Deubiquitinating Enzyme CYLD Regulates the Peripheral Development and Naive Phenotype Maintenance of B Cells
2007; Elsevier BV; Volume: 282; Issue: 21 Linguagem: Inglês
10.1074/jbc.m609952200
ISSN1083-351X
AutoresWei Jin, William R. Reiley, Andrew Lee, Ato Wright, Xuefeng Wu, Minying Zhang, Shao‐Cong Sun,
Tópico(s)Immune Response and Inflammation
ResumoDeubiquitinating enzymes (DUB) form a family of cysteine proteases that digests ubiquitin chains and reverses the process of protein ubiquitination. Despite the identification of a large number of DUBs, their physiological functions remain poorly defined. Here we provide genetic evidence that CYLD, a recently identified DUB, plays a crucial role in regulating the peripheral development and activation of B cells. Disruption of the CYLD gene in mice results in B cell hyperplasia and lymphoid organ enlargement. The CYLD-deficient B cells display surface markers indicative of spontaneous activation and are hyperproliferative upon in vitro stimulation. When challenged with antigens, the CYLD-/- mice develop exacerbated lymphoid organ abnormalities and abnormal B cell responses. Although the loss of CYLD has only a minor effect on B cell development in bone marrow, this genetic deficiency disrupts the balance of peripheral B cell populations with a significant increase in marginal zone B cells. In keeping with these functional abnormalities, the CYLD-/- B cells exhibit constitutive activation of the transcription factor NF-κB due to spontaneous activation of IκB kinase β and degradation of the NF-κB inhibitor IκBα. These findings demonstrate a critical role for CYLD in regulating the basal activity of NF-κB and maintaining the naive phenotype and proper activation of B cells. Deubiquitinating enzymes (DUB) form a family of cysteine proteases that digests ubiquitin chains and reverses the process of protein ubiquitination. Despite the identification of a large number of DUBs, their physiological functions remain poorly defined. Here we provide genetic evidence that CYLD, a recently identified DUB, plays a crucial role in regulating the peripheral development and activation of B cells. Disruption of the CYLD gene in mice results in B cell hyperplasia and lymphoid organ enlargement. The CYLD-deficient B cells display surface markers indicative of spontaneous activation and are hyperproliferative upon in vitro stimulation. When challenged with antigens, the CYLD-/- mice develop exacerbated lymphoid organ abnormalities and abnormal B cell responses. Although the loss of CYLD has only a minor effect on B cell development in bone marrow, this genetic deficiency disrupts the balance of peripheral B cell populations with a significant increase in marginal zone B cells. In keeping with these functional abnormalities, the CYLD-/- B cells exhibit constitutive activation of the transcription factor NF-κB due to spontaneous activation of IκB kinase β and degradation of the NF-κB inhibitor IκBα. These findings demonstrate a critical role for CYLD in regulating the basal activity of NF-κB and maintaining the naive phenotype and proper activation of B cells. Ubiquitination is a posttranslational mechanism that regulates the degradation and biological function of diverse proteins (1Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1066) Google Scholar, 2Haglund K. Dikic I. EMBO J. 2005; 24: 3353-3359Crossref PubMed Scopus (616) Google Scholar). Protein ubiquitination is catalyzed by well defined enzymatic machinery, composed of an ubiquitin-activating enzyme (E1), 4The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligases; DUB, deubiquitinating enzyme; MAP, mitogen-activated protein; MAPK, MAP kinase; TLR, Toll-like receptors; BAFF, B cell activation factor of the tumor necrosis factor family; MLN, mesenteric lymph node; CFSE, carboxyl fluorescent succinimidyl ester; SRBC, sheep red blood cells; LPS, lipopolysaccharide; NP-LPS, nitro-phenol-conjugated LPS; KLH, keyhole limpet hemocyanin; nitro-phenol-conjugated KLH, KLH; IB, immunoblotting; EMSA, electrophoresis mobility shift assay; IKKγ, IκB kinase; FITC, fluorescein isothiocyanate; PE,; phosphatidylethanolamine; PBS, phosphate-buffered saline; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; TCR, T cell receptor; BCR, B cell receptor. ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3). Recent studies on the E3 ubiquitin ligases demonstrate an important role for protein ubiquitination in the regulation of immune responses (3Liu Y.C. Penninger J. Karin M. Nat. Rev. Immunol. 2005; 5: 941-952Crossref PubMed Scopus (213) Google Scholar). In particular, ubiquitination is involved in the development, activation, and differentiation of lymphocytes. Defects in E3 ubiquitin ligases are associated with severe immunological disorders, such as the loss of immunological tolerance and development of autoimmunity (3Liu Y.C. Penninger J. Karin M. Nat. Rev. Immunol. 2005; 5: 941-952Crossref PubMed Scopus (213) Google Scholar). Emerging evidence suggests that protein ubiquitination is a tightly controlled and reversible process that is counter-regulated by deubiquitinating enzymes (DUBs), a family of cysteine proteases digesting ubiquitin chains (4Nijman S.M. Luna-Vargas M.P. Velds A. Brummelkamp T.R. Dirac A.M. Sixma T.K. Bernards R. Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (1464) Google Scholar). Like the E3s, the DUBs exist in large numbers, thus suggesting a high level of functional diversity and substrate specificity in their functions (4Nijman S.M. Luna-Vargas M.P. Velds A. Brummelkamp T.R. Dirac A.M. Sixma T.K. Bernards R. Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (1464) Google Scholar). However, despite the extensive studies on E3s, the physiological functions of DUBs are poorly defined. We have recently described the function of a DUB, CYLD, in regulating thymocyte development (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). CYLD positively regulates thymic TCR signaling and is required for the generation of CD4 and CD8 mature thymocytes (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). These findings provide the first example for how a DUB can function in the adaptive immune system. However, it is unclear whether CYLD also regulates other aspects of immune function, particularly the activation and homeostasis of lymphocytes. CYLD was originally identified as a tumor suppressor mutated in familial cylindromatosis (6Bignell G.R. Warren W. Seal S. Takahashi M. Rapley E. Barfoot R. Green H. Brown C. Biggs P.J. Lakhani S.R. Jones C. Hansen J. Blair E. Hofmann B. Siebert R. Turner G. Evans D.G. Schrander-Stumpel C. Beemer F.A. van Den Ouweland A. Halley D. Delpech B. Cleveland M.G. Leigh I. Leisti J. Rasmussen S. Nat. Genet. 2000; 25: 160-165Crossref PubMed Scopus (599) Google Scholar), an autosomal dominant predisposition to benign tumors of the skin appendages (7Lian F. Cockerell C.J. Adv. Dermatol. 2005; 21: 217-234Crossref PubMed Scopus (14) Google Scholar). More recent in vitro work suggests that CYLD functions as a DUB of tumor necrosis factor receptor-associated factors and the regulatory subunit of IκB kinase (IKKγ) (8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (842) Google Scholar, 9Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (866) Google Scholar, 10Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (822) Google Scholar, 11Yoshida H. Jono H. Kai H. Li J.D. J. Biol. Chem. 2005; 280: 41111-41121Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Ubiquitination of these signaling molecules appears to serve as a mechanism that activates their signal transduction functions (12Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1038) Google Scholar). Consistently, in vitro work demonstrates that CYLD inhibits the activation of NF-κB and MAP kinases (MAPKs) by Toll-like receptors (TLRs) and tumor necrosis factor receptors (8Brummelkamp T.R. Nijman S.M. Dirac A.M. Bernards R. Nature. 2003; 424: 797-801Crossref PubMed Scopus (842) Google Scholar, 9Kovalenko A. Chable-Bessia C. Cantarella G. Israel A. Wallach D. Courtois G. Nature. 2003; 424: 801-805Crossref PubMed Scopus (866) Google Scholar, 10Trompouki E. Hatzivassiliou E. Tsichritzis T. Farmer H. Ashworth A. Mosialos G. Nature. 