lymphocyte developmental, nor is p38 essential for the proliferation of mature B and T cells.
Introduction
The MAPKs include ERK-1, ERK-2, JNK-1, JNK-2, JNK-3, and the p38 kinases (p38 , , , and ) (1). These kinases are broadly expressed and participate in signaling cascades initiated by a host of cellular stimuli, leading to cellular differentiation and the activation of effector responses (2). The activation of specific MAPK family members is determined by the activity of the upstream MAPK kinases (MKKs)3 (3). These dual tyrosine and threonine kinases phosphorylate the MAPK TXY motif required for activation (4). Specific MKKs activate p38, JNK, and ERK kinases, although cross-activation can occur (5, 6, 7, 8).
The p38 MAPK was first identified as a mediator of LPS signaling in B cells and was found to be homologous to the high osmolarity glycerol 1 kinase that is involved in osmotic stress responses in yeast (9). Subsequent studies implicated p38 in mediating signals induced by a wide variety of inflammatory mediators, including TNF- and IL-1 (9, 10, 11). In addition, p38 is involved in signaling from growth factor and G protein-coupled receptors that promote a wide range of biologic processes, including developmental patterning, proliferation, and tissue differentiation (12, 13, 14, 15, 16). There are three other p38 isoforms (, , and ) encoded by distinct genes (9, 17, 18, 19). The activity of the p38 MAPKs is regulated by MKK3, MKK4, and MKK6 (7, 8, 20, 21). All of the p38 kinases have a relatively broad tissue distribution, except for p38, which appears to be expressed primarily in muscle (17, 22, 23). Although p38 is the major isoform expressed in T lymphocytes, these cells also express p38 and (24, 25).
Four independently generated p38-deficient mice have been described (26, 27, 28, 29). In each case, p38 deficiency led to embryonic lethality, although there was some variability in the gestational age of death. This embryonic lethality prohibits the assessment of p38 deficiency in adult mouse tissues, including lymphocytes. Nonetheless, p38 has been implicated as an important signaling component in many processes required for T cell development and function. A host of experimental modalities has been used to modulate p38 activity. Pharmacologic inhibitors of p38, such as the SB203580 compound, can affect the activity of p38 and and, at high concentrations, may affect the activity of other kinases (30, 31, 32). Transgenic and retrovirally mediated overexpression of the wild-type or constitutively active forms of MKK3 and MKK6 or a dominant-negative form of p38 has also been used to assess p38 function (33, 34, 35). Together, these studies have implicated p38 in T cell developmental and effector functions, including, but not limited to, signaling from the pre-TCR, TCR, and several cytokine receptors, including IL-2R, IL-7R, and IL-18R (30, 31, 34, 35, 36, 37, 38, 39). Furthermore, as might be expected, analyses using different experimental approaches have at times led to conflicting conclusions (30, 31, 33, 34). In contrast to T cells, few studies have attempted to assess the requirements for p38 in B cell development and function.
We generated a series of low passage p38+/+, p38+/–, and p38–/– embryonic stem (ES) cells through the intercrossing of p38+/– mice (28). These ES cells were used to generate chimeric mice by RAG-deficient blastocyst complementation (RDBC) (40). These mice develop normally as cells derived from the RAG-2–/– blastocyst express p38. However, the lymphocytes in these mice are derived entirely from the ES cells as the RAG-2-deficient blastocyst is not able to give rise to lymphocytes. These mice were directly analyzed to assess the requirement for p38 in B and T cell development and function.
Materials and Methods
Generation of ES cell lines
The p38+/– mice were intercrossed, and blastocysts were harvested by flushing oviducts from female mice at 72 h postcoitum. Blastocysts were individually cultured in wells of a 24-well plate containing a mouse embryo fibroblast monolayer in DMEM containing 15% ES-tested FCS (Invitrogen Life Technologies) and 1000 U/ml LIF (Chemicon International), and supplemented with nonessential amino acids and penicillin-streptomycin. After 7–10 days, the cells in individual wells were dispersed with 0.25% trypsin:1 mM EDTA and replated in individual wells of a 24-well plate with a mouse embryo fibroblast monolayer. These cells were redispersed and expanded in culture once ES colonies were visible (usually 7–10 days). ES cell lines were expanded to 5 x 106 cells before being frozen for later use in RDBC.
Southern blotting
Southern blotting of ES cell genomic DNA was conducted, as previously described (41). The p38 probe is a 1.3-kb fragment from murine p38 intron 2, generated by EcoRI digestion of PCR product generated with forward primer 5'-ttgtgattattggggactgtaggg-3' and reverse primer 5'-ggacatacacatggacacacatcg-3'.
Flow cytometry
Single cell suspensions were prepared from thymus, bone marrow, spleen, and lymph nodes, as previously described (40). Cells were stained with FITC- or PE-conjugated Abs or biotinylated Abs, followed by streptavidin-allophycocyanin, and were analyzed by a FACScan (BD Biosciences). Live cell gating was performed by propidium iodide exclusion. The following Abs from BD Pharmingen were used in these studies: CD4 PE, CD8 FITC, B220 PE, CD43 FITC, and IgM biotin. Stained cells were analyzed on a FACSCalibur and plotted using CellQuest software (BD Biosciences).
