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Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17β-estradiol (E 2 ) has been shown to promote the differentiation ofDCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERa- or ERβ-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERa, but not ERβ, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERa activation and provide evidence for a cell-autonomous function of ERa signaling in DC differentiation. ERa-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERa-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4 + T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERa-deficient DCs were defective in their ability to secrete such cytokines in response to CD40-CD40L interactions. Taken together, these results provide the first genetic evidence that ERa is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.

Cross-reactive idiotypes (CRId) borne on autoanti-glomerular basement membrane antibodies of Brown-Norway (BN) rats with mercury-induced glomerulonephritis have been described in the preceding study (Guery, J.-C. et al., Eur. J. Immunol. 1990. 20:93). BN rats treated with sodium aurothiopropanol sulfonate or D-penicillamine, as well as (LEW X BN)F1 hybrids transferred with BN rat spleen cells, developed quite similar autoimmune abnormalities. In the present study, it is shown that immunoglobulins bearing such "public" idiotypes are also produced and deposited in the kidney in these three models. The CRId here described may, therefore, be considered as a marker of sets of recurrently expressed V region genes during the course of these autoimmune disorders. Anti-self class II T cells are present in the three models of toxin-induced autoimmunity and anti-allo class II T cells are responsible for the chronic graft-vs.-host reaction. The same B cell clones are probably triggered during these processes as a consequence of a polyclonal B cell activation mediated by anti-class II T cells.

Calcium influx into lymphocytes is essential for activation, differentiation, and effector functions. While several channel- and receptor-types contribute to calcium influx, voltage-gated calcium channels (VGCC) mediate a well-characterized calcium influx pathway that is most exclusively identified in excitable cells. The role of L-type VGCCs, which belong to high-voltage activated calcium channels and are defined as dihydropyridine (DHP) receptors in excitable cells, is well documented. Interestingly, while lymphocytes do not range in the excitable cell category, the modulatory role of DHP agonists and antagonists and the identification of L-type VGCC-related molecules in B and T lymphocytes, mainly in Th2 cells, suggest these proteins are involved in the calcium response of these cells. Because the identity and the regulation of DHP receptors/channels in lymphocytes is far from being solved, we will discuss the challenging issues of demonstrating a role of L-type VGCCs in nonexcitable cells and the arguments supporting their role in lymphocytes. We will comment on the limitation of the use of DHP agonists and antagonists to ascertain a specific involvement of L-type VGCCs in lymphocyte calcium signaling. Finally, we will provide new clues on the interest of a potential use of DHP antagonists in Th2-cell-mediated pathology.

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the β chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-γ and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

Short-term exposure to 17β-estradiol (E2) in vitro has been reported to decrease the production of proinflammatory cytokines by LPS-activated macrophages through estrogen receptor α (ERα)-dependent activation of the PI3K pathway. In the present study, we confirm that in vitro exposure of mouse peritoneal macrophages to E2 enhanced Akt phosphorylation and slightly decreased LPS-induced cytokine production. In striking contrast, we show that chronic administration of E2 to ovariectomized mice markedly increases the expression of IL-1β, IL-6, IL-12p40, and inducible NO synthase by resident peritoneal macrophages in response to LPS ex vivo. These results clearly indicate that short-term E2 treatment in vitro does not predict the long-term effect of estrogens in vivo on peritoneal macrophage functions. We show that this in vivo proinflammatory effect of E2 was mediated through ERα. Although the expression of components of the LPS-recognition complex remained unchanged, we provided evidences for alterations of the TLR4 signaling pathway in macrophages from E2-treated mice. Indeed, E2 treatment resulted in the inhibition of PI3K activity and Akt phosphorylation in LPS-activated macrophages, whereas NF-κB p65 transcriptional activity was concomitantly increased. Incubation of macrophages with the PI3K inhibitor wortmanin enhanced proinflammatory cytokine gene expression in response to TLR4 activation, and abolishes the difference between cells from placebo- or E2-treated mice, demonstrating the pivotal role of the PI3K/Akt pathway. We conclude that the macrophage activation status is enhanced in vivo by E2 through ERα and, at least in part, by the down-modulation of the PI3K/Akt pathway, thereby alleviating this negative regulator of TLR4-signaling.

