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Defective proliferative responses in B lymphocytes and thymocytes that lack neurofibromin
Molecular Immunology 38 (2001) 701–708
Defective proliferative responses in B lymphocytes and thymocytes that lack neurofibromin Tae Jin Kim a,1,2 , Annaiah Cariappa a,2 , John Iacomini b , Mei Tang a , Shane Shih c , Andre Bernards a , Tyler Jacks c , Shiv Pillai a,∗ a
c
Cancer Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown Navy Yard, Boston, MA 02129, USA b Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA Center for Cancer Research and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 28 May 2001; received in revised form 10 October 2001; accepted 24 October 2001
Abstract Nf1−/− fetal liver cells were used to reconstitute B and T cells in Rag-1−/− mice. Lymphocyte development was largely unimpaired in the absence of neurofibromin. However antigen-receptor induced proliferation was defective in neurofibromin deficient peripheral B cells and CD4+ single positive thymocytes. In contrast to its role as a negative regulator of proliferation in many other cell types, neurofibromin may be a positive regulator of lymphocyte proliferation. Peripheral B cells exhibited circumscribed defects in anti-IgM induced protein tyrosine phosphorylation, which may contribute to the unexpected proliferative defect seen in these cells. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: B lymphocytes; Signal transduction; Cellular activation
1. Introduction GTPase activating proteins play important roles in catalyzing the conversion of GTP bound to small G proteins such as Ras to GDP. These enzymes bind to the effector domains of these small G proteins and enhance their intrinsic GTPase activity. The Nf1 gene is mutated in patients with type 1 neurofibromatosis and encodes a RasGAP known as neurofibromin (Viskochil et al., 1990; Xu et al., 1990; Martin et al., 1990; Ballester et al., 1990; Bollag and McCormick, 1991). Although neurofibromin shares a GAP domain with p120GAP, outside of this homologous segment the proteins are quite dissimilar. In particular neurofibromin lacks the SH2, SH3, pleckstrin-homology (PH) and calcium/phospholipid binding (CaLB) domains seen in p120GAP. Neurofibromin, however, closely resembles the yeast rasGAPs, IRA1 and IRA2, and can complement their activity in the appropriate ira− S. cerevisiae mutants ∗ Corresponding author. Tel.: +1-617-726-5619; fax: +1-617-724-9648. E-mail address: [email protected] (S. Pillai). 1 Present address: Department of Pathology, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea. 2 Equal contributors.
(Viskochil et al., 1990; Xu et al., 1990; Martin et al., 1990). Consistent with its role as a RasGAP, neurofibromin is a negative regulator of proliferation in several cell types including astrocytes, melanocytes, and myeloid cells (Shannon et al., 1994; Largaespada et al., 1996; Bollag et al., 1996; Gutmann et al., 1999; Ingram et al., 2000). Crosslinking of membrane IgM on splenic B cells leads to the cocapping of this protein with neurofibromin but not with p120GAP (Boyer et al., 1994). The biological implications of the co-capping of neurofibromin (Boyer et al., 1994) and Ras (Graziadei et al., 1990) with membrane IgM are poorly understood. We were interested in examining whether neurofibromin is required for B cell development and to determine if signaling downstream of the antigen receptor is enhanced in B cells that lack neurofibromin. Nf1−/− mice die around day 14 of embryogenesis primarily because of a cardiac defect (Jacks et al., 1994; Brannan et al., 1994). We reconstituted Rag-1−/− mice with fetal liver cells from Nf1−/− embryos. While B and T cell development in the absence of NF1 was grossly normal, antigen-receptor induced proliferation was defective in peripheral B cells lacking neurofibromin, and in neurofibromin deficient CD4+ single positive thymocytes.
