We have shown that male and female mice differ significantly with respect to their immune response genes in post-pubertal life. The innate immune response genes are highly up-regulated in post-pubertal male but not female mice. Post-pubertal male mice also produce higher levels of IL-1α and IL-1β in response to the TLR-2 ligand (Table ). The biological relevance of these findings can be seen in both infectious and autoimmune disease conditions. Although males are more susceptible than females to many parasitic infections, there are some parasites for which males are more resistant than females and differences in innate and adaptive arms of the immune system may explain this sex reversal. For example, the innate immune response plays a critical role in offering males protection against Toxoplasma gondii
]. Our data are consistent with the relative deficiency of innate immune response genes in female mice, as evidenced by their enhanced susceptibility to and higher mortality associated with certain parasitic infections (e.g., T. gondii
). Thus, the relative resistance of the males to T. gondii
infection is likely explained by their high levels of innate immunity-related proteins. Furthermore, it is also known that the 5-lipoxygenase pathway and leukotrienes are integral components of innate immune cells such as macrophages, mast cells and eosinophils [24
]. Recent experiments have clearly demonstrated that 5-lipoxygenase-deficient male mice on an MRL lpr
background show a marked decrease in survival, further supporting a protective role for innate immune response genes in autoimmune diseases [25
In contrast, adaptive immune response genes are highly up-regulated in post-pubertal female mice. These mice also produce significantly higher levels of cytokines and chemokine that influence antibody production than do post-pubertal males (Table ). These findings are particularly relevant to autoimmune diseases, in which the adaptive immune system attacks normal self tissue. We propose that enhanced susceptibility to autoimmune disease in post-pubertal life is the result of an altered ratio of adaptive and innate immune response genes. This hypothesis is in fact supported by the finding that genetic defects in innate immune response genes (complement C1q and serum amyloid P) in mice result in spontaneous autoimmune disease [26
]. It is known that females produce higher levels of Igs than do male mice in response to a variety of antigens, and these effects have been attributed to sex steroids [29
]. Our results confirm these findings and further indicate that even non-immunized female mice show significantly elevated levels of various Ig isotype genes, and that the levels are even more enhanced in post-pubertal life.
Fas and FasL genes showed spatial and temporal expression patterns similar to those of immunoglobulin genes. The preferential expression of Fas and FasL in post-pubertal females suggested a role for this pathway in generating sexual dimorphism in immunoglobulin gene expression. The observed post-pubertal sex differences in Ig levels in B6 mice were abolished in B6 lpr and B6 gld mice, indicating that the post-pubertal levels of specific Ig isotypes are regulated through Fas/FasL pathway.
Genetic defects in both Fas and FasL are known to cause severe lupus like autoimmune disease on the MRL/Mp genetic background. The gender differences in disease severity (mortality, pancreatitis and autoantibodies) in MRL/Mp mice are abolished when Fas (lpr
) mutation is transferred onto this background, suggesting that MRL lpr
mice are gender-neutral [32
]. It is important to note that in a previous study, transferring the C1q deficiency onto the MRL background did not abolish the gender differences [34
]. Thus, the defects in the Fas-FasL signaling pathway alone abolish the gender differences in lupus-like autoimmune disease in MRL mice. Further supporting this observation is the finding that lpr
mice show spontaneous polyclonal B cell activation and lymphadenopathy [35
]. The male lpr
mice showed significant increases in Ig levels, similar to those seen in females (Figure ). These results are interesting, especially when correlated with the disease-prone MRL lpr
mouse model of lupus, in which male mice die as early as female mice (50% mortality in both male and female mice by 5.5 months of age). This finding suggests that increased IgG levels in males lead to increases in immune complex-mediated disease, similar to those in female mice.
This hypothesis is further supported by another model of autoimmunity: MRL-Fas lprcg
mice have a phenotype similar to that of MRL lpr
mice because of a defect in Fas-mediated apoptotic signaling (a single amino acid mutation in the cytoplasmic death domain) [36
]. The reverse signaling pathway through FasL is functional because of the intact extracellular domain that interacts with FasL. In fact, the MRL-Faslprcg
mice exhibit sex differences in disease severity [37
]. These observations suggest that reverse signaling through FasL is involved in generating sex differences in IgG isotypes, and consequently in the frequency of severe disease in female mice.
It has been shown that FasL expression in ovaries is closely correlated with estrogen levels, which vary at different phases of the female estrus cycle. This result suggests that estrogen dynamically controls FasL expression on various cells and may enhance Ig levels only once during each cycle [38
]. To directly establish the role of estrogen in this reverse signaling pathway, we carried out in vitro
stimulation of CD8+
T cells and assessed Ig isotype levels. We have shown here that FasL expression on activated CD8+
T cells is influenced by estrogen and have further demonstrated that the culture supernatants from estrogen-activated CD8+
T cells produce growth factors that enhance in vitro
immunoglobulin levels. These data suggest that reverse signaling through FasL in CD8+
T cells leads to the production of growth factors that enhance the expression of Ig isotypes and that females are expected to have enhanced Ig switching because of their elevated post-pubertal estrogen levels. It is likely that some of the growth factors secreted by the activated CD8+
T cells also influence B cell growth, maturation and differentiation.
In addition to their effects on CD8+
T cells, estrogens affect the production of IFN-γ [39
], which is known to enhance IgG2a responses [41
]. These activated CD8+
T cells would be expected to secrete growth factors and cytokines, which in turn would affect B cell growth and differentiation, leading to the enhanced immunoglobulin isotype expression in post-pubertal female mice. We therefore assessed the effect of IFN-γ on IgG2a levels in B6 IFN-γ knockout mice. These data suggested that increases in post-pubertal Ig isotype levels may be due to differential expression of cytokines (e.g., IFN-γ) produced by CD8+
T cells activated through Fas-FasL reverse signaling. Recently, it has been shown that IgG2a-chromatin immune complexes, together with TLR 9 are very efficient in activating autoreactive B cells [42
]. Our findings suggest that the increased IgG2a induced by the estrogen-Fas/FasL- IFN-γ pathway in post-pubertal female mice is one of the susceptibility factors enhancing autoimmunity in females. We speculate that differential expression of cytokines such as TGF-β may be involved in generating IgG2b differences in post-pubertal life.
Ig genes are transiently increased at the time of puberty in male mice (Figure ). The exact mechanism by which this increase occurs is not known. It is likely that the transiently elevated levels of estrogen at the time of puberty in males [43
] may enhance FasL expression on CD8+
T cells. Reverse signaling through FasL may also be responsible for this transient increase in Ig gene expression in male pubertal mice. The molecular basis for the large increase in innate immune response genes in males as compared to the adaptive immune response genes in females is not clear. It is possible that male hormones may regulate some of the innate immune response genes directly.
While the pathway analysis presented here has focused on the estrogen-Fas/FasL- IFN-γ pathway, our data also have implications with regard to male-related immunity. It has been observed that males have a higher mortality due to infectious diseases than do females [45
], in part because of testosterone-induced immunosuppression in post-pubertal males [46
]. The exact molecular mechanisms by which testosterone suppresses the acquired immune system are not yet understood. The data presented here suggest that males have an adequate innate immune response (first line of defense) but a relatively diminished adaptive immune response, which is critical for the elimination of the microorganisms. Thus, the documented higher mortality rates in males worldwide may be due in part to this relatively deficient adaptive immune response.