Danqing Zhang

Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility

Abstract Autoimmune diseases involve multiple genes. While functions of these genes are largely unknown, some may be related to an intrinsic hyperresponsiveness of B cells. B-cell responses are controlled by signaling thresholds through the B-cell antigen receptor (BCR) complex. The B1 isoform of type II IgG Fc receptors (FcγRIIB1) is exclusively expressed on B cells and serves as a negative regulator for inhibiting BCR-elicited activation. Thus, its allelic variants associated with functional deficits could be examined for possible associations with susceptibility to autoimmune diseases. We found that there are three types of polymorphisms in the reported FcγRIIB transcription regulatory regions in mouse strains. Compared to normal healthy mouse strains (group III), autoimmune disease-prone strains (group I) share three deletion sites: two in the promoter region and one in the third intron. Strains (group II) that per se are not autoimmune-prone, but have potentials to accelerate autoimmune diseases share two deletion sites in the third intron: one identical to that in group I and the other unique to group II. These polymorphisms correlated well with extents of down-regulation of FcγRIIB1 expression in germinal-center B cells upon stimulation with antigens and up-regulation of IgG antibody responses. Our data imply that these FcγRIIB polymorphisms are selected evolutionarily for natural defense against pathogens, and that such polymorphisms may, in turn, form the basis of one aspect of autoimmune susceptibility.

Transcriptional Regulation of Fcgr2b Gene by Polymorphic Promoter Region and Its Contribution to Humoral Immune Responses

FcγRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcγRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down-regulates FcγRIIB1 expression levels in germinal center B cells and up-regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position −302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB-derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up-regulation of IgG Ab responses through down-regulation of FcγRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes.

Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein.

Birt-Hogg-Dubé (BHD) syndrome is characterized by the development of pneumothorax, hair folliculomas and renal tumors and the responsible BHD gene is thought to be a tumor suppressor. The function of folliculin (Flcn), encoded by BHD, is totally unknown, although its interaction with Fnip1 has been reported. In this study, we identified a novel protein binding to Flcn, which is highly homologous to Fnip1, and which we named FnipL (recently reported in an independent study as Fnip2). The interaction between FnipL/Fnip2 and Flcn may be mediated mainly by the C-terminal domains of each protein as is the case for the Flcn-Fnip1 interaction. FnipL/Fnip2 and Flcn were located together in the cytoplasm in a reticular pattern, although solely expressed Flcn was found mainly in the nucleus. Cytoplasmic retention of Flcn was canceled with C-terminal truncation of FnipL/Fnip2, suggesting that FnipL/Fnip2 regulates Flcn distribution through their complex formation. By the employment of siRNA, we observed a decrease in S6K1 phosphorylation in the BHD-suppressed cell. We also observed a decrease in S6K1 phosphorylation in FNIP1- and, to a lesser extent, in FNIPL/FNIP2-suppressed cells. These results suggest that Flcn-FnipL/Fnip2 and Flcn-Fnip1 complexes positively regulate S6K1 phosphorylation and that FnipL/Fnip2 provides an important clue to elucidating the function of Flcn and the pathogenesis of BHD.

C1q Regulatory Region Polymorphism Down-Regulating Murine C1q Protein Levels with Linkage to Lupus Nephritis

Much of the pathology of systemic lupus erythematosus (SLE) is caused by deposition of immune complexes (ICs) into various tissues, including renal glomeruli. Because clearance of ICs depends largely on early complement component C1q, homozygous C1q deficiency is a strong genetic risk factor in SLE, although it is rare in SLE patients overall. In this work we addressed the issue of whether genetic polymorphisms affecting C1q levels may predispose to SLE, using the (NZB × NZW)F1 model. C1q genes are composed of three genes, C1qa, C1qc, and C1qb, arranged in this order, and each gene consists of two exons separated by one intron. Sequence analysis of the C1q gene in New Zealand Black (NZB), New Zealand White (NZW), and BALB/c mice showed no polymorphisms in exons and introns of three genes. However, Southern blot analysis revealed unique insertion polymorphism of a total of ∼3.5 kb in the C1qa upstream region of NZB mice. C1q levels in sera and culture supernatants of LPS-stimulated peritoneal macrophages and C1q messages in spleen cells were all lower in disease-free young NZB and (NZB × NZW)F1 mice than in age-matched non-autoimmune NZW and BALB/c mice. Quantitative trait loci analysis using (NZB × NZW)F1 × NZW backcrosses showed that NZB microsatellites in the vicinity of the C1q allele on chromosome 4 were significantly linked to low serum C1q levels and the development of nephritis. These data imply that not only C1q deficiency but also regulatory region polymorphisms down-regulating C1q levels may confer the risk for lupus nephritis by reducing IC clearance and thus promoting IC deposition in glomeruli.