Although much progress has been made in understanding the role of NK cells in bone marrow transplantation, little is known about their function in CD4 T cell-mediated allograft rejection. We have previously shown that in the absence of CD8 T lymphocyte priming, the in vivo default development pathway of alloreactive CD4 T cells was strongly biased toward Th2 phenotype acquisition. In this study, we investigate the impact of NK cells on the activation and differentiation of alloreactive CD4 T cells in various donor/recipient combinations. Our data demonstrate that defective inhibition of host NK cells by donor APCs including dendritic cells (DCs) results in diminished allospecific Th cell responses associated with the development of effector Th cells producing IFN-γ rather than type 2 cytokines. Turning host NK cells off was sufficient to restore strong alloreactive CD4 T cell priming and Th2 cell development. Similar results were obtained by analyzing the effect of NK cell activation on CD4 T cell responses to skin allografts. However, despite the dramatic effect of NK cells on alloreactive Th1/Th2 cell development, the kinetics of skin graft rejection were not affected. Thus, Th2 differentiation is a major pathway of alloreactive CD4 T cell development during solid organ transplant rejection, as long as host NK and CD8 T cells are not activated. We propose the hypothesis that MHC class I-driven interactions between donor DCs and host NK cells or CD8 T cells might result in DC-carried signals controlling the dynamics of alloreactive CD4 T cell priming and polarization.

The pathogenesis of gold-induced autoimmunity and membranous glomerulopathy is not well understood. HgCl2 and D-penicillamine, other chemicals known to trigger membranous glomerulopathy in humans, induce autoimmune manifestations in Brown-Norway (BN) rats but not in Lewis (LEW) rats. These chemicals trigger T-cell clones which are specific for self class II molecules from the major histocompatibility complex and are probably responsible for the polyclonal B-cell activation observed. The aim of this work was to test the effects of aurothiopropanolsulphonate (ATPS) in BN and LEW rats. In BN rats, ATPS induced a polyclonal B-cell activation marked by lymphoproliferation, hyperimmunoglobulinaemia affecting mainly IgE, and by the production of numerous autoantibodies. A glomerulonephritis occurred, initially due to anti-glomerular basement membrane antibody deposition, and later to the formation of granular deposits, occasionally resulting in a typical membranous glomerulopathy. Self class-II-specific T-cells were found that might be responsible for the polyclonal B-cell activation. Lewis rats were free of glomerulopathy but, like BN rats, exhibited an interstitial nephritis and some degree of polyclonal B-cell activation. These findings demonstrate that, depending on the strain, ATPS triggers different B-cell clones inducing different degrees of autoimmunity.

Background Although estrogen administration to hysterectomized menopausal women did not prevent the occurence of myocardial infarction in a randomized controlled trial (WHI 2004), epidemiological studies suggest and experimental results clearly demonstrate a major atheroprotective action of estrogens. The goal of the present study was to identify the cellular target(s) accounting for the estradiol (E2) beneficial action on fatty streak development. Methods and Results We first confirmed the key role of estrogen receptor α (ERα) in atheroprotective effect of E2 as this action was completely abolished in mice deficient both in Low Density Lipoprotein receptor (LDLr) and in ERα. Comparison of LDLr-/- mice transplanted with either ERα+/+ or ERα-/- bone marrow showed that functional ERα in the hematopoietic lineage is not required for E2 atheroprotection. We then showed that ERα floxed mice (ERαflox/flox) bred with the Tie2-Cre mice on the LDLr-/- background had a complete inactivation of ERα both in bone marrow and in endothelial cells. Remarkably, in this mouse model, the E2 atheroprotective action was completely abolished. Conclusions Altogether, this is the first in vivo demonstration that endothelial ERα represents a key target of the atheroprotective effect of E2, whereas the hematopoietic ERα is dispensable for the protective action. Selective estrogen receptor modulators that mimic this endothelial action of E2 should now be considered in hormonal treatment as well as in atheroprotection.