A low level of TNF expression is associated with a significant proportion of human SLE and with the classic (NZB × NZW)F1
murine model (17
). Although a protective role for TNF administration in (NZB × NZW)F1
mice has been established, it had not been known whether this effect segregates at the level of the individual TNFR. We have now demonstrated that the effect of TNF in closely related SLE-prone NZM 2328 mice does not segregate uniquely to either of the two TNFR. That is, disease development in TNFR1 or TNFR2 singly-deficient NZM 2328 mice was similar to that in WT mice (intact for both TNFR1 and TNFR2), whereas disease was unequivocally accelerated in DKO mice.
Unlike previous studies that focused on the biological consequences of TNFR deletion by evaluating only single receptor-deficient mice (30
), our work emphasizes that to gain insight into the full spectrum of TNF/TNFR system, one must study model systems in which deletion of both receptors has occurred, as well as evaluate those systems in which the deletion of only a single individual receptor has occurred.
Our data point to a certain degree of functional redundancy for the two TNFR in NZM 2328 mice. In the complete absence of one, signaling through the other can sufficiently compensate and permit persistence of the WT clinical and pathological phenotypes. This may entail a convergence of the signaling pathways for T cell death as it occurs for cell survival. Indeed, TNFR1 induces TNFR-associated factor 2 (TRAF2)-receptor interacting protein-IKK-dependent, NF-κ
B-dependent antiapoptotic pathways, and also two distinct apoptotic pathways: TNFR1-TRADD-FADD-caspase-8 and TRAF2-AIP1-ASK1-JNK/p38 kinase cascade (40
). Thus, recruitment of TRAF2 by TNFR1 for the formation of the signaling complex is essential for activation of several distinct signaling cascades. Although much less is known about the proteins recruited to TNFR2 and downstream signaling, TNFR2, like TNFR1, can also recruit TRAF2 and use the two cellular inhibitors of apoptotic proteins (cIAP1 and cIAP2) (41
). Perhaps a similar convergence for the cell death signaling pathways occurs, if not through TRAF2, then by some other means. Consequently, when both receptors are deleted, a complete loss of regulatory functions of the TNF/TNFR pathway takes place. Investigation into the relevant biochemical pathways is needed to delineate the signaling cascades triggered by the individual TNFRs.
Anti-TNF agents have successfully been used to treat patients with a variety of chronic inflammatory diseases (42
), and such agents have recently been suggested for use in SLE. This advice must be taken with caution given our results in the NZM 2328 model, which shows that abrogating the proinflammatory effects of TNF by deleting the two receptors leads to a heightened, distinct inflammatory pathway, probably resulting from the loss of immunoregulatory functions of TNF/TNFR system.
The recent anecdotal reports of the benefit following anti-TNF treatment in a few SLE patients (26
) might be explained by an incomplete blockade of TNF. That is, there is still free TNF available to provide certain protective effects. A nonmutually exclusive possibility is that those patients treated to date are Caucasians possessing HLA class II genotype of DR3 and/or DR4, who have been shown to be capable of producing higher levels of TNF in response to activation (38
). Consequently, reducing TNF levels in such patients might mitigate the inflammatory response while preserving sufficient TNF levels to provide protective regulation. In contrast, DR2/DQw1-positive SLE patients (mostly non-Caucasians), who have lower TNF inducibility (38
), might be more prone to the harmful consequences of TNF blockade. Our findings, therefore, suggest much circumspection before using TNF antagonists in SLE. Furthermore, the accumulating data on the involvement of the TNF/TNFR pathway in the normal development of the lymphoid microarchitecture suggest that TNF antagonists might be contraindicated during pregnancy due to potential defects in embryonic development of the lymphoid system in the fetus.
In terms of a mechanistic explanation for our findings at the cellular level, NZM 2328 mice deficient in one or both TNFRs underwent important phenotypic changes. Follicular B cell organization and GC formation in the spleen were altered in these mice. These microstructural changes complement previous studies that documented similar changes in TNF- or TNFR1-deficient nonautoimmune-prone mice (43
) and are consistent with the established role of the TNF/TNFR system in the development of normal lymphoid architecture (45
). Of note, the impaired ability of DKO mice to form organized follicles in the spleen () did not compromise their ability to produce high levels of autoantibodies (). Moreover, attenuated spontaneous GC formation in the various TNFR-deficient mice (especially in p55−/−
mice) did not affect their ability to generate autoantibody levels identical with those in WT mice (). Most importantly, these impairments in the organization of the B cell compartment in the DKO mice do not provide an explanation for their accelerated clinical disease, because single-receptor-deficient mice share the same B cell phenotype, but do not develop accelerated disease.