Western blotting
Thymocyte cell lysates were prepared using 1x Nonidet P-40 supplemented with 1 mM EDTA, 0.5 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Cell extracts were centrifuged for 15 min at 4°C at 15,000 rpm. Soluble lysates were subjected to SDS-PAGE and immunoblotted with an anti-p38 polyclonal rabbit antisera (C-20; Santa Cruz Biotechnology) or anti-p85 polyclonal rabbit antisera (06-195; Upstate Biotechnology).
Proliferation assays
Total splenocytes from p38+/+, p38+/–, p38–/– chimeric mice were cultured in complete IMDM. Triplicate samples of 1 x 105 cells were plated in a single well of a flat-bottom 96-well Costar plate and stimulated with plate-bound anti-CD3 (2C11), anti-CD40 (3/23; BD Pharmingen), anti-IgM (Jackson ImmunoResearch Laboratories), or PMA with ionomycin. After 48-h stimulation, cells were pulsed with 2 μCi of [3H]thymidine/well and incubated for an additional 12 h. Proliferation was measured by [3H]thymidine incorporation using a Molecular Devices Micro96 Harvester and counted with a Beckman 6500 scintillation counter.
ELISAs
Serum was collected from 6- to 8-wk-old p38+/+, p38+/–, p38–/– chimeric mice. Goat anti-mouse Ig capture Ab in bicarbonate buffer (pH 8.8) was coated overnight at 4°C in Immulon 96-well plates. Plates were washed with 50 mM Tris (pH 7.6) containing 2% Triton X-100, and blocked in 10% FCS tissue culture medium for 3 h at room temperature. Serum was initially diluted 2000-fold for IgM; 1000-fold for IgG1, IgG2a, and IgG2b; and 500-fold for IgG3 and IgA. Diluted serum was subjected to 3-fold serial dilutions and was plated in triplicate at 4°C overnight. Samples were extensively washed, and HRP-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA (Southern Biotechnology Associates) were applied for 1 h. Samples were developed with 100 mM sodium citrate, 1 mM ABTS, and 0.016% H2O2. OD was read at 405 nm.
Results
Generation of p38–/–, p38+/–, and p38+/+ ES cells
A large panel of low passage p38+/+, p38+/–, and p38–/– ES cells was generated through the intercrossing of p38+/– mice (28). Blastocysts isolated from these intercrosses were independently cultured for ES cell isolation, as described in Materials and Methods (Fig. 1). A total of 35 ES cell lines was isolated from 77 independently cultured blastocysts, with Southern blot analyses revealing near Mendelian ratios of p38+/+, p38+/–, and p38–/– ES cell lines (Fig. 1). Chimeric mice were generated by RDBC using 3 p38+/+, 3 p38+/–, and 7 p38–/– ES cell lines.
FIGURE 1. Generation of p38+/+, p38+/–, and p38–/– ES cells. Shown is a flow diagram for the development of ES cells through the intercrossing of p38+/– male and female mice, as described in Materials and Methods. Southern blot analysis of BamHI-digested genomic DNA from 10 ES clones using the p38 probe is shown. The band generated by the wild-type (+) and p38– (–) alleles is indicated. The number of p38+/+, p38+/–, and p38–/– ES cells generated from 77 cultured blastocysts is indicated. The percentages of each genotype are shown in parentheses.
Development of p38-deficient T and B cells
Lymphocyte development in p38+/+, p38+/–, and p38–/– chimeric mice was assessed by flow cytometric analyses of thymocytes and bone marrow cells (Figs. 2 and 3). Surprisingly, chimeric mice generated with p38–/– ES cells exhibited similar total thymocyte numbers as compared with chimeric mice generated with either p38+/– or p38+/+ ES cells (Fig. 2A). Furthermore, the CD4–/CD8– (double-negative), CD4+/CD8+ (double-positive (DP)), and CD4+ or CD8+ (single-positive (SP)) thymocyte compartments also appeared intact in p38–/– chimeric mice (Fig. 2).
FIGURE 2. Thymocyte development in p38–/– chimeras. A, Quantitation of total thymic cellularity and CD4+CD8+ (DP), CD4+ (SP), and CD8+ (SP) thymocytes. Results from 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old are shown. B, Representative flow cytometric analysis of thymocytes from p38+/+, p38+/–, and p38–/– chimeric mice using anti-CD4 and anti-CD8 mAbs.
FIGURE 3. Flow cytometric analysis of B cell development in p38–/– chimeras. Bone marrow cells from p38+/+, p38+/–, and p38–/– chimeric mice were analyzed by flow cytometry using anti-B220, anti-CD43, and anti-IgM Abs.