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2. Materials and methods 2.1. Mice The generation of Nf1−/− mice has been previously described (Jacks et al., 1994). Rag-1/− mice were obtained from Jackson laboratories. 2.2. Reconstitution of Rag-1−/− mice with Nf1−/− fetal liver cells Nf1+/− mice were mated, and at day 12.5 or day 13.5%, embryos were harvested and suspensions of fetal liver cells were made. Cells from a single fetal liver were injected into the tail vein of one recipient Rag-1−/− mouse. Three hours prior to the cell transfer, Rag-1−/− mice were mildly irradiated (300 rad). Carcasses were genotyped by PCR to determine if embryos were Nf1+/+ , Nf1+/− or Nf1−/− (Jacks et al., 1994). 2.3. Flow cytometry Single cell suspensions were made from spleen, bone marrow, thymus, mesenteric lymph nodes, and peritoneum. For surface staining, 1 × 106 cells were reacted with 2.4G2 (anti-CD16/CD32 {Fc␥ III/II receptor}, culture supernatant), prior to staining with the following antibodies: anti CD5-biotin clone 57-7.3, anti-CD11b-allophycocyanin (APC) clone M1/70, anti CD4 PE clone RM4-5, anti-CD8␣ FITC clone 53–67, anti-CD45R-FITC (or APC) B220 clone RA36B2, antiCD43/leukosialin-FITC clone S7, anti-BP1-PE clone BP1, anti-CD24-biotin clone 30F1, and anti-IgM-PE clone R6-60. All of the above antibodies were obtained from Pharmingen. Anti-IgD-biotin clone 11–26 and goat anti-mouse IgM PE was from Southern
Biotechnology. Biotinylated antibodies were revealed using streptavidin-APC (Pharmingen) or Streptavidin RED 613 (GIBCO BRL). Viable lymphoid cells as determined by forward and side scatter were gated on and 30,000 events collected. Flow cytometric analysis was performed on an Epics Elite ESP (Coulter) flow cytometer equipped with an ultraviolet enhanced argon ion blue laser and a helium/neon red laser. In general, negative controls were used to set voltage and single color positive controls were used for electronic compensation. Processed samples were analyzed using Epics Elite analysis software and FloJo version 3.1.1 (Tree Star Corp.). 2.4. Immunoblot assays and assay for ERK activity Immunoblot assays using anti-phosphotyrosine antibodies and antibodies to c-Cbl were performed as described earlier (Kim et al., 1995a). For ERK kinase assays, 107 splenic B cells were either left untreated or stimulated with anti-IgM (25 g/ml) and lysed in 1 ml of 1% NP-40, 20 mM Tris pH 8, 120 mM NaCl, 20 mM sodium vanadate, and 200 M PMSF. ERK was immunoprecipitated with 5 g of anti-ERK antibody (Santa Cruz) and protein A-Sepharose and washed twice in lysis buffer and twice with 1×kinase buffer (20 mM HEPES pH 7.2, 0.5 mM EGTA, 10 mM ATP, 0.5 mM DTT, 0.005% Triton X-100, 10 mM MgCl2 ). Beads were resuspended in 50 l of kinase buffer with 5 g of MBP and 5 Ci of ␥32 P-ATP and incubated at 30 ◦ C for 15 min. The reaction was stopped by the addition of sample buffer and subjected to 10% SDS-PAGE. 2.5. Proliferation assays A 105 cells in RPMI1640 with 10% FBS were plated on 96-well plates and stimulated with anti-IgM (25 g/ml) or
Fig. 1. Reconstitution of the thymus in Rag-1 deficient mice with Nf1−/− fetal liver cells. CD4 and CD8 staining of thymocytes from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells was performed.
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Fig. 2. Reconstitution of lymphoid populations in the bone marrow of Rag-1 deficient mice with Nf1−/− fetal liver cells. Characterization of bone marrow B lineage cells in Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells. Fractions A, B, C, C , D, E, and F were delineated according to Hardy et al. (1991).
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previously bound anti-CD3. For anti-CD3 stimulation, plates were incubated with 10 g/ml anti-CD3 in 50 mM Tris pH 9.5 for 1 h and then washed three times with PBS. After 54 h of incubation, 0.2 Ci 3 H-thymidine was added to each well. Cells were harvested at 72 h and the level of the 3 H uptake was measured by scintillation counting.
3. Results Nf1−/− mice die in utero around embryonic day 13.5 (Jacks et al., 1994; Brannan et al., 1994). We reconstituted the lymphoid compartment in Rag-1−/− mice with fetal liver cells from day 12.5 or day 13.5 Nf1+/− or Nf1−/− fetuses.
Fig. 3. Peripheral B cell development in the absence of neurofibromin. IgM and IgD expressing B cells from the spleens (a) and mesenteric lymph nodes (b) of Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells were examined. Fractions I, II, and III were delineated based on IgM and IgD staining (Hardy et al., 1982). B220+ CD5+ CD11b+ B-1 cells in the peritoneum were also analyzed (c).