2003; 424: 793-796Crossref PubMed Scopus (822) Google Scholar, 11Yoshida H. Jono H. Kai H. Li J.D. J. Biol. Chem. 2005; 280: 41111-41121Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13Regamey A. Hohl D. Liu J.W. Roger T. Kogerman P. Toftgard R. Huber M. J. Exp. Med. 2003; 198: 1959-1964Crossref PubMed Scopus (102) Google Scholar, 14Reiley W. Zhang M. Sun S-C. J. Biol. Chem. 2004; 279: 55161-55167Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). However, how CYLD regulates signal transduction under physiological conditions is still poorly understood. Recent studies using CYLD knock-out (CYLD-/-) mice suggest that the signaling function of CYLD is complex, which may involve distinct target proteins in different cell types and signaling pathways (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar, 15Massoumi R. Chmielarska K. Hennecke K. Pfeifer A. Fassler R. Cell. 2006; 125: 665-677Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). For example, the loss of CYLD in primary macrophages has no significant effect on the activation of NF-κB induced by tumor necrosis factor-α and TLR ligands (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). On the other hand, CYLD modulates the signaling function of a protein tyrosine kinase, Lck, in thymocytes (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar) and the nuclear translocation of an NF-κB coactivator protein, Bcl-3, in kerotinocytes (15Massoumi R. Chmielarska K. Hennecke K. Pfeifer A. Fassler R. Cell. 2006; 125: 665-677Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Clearly, the precise signaling role of CYLD, especially that in the regulation of NF-κB, warrants further studies. NF-κB represents a family of transcription factors that regulates diverse genes involved in the activation and survival of lymphocytes (16Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3408) Google Scholar). In mammals, the NF-κB family includes RelA, RelB, c-Rel, NF-κB1 (or p50), and NF-κB2 (or p52), which form different homo- and heterodimers. The NF-κB members are normally sequestered in the cytoplasm as inactive complexes by physical interaction with specific inhibitors, including IκBα and related proteins (17Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2139) Google Scholar). Activation of NF-κB involves phosphorylation-triggered degradation of IκBα and nuclear translocation of NF-κB complexes, particularly the p50/RelA and p50/c-Rel dimers. A multisubunit IKK complex responds to diverse cellular stimuli and mediates the phosphorylation of IκBα (18Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4155) Google Scholar). In addition to this canonical pathway of NF-κB activation, a noncanonical pathway exists to mediate activation of two specific NF-κB members, RelB and NF-κB2 (17Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2139) Google Scholar). Accumulating evidence suggests that the deregulated activation of NF-κBs can cause severe immunological disorders, such as lymphoid malignancies and autoimmunity (19Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (256) Google Scholar, 20Sun S.C. Yamaoka S. Oncogene. 2005; 24: 5952-5964Crossref PubMed Scopus (196) Google Scholar, 21Kalled S.L. Immunol. Rev. 2005; 204: 43-54Crossref PubMed Scopus (117) Google Scholar). As such, both the basal and the inducible activity of NF-κB are likely subject to negative mechanism of regulation, although the physiological negative regulators of NF-κB remain poorly defined. In the present study, we show that CYLD plays a critical role in preventing uncontrolled NF-κB activation in B cells. Consistently, CYLD-deficient B cells are hyperproliferative when stimulated in vitro and display elevated levels of antigen responses in vivo. The CYLD-/- mice develop B cell hyperplasia and lymphoid organ abnormalities, which can be further exacerbated when these animals are challenged with antigens. We further show that CYLD also regulates peripheral B cell development since the loss of CYLD results in abnormal