Abstract
B-1 cells are innate-like lymphocytes that generate natural, polyreactive antibodies with important functions in tissue homeostasis and immune defense. While B-1-cell frequencies in secondary lymphoid tissues are low, relative high frequencies are found within peritoneal and pleural cavities of mice, including both CD5+ B-1a and CD5− B-1b cells. They represent reservoirs of B-1 cells that can be activated for migration to lymphoid tissues to secrete antibodies and/or cytokines. Here, we outline efficient methods for the extraction and magnetic isolation of B-1a cells from the peritoneal and pleural cavities and the separation and phenotypic characterization of B-1a and B1-b cells by flow cytometry.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Baumgarth N (2011) The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol 11:34–46
Baumgarth N, Herman OC, Jager GC, Brown L, Herzenberg LA (1999) Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system. Proc Natl Acad Sci U S A 96:2250–2255
Hooijkaas H, Benner R, Pleasants JR, Wostmann BS (1984) Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered “antigen-free” diet. Eur J Immunol 14:1127–1130
Bos NA, Kimura H, Meeuwsen CG, De Visser H, Hazenberg MP, Wostmann BS, Pleasants JR, Benner R, Marcus DM (1989) Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet. Eur J Immunol 19:2335–2339
Haury M, Sundblad A, Grandien A, Barreau C, Coutinho A, Nobrega A (1997) The repertoire of serum IgM in normal mice is largely independent of external antigenic contact. Eur J Immunol 27:1557–1563
Tornberg UC, Holmberg D (1995) B-1a, B-1b and B-2 B cells display unique VHDJH repertoires formed at different stages of ontogeny and under different selection pressures. EMBO J 14:1680–1689
Kantor AB, Merrill CE, Herzenberg LA, Hillson JL (1997) An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells. J Immunol 158: 1175–1186
Notkins AL (2004) Polyreactivity of antibody molecules. Trends Immunol 25:174–179
Stewart J (1992) Immunoglobulins did not arise in evolution to fight infection. Immunol Today 13:396–399, discussion 399–400
Bouvet JP, Dighiero G (1998) From natural polyreactive autoantibodies to a la carte monoreactive antibodies to infectious agents: is it a small world after all? Infect Immun 66:1–4
Baumgarth N, Tung JW, Herzenberg LA (2005) Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion. Springer Semin Immunopathol 26:347–362
Chen Y, Park YB, Patel E, Silverman GJ (2009) IgM antibodies to apoptosis-associated determinants recruit C1q and enhance dendritic cell phagocytosis of apoptotic cells. J Immunol 182:6031–6043
Chen GY, Tang J, Zheng P, Liu Y (2009) CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science 323:1722–1725
Zhang M, Alicot EM, Chiu I, Li J, Verna N, Vorup-Jensen T, Kessler B, Shimaoka M, Chan R, Friend D, Mahmood U, Weissleder R, Moore FD, Carroll MC (2006) Identification of the target self-antigens in reperfusion injury. J Exp Med 203:141–152
Kulik L, Fleming SD, Moratz C, Reuter JW, Novikov A, Chen K, Andrews KA, Markaryan A, Quigg RJ, Silverman GJ, Tsokos GC, Holers VM (2009) Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury. J Immunol 182:5363–5373
Choi YS, Baumgarth N (2008) Dual role for B-1a cells in immunity to influenza virus infection. J Exp Med 205:3053–3064
Martin F, Oliver AM, Kearney JF (2001) Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617–629
Alugupalli KR, Leong JM, Woodland RT, Muramatsu M, Honjo T, Gerstein RM (2004) B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity 21:379–390
Baumgarth N, Herman OC, Jager GC, Brown LE, Herzenberg LA, Chen J (2000) B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J Exp Med 192:271–280
Jayasekera JP, Moseman EA, Carroll MC (2007) Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity. J Virol 81:3487–3494
Briles DE, Nahm M, Schroer K, Davie J, Baker P, Kearney J, Barletta R (1981) Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae. J Exp Med 153:694–705
Boes M, Prodeus AP, Schmidt T, Carroll MC, Chen J (1998) A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J Exp Med 188: 2381–2386
Ochsenbein AF, Fehr T, Lutz C, Suter M, Brombacher F, Hengartner H, Zinkernagel RM (1999) Control of early viral and bacterial distribution and disease by natural antibodies. Science 286:2156–2159
Zhou ZH, Zhang Y, Hu YF, Wahl LM, Cisar JO, Notkins AL (2007) The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies. Cell Host Microbe 1:51–61
O’Garra A, Chang R, Go N, Hastings R, Haughton G, Howard M (1992) Ly-1 B (B-1) cells are the main source of B cell-derived interleukin 10. Eur J Immunol 22:711–717
