Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells

Nature Immunology volume 15, pages 275–282 (2014)Cite this article

Subjects

A Corrigendum to this article was published on 16 January 2015

Abstract

While a number of extrinsic factors are known to promote the survival of plasma cells (PCs), the signaling intermediates involved remain poorly characterized. Here we identified inducible nitric oxide synthase (iNOS) as an intermediate that supported the survival of PCs. PCs deficient in iNOS (Nos2−/− PCs) showed enhanced death in vitro, after transfer into congenic adoptive hosts, and in chimeras made with wild-type and Nos2−/− bone marrow. The iNOS-mediated protection involved activation of protein kinase G and modulation of endoplasmic reticulum stress components. Activation of caspases was also diminished. We found that iNOS was required for PCs to respond to some prosurvival mediators associated with bone marrow stromal cells and that at least one mediator, interleukin 6, fed directly into this pathway by inducing iNOS.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Deficiency in iNOS enhances the loss of PCs.
Figure 2: Effect of iNOS inhibitors and an NO donor on loss of PCs.
Figure 3: Response of PCs to survival signals.
Figure 4: The prosurvival effect of IL-6 is related to the induction of iNOS and not to expression of the receptor for IL-6 (CD126).
Figure 5: Involvement of PKG downstream of iNOS in promoting PC survival.
Figure 6: Effect of iNOS deficiency on ER-stress components and caspase activation.

Similar content being viewed by others

References

  1. Vieira, P. & Rajewsky, K. The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18, 313–316 (1988).

    Article  CAS  PubMed  Google Scholar 

  2. van Anken, E. et al. Sequential waves of functionally related proteins are expressed when B cells prepare for antibody secretion. Immunity 18, 243–253 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Gass, J.N., Jiang, H.Y., Wek, R.C. & Brewer, J.W. The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells. Mol. Immunol. 45, 1035–1043 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Ho, F., Lortan, J.E., MacLennan, I.C. & Khan, M. Distinct short-lived and long-lived antibody-producing cell populations. Eur. J. Immunol. 16, 1297–1301 (1986).

    Article  CAS  PubMed  Google Scholar 

  5. Manz, R.A., Thiel, A. & Radbruch, A. Lifetime of plasma cells in the bone marrow. Nature 388, 133–134 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Slifka, M.K. & Ahmed, R. Long-lived plasma cells: a mechanism for maintaining persistent antibody production. Curr. Opin. Immunol. 10, 252–258 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Hetz, C. et al. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1α. Science 312, 572–576 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Minges Wols, H.A., Underhill, G.H., Kansas, G.S. & Witte, P.L. The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity. J. Immunol. 169, 4213–4221 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Cassese, G. et al. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J. Immunol. 171, 1684–1690 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. O'Connor, B.P. et al. BCMA is essential for the survival of long-lived bone marrow plasma cells. J. Exp. Med. 199, 91–98 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Belnoue, E. et al. APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells. Blood 111, 2755–2764 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Chevrier, S. et al. CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche. Proc. Natl. Acad. Sci. USA 106, 3895–3900 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rozanski, C.H. et al. Sustained antibody responses depend on CD28 function in bone marrow-resident plasma cells. J. Exp. Med. 208, 1435–1446 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xiang, Z. et al. FcgammaRIIb controls bone marrow plasma cell persistence and apoptosis. Nat. Immunol. 8, 419–429 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Rodriguez Gomez, M. et al. Basophils support the survival of plasma cells in mice. J. Immunol. 185, 7180–7185 (2010).

    Article  PubMed  Google Scholar 

  16. Chu, V.T. et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat. Immunol. 12, 151–159 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Jang, E. et al. Foxp3+ regulatory T cells control humoral autoimmunity by suppressing the development of long-lived plasma cells. J. Immunol. 186, 1546–1553 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Salgo, M.G., Squadrito, G.L. & Pryor, W.A. Peroxynitrite causes apoptosis in rat thymocytes. Biochem. Biophys. Res. Commun. 215, 1111–1118 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Loweth, A.C., Williams, G.T., Scarpello, J.H. & Morgan, N.G. Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15. FEBS Lett. 400, 285–288 (1997).