production of marginal zone B cells. These findings establish CYLD as a key regulator of B cell activation and development and reveal a physiological function of CYLD in NF-κB regulation. Mice—Cyld knock-out mice were generated as described (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). Cyld+/- mice were intercrossed to generate Cyld-/- and Cyld+/+ littermates. Genotyping was performed by PCR using tail DNA and the following primers: Cyld forward primer 1, 5′-CCA GGC ACT TTG AAT TGC TGT C-3′; Cyld reverse primer 1, 5′-CGT TCT TCC CAG TAG GGT GAA G-3′; Cyld reverse primer 2, 5′-GCA TGC TCC AGA CTG CCT TGG-3′. When the three primers were used together, the PCR yielded a 209-bp product for Cyld+/+ mice, a 209- and a 255-bp product for Cyld+/- mice, and a 255-bp product for Cyld-/- mice. Unless specified, mice were housed in specific pathogen-free cages and monitored periodically for the lack of common pathogens. For studies that involved housing of mice under conventional conditions, age- and sex-matched CYLD-/- and wild-type mice were transferred from ventilated cages to conventional cages and housed for 6 weeks. Animal experiments were in accordance with protocols approved by the Pennsylvania State University College of Medicine Institutional Animal Care and Use Committee. Antibodies and Reagents—The anti-CYLD antibody was generated by injecting rabbits with a glutathione S-transferase fusion protein containing an N-terminal region of human CYLD (amino acid 136-301). Phospho-IκBα (Ser-32) antibody was from Cell Signaling. Antibodies for actin (C-2), IKKβ (H470), tubulin (Tu-02), p50 (C-19), c-Rel (sc-70), and RelB (C-19) were purchased from Santa Cruz Biotechnology, Inc. Fluorescence-labeled anti-mouse antibodies used in flow cytometry included activated protein C-anti-C19Rp (AA4.1), APC-anti-CD3 (145-2C11), PE.CY7-anti-CD19 (1D3), FITC-anti-CD21 (7G6), PE-anti-CD23 (B3B4), FITC-anti-CD80 (16-10A1), PE-anti-CD86 (GL1), FITC-anti-IgD (11-26c.2a), and PerCP-Cy5.5-anti-IgM (R6-60.2). Anti-C19Rp, anti-CD80, and anti-CD86 were purchased from eBioscience, and the rest of the conjugated antibodies were from BD Biosciences. Unconjugated anti-IgM and anti-CD40, used for B cell stimulation, were purchased from Jackson ImmunoResearch and BD Biosciences, respectively. Sheep red blood cells (SRBC) and human recombinant BAFF were purchased from Cocalico Biologicals, Inc. and BIOSOURCE, respectively. GST-IKKβ was cloned by inserting a cDNA fragment encoding amino acids 166-197 of human IKKβ into pGEX-4T vector (Amersham Biosciences). Recombinant protein was produced in Escherichia coli and purified using GST-Sepharose. Cycloheximide was obtained from Sigma, and all other antibodies and reagents have been described previously (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar, 22Waterfield M. Wei J. Reiley W. Zhang M.Y. Sun S-C. Mol. Cell. Biol. 2004; 24: 6040-6048Crossref PubMed Scopus (118) Google Scholar, 23Morrison M.D. Reiley W. Zhang M. SC S. J. Biol. Chem. 2005; 280: 10018-10024Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Flow Cytometry—Bone marrow cells were prepared as described previously (24Waterfield M. Zhang M. Norman L.P. Sun S.C. Mol. Cell. 2003; 11: 685-694Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Spleen and mesenteric lymph node (MLN) cell suspensions were prepared by gentle homogenization using a tissue homogenizer. Mononuclear cells were isolated by centrifugation over lymphocyte separation medium (Cellgro). Peritoneal cells were isolated by flushing the peritoneal cavity using 10 ml of PBS. Flow cytometry was performed as described previously (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). The data shown in Fig. 3D were collected using FACSCalibur, and all the other data were generated using FACSCanto. For analyses of in vitro cultured B cells, the cells were incubated for 48 h in Iscove's media either in the presence or in the absence of BAFF (100 ng/ml) and then subjected to flow cytometry. Cell Proliferation Assays—B cells were purified from splenocytes using anti-B220-conjugated magnetic