The p38– allele was generated by insertion of the neomycin resistance gene into exon 3 of the p38 locus (28). Importantly, polyclonal rabbit antisera raised to the C terminus of p38 failed to detect a p38 protein in p38–/– thymocytes (Fig. 4). Moreover, analysis of chimeric mice generated from p38–/– ES cells in which the p38– allele was generated through the deletion of eight exons, including the exon encoding the TGY motif, also failed to reveal a perturbation in thymocyte development (data not shown) (27). Together, these data make it unlikely that the robust thymocyte development observed in the p38–/– chimeric mice is due to the production of a truncated p38 protein from the p38– allele.
FIGURE 4. The p38 expression by p38+/+, p38+/–, and p38–/– thymocytes. Lysates from p38+/+, p38+/–, and p38–/– thymocytes were subjected to Western blot analyses using rabbit polyclonal p38 antisera and p85-specific antisera. Molecular weight markers are indicated.
Flow cytometric analyses of bone marrow from p38+/+, p38+/–, and p38–/– chimeric mice revealed relatively similar fractions of pro-B (B220+CD43+), pre-B (B220lowCD43–), and immature B (IgM+) cells (Fig. 3). Together, these findings demonstrate that, in the absence of p38, B and T cell development is generally unperturbed.
Proliferation of mature p38–/– T cells
Flow cytometric analyses revealed similar numbers of mature CD4+ and CD8+ splenic T cells in p38+/+, p38+/–, and p38–/– chimeric mice (Fig. 5). Furthermore, p38–/– T cells express similar TCR levels as compared with p38+/+ and p38+/– T cells (data not shown). Proliferation of p38–/– T cells was assayed upon stimulation of these cells with plate-bound anti-CD3 Abs. The p38–/– T cells exhibited robust proliferation over a broad range of anti-CD3 concentrations (Fig. 6). Moreover, this proliferation was similar in magnitude to that observed for p38+/+ and p38+/– and T cells. Together, these data demonstrate that neither the generation and maintenance of normal numbers of mature peripheral T cells nor the ability of these cells to undergo TCR-driven cellular expansion is dependent on p38.
FIGURE 5. Mature T cells in p38–/– chimeras. A, Total number of splenic T cells in 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old. B, Representative flow cytometric analysis of total splenocytes using anti-CD4 and anti-CD8. The percentage of CD4+ and CD8+ T cells is indicated.
FIGURE 6. Proliferative capacity of p38–/– T cells. Triplicate samples of splenocytes from p38+/+, p38+/–, and p38–/– chimeras were stimulated with medium alone or the indicated concentrations of plate-bound anti-CD3 Abs, as described in Materials and Methods. The data shown are representative of three experiments.
Mature p38–/– B cell function
Flow cytometric analyses of splenocytes from p38+/+, p38+/–, and p38–/– chimeric mice revealed similar numbers of B220+ IgM+ mature B cells and similar fractions of IgM+ IgD+ mature B cells (Fig. 7). When stimulated with either anti-IgM or anti-CD40, mature p38–/– B cells exhibited similar levels of proliferation as compared with p38+/+ or p38+/– B cells (Fig. 8, A and B). Furthermore, serum Ig isotype analyses revealed no significant differences in IgM, IgG1, IgG3, IgG2b, and IgA levels when comparing p38+/+, p38+/–, and p38–/– chimeric mice (Fig. 9). Together, these data demonstrate that p38 is not essential for CD40- or BCR-driven proliferation of mature B cells, nor is it required for the generation and maintenance of normal serum Ig levels.
FIGURE 7. Mature B cells in p38–/– chimeras. A, Total number of splenic B cells in 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old. B, Representative flow cytometric analysis of total splenocytes using anti-B220, anti-IgM, and anti-IgD. The percentage of B220+ IgM+ cells is indicated.
FIGURE 8. Proliferative capacity of p38–/– B cells. Triplicate samples of splenocytes from p38+/+, p38+/–, and p38–/– chimeras were stimulated with different concentrations of anti-IgM (A), anti-CD40 or PMA and ionomycin (B), or different concentrations of LPS (C). The data shown are representative of at least three experiments.
FIGURE 9. Serum Ig levels in p38–/– chimeric mice. Serum levels of IgM, IgA, IgG3, IgG1, and IgG2b were measured by ELISA from three p38+/+, five p38+/–, and seven p38–/– chimeric mice that were 6–8 wk old, as described in Materials and Methods.
The p38 was initially identified as a mediator of endotoxin signaling in B cells (9). B lymphocytes undergo proliferation in response to stimulation with endotoxin (42). Surprisingly, proliferation in response to LPS was indistinguishable when comparing p38–/–, p38+/+, or p38+/– B cells (Fig. 8C). Thus, p38 was also dispensable for the endotoxin-mediated proliferation of B cells.