    Article  CAS  PubMed  Google Scholar 

  20. Choi, B.M., Pae, H.O., Jang, S.I., Kim, Y.M. & Chung, H.T. Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. J. Biochem. Mol. Biol. 35, 116–126 (2002).

    CAS  PubMed  Google Scholar 

  21. Genaro, A.M., Hortelano, S., Alvarez, A., Martinez, C. & Bosca, L. Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J. Clin. Invest. 95, 1884–1890 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu, L. & Stamler, J.S.N.O. an inhibitor of cell death. Cell Death Differ. 6, 937–942 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Kim, Y.M. et al. Nitric oxide prevents tumor necrosis factor α-induced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8. Hepatology 32, 770–778 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Kitiphongspattana, K. et al. Protective role for nitric oxide during the endoplasmic reticulum stress response in pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 292, E1543–E1554 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Guo, F., Lin, E.A., Liu, P., Lin, J. & Liu, C. XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response. Cell. Signal. 22, 1818–1828 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Yu, X., Kennedy, R.H. & Liu, S.J. JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J. Biol. Chem. 278, 16304–16309 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Kolb, J.P. Mechanisms involved in the pro- and anti-apoptotic role of NO in human leukemia. Leukemia 14, 1685–1694 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Xu, W., Liu, L., Charles, I.G. & Moncada, S. Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. Nat. Cell Biol. 6, 1129–1134 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Matsuyama, S., Llopis, J., Deveraux, Q.L., Tsien, R.Y. & Reed, J.C. Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat. Cell Biol. 2, 318–325 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Li, J., Billiar, T.R., Talanian, R.V. & Kim, Y.M. Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem. Biophys. Res. Commun. 240, 419–424 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Li, J., Yang, S. & Billiar, T.R. Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation. J. Biol. Chem. 275, 13026–13034 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Kim, Y.M. et al. Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling. J. Neurosci. 19, 6740–6747 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Auner, H.W., Beham-Schmid, C., Dillon, N. & Sabbattini, P. The life span of short-lived plasma cells is partly determined by a block on activation of apoptotic caspases acting in combination with endoplasmic reticulum stress. Blood 116, 3445–3455 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Earnshaw, W.C., Martins, L.M. & Kaufmann, S.H. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Hotamisligil, G.S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140, 900–917 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shaffer, A.L. et al. XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity 21, 81–93 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Rutkowski, D.T. et al. Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS Biol. 4, e374 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Gunn, K.E. & Brewer, J.W. Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity. J. Immunol. 177, 3791–3798 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Skalet, A.H. et al. Rapid B cell receptor-induced unfolded protein response in nonsecretory B cells correlates with pro- versus antiapoptotic cell fate. J. Biol. Chem. 280, 39762–39771 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Ron, E. et al. Bypass of glycan-dependent glycoprotein delivery to ERAD by up-regulated EDEM1. Mol. Biol. Cell 22, 3945–3954 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gotoh, T. & Mori, M. Nitric oxide and endoplasmic reticulum stress. Arterioscler. Thromb. Vasc. Biol. 26, 1439–1446 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Hsieh, Y.H. et al. Differential endoplasmic reticulum stress signaling pathways mediated by iNOS. Biochem. Biophys. Res. Commun. 359, 643–648 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Zong, W.X. et al. Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J. Cell Biol. 162, 59–69 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Pelletier, N. et al. The endoplasmic reticulum is a key component of the plasma cell death pathway. J. Immunol. 176, 1340–1347 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Chatterjee, P., Tiwari, R.K., Rath, S., Bal, V. & George, A. Modulation of antigen presentation and B cell receptor signaling in B cells of beige mice. J. Immunol. 188, 2695–2702 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Mukhopadhyay, S., George, A., Bal, V., Ravindran, B. & Rath, S. Bruton's tyrosine kinase deficiency in macrophages inhibits nitric oxide generation leading to enhancement of IL-12 induction. J. Immunol. 163, 1786–1792 (1999).

    CAS  PubMed  Google Scholar 

  47. Chang, S., Chen, W. & Yang, J. Another formula for calculating the gene change rate in real-time RT-PCR. Mol. Biol. Rep. 36, 2165–2168 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank R. Kumar for assistance with flow cytometry, and I. Singh for help with breeding and maintenance of mouse strains. Supported by the Department of Biotechnology of the Government of India (BT/PR-14592/BRB/10/858/2010 to S.R.; BT/PR10954/BRB/10/625/2008 and BT/PR12849/MED/15/35/2009 to A.G.; and support for the National Institute of Immunology) and the Department of Science and Technology of the Government of India (SR/SO/HS-0005/2011 to V.B.).