beads (Miltenyl Biotec) and were stimulated in 4 replicate wells of 96-well plates (1 × 105 cells/well) with anti-IgM (10 μg/ml), anti-CD40 (2 μg/ml), or LPS (3 μg/ml). After the indicated times of stimulation, the cells were labeled for 5 h with [3H]thymidine for proliferation assays based on thymidine incorporation. For the carboxyl fluorescent succinimidyl ester (CFSE) cell proliferation assay, purified splenic B cells were washed once with PBS (prewarmed to 37 °C) and incubated with CFSE (1.25 μg/ml in PBS) for 10 min at 37 °C. After two washes with Iscove's medium, the cells were stimulated as described above followed by flow cytometry to measure the CFSE intensity. Mouse Immunization, Immunohistochemistry, and Antibody Analyses—Mice were injected intraperitoneally with 0.2 ml of SRBC (1 × 109/ml in PBS) and sacrificed 6 days later. Spleens were frozen in Tissue-Tec OCT compound (VWR International) using liquid nitrogen prechilled 2-methlbutane. The frozen tissues were stored at -70 °C until processed to produce 6-8-μm cryostat sections. The sections were stained with rat anti-mouse B220 (eBioScience) followed by biotinylated anti-rat immunoglobulin (Vector Laboratories) or with biotin-conjugated hamster anti-mouse CD3 (eBioScience), biotin-conjugated peanut agglutinin (Vector Laboratories). The immunostaining were then detected with peroxidase-conjugated streptavidin using diaminobenzidine as chromagen (VECTASTAIN Elite ABC kit, Vector Laboratories). For analyses of antibody responses, mice were injected intra-peritoneally with 0.2 ml of nitro-phenol-conjugated keyhole limpet hemocyanin (NP-KLH) or nitro-phenol-conjugated LPS (NP-LPS) (0.1 mg/ml in PBS). Sera were collected at the indicated times after immunization and subjected to ELISA to detect NP-specific antibodies using the SBA Clonotyping system (Southern Biotechnology, Inc.). IB and EMSA—Purified B cells were stimulated with anti-IgM (2.5 μg/ml) or LPS (2.5 μg/ml) for the indicated times. Total and subcellular extracts were prepared from the cells and subjected to immunoblotting (IB) and EMSA as described previously (25Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 176419Crossref Scopus (4013) Google Scholar, 26Ganchi P.A. Sun S-C. Greene W.C. Ballard D.W. Mol. Biol. Cell. 1992; 3: 1339-1352Crossref PubMed Scopus (204) Google Scholar). In the case of protein phosphorylation analyses, cells were lysed in a kinase cell lysis buffer supplemented with phosphatase inhibitors (27Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S-C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). For antibody supershift assays, the nuclear extracts were premixed with 0.5 μl of the indicated antibodies for 8 min at room temperature and then mixed with the 32P-radiolabeled κB oligonucleotide in EMSA buffer. In Vitro Kinase Assays—IKKβ was isolated by immunoprecipitation from untreated MLN B cells followed by analyzing its catalytic activity by in vitro kinase assays (27Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S-C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) using GST-IKKβ as substrate. RT-PCR—Total cellular RNA was isolated from purified MLN B cells using the TRI reagent (Molecular Research Center, Inc.). Semiquantitative RT-PCR was performed using the following primers to amplify murine CD23, Iκbα and Gapdh, CD23 forward, 5′-GTG AGG ACT GTG TGA TGA TGC-3′; CD23 reverse, 5′-GAG GAG AAA TCC AGA AGA GTG-3′; Iκbα forward, 5′-CTG TTT GTG AAA CTG AAG AGC TG-3′; Iκbα reverse, 5′-CTT CAC AAA AGC AAC ATA GTG GC-3′; Gapdh forward, 5′-CTC ATG ACC ACA GTC CAT GCC ATC-3′; Gapdh reverse, 5′-CTG CTT CAC CAC CTT CTT GAT GTC-3′. CYLD-/- Mice Display Lymphoid Organ Abnormalities and B Cell Hyperplasia—To investigate the role of CYLD in regulating immune system function, we began by analyzing the peripheral lymphoid organs of the CYLD-/- and wild-type mice. As early as 8 weeks of age, the CYLD-/- mice displayed striking enlargement of the MLNs, and this abnormality became even more profound at older ages (Fig. 1A). On the other hand, the CYLD-/- and wild-type mice did