T.J. Kim et al. / Molecular Immunology 38 (2001) 701–708
Studies included here are based on the analysis of lymphoid populations in mice in which reconstitution by Nf1+/− and Nf1−/− fetal liver cells led to the generation of normal levels of splenic B cells as assessed by cell counts and flow cytometry. Analysis in parallel of smaller numbers of wild-type mice revealed no phenotypic differences between wild-type Nf1+/+ and heterozygous Nf1+/− mice. Examination of the thymus revealed no differences in the proportions of double positive (DP; CD4+ CD8+ ) and CD4+ and CD8+ single positive (SP) T cell populations (Fig. 1) in mice reconstituted with Nf1+/− or Nf1−/− fetal liver cells. Bone marrow B lineage populations in mice reconstituted with Nf1+/− or Nf1−/− fetal liver cells were broadly similar. A small decrease in the number of C cells was noted in mice reconstituted with Nf1−/− fetal liver cells (Fig. 2). C cells represent pre-B cells that have successfully negotiated the pre-B receptor dependent checkpoint. Six pairs of Nf1+/− and Nf1−/− mice were compared in Figs. 1 and 2. Examination of peripheral B cells in reconstituted mice revealed no major differences in the IgM and IgD expressing B cell populations in the spleen and mesenteric lymph nodes (Fig. 3a and b). Of particular note is the absence of any reduction in fraction I (IgDhi IgMlo ) follicular Nf1−/− B cells whose numbers decline in certain mutant mice in which antigen receptor mediated B cell proliferation and peripheral B cell viability are compromised (Cariappa et al., 1999; Byth et al., 1996). Small differences in lymphoid compartment size in reconstituted mice cannot be evaluated with certainty, but in addition to the possible defect in C cells in the bone marrow, a small reduction in peritoneal B-1a cells in Rag-1−/− mice was observed in mice reconstituted with Nf1−/− fetal liver cells (Fig. 3c). This result should be interpreted with caution since analyses of peritoneal B cells were performed only on two Nf1+/− and two Nf1−/− mice. B-1a cells are diminished in mice harboring mutations in genes that encode signaling components required for B cell antigen receptor induced proliferation (Pillai, 1999). Given that neurofibromin functions as a RasGAP and has been shown to be a negative regulator of proliferation in astrocytes, myeloid cells, and melanocytes (Shannon et al., 1994; Largaespada et al., 1996; Bollag et al., 1996; Gutmann et al., 1999; Ingram et al., 2000), we wished to examine proliferative responses in B cells that lack neurofibromin. In a number of experiments, Nf1−/− splenic B cells proliferated fairly well in response to anti-CD40 and IL-4, sometimes more readily than control B cells (see for example, Fig. 4a). However, while responses to anti-CD40 and IL-4 varied and were not markedly reduced in any experiments, Nf1−/− B cells consistently proliferated very poorly in response to anti-IgM crosslinking as compared to Nf1+/− B cells (see Fig. 4a and b). When thymocyte proliferation was examined in response to plate bound anti-CD3, Nf1−/− thymocytes proliferated markedly less well than Nf1+/− thymocytes (Fig. 4c). Since DP thymocytes proliferate very poorly in comparison to SP thymocytes, we purified flow-sorted CD4+ thymocytes from thymuses reconstituted
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Fig. 4. Impaired antigen receptor induced proliferation of neurofibromin deficient lymphocytes (a) and (b). Impaired antigen receptor induced proliferation of neurofibromin deficient B cells. Results of two separate experiments are shown. Splenocytes from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells were stimulated with anti-IgM (a and b), or with CD40 and IL-4, or with bacterial lipopolysaccharide (LPS) (a). Similar deficits in anti-IgM induced proliferation were seen in separate experiments involving cells from eight matched pairs of mice (c). Impaired antigen receptor induced proliferation of neurofibromin deficient single positive thymocytes. Total thymocytes (TT), flow sorted CD4+ CD8+ double positive T cells (DP), and flow sorted single positive (SP) CD4+ T cells (from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells) were triggered using plate bound anti-CD3 antibodies. Similar results were observed in two separate experiments.
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with Nf1+/− and Nf1−/− fetal liver cells. As expected, double positive thymocytes of wild-type and mutant origin proliferated very poorly in response to anti-CD3 crosslinking. However, anti-CD3 driven proliferation of purified CD4+ single positive Nf1−/− thymocytes was defective when compared with purified Nf1+/− CD4+ thymocytes (Fig. 4c). Both p120GAP and neurofibromin are expressed in B lymphocytes. In keeping with the absence of any increase in antigen-receptor induced proliferation in the absence of
neurofibromin, the RasGAP activity of neurofibromin is likely to be redundant in B cells, and baseline and anti-IgM induced ERK activity is not enhanced in Nf1−/− B cells (data not shown). In the course of biochemical studies in which we attempted to address why anti-IgM induced B cell proliferation is impaired in Nf1−/− B cells, we discovered major deficits in anti-IgM induced tyrosine phosphorylation in B cells that lack neurofibromin. As seen in Fig. 5a and b, a number of proteins phosphorylated on tyrosine that are
Fig. 5. Defective tyrosine phosphorylation of antigen receptor triggered B cells that lack neurofibromin (a) and (b). Discrete phosphoprotein deficits were observed in anti-IgM triggered purified splenic B cells from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells. Lysates from non-stimulated (N) and stimulated (anti-IgM) cells were separated on a 9% polyacrylamide/SDS gel and tyrosine phosphorylated proteins revealed on an immunoblot using anti-PY 20 antibodies. Shc was analyzed as a loading control in (a). A separate experiment is depicted in (b). Similar results were observed in a total of six separate experiments (c). At least one of the phosphoproteins not seen after antigen receptor ligation of B cells is deficient even in non-stimulated Nf1−/− B cells. The upper panel shows an anti-c-Cbl western blot. Phospho p38 was revealed in the lower panel to demonstrate that at least equivalent amounts of cell lysates from Nf1−/− lymphocytes were loaded.
