In this study, we explore the abnormalities of cytokine production in lupus, and examine their role in the development and progression of lupus nephritis. Previous attempts to identify cytokine abnormalities in lupus and other diseases have generally required stimulation of immune cells, which may not reflect the intrinsic abnormality of a cytokine. We therefore used an in vivo cytokine capture assay, which does not require any exogenous stimulation, and allows detection of physiological and quite small amounts of cytokines. Using this assay, we identified two strains of lupus-prone mice, one (NZM.2410) that overexpresses IL-4 and the other (MRL-lpr) that overexpresses IFN-γ (). The use of these mice should help to define the roles of these cytokines in the development and progression of lupus.
IL-4 can rescue B cells from apoptosis and enhance their survival (6
). The increased expression of IL-4, therefore, may result in the expansion and activation of autoreactive B cells, and thus may contribute to the development or aggravation of autoantibody-mediated disease. Indeed, IL-4 transgenic C3H mice develop an autoimmune-type disorder that resembles lupus (17
). Thus, IL-4 may exacerbate B cell-mediated autoimmunity. IL-4 can also inhibit T cell activation in some in vivo systems (35
). Consistent with this role, NZM.2410 mice that overexpress IL-4 have less renal inflammation compared with MRL-lpr
mice that overexpress IFN-γ
(). NZM.2410 mice, however, have more glomerulosclerosis than MRL-lpr
mice (). Strikingly, the in vivo depletion of IL-4 or STAT6
gene deletion markedly inhibits chronic renal lesions and glomerulosclerosis (, and ). These observations suggest a correlation between increased IL-4 levels and the development of glomerulosclerosis, and elevated IFN-γ
levels with inflammatory cell infiltration. Thus, two different subsets or stages of disease may characterize lupus nephritis; one subset or stage of nephritis may be exemplified by MRL-lpr
-type nephritis with marked renal inflammation, and the other subset or stage may be exemplified by the NZM.2410-type nephritis, which presents with glomerulosclerosis and less marked renal inflammation ().
Type 1 cytokines have been implicated in the development of anti-DNA Ab and lupus in MRL-lpr
, and BXSB mouse models of lupus (4
). Treatment of young, prenephritic BWF1
mice with an anti-IL-12 mAb decreases IgG anti-dsDNA Ab levels (36
). Our results in STAT4-deficient NZM.2410 mice substantiate the importance of type 1 cytokines in autoantibody production (). The STAT4-deficient mice, however, experienced accelerated nephritis in our study. Studies are underway to examine the following possibilities to explain this finding: 1) increased type 2 cytokine production in STAT4-deficient mice aggravates glomerulosclerosis in lupus-prone mice; 2) autoantibodies are not critical for the development of lupus nephritis; and 3) autoantibodies other than anti-dsDNA Ab may cause nephritis in NZM.2410 mice.
Our results show that STAT6 deficiency or anti-IL-4 mAb treatment markedly inhibits the progression of lupus nephritis in NZM.2410 mice (, and ), even though IgG anti-dsDNA Ab levels are unchanged or slightly increased ( and ). STAT4-deficient NZM.2410 mice, by contrast, exhibited increased renal disease, despite a decrease in IgG anti-dsDNA Ab levels. These results appear to contradict a direct cause-effect relationship between the presence of autoantibodies and nephritis in NZM.2410 mice. One possibility is that some genes control the production and renal deposition of autoantibodies, while others contribute to the development and progression of renal disease (37
). Consistent with this idea, mouse chromosome 11, which harbors the genes for IL-4, IL-5, and IL-13, also contains two loci, D11 Mit23
and D11 Mit164
, which are linked to glomerulonephritis, but not to IgG anti-dsDNA Ab production in (NZM.2410 × C57BL/6)F2
). Another possibility is that renal deposition of autoantibodies in SLE may represent an early event, which later triggers renal cells to secrete extra cytokines and growth factors, such as IL-4 and TGF-β
, which may perpetuate glomerulosclerosis and chronic renal fibrosis. Finally, it is also possible that observed effects of the STAT4 or STAT6 knockout reflect the removal of lupus susceptibility or resistance genes during backcross of the mutated locus from the Sv129 onto the NZM.2410 genetic background. STAT4
genes, however, are on mouse chromosome 1 (25.9 cM) and mouse chromosome 10 (70.0 cM), respectively; thus, both genes are clearly outside any known lupus susceptibility or resistance region in NZM2410 mice. Therefore, it is unlikely that the phenotypes presented by these mice are due to any interference of the mutated locus with potential lupus susceptibility genes.
Our results raise the possibility that IL-4 and STAT6 may be directly involved in the development of lupus nephritis, particularly glomerulosclerosis and chronic renal fibrosis. IL-4 is known to promote fibroblast proliferation, collagen gene expression, and collagen synthesis in mouse models of pulmonary fibrosis (39
). Another type 2 cytokine, IL-13, which is also significantly increased in NZM.2410 mice as compared with BALB/c mice (our unpublished data), can increase type 1 procollagen synthesis in vitro (42
). Furthermore, IL-4 serves as a growth factor for cells that secrete TGF-β
), a cytokine known to cause tissue fibrosis (43
). IL-4 transgenic mice exhibit increased renal TGF-β
expression and develop glomerulosclerosis, which is independent of Ig deposition (44
). Studies are underway to examine these possibilities.
Finally, the cause of dysregulated IL-4 expression in NZM.2410 mice is not known. NZM.2410 mice have significantly increased numbers of IL-4-secreting CD4+
T cells (our unpublished data), which may be committed to produce IL-4 through a genetic regulation (45
). Also, increased IL-4 expression may be regulated at the level of CD1d-restricted T cells (46
), as CD1d null NZM.2410 mice have decreased IL-4 production (our unpublished data). Thus, CD1d-regulated events may contribute, at least in part, to the elevated IL-4 levels in NZM.2410 mice.
In summary, there may be several distinct immune pathways that can lead to the development of renal disease in mice (or probably humans) who have SLE. In the MRL-lpr mice, autoantibody and immune complex deposition probably have an important, although not exclusive role in the development of glomerulonephritis, proteinuria, and loss of renal function. In the NZM.2410, it is not at all clear that autoantibody and immune complex deposition is important; IL-4 and perhaps other cytokines induced by IL-4, such as IL-13, may be acting directly on glomerular cells to induce glomerulosclerosis. Thus, the immune system would be important in both strains for the induction of autoantibody production and cytokine production, but the autoantibodies would contribute importantly to disease in the MRL-lpr, while the cytokines would contribute more directly to disease in the NZM.2410. If these differences can be observed in mouse models of SLE, they may also subset human SLE patients, with the implication that different subsets of patients might have different prognoses and benefit from different therapies.