Discussion
MAPK p38 deficiency leads to placental defects and embryonic lethality (26, 27, 28, 29). Several different approaches have been used to assess the development and function of lymphocytes in the absence of proteins required for embryonic development. If the embryos survive until a gestational stage at which bone marrow or fetal liver can be harvested, these cells can be used to reconstitute the lymphoid compartment in RAG-deficient mice. Although in one report p38–/– mice survived under unique circumstances until a gestational age that permitted fetal liver harvest (29), the other reported p38–/– mice, including the one used in this study, did not (26, 27, 28).
RAG-deficient blastocyst complementation is an alternate approach for assessing lymphoid development and function in the absence of proteins required for embryonic development (40). This approach requires the generation of ES cells with homozygous mutations in autosomal genes of interest. Traditionally, this has been done through serial allele targeting or high drug selection approaches. Both of these approaches require long-term ES cell culture that can, on occasion, render these cells incapable of contributing to the lymphoid compartment due to ill-defined effects that are independent of the gene-targeted mutation. To circumvent this potential problem, we generated p38+/+, p38+/–, and p38–/– ES cells through the intercrossing of p38+/– mice and expanded these cells in culture for minimal periods of time (<1 mo) before use in RDBC. Approximately 40% of cultured blastocysts gave rise to ES cells, and most of these ES gave robust reconstitution of the lymphoid compartment in chimeric mice generated by RDBC. Thus, the de novo generation of ES cells for use in RDBC is an efficient and effective way to generate chimeric mice for analyses of lymphocyte development and function.
Surprisingly, p38–/– chimeric mice generated by RDBC revealed no significant perturbations in T cell development. Total thymocyte cellularity and the number of DP and SP thymocytes were not significantly different when comparing chimeric mice generated with p38+/+, p38+/–, and p38–/– ES cells. These findings do not exclude a potential role for p38 in thymocyte-negative selection (31). Furthermore, other p38 isoforms could compensate for p38 deficiency during lymphocyte development. In this regard, p38 and p38, but not p38, are expressed in thymocytes (24, 25). Notably, p38 and do not exhibit significant sequence homology and have few known substrates in common (19, 43, 44). In contrast, p38 has significant sequence homology to p38 and shares several known substrates with p38 (18, 19, 43, 44). Thus, it is possible that p38 and/or could compensate for p38 deficiency. Nevertheless, our findings demonstrate that p38 signals per se are not essential for the robust development and maintenance of the different thymocyte subsets.
The generation and maintenance of mature CD4+ and CD8+ T cells in the periphery were also unaffected by p38 deficiency. Studies using pharmacologic inhibitors have implicated p38 signaling in the TCR-mediated proliferation of mature T cells (45). However, we found no significant differences in the ability of p38+/+, p38+/–, and p38–/– T cells to proliferate in response to anti-CD3 treatment. Together, these findings demonstrate that p38 is not an essential component of TCR or cytokine receptor, such as IL-2R, signaling required for the maintenance and/or proliferation of mature T cells.
Similarly, the development and maintenance of mature B cells were unperturbed in p38–/– chimeric mice. The ability of these cells to produce Abs was sufficient to generate normal serum levels of IgM, IgG1, IgG3, IgG2b, and IgA. Proliferative responses of these B cells upon stimulation through the BCR or CD40 were also intact. Furthermore, and surprisingly, p38–/– B cells proliferated robustly in response to LPS.
Together, our findings demonstrate that p38 is not an essential component of B and TCR or cytokine receptor signaling pathways required for the robust development and expansion of B and T cells. However, p38 is an essential component of the signaling pathways of inflammatory cytokines. For example, p38 signals are required for the production of IL-6 in response to IL-1 (27). Thus, our findings imply that specific pharmacologic inhibitors of p38 may impact the inflammatory cytokine-mediated responses without impacting lymphocyte development or proliferation.
Acknowledgments
We thank Dr. John McNeish for providing us with p38–/– ES cells, and Dr. John S. Mudgett for p38+/– mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Cancer Research Institute (to B.P.S.). Mice were produced by a transgenic mouse core facility supported by the Rheumatic Diseases Core Center at Washington University (National Institutes of Health Grant P30-AR48335).
2 Address correspondence and reprint requests to Dr. Barry P. Sleckman, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110-1093. E-mail address: sleckman@pathbox.wustl.edu
3 Abbreviations used in this paper: MKK, MAPK kinase; DP, double positive; ES, embryonic stem; RDBC, RAG-deficient blastocyst complementation; SP, single positive.
Received for publication September 27, 2004. Accepted for publication November 2, 2004.
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Signals mediated by the p38 MAPK have been implicated in many processes required for the development and effector functions of innate and adaptive immune responses. As mice deficient in p38 exhibit embryonic lethality, most analyses of p38 function in lymphocytes have relied on the use of pharmacologic inhibitors and dominant-negative or constitutively active transgenes. In this study, we have generated a panel of low passage p38+/+, p38+/–, and p38–/– embryonic stem (ES) cells through the intercrossing of p38+/– mice. These ES cells were used to generate chimeric mice by RAG-deficient blastocyst complementation, with the lymphocytes in these mice being derived entirely from the ES cells. Surprisingly, B and T cell development were indistinguishable when comparing chimeric mice generated with p38+/+, p38+/–, and p38–/– ES cell lines. Moreover, proliferation of p38–/– B and T cells in response to Ag receptor and non-Ag receptor stimuli was intact. Thus, p38 is not an essential component of signaling pathways required for robust B and T lymphocyte developmental, nor is p38 essential for the proliferation of mature B and T cells.