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observed after anti-IgM crosslinking in wild-type B cells are not seen in receptor ligated Nf1−/− B cells. Similar deficits have been consistently observed in numerous experiments. Defective tyrosine phosphorylation of selected signaling molecules may explain why antigen receptor ligation fails to induce optimal lymphocyte proliferation in the absence of neurofibromin. Why exactly these deficits in tyrosine phosphorylation are observed is unclear. The inability to detect subsets of phosphoproteins following antigen receptor ligation of activated Nf1−/− B cells might be attributed to deficits in tyrosine kinase activity, enhanced activation of tyrosine phosphatases, decreased degradation and proteasomal clearance of protein tyrosine phosphatases, the enhanced proteasomal degradation of activated tyrosine kinases, or possibly the increased proteasomal degradation specifically of the phosphoproteins that make up the missing subset. None of these putative mechanisms however apply to a prominent missing phosphoprotein, c-Cbl. This protein can both negatively and positively influence antigen receptor induced signaling and can function as an E3 for the ubiquitin mediated degradation of tyrosine phosphorylated proteins (Joazeiro et al., 1999). c-Cbl cannot be detected even in resting Nf1−/− B cells (Fig. 5c), and the mechanisms by which the levels of this protein are altered in the absence of neurofibromin have not yet been established.
4. Discussion Peripheral B lymphocytes and single positive T cells that lack neurofibromin proliferate very poorly in response to antigen receptor crosslinking. While small changes in the sizes of cellular compartments in reconstituted mice should be interpreted with caution, the reduction in C cells in the bone marrow and the reduction in B-1a cells in the peritoneum may be the consequence of defective signaling in vivo via the pre-BCR and the BCR, respectively. Because of the variability of reconstitution, the analysis of immune responses of heterozygous and neurofibromin deficient reconstituted Rag-1−/− mice immunized with T-dependent and T-independent antigens was not pursued. Given that neurofibromin is a negative regulator of proliferation in a number of cell types, the proliferative defects seen in lymphoid cells were unexpected. Apart from its known RasGAP activity, another potential biochemical function of NF1 has emerged from studies on Drosophila NF1 (The et al., 1997; Guo et al., 1997). The phenotypes of flies in which NF1 is mutated can be rescued by increasing signaling through the cAMP-PKA pathway (The et al., 1997). While there is no evidence to suggest that PKA signaling is regulated by NF1 in vertebrate cells, it should be noted that cAMP inhibits anti-IgM induced murine B cell proliferation (Cohen and Rothstein, 1989). If indeed NF1 is actually a positive regulator of PKA signaling in B lymphocytes, we would have expected to see enhanced proliferation
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of Nf1−/− B cells in response to anti-IgM crosslinking. The marked reduction of B cell and thymocyte proliferation suggests a novel function of NF1 in lymphoid cells which may or may not be related to its known function as a RasGAP or its potential, but as yet unproven, function as a regulator of PKA signaling. It is possible that the apparent defect in PKA signaling in Drosophila NF1 mutants is not directly linked to NF1 function but may result from compensatory alterations secondary to the absence of NF1. We believe that the defects in tyrosine phosphorylation in neurofibromin deficient lymphocytes may also result from poorly understood indirect changes downstream of the loss of NF1. The absence of c-Cbl in B cells probably represents only one of a number of complex compensatory alterations in the levels of key regulators in lymphoid cells that lack neurofibromin; we have presented data on this protein in order to emphasize that complex changes may be responsible for the discrete alterations observed in antigen receptor induced tyrosine phosphorylation in Nf1−/− B cells. These changes might result from increased Ras activity at some early stage of B cell development, or may occur in a RasGAP independent manner. Neurofibromin negatively regulates signals that contribute to the survival of sensory and sympathetic neurons (Vogel et al., 1995). On the other hand, although Ras-GTP levels are elevated in murine Schwann cells that lack neurofibromin, proliferative responses to glial growth factor 2 are impaired in these cells (Kim et al., 1995b). In some respects therefore, the alterations seen in lymphoid cells that lack neurofibromin may not be unique, and the absence of neurofibromin may contribute to a defect in proliferation downstream of different receptors in different cell types. The biochemical basis for the altered tyrosine phosphorylation seen in Nf1−/− lymphoid cells remains to be established. A detailed mechanistic understanding of the changes that occur in Nf1−/− B cells would be facilitated by the more efficient generation of neurofibromin deficient B cells, possibly by a Cre-lox approach.
Acknowledgements This work was supported by Grants CA 69618 and AI 33507 from the NIH, and by an award from the Massachusetts Chapter of the Arthritis Foundation.