Rauch PJ, Chudnovskiy A, Robbins CS, Weber GF, Etzrodt M, Hilgendorf I, Tiglao E, Figueiredo JL, Iwamoto Y, Theurl I, Gorbatov R, Waring MT, Chicoine AT, Mouded M, Pittet MJ, Nahrendorf M, Weissleder R, Swirski FK (2012) Innate response activator B cells protect against microbial sepsis. Science 335:597–601
Hayakawa K, Hardy RR, Herzenberg LA (1985) Progenitors for Ly-1 B cells are distinct from progenitors for other B cells. J Exp Med 161:1554–1568
Kawahara T, Ohdan H, Zhao G, Yang YG, Sykes M (2003) Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells. J Immunol 171:5406–5414
Tumang JR, Frances R, Yeo SG, Rothstein TL (2005) Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors. J Immunol 174:3173–3177
Yang Y, Tung JW, Ghosn EE, Herzenberg LA (2007) Division and differentiation of natural antibody-producing cells in mouse spleen. Proc Natl Acad Sci U S A 104:4542–4546
McIntyre TM, Holmes KL, Steinberg AD, Kastner DL (1991) CD5+ peritoneal B cells express high levels of membrane, but not secretory, C mu mRNA. J Immunol 146:3639–3645
Choi YS, Dieter JA, Rothaeusler K, Luo Z, Baumgarth N (2012) B-1 cells in the bone marrow are a significant source of natural IgM. Eur J Immunol 42:120–129
Ohdan H, Swenson KG, Kruger Gray HS, Yang YG, Xu Y, Thall AD, Sykes M (2000) Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice. J Immunol 165:5518–5529
Ha SA, Tsuji M, Suzuki K, Meek B, Yasuda N, Kaisho T, Fagarasan S (2006) Regulation of B1 cell migration by signals through Toll-like receptors. J Exp Med 203:2541–2550
Cole LE, Yang Y, Elkins KL, Fernandez ET, Qureshi N, Shlomchik MJ, Herzenberg LA, Vogel SN (2009) Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge. Proc Natl Acad Sci U S A 106:4343–4348
Nisitani S, Tsubata T, Murakami M, Honjo T (1995) Administration of interleukin-5 or -10 activates peritoneal B-1 cells and induces autoimmune hemolytic anemia in anti-erythrocyte autoantibody-transgenic mice. Eur J Immunol 25:3047–3052
Murakami M, Tsubata T, Shinkura R, Nisitani S, Okamoto M, Yoshioka H, Usui T, Miyawaki S, Honjo T (1994) Oral administration of lipopolysaccharides activates B-1 cells in the peritoneal cavity and lamina propria of the gut and induces autoimmune symptoms in an autoantibody transgenic mouse. J Exp Med 180:111–121
Haas KM, Poe JC, Steeber DA, Tedder TF (2005) B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 23:7–18
Gil-Cruz C, Bobat S, Marshall JL, Kingsley RA, Ross EA, Henderson IR, Leyton DL, Coughlan RE, Khan M, Jensen KT, Buckley CD, Dougan G, MacLennan IC, Lopez-Macias C, Cunningham AF (2009) The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response. Proc Natl Acad Sci U S A 106:9803–9808
Kroese FG, Butcher EC, Stall AM, Lalor PA, Adams S, Herzenberg LA (1989) Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int Immunol 1:75–84
Rosado MM, Aranburu A, Capolunghi F, Giorda E, Cascioli S, Cenci F, Petrini S, Miller E, Leanderson T, Bottazzo GF, Natali PG, Carsetti R (2009) From the fetal liver to spleen and gut: the highway to natural antibody. Mucosal Immunol 2:351–361
Baumgarth N, Roederer M (2000) A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 243: 77–97
Baumgarth N (2004) B-cell immunophenotyping. Methods Cell Biol 75:643–662
Wells SM, Kantor AB, Stall AM (1994) CD43 (S7) expression identifies peripheral B cell subsets. J Immunol 153:5503–5515
Herzenberg LA, Stall AM, Braun J, Weaver D, Baltimore D, Grosschedl R (1987) Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice. Nature 329:71–73
Stall AM, Adams S, Herzenberg LA, Kantor AB (1992) Characteristics and development of the murine B-1b (Ly-1 B sister) cell population. Ann N Y Acad Sci 651:33–43
Kim KJ, Kanellopoulos-Langevin C, Merwin RM, Sachs DH, Asofsky R (1979) Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol 122:549–554
Pattengale PK, Frith CH (1983) Immunomorphologic classification of spontaneous lymphoid cell neoplasms occurring in female BALB/c mice. J Natl Cancer Inst 70:169–179
Acknowledgments
Data for the B-1-cell FACS sorts were kindly provided by Elizabeth E. Waffarn (UC Davis). This work was supported in part by NIH AI051354 and AI085568.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Yenson, V., Baumgarth, N. (2014). Purification and Immune Phenotyping of B-1 Cells from Body Cavities of Mice. In: Vitale, G., Mion, F. (eds) Regulatory B Cells. Methods in Molecular Biology, vol 1190. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1161-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1161-5_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1160-8
Online ISBN: 978-1-4939-1161-5
eBook Packages: Springer Protocols