Introduction
The MAPKs include ERK-1, ERK-2, JNK-1, JNK-2, JNK-3, and the p38 kinases (p38 , , , and ) (1). These kinases are broadly expressed and participate in signaling cascades initiated by a host of cellular stimuli, leading to cellular differentiation and the activation of effector responses (2). The activation of specific MAPK family members is determined by the activity of the upstream MAPK kinases (MKKs)3 (3). These dual tyrosine and threonine kinases phosphorylate the MAPK TXY motif required for activation (4). Specific MKKs activate p38, JNK, and ERK kinases, although cross-activation can occur (5, 6, 7, 8).
The p38 MAPK was first identified as a mediator of LPS signaling in B cells and was found to be homologous to the high osmolarity glycerol 1 kinase that is involved in osmotic stress responses in yeast (9). Subsequent studies implicated p38 in mediating signals induced by a wide variety of inflammatory mediators, including TNF- and IL-1 (9, 10, 11). In addition, p38 is involved in signaling from growth factor and G protein-coupled receptors that promote a wide range of biologic processes, including developmental patterning, proliferation, and tissue differentiation (12, 13, 14, 15, 16). There are three other p38 isoforms (, , and ) encoded by distinct genes (9, 17, 18, 19). The activity of the p38 MAPKs is regulated by MKK3, MKK4, and MKK6 (7, 8, 20, 21). All of the p38 kinases have a relatively broad tissue distribution, except for p38, which appears to be expressed primarily in muscle (17, 22, 23). Although p38 is the major isoform expressed in T lymphocytes, these cells also express p38 and (24, 25).
Four independently generated p38-deficient mice have been described (26, 27, 28, 29). In each case, p38 deficiency led to embryonic lethality, although there was some variability in the gestational age of death. This embryonic lethality prohibits the assessment of p38 deficiency in adult mouse tissues, including lymphocytes. Nonetheless, p38 has been implicated as an important signaling component in many processes required for T cell development and function. A host of experimental modalities has been used to modulate p38 activity. Pharmacologic inhibitors of p38, such as the SB203580 compound, can affect the activity of p38 and and, at high concentrations, may affect the activity of other kinases (30, 31, 32). Transgenic and retrovirally mediated overexpression of the wild-type or constitutively active forms of MKK3 and MKK6 or a dominant-negative form of p38 has also been used to assess p38 function (33, 34, 35). Together, these studies have implicated p38 in T cell developmental and effector functions, including, but not limited to, signaling from the pre-TCR, TCR, and several cytokine receptors, including IL-2R, IL-7R, and IL-18R (30, 31, 34, 35, 36, 37, 38, 39). Furthermore, as might be expected, analyses using different experimental approaches have at times led to conflicting conclusions (30, 31, 33, 34). In contrast to T cells, few studies have attempted to assess the requirements for p38 in B cell development and function.
We generated a series of low passage p38+/+, p38+/–, and p38–/– embryonic stem (ES) cells through the intercrossing of p38+/– mice (28). These ES cells were used to generate chimeric mice by RAG-deficient blastocyst complementation (RDBC) (40). These mice develop normally as cells derived from the RAG-2–/– blastocyst express p38. However, the lymphocytes in these mice are derived entirely from the ES cells as the RAG-2-deficient blastocyst is not able to give rise to lymphocytes. These mice were directly analyzed to assess the requirement for p38 in B and T cell development and function.
Materials and Methods
Generation of ES cell lines
The p38+/– mice were intercrossed, and blastocysts were harvested by flushing oviducts from female mice at 72 h postcoitum. Blastocysts were individually cultured in wells of a 24-well plate containing a mouse embryo fibroblast monolayer in DMEM containing 15% ES-tested FCS (Invitrogen Life Technologies) and 1000 U/ml LIF (Chemicon International), and supplemented with nonessential amino acids and penicillin-streptomycin. After 7–10 days, the cells in individual wells were dispersed with 0.25% trypsin:1 mM EDTA and replated in individual wells of a 24-well plate with a mouse embryo fibroblast monolayer. These cells were redispersed and expanded in culture once ES colonies were visible (usually 7–10 days). ES cell lines were expanded to 5 x 106 cells before being frozen for later use in RDBC.
Southern blotting
Southern blotting of ES cell genomic DNA was conducted, as previously described (41). The p38 probe is a 1.3-kb fragment from murine p38 intron 2, generated by EcoRI digestion of PCR product generated with forward primer 5'-ttgtgattattggggactgtaggg-3' and reverse primer 5'-ggacatacacatggacacacatcg-3'.