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Defective proliferative responses in B lymphocytes and thymocytes that lack neurofibromin Tae Jin Kim a,1,2 , Annaiah Cariappa a,2 , John Iacomini b , Mei Tang a , Shane Shih c , Andre Bernards a , Tyler Jacks c , Shiv Pillai a,∗ a
c
Cancer Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown Navy Yard, Boston, MA 02129, USA b Transplantation Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA Center for Cancer Research and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 28 May 2001; received in revised form 10 October 2001; accepted 24 October 2001
Abstract Nf1−/− fetal liver cells were used to reconstitute B and T cells in Rag-1−/− mice. Lymphocyte development was largely unimpaired in the absence of neurofibromin. However antigen-receptor induced proliferation was defective in neurofibromin deficient peripheral B cells and CD4+ single positive thymocytes. In contrast to its role as a negative regulator of proliferation in many other cell types, neurofibromin may be a positive regulator of lymphocyte proliferation. Peripheral B cells exhibited circumscribed defects in anti-IgM induced protein tyrosine phosphorylation, which may contribute to the unexpected proliferative defect seen in these cells. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: B lymphocytes; Signal transduction; Cellular activation
1. Introduction GTPase activating proteins play important roles in catalyzing the conversion of GTP bound to small G proteins such as Ras to GDP. These enzymes bind to the effector domains of these small G proteins and enhance their intrinsic GTPase activity. The Nf1 gene is mutated in patients with type 1 neurofibromatosis and encodes a RasGAP known as neurofibromin (Viskochil et al., 1990; Xu et al., 1990; Martin et al., 1990; Ballester et al., 1990; Bollag and McCormick, 1991). Although neurofibromin shares a GAP domain with p120GAP, outside of this homologous segment the proteins are quite dissimilar. In particular neurofibromin lacks the SH2, SH3, pleckstrin-homology (PH) and calcium/phospholipid binding (CaLB) domains seen in p120GAP. Neurofibromin, however, closely resembles the yeast rasGAPs, IRA1 and IRA2, and can complement their activity in the appropriate ira− S. cerevisiae mutants ∗ Corresponding author. Tel.: +1-617-726-5619; fax: +1-617-724-9648. E-mail address: [email protected] (S. Pillai). 1 Present address: Department of Pathology, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea. 2 Equal contributors.
(Viskochil et al., 1990; Xu et al., 1990; Martin et al., 1990). Consistent with its role as a RasGAP, neurofibromin is a negative regulator of proliferation in several cell types including astrocytes, melanocytes, and myeloid cells (Shannon et al., 1994; Largaespada et al., 1996; Bollag et al., 1996; Gutmann et al., 1999; Ingram et al., 2000). Crosslinking of membrane IgM on splenic B cells leads to the cocapping of this protein with neurofibromin but not with p120GAP (Boyer et al., 1994). The biological implications of the co-capping of neurofibromin (Boyer et al., 1994) and Ras (Graziadei et al., 1990) with membrane IgM are poorly understood. We were interested in examining whether neurofibromin is required for B cell development and to determine if signaling downstream of the antigen receptor is enhanced in B cells that lack neurofibromin. Nf1−/− mice die around day 14 of embryogenesis primarily because of a cardiac defect (Jacks et al., 1994; Brannan et al., 1994). We reconstituted Rag-1−/− mice with fetal liver cells from Nf1−/− embryos. While B and T cell development in the absence of NF1 was grossly normal, antigen-receptor induced proliferation was defective in peripheral B cells lacking neurofibromin, and in neurofibromin deficient CD4+ single positive thymocytes.