Transgene-mediated hyper-expression of IL-5 inhibits autoimmune disease but increases the risk of B cell chronic lymphocytic leukemia in a model of murine lupus

IL‐5 preferentially activates B1 cells to produce natural antibodies cross‐reactive to self antigens. To determine the role of IL‐5 in antibody‐mediated autoimmune disease, we generated systemic lupus erythematosus (SLE)‐prone (NZB×NZW)F1 mice congenic for IL‐5 transgene (TG‐F1). The transgene unexpectedly reduced the incidence of lupus nephritis. Anti‐DNA antibodies in sera and those produced by splenic B cells in vitro were markedly decreased in TG‐F1 mice, while total polyclonal Ig levels were comparable to those in IL‐5 transgene‐negative (NZB×NZW)F1 (non‐TG‐F1) littermates. Flow cytometry‐sorted splenic B1 cells showed a significant reduction of anti‐DNA antibody synthesis in response to IL‐5, while proliferative responses to IL‐5 did not significantly differ between TG‐F1 and non‐TG‐F1 mice. As TG‐F1 mice aged, frequencies of peripheral B1 cells progressively increased, and the mice frequently developed B cell chronic lymphocytic leukemia (B‐CLL). Our results suggest that dysregulated, continuous high expression of IL‐5 in SLE‐prone mice may directly or indirectly mediate a skewed signaling of proliferation/differentiation of self‐antigen‐activated B1 cells, leading to suppression of autoimmune disease, but instead to aberrant expansion of B1 cells, giving rise to B‐CLL. Thus, this model may provide a clue to the pathogenesis of both SLE and B‐CLL.

Heterozygosity of the major histocompatibility complex controls the autoimmune disease in (NZW × BXSB) F1 mice☆

In the F1 hybrid of phenotypically normal NZW (H-2z) and systemic lupus erythematosus (SLE)-prone BXSB mice (H-2b), features of the disease became more severe than those seen in the BXSB mice, regardless of the presence or absence of the Yaa (Y-chromosome-linked autoimmune acceleration) mutant gene. To determine whether the gene(s) linked to the major histocompatibility complex (MHC) of NZW mice is involved in this event, we developed the H-2-congenic NZW.H-2d strain and compared the severity of autoimmune disease between (NZW x BXSB) F1 (H-2z/b) and (NZW.H-2d x BXSB) F1 mice (H-2d/b). The H-2z/b, but not H-2d/b, heterozygous F1 mice of both sexes showed an accelerated, higher incidence of proteinuria and a more severe thrombocytopenia than did the BXSB mice. In NZW x (NZW x BXSB) F1 backcross mice, the H-2z/b heterozygous progeny showed more severe disease than did the H-2z/z homozygotes. Thus, disease-accelerating events in (NZW x BXSB) F1 mice are linked to the H-2z/b heterozygosity. Because H-2d/z heterozygosity plays a crucial role for SLE in (NZB x NZW) F1 mice, in which SLE features differ from those in (NZW x BXSB) F1 mice, the present observations may imply that the different but related MHC heterozygosity acts as a predisposing genetic element in these different SLE syndromes.

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product

MINT‐7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)

Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway.