Flow cytometry
Single cell suspensions were prepared from thymus, bone marrow, spleen, and lymph nodes, as previously described (40). Cells were stained with FITC- or PE-conjugated Abs or biotinylated Abs, followed by streptavidin-allophycocyanin, and were analyzed by a FACScan (BD Biosciences). Live cell gating was performed by propidium iodide exclusion. The following Abs from BD Pharmingen were used in these studies: CD4 PE, CD8 FITC, B220 PE, CD43 FITC, and IgM biotin. Stained cells were analyzed on a FACSCalibur and plotted using CellQuest software (BD Biosciences).
Western blotting
Thymocyte cell lysates were prepared using 1x Nonidet P-40 supplemented with 1 mM EDTA, 0.5 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Cell extracts were centrifuged for 15 min at 4°C at 15,000 rpm. Soluble lysates were subjected to SDS-PAGE and immunoblotted with an anti-p38 polyclonal rabbit antisera (C-20; Santa Cruz Biotechnology) or anti-p85 polyclonal rabbit antisera (06-195; Upstate Biotechnology).
Proliferation assays
Total splenocytes from p38+/+, p38+/–, p38–/– chimeric mice were cultured in complete IMDM. Triplicate samples of 1 x 105 cells were plated in a single well of a flat-bottom 96-well Costar plate and stimulated with plate-bound anti-CD3 (2C11), anti-CD40 (3/23; BD Pharmingen), anti-IgM (Jackson ImmunoResearch Laboratories), or PMA with ionomycin. After 48-h stimulation, cells were pulsed with 2 μCi of [3H]thymidine/well and incubated for an additional 12 h. Proliferation was measured by [3H]thymidine incorporation using a Molecular Devices Micro96 Harvester and counted with a Beckman 6500 scintillation counter.
ELISAs
Serum was collected from 6- to 8-wk-old p38+/+, p38+/–, p38–/– chimeric mice. Goat anti-mouse Ig capture Ab in bicarbonate buffer (pH 8.8) was coated overnight at 4°C in Immulon 96-well plates. Plates were washed with 50 mM Tris (pH 7.6) containing 2% Triton X-100, and blocked in 10% FCS tissue culture medium for 3 h at room temperature. Serum was initially diluted 2000-fold for IgM; 1000-fold for IgG1, IgG2a, and IgG2b; and 500-fold for IgG3 and IgA. Diluted serum was subjected to 3-fold serial dilutions and was plated in triplicate at 4°C overnight. Samples were extensively washed, and HRP-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA (Southern Biotechnology Associates) were applied for 1 h. Samples were developed with 100 mM sodium citrate, 1 mM ABTS, and 0.016% H2O2. OD was read at 405 nm.
Results
Generation of p38–/–, p38+/–, and p38+/+ ES cells
A large panel of low passage p38+/+, p38+/–, and p38–/– ES cells was generated through the intercrossing of p38+/– mice (28). Blastocysts isolated from these intercrosses were independently cultured for ES cell isolation, as described in Materials and Methods (Fig. 1). A total of 35 ES cell lines was isolated from 77 independently cultured blastocysts, with Southern blot analyses revealing near Mendelian ratios of p38+/+, p38+/–, and p38–/– ES cell lines (Fig. 1). Chimeric mice were generated by RDBC using 3 p38+/+, 3 p38+/–, and 7 p38–/– ES cell lines.
FIGURE 1. Generation of p38+/+, p38+/–, and p38–/– ES cells. Shown is a flow diagram for the development of ES cells through the intercrossing of p38+/– male and female mice, as described in Materials and Methods. Southern blot analysis of BamHI-digested genomic DNA from 10 ES clones using the p38 probe is shown. The band generated by the wild-type (+) and p38– (–) alleles is indicated. The number of p38+/+, p38+/–, and p38–/– ES cells generated from 77 cultured blastocysts is indicated. The percentages of each genotype are shown in parentheses.
Development of p38-deficient T and B cells
Lymphocyte development in p38+/+, p38+/–, and p38–/– chimeric mice was assessed by flow cytometric analyses of thymocytes and bone marrow cells (Figs. 2 and 3). Surprisingly, chimeric mice generated with p38–/– ES cells exhibited similar total thymocyte numbers as compared with chimeric mice generated with either p38+/– or p38+/+ ES cells (Fig. 2A). Furthermore, the CD4–/CD8– (double-negative), CD4+/CD8+ (double-positive (DP)), and CD4+ or CD8+ (single-positive (SP)) thymocyte compartments also appeared intact in p38–/– chimeric mice (Fig. 2).
FIGURE 2. Thymocyte development in p38–/– chimeras. A, Quantitation of total thymic cellularity and CD4+CD8+ (DP), CD4+ (SP), and CD8+ (SP) thymocytes. Results from 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old are shown. B, Representative flow cytometric analysis of thymocytes from p38+/+, p38+/–, and p38–/– chimeric mice using anti-CD4 and anti-CD8 mAbs.