not show obvious size differences in other lymph nodes or the Peyer's patches, and only a small percentage of the CYLD-/- mice had slightly enlarged spleens (data not shown). Thus, a prominent lymphoid abnormality of the CYLD-/- mice is the enlargement of MLNs. To examine the effect of CYLD on lymphocyte homeostasis, we performed flow cytometry analyses to measure the frequency of B and T cells in MLNs. The CYLD-/- MLNs exhibited a profound increase in the percentage of B cells and a reduction in the percentage of T cells (Fig. 1B). The absolute number of MLN B cells was even more drastically increased in the CYLD-/- animals (Fig. 1C) due to the severe lymphadenopathy (Fig. 1A). We previously reported that the spleen of CYLD-/- mice contained more B cells and reduced numbers of T cells (5Reiley W.W. Zhang M. Jin W. Losiewicz M. Donohue K.B. Norbury C.C. Sun S.C. Nat. Immunol. 2006; 7: 411-417Crossref PubMed Scopus (187) Google Scholar). Consistently, flow cytometry analyses of multiple animals revealed significantly higher frequency and numbers of B cells and reduced frequency and numbers of T cells in the spleens of CYLD-/- mice (Fig. 1D and data not shown). In addition to the mainstream B cells (B2 cells), we also analyzed the frequency of B1 cells, which are predominantly located in the peritoneal cavity. The CYLD-/- mice only showed a slight increase in this population of B cells in the peritoneal cavity (Fig. 1E) and no difference in spleen and MLNs (data not shown). Taken together, these results suggest that the loss of CYLD causes hyperplasia of mainstream B cells and abnormalities of peripheral lymphoid organs, especially MLNs. CYLD Plays a Minor Role in Regulating B Cell Development in the Bone Marrow—Peripheral B cells are derived from immature B cells generated in the bone marrow. Because of the peripheral B cell hyperplasia in CYLD-/- mice, we examined whether the loss of CYLD resulted in elevated generation of immature B cells in the bone marrow. Flow cytometry analyses of bone marrow CD19+ cells (B cells) detected three major populations: the early stages of developing B cells (ProPre B cells, IgM-IgD-), the immature B cells (IgM+IgD-), and the recirculating mature B cells (IgM+IgD+). The CYLD-/- mice did not produce more immature B cells but rather had a moderate reduction in this population of B cells (Fig. 2, A and B). This result suggests that CYLD plays a minor and positive role in B cell development at the immature B stage. Thus, the peripheral B cell hyperplasia of CYLD-/- mice was not due to the overproduction of immature B cells within the bone marrow. CYLD Regulates Marginal Zone B Cell Development—In the spleen, immature B cells go through transitional stages and eventually become follicular mature B cells or marginal zone B cells (28Cancro M.P. Immunol. Rev. 2004; 197: 89-101Crossref PubMed Scopus (90) Google Scholar). To examine how the loss of CYLD affects peripheral B cell maturation, we analyzed the splenic B cell populations based on their defined surface markers (28Cancro M.P. Immunol. Rev. 2004; 197: 89-101Crossref PubMed Scopus (90) Google Scholar). Young CYLD-/- mice (8 week) did not display profound alterations in B cell maturation (Fig. 2, C and D), although they had a slight reduction in the CD21loCD23lo T1 cells (Fig. 2D). Additionally, we detected a small increase in the CD21hiCD23lo marginal zone B cell population in these mutant animals (Fig. 2D). Interestingly, the increase in marginal zone B cells became much more prominent in older CYLD-/- mice (14 weeks), as assessed based on the staining of both CD21/CD23 (Fig. 2E) and another marginal zone B cell marker, CD1d (Fig. 2F). These results suggest a role for CYLD in regulating the peripheral development of B cells to marginal zone population. Spontaneous Activation of B Cells in CYLD-/- Mice—We next examined whether the B cell hyperplasia in CYLD-/- mice was associated with abnormal B cell activation. This possibility was first indicated in our analyses of B cell maturation markers. Although the CYLD deficiency in young mice did not