Among the individual B or T cell populations analyzed, the closest association with development of clinical disease was with CD4+
(activated memory) T cells, suggesting that these cells might have a causal role. Our results agree well with previous studies that also documented an accumulation of activated memory T cells in other SLE-prone mice (46
). Of note, such cells are refractory to anti-CD3-induced apoptosis in vitro and express high levels of apoptosis-inhibiting genes such as Bcl-xL
), perhaps explaining why they steadily accumulate in SLE-prone mice.
To eludicate how CD4+
T cells might drive accelerated disease, we focused on their cytokine profile. There is considerable evidence that a skewing toward a Th2-type response can contribute to murine SLE (48
T cells from (NZB × NZW)F1
mice secrete less IL-2 as the mice age and as the disease becomes more clinically evident (49
). Furthermore, IL-4 mRNA expression increases with age and disease development in (NZB × NZW)F1
mice, whereas IL-12 mRNA expression decreases (50
). Additionally, mitogen-stimulated T cells from these mice produce high levels of Th2-type cytokines (IL-4, IL-5, and IL-10), whereas Th1 cytokine (IL-2, IFN-γ
) production is low (51
). Moreover, the administration of anti-IL-10 to (NZB × NZW)F1
mice delays disease onset (19
), and treatment with IL-4 antagonists decreases anti-DNA Ab levels and associated renal disease (52
). Finally, transgenic expression of IL-4 under a MHC class I promoter induces SLE-like autoimmunity in a nonautoimmune background (53
). In contrast, Th1 responses may contribute as well. For example, the major isotype eluted from glomerular lesions of SLE mice is IgG2a (36
), which depends on IFN-γ
for its synthesis. Indeed, neutralization of IFN-γ
by a mAb (54
) or a soluble IFN-γ
) prevents glomerulonephritis in (NZB × NZW)F1
mice, whereas administration of exogenous IFN-γ
accelerates renal disease. In addition, renal disease and anti-DNA Ab levels are attenuated in IFN-γ
receptor-deficient (NZB × NZW)F1
Unexpectedly, we found that CD4+
T cells in DKO NZM 2328 mice expressed neither a Th1 nor a Th2 profile. Rather, they expressed a Th17 profile, with significantly increased expression of IL-17A, IL-17F, IL-23A, IL-23R, Socs3, Jak1, Jak2, Mmp13, ICOS, and RORγt
, along with decreased expression of IL-4, IL-25, and GATA3. Th17 cells have recently emerged as an independent subset that is highly pathogenic in the development of organ-specific autoimmunity (57
). The association of Th17 cells with systemic autoimmunity (SLE) suggests that the pathogenic role of these cells may be broader than initially appreciated.
Our finding of increased expression of Jak1 and Jak2 in CD4+
T cells is consistent with other reports on Th17 gene expression profiles (59
). Although Th1 and Th17 are distinct subsets, cells producing both IL-17 and IFN-γ
have been identified (61
), so the up-regulated expression of IFN-γ
in the present study is not inconsistent with a Th17 response. In addition, our finding of increased expression of Mmp13 is also consistent with a Th17 profile. The induction of matrix metalloproteinases that are a component of chronic inflammatory responses has been suggested as a function of Th17 cells (64
). Indeed, deletion of IL-17R in mice results in diminished synovial expression of Mmp 3, 9, and 13, and prevents cartilage destruction in streptococcal cell wall-induced arthritis (65
We do not claim that activated memory T cells universally have a Th17 profile. Rather, our data suggest that within the CD4+
T cell population in DKO NZM 2328 mice (and 8- to 9-mo-old overtly sick WT NZM 2328 mice), there is a subset of Th17 cells. Indeed, CD4+
T cells from age- and sex-matched, nonautoimmune C57BL/6 mice do not manifest a Th17 profile. We cannot exclude the possibility that certain CD4+
T cells are non-Th17 cells even in DKO NZM 2328 mice because, at present, we have no way to delineate subsets within this activated memory phenotype other than by the cell surface markers used in flow cytometry. Accelerated disease in these mice may be promoted not only by such Th17 cells, but by other pathways as well. The existence of a Th17-mediated pathogenic pathway does not preclude the concurrent existence of other pathogenic pathways. Nevertheless, our results provide novel evidence for the association of IL-17/Th17 pathway with an animal model of SLE, the importance of which is underscored by the very recent demonstration of IL-17 production in human SLE (66