FIGURE 3. Flow cytometric analysis of B cell development in p38–/– chimeras. Bone marrow cells from p38+/+, p38+/–, and p38–/– chimeric mice were analyzed by flow cytometry using anti-B220, anti-CD43, and anti-IgM Abs.
The p38– allele was generated by insertion of the neomycin resistance gene into exon 3 of the p38 locus (28). Importantly, polyclonal rabbit antisera raised to the C terminus of p38 failed to detect a p38 protein in p38–/– thymocytes (Fig. 4). Moreover, analysis of chimeric mice generated from p38–/– ES cells in which the p38– allele was generated through the deletion of eight exons, including the exon encoding the TGY motif, also failed to reveal a perturbation in thymocyte development (data not shown) (27). Together, these data make it unlikely that the robust thymocyte development observed in the p38–/– chimeric mice is due to the production of a truncated p38 protein from the p38– allele.
FIGURE 4. The p38 expression by p38+/+, p38+/–, and p38–/– thymocytes. Lysates from p38+/+, p38+/–, and p38–/– thymocytes were subjected to Western blot analyses using rabbit polyclonal p38 antisera and p85-specific antisera. Molecular weight markers are indicated.
Flow cytometric analyses of bone marrow from p38+/+, p38+/–, and p38–/– chimeric mice revealed relatively similar fractions of pro-B (B220+CD43+), pre-B (B220lowCD43–), and immature B (IgM+) cells (Fig. 3). Together, these findings demonstrate that, in the absence of p38, B and T cell development is generally unperturbed.
Proliferation of mature p38–/– T cells
Flow cytometric analyses revealed similar numbers of mature CD4+ and CD8+ splenic T cells in p38+/+, p38+/–, and p38–/– chimeric mice (Fig. 5). Furthermore, p38–/– T cells express similar TCR levels as compared with p38+/+ and p38+/– T cells (data not shown). Proliferation of p38–/– T cells was assayed upon stimulation of these cells with plate-bound anti-CD3 Abs. The p38–/– T cells exhibited robust proliferation over a broad range of anti-CD3 concentrations (Fig. 6). Moreover, this proliferation was similar in magnitude to that observed for p38+/+ and p38+/– and T cells. Together, these data demonstrate that neither the generation and maintenance of normal numbers of mature peripheral T cells nor the ability of these cells to undergo TCR-driven cellular expansion is dependent on p38.
FIGURE 5. Mature T cells in p38–/– chimeras. A, Total number of splenic T cells in 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old. B, Representative flow cytometric analysis of total splenocytes using anti-CD4 and anti-CD8. The percentage of CD4+ and CD8+ T cells is indicated.
FIGURE 6. Proliferative capacity of p38–/– T cells. Triplicate samples of splenocytes from p38+/+, p38+/–, and p38–/– chimeras were stimulated with medium alone or the indicated concentrations of plate-bound anti-CD3 Abs, as described in Materials and Methods. The data shown are representative of three experiments.
Mature p38–/– B cell function
Flow cytometric analyses of splenocytes from p38+/+, p38+/–, and p38–/– chimeric mice revealed similar numbers of B220+ IgM+ mature B cells and similar fractions of IgM+ IgD+ mature B cells (Fig. 7). When stimulated with either anti-IgM or anti-CD40, mature p38–/– B cells exhibited similar levels of proliferation as compared with p38+/+ or p38+/– B cells (Fig. 8, A and B). Furthermore, serum Ig isotype analyses revealed no significant differences in IgM, IgG1, IgG3, IgG2b, and IgA levels when comparing p38+/+, p38+/–, and p38–/– chimeric mice (Fig. 9). Together, these data demonstrate that p38 is not essential for CD40- or BCR-driven proliferation of mature B cells, nor is it required for the generation and maintenance of normal serum Ig levels.
FIGURE 7. Mature B cells in p38–/– chimeras. A, Total number of splenic B cells in 3 p38+/+, 8 p38+/–, and 13 p38–/– chimeric mice 4–6 wk old. B, Representative flow cytometric analysis of total splenocytes using anti-B220, anti-IgM, and anti-IgD. The percentage of B220+ IgM+ cells is indicated.
FIGURE 8. Proliferative capacity of p38–/– B cells. Triplicate samples of splenocytes from p38+/+, p38+/–, and p38–/– chimeras were stimulated with different concentrations of anti-IgM (A), anti-CD40 or PMA and ionomycin (B), or different concentrations of LPS (C). The data shown are representative of at least three experiments.
FIGURE 9. Serum Ig levels in p38–/– chimeric mice. Serum levels of IgM, IgA, IgG3, IgG1, and IgG2b were measured by ELISA from three p38+/+, five p38+/–, and seven p38–/– chimeric mice that were 6–8 wk old, as described in Materials and Methods.