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2. Materials and methods 2.1. Mice The generation of Nf1−/− mice has been previously described (Jacks et al., 1994). Rag-1/− mice were obtained from Jackson laboratories. 2.2. Reconstitution of Rag-1−/− mice with Nf1−/− fetal liver cells Nf1+/− mice were mated, and at day 12.5 or day 13.5%, embryos were harvested and suspensions of fetal liver cells were made. Cells from a single fetal liver were injected into the tail vein of one recipient Rag-1−/− mouse. Three hours prior to the cell transfer, Rag-1−/− mice were mildly irradiated (300 rad). Carcasses were genotyped by PCR to determine if embryos were Nf1+/+ , Nf1+/− or Nf1−/− (Jacks et al., 1994). 2.3. Flow cytometry Single cell suspensions were made from spleen, bone marrow, thymus, mesenteric lymph nodes, and peritoneum. For surface staining, 1 × 106 cells were reacted with 2.4G2 (anti-CD16/CD32 {Fc␥ III/II receptor}, culture supernatant), prior to staining with the following antibodies: anti CD5-biotin clone 57-7.3, anti-CD11b-allophycocyanin (APC) clone M1/70, anti CD4 PE clone RM4-5, anti-CD8␣ FITC clone 53–67, anti-CD45R-FITC (or APC) B220 clone RA36B2, antiCD43/leukosialin-FITC clone S7, anti-BP1-PE clone BP1, anti-CD24-biotin clone 30F1, and anti-IgM-PE clone R6-60. All of the above antibodies were obtained from Pharmingen. Anti-IgD-biotin clone 11–26 and goat anti-mouse IgM PE was from Southern
Biotechnology. Biotinylated antibodies were revealed using streptavidin-APC (Pharmingen) or Streptavidin RED 613 (GIBCO BRL). Viable lymphoid cells as determined by forward and side scatter were gated on and 30,000 events collected. Flow cytometric analysis was performed on an Epics Elite ESP (Coulter) flow cytometer equipped with an ultraviolet enhanced argon ion blue laser and a helium/neon red laser. In general, negative controls were used to set voltage and single color positive controls were used for electronic compensation. Processed samples were analyzed using Epics Elite analysis software and FloJo version 3.1.1 (Tree Star Corp.). 2.4. Immunoblot assays and assay for ERK activity Immunoblot assays using anti-phosphotyrosine antibodies and antibodies to c-Cbl were performed as described earlier (Kim et al., 1995a). For ERK kinase assays, 107 splenic B cells were either left untreated or stimulated with anti-IgM (25 g/ml) and lysed in 1 ml of 1% NP-40, 20 mM Tris pH 8, 120 mM NaCl, 20 mM sodium vanadate, and 200 M PMSF. ERK was immunoprecipitated with 5 g of anti-ERK antibody (Santa Cruz) and protein A-Sepharose and washed twice in lysis buffer and twice with 1×kinase buffer (20 mM HEPES pH 7.2, 0.5 mM EGTA, 10 mM ATP, 0.5 mM DTT, 0.005% Triton X-100, 10 mM MgCl2 ). Beads were resuspended in 50 l of kinase buffer with 5 g of MBP and 5 Ci of ␥32 P-ATP and incubated at 30 ◦ C for 15 min. The reaction was stopped by the addition of sample buffer and subjected to 10% SDS-PAGE. 2.5. Proliferation assays A 105 cells in RPMI1640 with 10% FBS were plated on 96-well plates and stimulated with anti-IgM (25 g/ml) or
Fig. 1. Reconstitution of the thymus in Rag-1 deficient mice with Nf1−/− fetal liver cells. CD4 and CD8 staining of thymocytes from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells was performed.
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Fig. 2. Reconstitution of lymphoid populations in the bone marrow of Rag-1 deficient mice with Nf1−/− fetal liver cells. Characterization of bone marrow B lineage cells in Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells. Fractions A, B, C, C , D, E, and F were delineated according to Hardy et al. (1991).
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previously bound anti-CD3. For anti-CD3 stimulation, plates were incubated with 10 g/ml anti-CD3 in 50 mM Tris pH 9.5 for 1 h and then washed three times with PBS. After 54 h of incubation, 0.2 Ci 3 H-thymidine was added to each well. Cells were harvested at 72 h and the level of the 3 H uptake was measured by scintillation counting.
3. Results Nf1−/− mice die in utero around embryonic day 13.5 (Jacks et al., 1994; Brannan et al., 1994). We reconstituted the lymphoid compartment in Rag-1−/− mice with fetal liver cells from day 12.5 or day 13.5 Nf1+/− or Nf1−/− fetuses.
Fig. 3. Peripheral B cell development in the absence of neurofibromin. IgM and IgD expressing B cells from the spleens (a) and mesenteric lymph nodes (b) of Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells were examined. Fractions I, II, and III were delineated based on IgM and IgD staining (Hardy et al., 1982). B220+ CD5+ CD11b+ B-1 cells in the peritoneum were also analyzed (c).