Author information

Author notes

  1. Gautam N Shenoy

    Present address: Present address: Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, New York, USA.,

  2. Ankur S Saini and Gautam N Shenoy: These authors contributed equally to this work.

  3. Satyajit Rath, Vineeta Bal and Anna George: These authors jointly directed this work.

Authors and Affiliations

  1. National Institute of Immunology, New Delhi, India

    Ankur S Saini, Gautam N Shenoy, Satyajit Rath, Vineeta Bal & Anna George

Authors
  1. Ankur S Saini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gautam N Shenoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satyajit Rath
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vineeta Bal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anna George
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.S.S. and G.N.S. did experiments and helped in their design and analysis; A.S.S. contributed to manuscript preparation; and S.R., V.B. and A.G. designed and planned the experiments, analyzed and interpreted data and wrote the manuscript.

Corresponding author

Correspondence to Anna George.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Effect of iNOS deficiency on B cell differentiation.

(a) Frequency of plasma cells 96 h after stimulation of B cells with anti-IgM. Representative of 3 experiments (b) Upregulation of activation markers by wild-type (black) and Nos2−/− (red) B cells stimulated with 10 μg/ml LPS. Dotted lines: unstimulated cells. Shaded histogram: unstained cells. (c) Proliferation of B cells in response to LPS and anti-IgM stimulation for 48 h. Representative of 4 experiments. (d) Identification of plasma cells in bone marrow (BM, representative of 3 plots) and in spleens of mice 96 h after intraperitoneal injection of 50 μg LPS (representative of 15 plots), for sorting. WT: wild-type, KO: Nos2−/−

Source data

Supplementary Figure 2 iNOS deficiency does not affect B cell proliferation.

(a) CFSE dilution profiles of live-gated B cells that were stimulated for 48, 72 or 96 h with 10 μg/ml LPS (representative of 3 independent experiments). (b) Number of cells in each division cycle (mean +/- SE of 3 independent experiments). WT: wild-type, KO: Nos2−/−

Source data

Supplementary Figure 3 iNOS deficiency does not affect B cell differentiation.

Modulation of transcript abundance of genes known to be involved in terminal differentiation of B cells (a) and kinetics of plasma cell generation (b) scored over time following stimulation of IgD+ cells with 10 μg/ml LPS. Fold expression in (a) is calculated relative to wild-type at 0 h. Numbers in (b) represent frequency of plasma cells. Data are mean +/- SE of 3 experiments. WT: wild-type, KO: Nos2−/−

Source data

Supplementary Figure 4 Estimation of plasma cell viability by flow cytometry.

Sort-purified plasma cells from wild-type (WT) and Nos2−/− (KO) mice were cultured in vitro and death estimated over time by Sytox positivity. Data are representative of 2 experiments. Numbers above gates indicate percentage of dead cells.

Supplementary Figure 5 iNOS deficiency does not affect ROS levels or mitochondrial potential during B cell differentiation.

(a) Effect of addition of N-acetyl cysteine on plasma cell frequencies in vitro. Numbers in plots represent frequency. Plots are representative of 2 experiments (b) Estimation of intracellular ROS levels in wild-type (black line) and Nos2−/− (red line) plasma cells. Shaded histogram: Unstained cells. Data are representative of 3 experiments (c) Estimation of mitochondrial mass and potential in wild-type (WT) and Nos2−/− (KO) plasma cells. Data are representative of 5 experiments.

Supplementary Figure 6 Activated caspases in live-gated B220loCD138+ cells following stimulation of wild-type (WT) and Nos2−/− (KO) splenocytes with 10 μg/ml LPS for 96 h.

Plots are representative of 4 experiments. Numbers above gates indicate the proportion of cells expressing activated caspases.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 10487 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saini, A., Shenoy, G., Rath, S. et al. Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol 15, 275–282 (2014). https://doi.org/10.1038/ni.2806

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2806

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing