Modulation of human B lymphocyte differentiation by therapeutic immunoglobulins: from protein to mRNA levels

Nathalie Dussault, Nellie Dumont, Sonia Néron
DOI: 10.4236/oji.2011.13008                

Abstract

Several groups are investigating the mechanisms of action of therapeutic immunoglobulins (IVIg) in order to improve their use. In vitro models such as CD40-CD154 interaction are necessary to study the physiological response of human B lymphocytes to IVIg. Human B lymphocytes treated with IVIg triggers a rapid phospho-rylation (<1 h) of extracellular-regulated kinases 1 and 2 (ERK1/2), which subsequently results in increased differentiation and decreased pro-liferation. However, the modulation of human lymphocyte physiology by IVIg is a gradual and cumulative process and requires long-term experimentation. Differentiation of human B lymphocytes into Ig-secreting cells can be evaluated both at the transcription and translation levels. The secretion of immunoglobulins can be assessed using ELISA or ELISPOTS whereas expression of immunoglobulin genes can be measured using semi-quantitative or quantitative PCR methods. We hereby report a comparison of these methods to explain how contradictory observations towards IVIg effects could result from their use. Our results indicate that ELISA and ELISPOTS will provide consistent observations by opposition to real-time PCR quantification. Besides, the reliability of each of these me-thods remained dependent on the stimulation period as well as the preparation of cellular extracts or cell samples following IVIg-treatment.

