Estrogen & the Immune System: Of mice and women (and nitric oxide)

Being female can be both an advantage and a disadvantage, immunologically speaking. It has long been known that both innate and adaptive immune responses of females are somewhat more robust than males. On the other hand, females are more susceptible to autoimmune disorders such as lupus, rheumatoid arthritis, and multiple sclerosis. These gender differences are largely attributed to estrogen levels.1,2 Estrogen-related regulation of nitric oxide (NO), and the enzymes that produce it, increasingly appear to play a key role.1

NO synthases (NOS) catalyze the conversion of L-arginine to citrulline and NO. Macrophages, dendritic cells (DCs), and NK cells produce both endothelial and inducible types, eNOS and iNOS, with iNOS generating the highest NO levels.3 NO production is modulated by many mechanisms, including control of arginases, iNOS induction, co-factors (FAD, FMN, heme, and calmodulin), calcium, dimerization, feedback regulation, and protein stability.3 The actions of NO are also difficult to determine, since NO is a labile and highly diffusible gas that takes many forms. However, among other reported actions, NO has concentration-dependent anti-microbial, anti-inflammatory, and anti-apoptotic activities, and modulates cytokine and chemokine production in specific cell types and settings.1,3,5

Recent data explain one way that estrogen stimulates NO production in mouse spleen.4 Estrogen treatment in vivo allows profound NO production by isolated splenocytes, provided three conditions are met. First, T cells need stimulation, for example by the T cell mitogen ConA or by CD3 antibodies. Second, costimulation by an antigen-presenting cell is required, since CTLA-4 Ig blocks both the B7/CD28 interaction and NO production. Third, T cell production of IFN-gamma is required, since lack of NO production by IFN-gamma-/- splenocytes is restored by exogenous IFN-gamma. Splenocytes from control mice also produce IFN-gamma, iNOS, and NO when stimulated, but estrogen-treated mice require considerably less stimulation. This may be a significant reason that the female immune system appears to be “primed.”1

Figure 1
Figure 1. RANKL binding (shown in blue) promotes macrophage osteoclast differentiation. The pathway from estrogen through IFN-gamma, iNOS, and NO (shown in red) generates RANKL-blocking osteoprotegerin (OPG) and down-regulates osteoclast formation.

Estrogen maintains the balance between bone deposition and resorption, an activity that also involves NO (Figure 1).4,5 RANKL/TRANCE (the receptor activator of NFkB ligand) and M-CSF promote differentiation of bone-resorbing osteoclasts (OC), while osteoprotegerin (OPG) acts as a dummy receptor for RANKL, down-regulating OC formation and promoting bone-replenishing osteoblasts (OB). Both bone-resorptive and retentive activities of IFN-gamma have been reported, and strong stimulation of T cells has been proposed to tip the balance against resorption.5 NO produced by estrogen-stimulated bone marrow cells decreases the OC/OB ratio primarily by activating the transcription factor RUNX2/CBFA1 to induce OPG production.6,7 Differentiation of the RAW264.7 mouse macrophage cell line to OC can be inhibited by co-culture with CD3e and CD28 antibody-activated T cells, an effect that is blocked by neutralizing IFN-gamma.8 Interestingly, IFN-gamma, or IFN-beta induced by RANKL, promote iNOS production and increases NO in OC-differentiating RAW cells, apparently as a self-limiting mechanism.9 Enhancement of IFN-gamma or other cytokines produced by costimulated T cells could well be involved in the efficacy of estrogen replacement in preventing osteoporosis. The full role of IFN-gamma-stimulated NO as a mediator of estrogen’s effects on the immune system is not yet clear. Other estrogen effects, such as modulation of TNF-alpha, TGF-beta, and IL-1 activity in the bone marrow, and effects on B cells, DCs, and CD4+CD25+ regulatory T cells involved in autoimmunity, are areas of active research.5,10


  1. Verthelyi, D. (2006) Endocrinol. 147:659.
  2. Carlsten, H. (2005) Immunol. Rev. 208:194.
  3. Bogdan, C. (2001) Nat. Immunol. 2:907.
  4. Karpuzoglu, E. et al. (2006) Endocrinol. 147:662.
  5. Weitzmann, M. N. & R. Pacifici (2005) Immunol. Rev. 208:154.
  6. O’Shaughnessy, M. T. et al. (2000) Biochem. Biophys. Res. Comm. 277:604.
  7. Wang, F.-S. et al. (2004) Endocrinology 145:2148.
  8. Wyzga, N. et al. (2004) Bone 35:614.
  9. Zheng, H. et al. (2006) J. Biol. Chem. 281:15809.
  10. Wood, K. J. et al. (2006) Trends Immunol. 27:183.