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Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations

Abstract

B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5×106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1×106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.

Abbreviations

BCR:

B cell antigen receptor

DAG:

diacylglycerol

DMEM:

Dulbecco’s modified Eagle’s media

IP3 :

inositol triphosphate

FCM:

flow cytometry

MACS:

magnetic bead cell sorting

MFI:

mean fluorescence intensity

PKC:

protein kinase C

RBC:

red blood cell

References

  1. Allman D, Lindsley RC, DeMuth W, Rudd K, Shinton SA, Hardy RR. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J Immunol 2001;167(12):6834–40.

    PubMed  CAS  Google Scholar 

  2. Hoek KL, Antony P, Lowe J, Shinners N, Sarmah B, Wente SR, et al. Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement. J Immunol 2006;177(8):5405–13.

    PubMed  CAS  Google Scholar 

  3. Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, et al. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med 1999;190(1):75–89.

    Article  PubMed  CAS  Google Scholar 

  4. Srivastava B, Quinn WJ, 3rd, Hazard K, Erikson J, Allman D. Characterization of marginal zone B cell precursors. J Exp Med 2005;202(9):1225–34.

    Article  PubMed  CAS  Google Scholar 

  5. Su TT, Guo B, Wei B, Braun J, Rawlings DJ. Signaling in transitional type 2 B cells is critical for peripheral B-cell development. Immunol Rev 2004;197:161–78.

    Article  PubMed  CAS  Google Scholar 

  6. Antony P, Petro JB, Carlesso G, Shinners NP, Lowe J, Khan WN. B cell receptor directs the activation of NFAT and NFkappaB via distinct molecular mechanisms. Exp Cell Res 2003;291(1):11–24.

    Article  PubMed  CAS  Google Scholar 

  7. Coggeshall KM, Cambier JC. B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis. J Immunol 1984;133(6): 3382–6.

    PubMed  CAS  Google Scholar 

  8. Hikida M, Johmura S, Hashimoto A, Takezaki M, Kurosaki T. Coupling between B cell receptor and phospholipase Cgamma2 is essential for mature B cell development. J Exp Med 2003;198(4):581–9.

    Article  PubMed  CAS  Google Scholar 

  9. Wen R, Chen Y, Xue L, Schuman J, Yang S, Morris SW, et al. Phospholipase Cgamma2 provides survival signals via Bcl2 and A1 in different subpopulations of B cells. J Biol Chem 2003;278(44):43654–62.

    Article  PubMed  CAS  Google Scholar 

  10. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226(1):497–509.

    PubMed  CAS  Google Scholar 

  11. Antony P, Farooqui AA, Horrocks LA, Freysz L. Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA-N-1 neuroblastoma cells: a key role for diacylglycerol. FEBS Lett 2001;509(1):115–8.

    Article  PubMed  CAS  Google Scholar 

  12. Antony P, Petro JB, Carlesso G, Shinners NP, Lowe J, Khan WN. B-cell antigen receptor activates transcription factors NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB) via a mechanism that involves diacylglycerol. Biochem Soc Trans 2004;32(Pt 1):113–5.

    Article  PubMed  CAS  Google Scholar 

  13. York JD, Odom AR, Murphy R, Ives EB, Wente SR. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999;285(5424):96–100.

    Article  PubMed  CAS  Google Scholar 

  14. Chien MM, Cambier JC. Divalent cation regulation of phosphoinositide metabolism. Naturally occurring B lymphoblasts contain a Mg2(+)-regulated phosphatidylinositol-specific phospholipase C. J Biol Chem 1990;265(16):9201–7.

    PubMed  CAS  Google Scholar 

  15. Bijsterbosch MK, Meade CJ, Turner GA, Klaus GG. B lymphocyte receptors and polyphosphoinositide degradation. Cell 1985;41(3):999–1006.

    Article  PubMed  CAS  Google Scholar 

  16. Petro JB, Gerstein RM, Lowe J, Carter RS, Shinners N, Khan WN. Transitional type 1 and 2 B lymphocyte subsets are differentially responsive to antigen receptor signaling. J Biol Chem 2002;277(50):48009–19.

    Article  PubMed  CAS  Google Scholar 

  17. Su TT, Rawlings DJ. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J Immunol 2002;168(5):2101–10.

    PubMed  CAS  Google Scholar 

  18. Saito K, Tolias KF, Saci A, Koon HB, Humphries LA, Scharenberg A, et al. BTK regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. Immunity 2003;19(5):669–78.

    Article  PubMed  CAS  Google Scholar 

  19. Toker A. The synthesis and cellular roles of phosphatidylinositol 4,5-bisphosphate. Curr Opin Cell Biol 1998;10(2):254–61.

    Article  PubMed  CAS  Google Scholar 

  20. Preiss J, Loomis CR, Bishop WR, Stein R, Niedel JE, Bell RM. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem 1986;261(19):8597–8600.

    PubMed  CAS  Google Scholar 

  21. Kroesen BJ, Pettus B, Luberto C, Busman M, Sietsma H, de Leij L, et al. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J Biol Chem 2001;276(17): 13606–14.

    PubMed  CAS  Google Scholar 

  22. Fukuda M, Kojima T, Kabayama H, Mikoshiba K. Mutation of the pleckstrin homology domain of Bruton’s tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity. J Biol Chem 1996;271(48):30303–6.

    Article  PubMed  CAS  Google Scholar 

  23. Khan WN, Alt FW, Gerstein RM, Malynn BA, Larsson I, Rathbun G, et al. Defective B cell development and function in Btk-deficient mice. Immunity 1995;3(3):283–99.

    Article  PubMed  CAS  Google Scholar 

  24. Marechal Y, Pesesse X, Jia Y, Pouillon V, Perez-Morga D, Daniel J, et al. Inositol 1,3,4,5-tetrakisphosphate controls proapoptotic Bim gene expression and survival in B cells. Proc Natl Acad Sci U S A 2007;104(35):13978–13983.

    Article  PubMed  CAS  Google Scholar 

  25. Shinners NP, Carlesso G, Castro I, Hoek KL, Corn RA, Woodland RL, et al. Bruton’s Tyrosine Kinase Mediates NF-{kappa}B Activation and B Cell Survival by B Cell-Activating Factor Receptor of the TNF-R Family. J Immunol 2007;179(6):3872–80.

    PubMed  CAS  Google Scholar 

  26. Ives EB, Nichols J, Wente SR, York JD. Biochemical and functional characterization of inositol 1,3,4,5, 6-pentakisphosphate 2-kinases. J Biol Chem 2000;275(47): 36575–83.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Wasif N. Khan PhD..

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These authors contributed equally.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Antony, P., Hoek, K., Sarmah, B. et al. Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations. Biol. Proced. Online 9, 73–83 (2007). https://doi.org/10.1251/bpo135

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  • DOI: https://doi.org/10.1251/bpo135

Indexing terms

  • receptors, antigen, b-cell
  • immunomagnetic separation