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Studies included here are based on the analysis of lymphoid populations in mice in which reconstitution by Nf1+/− and Nf1−/− fetal liver cells led to the generation of normal levels of splenic B cells as assessed by cell counts and flow cytometry. Analysis in parallel of smaller numbers of wild-type mice revealed no phenotypic differences between wild-type Nf1+/+ and heterozygous Nf1+/− mice. Examination of the thymus revealed no differences in the proportions of double positive (DP; CD4+ CD8+ ) and CD4+ and CD8+ single positive (SP) T cell populations (Fig. 1) in mice reconstituted with Nf1+/− or Nf1−/− fetal liver cells. Bone marrow B lineage populations in mice reconstituted with Nf1+/− or Nf1−/− fetal liver cells were broadly similar. A small decrease in the number of C cells was noted in mice reconstituted with Nf1−/− fetal liver cells (Fig. 2). C cells represent pre-B cells that have successfully negotiated the pre-B receptor dependent checkpoint. Six pairs of Nf1+/− and Nf1−/− mice were compared in Figs. 1 and 2. Examination of peripheral B cells in reconstituted mice revealed no major differences in the IgM and IgD expressing B cell populations in the spleen and mesenteric lymph nodes (Fig. 3a and b). Of particular note is the absence of any reduction in fraction I (IgDhi IgMlo ) follicular Nf1−/− B cells whose numbers decline in certain mutant mice in which antigen receptor mediated B cell proliferation and peripheral B cell viability are compromised (Cariappa et al., 1999; Byth et al., 1996). Small differences in lymphoid compartment size in reconstituted mice cannot be evaluated with certainty, but in addition to the possible defect in C cells in the bone marrow, a small reduction in peritoneal B-1a cells in Rag-1−/− mice was observed in mice reconstituted with Nf1−/− fetal liver cells (Fig. 3c). This result should be interpreted with caution since analyses of peritoneal B cells were performed only on two Nf1+/− and two Nf1−/− mice. B-1a cells are diminished in mice harboring mutations in genes that encode signaling components required for B cell antigen receptor induced proliferation (Pillai, 1999). Given that neurofibromin functions as a RasGAP and has been shown to be a negative regulator of proliferation in astrocytes, myeloid cells, and melanocytes (Shannon et al., 1994; Largaespada et al., 1996; Bollag et al., 1996; Gutmann et al., 1999; Ingram et al., 2000), we wished to examine proliferative responses in B cells that lack neurofibromin. In a number of experiments, Nf1−/− splenic B cells proliferated fairly well in response to anti-CD40 and IL-4, sometimes more readily than control B cells (see for example, Fig. 4a). However, while responses to anti-CD40 and IL-4 varied and were not markedly reduced in any experiments, Nf1−/− B cells consistently proliferated very poorly in response to anti-IgM crosslinking as compared to Nf1+/− B cells (see Fig. 4a and b). When thymocyte proliferation was examined in response to plate bound anti-CD3, Nf1−/− thymocytes proliferated markedly less well than Nf1+/− thymocytes (Fig. 4c). Since DP thymocytes proliferate very poorly in comparison to SP thymocytes, we purified flow-sorted CD4+ thymocytes from thymuses reconstituted
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Fig. 4. Impaired antigen receptor induced proliferation of neurofibromin deficient lymphocytes (a) and (b). Impaired antigen receptor induced proliferation of neurofibromin deficient B cells. Results of two separate experiments are shown. Splenocytes from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells were stimulated with anti-IgM (a and b), or with CD40 and IL-4, or with bacterial lipopolysaccharide (LPS) (a). Similar deficits in anti-IgM induced proliferation were seen in separate experiments involving cells from eight matched pairs of mice (c). Impaired antigen receptor induced proliferation of neurofibromin deficient single positive thymocytes. Total thymocytes (TT), flow sorted CD4+ CD8+ double positive T cells (DP), and flow sorted single positive (SP) CD4+ T cells (from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells) were triggered using plate bound anti-CD3 antibodies. Similar results were observed in two separate experiments.
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with Nf1+/− and Nf1−/− fetal liver cells. As expected, double positive thymocytes of wild-type and mutant origin proliferated very poorly in response to anti-CD3 crosslinking. However, anti-CD3 driven proliferation of purified CD4+ single positive Nf1−/− thymocytes was defective when compared with purified Nf1+/− CD4+ thymocytes (Fig. 4c). Both p120GAP and neurofibromin are expressed in B lymphocytes. In keeping with the absence of any increase in antigen-receptor induced proliferation in the absence of
neurofibromin, the RasGAP activity of neurofibromin is likely to be redundant in B cells, and baseline and anti-IgM induced ERK activity is not enhanced in Nf1−/− B cells (data not shown). In the course of biochemical studies in which we attempted to address why anti-IgM induced B cell proliferation is impaired in Nf1−/− B cells, we discovered major deficits in anti-IgM induced tyrosine phosphorylation in B cells that lack neurofibromin. As seen in Fig. 5a and b, a number of proteins phosphorylated on tyrosine that are
Fig. 5. Defective tyrosine phosphorylation of antigen receptor triggered B cells that lack neurofibromin (a) and (b). Discrete phosphoprotein deficits were observed in anti-IgM triggered purified splenic B cells from Rag-1−/− mice reconstituted with Nf1−/− and Nf1+/− fetal liver cells. Lysates from non-stimulated (N) and stimulated (anti-IgM) cells were separated on a 9% polyacrylamide/SDS gel and tyrosine phosphorylated proteins revealed on an immunoblot using anti-PY 20 antibodies. Shc was analyzed as a loading control in (a). A separate experiment is depicted in (b). Similar results were observed in a total of six separate experiments (c). At least one of the phosphoproteins not seen after antigen receptor ligation of B cells is deficient even in non-stimulated Nf1−/− B cells. The upper panel shows an anti-c-Cbl western blot. Phospho p38 was revealed in the lower panel to demonstrate that at least equivalent amounts of cell lysates from Nf1−/− lymphocytes were loaded.
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observed after anti-IgM crosslinking in wild-type B cells are not seen in receptor ligated Nf1−/− B cells. Similar deficits have been consistently observed in numerous experiments. Defective tyrosine phosphorylation of selected signaling molecules may explain why antigen receptor ligation fails to induce optimal lymphocyte proliferation in the absence of neurofibromin. Why exactly these deficits in tyrosine phosphorylation are observed is unclear. The inability to detect subsets of phosphoproteins following antigen receptor ligation of activated Nf1−/− B cells might be attributed to deficits in tyrosine kinase activity, enhanced activation of tyrosine phosphatases, decreased degradation and proteasomal clearance of protein tyrosine phosphatases, the enhanced proteasomal degradation of activated tyrosine kinases, or possibly the increased proteasomal degradation specifically of the phosphoproteins that make up the missing subset. None of these putative mechanisms however apply to a prominent missing phosphoprotein, c-Cbl. This protein can both negatively and positively influence antigen receptor induced signaling and can function as an E3 for the ubiquitin mediated degradation of tyrosine phosphorylated proteins (Joazeiro et al., 1999). c-Cbl cannot be detected even in resting Nf1−/− B cells (Fig. 5c), and the mechanisms by which the levels of this protein are altered in the absence of neurofibromin have not yet been established.
4. Discussion Peripheral B lymphocytes and single positive T cells that lack neurofibromin proliferate very poorly in response to antigen receptor crosslinking. While small changes in the sizes of cellular compartments in reconstituted mice should be interpreted with caution, the reduction in C cells in the bone marrow and the reduction in B-1a cells in the peritoneum may be the consequence of defective signaling in vivo via the pre-BCR and the BCR, respectively. Because of the variability of reconstitution, the analysis of immune responses of heterozygous and neurofibromin deficient reconstituted Rag-1−/− mice immunized with T-dependent and T-independent antigens was not pursued. Given that neurofibromin is a negative regulator of proliferation in a number of cell types, the proliferative defects seen in lymphoid cells were unexpected. Apart from its known RasGAP activity, another potential biochemical function of NF1 has emerged from studies on Drosophila NF1 (The et al., 1997; Guo et al., 1997). The phenotypes of flies in which NF1 is mutated can be rescued by increasing signaling through the cAMP-PKA pathway (The et al., 1997). While there is no evidence to suggest that PKA signaling is regulated by NF1 in vertebrate cells, it should be noted that cAMP inhibits anti-IgM induced murine B cell proliferation (Cohen and Rothstein, 1989). If indeed NF1 is actually a positive regulator of PKA signaling in B lymphocytes, we would have expected to see enhanced proliferation
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of Nf1−/− B cells in response to anti-IgM crosslinking. The marked reduction of B cell and thymocyte proliferation suggests a novel function of NF1 in lymphoid cells which may or may not be related to its known function as a RasGAP or its potential, but as yet unproven, function as a regulator of PKA signaling. It is possible that the apparent defect in PKA signaling in Drosophila NF1 mutants is not directly linked to NF1 function but may result from compensatory alterations secondary to the absence of NF1. We believe that the defects in tyrosine phosphorylation in neurofibromin deficient lymphocytes may also result from poorly understood indirect changes downstream of the loss of NF1. The absence of c-Cbl in B cells probably represents only one of a number of complex compensatory alterations in the levels of key regulators in lymphoid cells that lack neurofibromin; we have presented data on this protein in order to emphasize that complex changes may be responsible for the discrete alterations observed in antigen receptor induced tyrosine phosphorylation in Nf1−/− B cells. These changes might result from increased Ras activity at some early stage of B cell development, or may occur in a RasGAP independent manner. Neurofibromin negatively regulates signals that contribute to the survival of sensory and sympathetic neurons (Vogel et al., 1995). On the other hand, although Ras-GTP levels are elevated in murine Schwann cells that lack neurofibromin, proliferative responses to glial growth factor 2 are impaired in these cells (Kim et al., 1995b). In some respects therefore, the alterations seen in lymphoid cells that lack neurofibromin may not be unique, and the absence of neurofibromin may contribute to a defect in proliferation downstream of different receptors in different cell types. The biochemical basis for the altered tyrosine phosphorylation seen in Nf1−/− lymphoid cells remains to be established. A detailed mechanistic understanding of the changes that occur in Nf1−/− B cells would be facilitated by the more efficient generation of neurofibromin deficient B cells, possibly by a Cre-lox approach.
Acknowledgements This work was supported by Grants CA 69618 and AI 33507 from the NIH, and by an award from the Massachusetts Chapter of the Arthritis Foundation.
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