The p38 was initially identified as a mediator of endotoxin signaling in B cells (9). B lymphocytes undergo proliferation in response to stimulation with endotoxin (42). Surprisingly, proliferation in response to LPS was indistinguishable when comparing p38–/–, p38+/+, or p38+/– B cells (Fig. 8C). Thus, p38 was also dispensable for the endotoxin-mediated proliferation of B cells.
Discussion
MAPK p38 deficiency leads to placental defects and embryonic lethality (26, 27, 28, 29). Several different approaches have been used to assess the development and function of lymphocytes in the absence of proteins required for embryonic development. If the embryos survive until a gestational stage at which bone marrow or fetal liver can be harvested, these cells can be used to reconstitute the lymphoid compartment in RAG-deficient mice. Although in one report p38–/– mice survived under unique circumstances until a gestational age that permitted fetal liver harvest (29), the other reported p38–/– mice, including the one used in this study, did not (26, 27, 28).
RAG-deficient blastocyst complementation is an alternate approach for assessing lymphoid development and function in the absence of proteins required for embryonic development (40). This approach requires the generation of ES cells with homozygous mutations in autosomal genes of interest. Traditionally, this has been done through serial allele targeting or high drug selection approaches. Both of these approaches require long-term ES cell culture that can, on occasion, render these cells incapable of contributing to the lymphoid compartment due to ill-defined effects that are independent of the gene-targeted mutation. To circumvent this potential problem, we generated p38+/+, p38+/–, and p38–/– ES cells through the intercrossing of p38+/– mice and expanded these cells in culture for minimal periods of time (<1 mo) before use in RDBC. Approximately 40% of cultured blastocysts gave rise to ES cells, and most of these ES gave robust reconstitution of the lymphoid compartment in chimeric mice generated by RDBC. Thus, the de novo generation of ES cells for use in RDBC is an efficient and effective way to generate chimeric mice for analyses of lymphocyte development and function.
Surprisingly, p38–/– chimeric mice generated by RDBC revealed no significant perturbations in T cell development. Total thymocyte cellularity and the number of DP and SP thymocytes were not significantly different when comparing chimeric mice generated with p38+/+, p38+/–, and p38–/– ES cells. These findings do not exclude a potential role for p38 in thymocyte-negative selection (31). Furthermore, other p38 isoforms could compensate for p38 deficiency during lymphocyte development. In this regard, p38 and p38, but not p38, are expressed in thymocytes (24, 25). Notably, p38 and do not exhibit significant sequence homology and have few known substrates in common (19, 43, 44). In contrast, p38 has significant sequence homology to p38 and shares several known substrates with p38 (18, 19, 43, 44). Thus, it is possible that p38 and/or could compensate for p38 deficiency. Nevertheless, our findings demonstrate that p38 signals per se are not essential for the robust development and maintenance of the different thymocyte subsets.
The generation and maintenance of mature CD4+ and CD8+ T cells in the periphery were also unaffected by p38 deficiency. Studies using pharmacologic inhibitors have implicated p38 signaling in the TCR-mediated proliferation of mature T cells (45). However, we found no significant differences in the ability of p38+/+, p38+/–, and p38–/– T cells to proliferate in response to anti-CD3 treatment. Together, these findings demonstrate that p38 is not an essential component of TCR or cytokine receptor, such as IL-2R, signaling required for the maintenance and/or proliferation of mature T cells.
Similarly, the development and maintenance of mature B cells were unperturbed in p38–/– chimeric mice. The ability of these cells to produce Abs was sufficient to generate normal serum levels of IgM, IgG1, IgG3, IgG2b, and IgA. Proliferative responses of these B cells upon stimulation through the BCR or CD40 were also intact. Furthermore, and surprisingly, p38–/– B cells proliferated robustly in response to LPS.
Together, our findings demonstrate that p38 is not an essential component of B and TCR or cytokine receptor signaling pathways required for the robust development and expansion of B and T cells. However, p38 is an essential component of the signaling pathways of inflammatory cytokines. For example, p38 signals are required for the production of IL-6 in response to IL-1 (27). Thus, our findings imply that specific pharmacologic inhibitors of p38 may impact the inflammatory cytokine-mediated responses without impacting lymphocyte development or proliferation.
Acknowledgments
We thank Dr. John McNeish for providing us with p38–/– ES cells, and Dr. John S. Mudgett for p38+/– mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Cancer Research Institute (to B.P.S.). Mice were produced by a transgenic mouse core facility supported by the Rheumatic Diseases Core Center at Washington University (National Institutes of Health Grant P30-AR48335).
2 Address correspondence and reprint requests to Dr. Barry P. Sleckman, Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110-1093. E-mail address: sleckman@pathbox.wustl.edu
3 Abbreviations used in this paper: MKK, MAPK kinase; DP, double positive; ES, embryonic stem; RDBC, RAG-deficient blastocyst complementation; SP, single positive.
Received for publication September 27, 2004. Accepted for publication November 2, 2004.
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