Keywords

Human B Lymphocytes; CD40-CD154; IVIg; Differentiation; Immunomodulation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Durandy, A., Kaveri, S. V., Kuijpers, T. W., Basta, M., Miescher, S., Ravetch, J. V. and Rieben, R. (2009) Intravenous immunoglobulins—Understanding properties and mechanisms. Clinical & Experimental Immunology, 158, 2-13. doi:10.1111/j.1365-2249.2009.04022.x
[2]	Wasserman, R.L., Irani, A.M., Tracy, J., Tsoukas, C., Stark, D., Levy, R., Chen, J., Sorrells, S., Roberts, R. and Gupta, S. (2010) Pharmacokinetics and safety of subcutaneous immune globulin (human), 10% caprylate/ chromatography purified in patients with primary immunodeficiency disease. Clinical & Experimental Immunology, 161, 518-526. doi:10.1111/j.1365-2249.2010.04195.x
[3]	Kreuz, W., Erdos, M., Rossi, P., Bernatowska, E., Espanol, T. and Marodi, L. (2010) A multi-centre study of efficacy and safety of Intratect((R)), a novel intravenous immunoglobulin preparation. Clinical & Experimental Immunology, 101, 512-517. doi:10.1111/j.1365-2249.2010.04187.x
[4]	Kuitwaard, K., de Gelder, J., Tio-Gillen, A. P., Hop, W.C., van Gelder, T., van Toorenenbergen, A.W., van Doorn, P.A. and Jacobs, B.C. (2009) Pharmacokinetics of intravenous immunoglobulin and outcome in Guillain-Barre syndrome. Annals of Neurology, 66, 597-603. doi:10.1002/ana.21737
[5]	Dussault, N., Ducas, E., Racine, C., Jacques, A., Pare, I., Cote S. and Neron, S. (2008) Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2. International Immunology, 20, 1369-1379. doi:10.1093/intimm/dxn090
[6]	de Grandmont, M.J., Racine, C., Roy, A., Lemieux, R. and Néron, S. (2003) Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG. Blood, 101, 3065-3073. doi:10.1182/blood-2002-06-1684
[7]	Néron, S., Boire, G., Dussault, N., Racine, C., de Brum-Fernandes, A. J., Cote S. and Jacques, A. (2009) CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro. Archivum Immunologiae et Therapiae Experimentalis, 57, 447-458. doi:10.1007/s00005-009-0048-3
[8]	Néron, S., Nadeau, P.J., Darveau, A. and Leblanc, J.F. (2011) Tuning of CD40-CD154 Interactions in Human B-Lymphocyte Activation: A Broad Array of In Vitro Models for a Complex in vivo Situation. Archivum Immunologiae et Therapiae Experimentalis, 59, 25-40.
[9]	Néron, S., Racine, C., Roy, A. and Guérin, M. (2005) Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction. Immunology, 116, 454-463. doi:10.1007/s00005-010-0108-8
[10]	Stewart, R., Wei, W.B., Challa, A., Armitage, R.J., Arrand J.R., Rowe, M., Young, L.S., Eliopoulos, A. and Gordon, J. (2007) CD154 tone sets the signaling pathways and transcriptome generated in model CD40-Pluricompetent L3055 Burkitt’s lymphoma cells. Journal of Immunology, 179, 2705-2712.
[11]	Luft, T., Maraskovsky, E., Schnurr, M., Knebel, K., Kirsch, M., Gorner, M., Skoda, R., Ho, A.D., Nawroth, P. and Bierhaus, A. (2004) Tuning the volume of the immune response: Strength and persistence of stimulation determine migration and cytokine secretion of Dendritic Cells. Blood, 104, 1066-1074. doi:10.1182/blood-2003-12-4146
[12]	Mathur, R.K., Awasthi, A., Wadhone, P., Ramanamurthy, B. and Saha, B. (2004) Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nature Medicine, 10, 540-544. doi:10.1038/nm1045
[13]	Ducas, E., Dussault, N., Roy, A., Dumont, N. and Neron, S. (2009) Estimation of the number of CD154 molecules in membrane extracts used as a source of CD40 stimulation of human B lymphocytes. Journal of Immunological Methods, 344, 133-137. doi:10.1016/j.jim.2009.03.009
[14]	Fecteau, J.F. and Néron, S. (2003) CD40 stimulation of human peripheral B lymphocytes: Distinct response from na?ve and memory cells. Journal of Immunology, 171, 4621-4629.
[15]	Fecteau, J.F., Roy, A. and Neron, S. (2009) Peripheral blood CD27(+) IgG(+) B cells rapidly proliferate and differentiate into immunoglobulin-secreting cells after exposure to low CD154 interaction. Immunology, 128, e353-e365. doi:10.1111/j.1365-2567.2008.02976.x
[16]	Néron, S., Thibault, L., Dussault, N., Cote, G., Ducas, E., Pineault, N. and Roy, A. (2007) Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: A new source of viable human blood cells. Transfusion, 47, 1042-1049. doi:10.1111/j.1537-2995.2007.01233.x
[17]	Néron, S., Pelletier, A., Chevrier, M. C., Monier, G., Lemieux, R. and Darveau, A. (1996) Induction of LFA-1 independent human B cell proliferation and differentiation by binding of CD40 with its ligand. Immunological Investigations, 25, 79-89. doi:10.3109/08820139609059292
[18]	Roy, A., Krzykwa, E., Lemieux, R. and Néron, S. (2001) Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures. Journal of Hematotherapy & Stem Cell Research, 10, 873-880. doi:10.1089/152581601317210962
[19]	Barbas, I.C.F., Burton, D.R. and Scott, J.K., Silverman, G.J. (2001) Phage display: A laboratory manual. Cold Spring Harbor Laboratory Press, New-York.
[20]	Heidt, S., Roelen, D.L., Eijsink, C., Eikmans, M., Claas, F.H.J. and Mulder, A. (2009) Intravenous immunoglobulin preparations have no direct effect on B cell proliferation and immunoglobulin production. Clinical & Experimental Immunology, 158, 99-105. doi:10.1111/j.1365-2249.2009.03996.x
[21]	de Kok, J.B., Roelofs, R.W., Giesendorf, B.A., Pennings, J.L., Waas, E.T., Feuth, T., Swinkels, D.W. and Span, P.N. (2005) Normalization of gene expression measurements in tumor tissues: Comparison of 13 endogenous control genes. Laboratory Investigation, 85, 154-159.
[22]	Giricz, O., Lauer-Fields, J.L. and Fields, G.B. (2008) The normalization of gene expression data in melanoma: investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR. Analytical Biochemistry, 380, 137-139. doi:10.1016/j.ab.2008.05.024
[23]	Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.
[24]	Proulx, D.P., Aubin, E., Lemieux, R. and Bazin, R. (2009) Spontaneous internalization of IVIg in activated B cells. Immunology Letters, 124, 18-26. doi:10.1016/j.imlet.2009.03.012
[25]	Fecteau, J.F. and Néron, S. (2004) Characterization of na?ve and memory B cell differentiation toward Plasma cells following low CD40 stimulation. Immunology Collection of Free Papers. Medimond Srl, Bologna, 303-307.
[26]	Bayry, J., Fournier, E.M., Maddur, M.S., Vani, J., Wootla, B., Siberil, S., Dimitrov, J.D., Lacroix-Desmazes, S., Berdah, M., Crabol, Y., Oksenhendler, E., Levy, Y., Mouthon, L., Sautes-Fridman, C., Hermine, O. and Kaveri, S.V. (2011) Intravenous immunoglobulin induces proliferation and immunoglobulin synthesis from B cells of patients with common variable immunodeficiency: A mechanism underlying the beneficial effect of IVIg in primary immunodeficiencies. Journal of Autoimmunity, 36, 9-15. doi:10.1016/j.
[27]	Padet, L., St-Amour, I., Aubin, E., Proulx, D. P., Bazin, R. and Lemieux, R. (2009) Dose-dependent inhibition of BrdU detection in the cell proliferation ELISA by culture medium proteins. Journal of Immunoassay and Immunochemistry, 30, 348-357. doi:10.1080/15321810903084863
[28]	van Laar, J.M., Melchers, M., Teng, Y.K.O., van der Zouwen, B., Mohammadi, R., Fischer, R., Margolis, L., Fitzgerald, W., Grivel, J.C., Breedveld, F.C., Lipsky, P.E. and Grammer, A.C. (2007) Sustained secretion of immunoglobulin by long-lived human tonsil plasma cells. The American Journal of Pathology, 171, 917-927. doi:10.2353/ajpath.2007.070005
[29]	Hommes, O.R., Haas, J., Soelberg-Sorenson, P. and Friedrichs, M. (2009) IVIG trials in MS. Is albumin a placebo? Journal of Neurology, 256, 268-270. doi:10.1007/s00415-009-0893-3
[30]	Ho-Pun-Cheung, A., Cellier, D. and Lopez-Crapez, E. (2008) Considerations for normalisation of RT-qPCR in oncology. Ann Biol Clin (Paris), 66, 121-129.
[31]	Mane, V.P., Heuer, M.A., Hillyer, P., Navarro, M.B. and Rabin, R.L. (2008) Systematic method for determining an ideal housekeeping gene for real-time PCR analysis. J Biomol Tech, 19, 342-347.