The Birt-Hogg-Dubé gene (BHD) encodes the tumor suppressor protein folliculin (FLCN). The function of FLCN has recently been implicated in the regulation of rapamycin-sensitive mTOR complex (mTORC1). Reciprocally, the mTORC1-dependent phosphorylation of FLCN was reported. However, precise mechanism of FLCN phosphorylation and functional interaction of FLCN with tuberin, the product of tuberous sclerosis 2 gene (TSC2) which is a negative regulator of mTORC1, are unclear. Here we report that multiple phosphorylation in FLCN are evoked by downregulation of tuberin as well as by Rheb expression. We found that phosphorylation at Ser62 and Ser302 are differently regulated by mTORC1-dependent pathway. Some unknown kinases downstream of tuberin-mTORC1 are thought to directly phosphorylate FLCN. Interestingly, our results also suggest that the complex formation of FLCN with AMPK is modulated by FLCN phosphorylation. These results suggest that FLCN is involved in a novel mechanism of signal transduction downstream of tuberin.

The E-linked subregion of the major histocompatability complex down-regulates autoimmunity in NZB x NZW F1 mice

The NZB x NZW F1 mxce spontaneously develop autoimmune disease resembling human systemic lupus erythematosus (SLE). This disease is characterized by severe lupus nephritis, which develops in close association with the production of IgG class autoantibodies to double-stranded (ds) DNA and histones (Shirai et al. 1991). The occurrence of NZB x NZW F1 disease is genetically determined and is restricted by H-2 evz heterozygosity of the major histocompatibility complex (MHC), H-2 d from NZB, and H-2 z from NZW strains (Hirose et al. 1986). Together with the finding that a locus or a cluster of loci linked to the T-cell receptor (TCR)[5 chain gene complex of the NZW strain in combination with H-2 d/z heterozygosity contributed to the disease (Hirose et al. 1991), we proposed that the interaction between unique structures of the TCR and the mixed haplotype hybrid class II molecule-autoantigen complex preferentially transduces the signals for pathogenic IgG autoantibody synthesis in these mice. Four possible Fl-specific mixed haplotype hybrid class II molecules could be expressed in NZB x NZW F1 mice, i. e., Aocd~ z, A(~z~ d, Eo~d~ z, and E~z~ d. Gotoh and co-workers (1993) have isolated from NZB x NZW F1 mice autoreactive T-cell clones restricted by the A~d[5 z. Because T-cell clones restricted by other mixed haplotype class II molecules have not been obtained, it was suggested that the Ac~a[3 z molecule may be a candidate for one predisposing genetic element to NZB x NZW F1 disease (Kimoto et al. 1993). However, the possibility remained that the E subregion is also involved in the control of SLE in these mice. In the present study, we established an H-2-congenic NZB.GD strain carrying the H-2g 2 haplotype, by selective backcrossing of the F1 hybrid of the NZB (/-/-2 d) and B10.GD (H-2g 2) strain to NZB mice for eight generations.

H-2z homozygous New Zealand mice as a model for B-cell chronic lymphocytic leukemia : elevated bcl-2 expression in CD5 B cells at premalignant and malignant stages

In New Zealand mice, the major histocompatibility complex (MHC) controls the development of both autoimmune disease and B cell chronic lymphocytic leukemia (B‐CLL). While H‐2d/H‐2z heterozygosity acts as one major predisposing genetic element for autoimmune disease, H‐2z/H‐2z homozygosity acts as an element for B‐CLL. In the H‐2z/H‐2z homozygotes, there was an age‐dependent increase in frequencies of CD5 B cells in the blood and spleen, and such CD5 B cells showed oligoclonal to monoclonal expansion, giving rise to B‐CLL. B‐CLL cells from these mice had surface phenotypes typical of CD5 B lineage cells, and expressed high levels of proto‐oncogene bcl‐2. Elevated bcl‐2 expression was also observed in premalignant B cells in the aged mice, thereby suggesting that apoptosis‐resistant, long‐surviving CD5 B cells with a self‐renewal capacity form the basis of malignant transformation. This model not only provides clues for analyzing multiple steps of genetic alterations involved in the generation of B‐CLL, but also sheds light on the correlation between B‐CLL and autoimmune disease.