profoundly alter the frequency of transitional and mature B cell subpopulations in the spleen (Fig. 2D), the CYLD-/- splenic B cells displayed considerably higher intensity of CD23 and CD21 (Fig. 3A, Total, dotted lines). This abnormality occurred primarily in follicular B cells (Fig. 3A, FO) but not marginal zone B cells (Fig. 3A, MZ). Since follicular B cells contain both mature and transitional populations, we further identified the CD21/CD23 overexpressing cells based on the expression of AA4.1. Loss of CYLD caused CD21/CD23 up-regulation in both transitional (AA4.1 positive) and mature (AA4.1 negative) B cells (Fig. 3A, bottom panel, and data not shown). Further, this abnormality was also detected on B cells isolated from the MLNs (Fig. 3B). Parallel RT-PCR analyses showed that the CYLD-/- B cells expressed substantially higher levels of CD23 mRNA than the wild-type B cells, suggesting a role for CYLD in regulating CD23 gene expression (Fig. 3C). Since CD21 and CD23 have been implicated in B cell activation and humoral immune responses (29Poe J.C. Hasegawa M. Tedder T.F. Int. Rev. Immunol. 2001; 20: 739-762Crossref PubMed Scopus (83) Google Scholar, 30Stief A. Texido G. Sansig G. Eibel H. Le Gros G. van der Putten H. J. Immunol. 1994; 152: 3378-3390Crossref PubMed Google Scholar, 31Militi S. Chiapparino C. Testa U. Carminati P. De Santis R. Serlupi-Crescenzi O. Cytokine. 2005; 31: 314-323Crossref PubMed Scopus (2) Google Scholar, 32Rickert R.C. Curr. Opin. Immunol. 2005; 17: 237-243Crossref PubMed Scopus (91) Google Scholar), the findings described above, together with the B cell hyperplasia, suggest the possibility that the loss of CYLD may lead to abnormal B cell activation. To further confirm this possibility, we analyzed the expression of two other known B cell activation markers, CD80 and CD86, which function as costimulatory molecules modulating the activation of T and B cells (33Greenwald R.J. Freeman G.J. Sharpe A.H. Annu. Rev. Immunol. 2005; 23: 515-548Crossref PubMed Scopus (1998) Google Scholar, 34Podojil J.R. Sanders V.M. Trends Immunol. 2005; 26: 180-185Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). As expected, wild-type B cells expressed low levels of CD80 and CD86 (Fig. 3D, solid lines). In contrast, the CYLD-/- B cells expressed markedly higher levels of CD80 and CD86 (Fig. 3D, dotted lines), thus further suggesting the activation phenotype of these mutant B cells. The spontaneous activation of CYLD-/- B cells was also indicated by their larger size, as demonstrated by the forward scatter analysis in flow cytometry (Fig. 3E). Taken together, these results suggest a key role for CYLD in maintaining the naive phenotype of B cells and provide an explanation for the B cell hyperplasia in CYLD-/- mice. Hyperreponsiveness of CYLD-/- B Cells—As a more direct approach to determine the effect of CYLD deficiency on B cell activation, we analyzed the proliferative response of the CYLD-/- B cells to stimulation via different receptors. Thymidine incorporation assays revealed that the CYLD-/- splenic B cells had significantly higher proliferative ability than the wild-type B cells when stimulated with the BCR inducer anti-IgM (Fig. 4A). The CYLD-/- B cells were also hyperresponsive to LPS (Fig. 4A), a bacterial cell wall component stimulating B cells via TLR4 and the TLR-related molecule RP105 (35Vos Q. Lees A. Wu Z.Q. Snapper C.M. Mond J.J. Immunol. Rev. 2000; 176: 154-170Crossref PubMed Scopus (359) Google Scholar). On the other hand, the CYLD-/- and wild-type B cells only exhibited a moderate difference in their responses to an agonistic antibody to CD40 (Fig. 4A), a key costimulatory molecule that mediates B cell activation by helper T cells (36Bishop G.A. Hostager B.S. Cytokine Growth Factor Rev. 2003; 14: 297-309Crossref PubMed Scopus (156) Google Scholar). To further confirm the hyperproliferative phenotype of CYLD-/- B cells, we analyzed the division rate of CYLD-/- and control B cells using the CFSE labeling technique (Fig. 4B). Consistent with the thymidine incorporation results, the CYLD-/- B
Referência(s)