Experimental Study On The Expression And Function Of HAX-1 In Peripheral Blood Mononuclear Cells Of Systemic Lupus Erythematosus

Background and objectivesSystemic lupus erythematosus (SLE), which develops predominantly in women of childbearing age, is a chronic autoimmune disease with a wide spectrum of clinical and immunological abnormalities. It is characterized by overactivation of autoreactive T and B lymphocytes, the presence of various autoantibodies, especially those directed to dsDNA,and circulating immune complexes deposit in tissues and organs. Both the accurate etiology and the pathogenesis of SLE are still unexplained.In recent years, insight into the pathogenesis of SLE has deepened. In particular studies on apoptosis disorders in lupus have shed a new and intriguing light on the development and course of the disease. In human lupus, the abnormality of apoptosis leads to the autoimmune disorder relating with apoptosis and the occurrence of disease. On one hand, several studies have demonstrated elevated levels of Fas/FasL in peripheral blood mononuclear cells (PBMC) of SLE patients. Levels of apoptotic lymphocytes in SLE patients in vitro as well as in vivo were higher than in healthy controls. Due to increased amount of apoptotic cells, together with disturbances of clearance of apoptotic cells and the presentation of autoantigens from apoptotic cells to the immune system, immune tolerance can be broken and autoimmunity occurs. On the other hand, the proto-oncogene bcl-2, which is known to inhibit cells from undergoing apoptosis, has been found to be overexpressed in peripheral blood lymphocytes of patients with SLE. Elevated levels of soluble Fas (sFas) were found to block Fas-induced apoptosis and correlated with the state of activation of B lymphocytes. The life of autoreactive T and B lymphocytes is prolonged and autoantibodies are produced increasingly. Clinically glucocorticosteroid therapy can induce the abnormally activated lymphocytes to apoptosis, which has been administrated to SLE patients and good therapeutic effects have been gotten. So, the abnormality of apoptosis may play an important role in the development of SLE. However, the reported disorders of apoptosis in PBMC seem not to explain all the apoptosis abnormality in SLE. There may be other factors which regulate the apoptosis by the way that we haven't known up to now. Therefore, apoptotic cell clearance and its mechanisms leading to immunogenicity are now the focus in SLE. Further investigations of apoptosis in SLE should open a new chapter in lupus research and may yield important new insights into the fundamental pathogenesis of the disease.HS1-associated protein X-1(HAX-1) is a new anti-apoptotic protein, but the mechanism of its action is still unknown. In our previous study, Dr. Deng found elevated levels of HAX-1 in T lymphocytes of an active SLE patient by long serial analysis of gene expression (LongSAGE) and generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification (GLGI). There is no more related report about HAX-1 and SLE now. We suppose that overexpression of HAX-1 in lymphocytes of SLE can inhibit lymphocytes from apoptosis and excessively activate them, and then promote the development of SLE. It is important to study the expression and function of HAX-1 in PBMC to deepen insight into apoptosis mechanisms of SLE. Methods and results1. Semiquantitative reverse transcriptase polymerase chain reaction method (RT-PCR) and Western blot were applied to detect expression of HAX-1 in PBMC of active SLE, and subcellular localization of HAX-1 in PBMC was observed by laser scanning confocal fluorescence microscope. The results showed that expression levels of HAX-1 in PBMC of active SLE patients are higher than in healthy controls (P<0.01), and it was correlated with the active state of SLE. This suggested that highly expressed HAX-1 in PBMC of active SLE could produce anti-apoptotic effects during the course of SLE.2. Eukaryotic expression plasmid vectors of pEGFP-C3-HAX-1 were designed and generated by molecular biological technology. The plasmid vectors were transfected into Jurkat cells by cation liposomes DMRIE-C. The expressions of HAX-1 mRNA and protein were detected by RT-PCR and Western blot respectively. The apoptosis conditions of the cells under dexamethasone (10μmol/L),β-irradiation and serum deprivation were analyzed by morphologic observation, transmission electron microscope and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). HAX-1 has greater anti-apoptotic function in Jurkat cells transfected by pEGFP-C3-HAX-1 than controls (P<0.01). The results establish a theoretical base for the further study of MRL/lpr murine model of lupus in vivo.3. Full length HAX-1 cDNA was obtained by RT-PCR. The HAX-1 cDNA was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further reconfirmed by sequencing. The recombinant vectors were transfected into AD293 cells with LipofectamineTM2000 for packaging and amplification of AdHAX-1 virus and the high-titer adenovirus supernatants were harvested. After transfection with adenovirus DNA, cytopathic effects were caused and infectious viruses were produced only in the permissive cell line AD293, but not in the non-permissive cell line HeLa. It was confirmed that only replication-defective but not wild type virus was generated. The high expression of mouse HAX-1 was verified by PCR in recombinant adenovirus AdHAX-1 but not in control virus AdEGFP.At the same time, a eukaryotic expression plasmid vector of short hairpin RNA (shRNA) specific for HAX-1 gene was designed and generated by molecular biological technology. The plasmid vector was transfected into HeLa cells by liposomes and the expressions of mRNA and protein of HAX-1 were detected by RT-PCR and Western blot. The HAX-1 levels were significantly decreased by 84.8% and 70.0% comparing with those of the controls (P<0.01). It was confirmed that the effective shRNA eukaryotic expression vector specific for the HAX-1 gene was generated successfully.The recombinant adenovirus AdHAX-1 and the effective shRNA expression vectors specific for the HAX-1 were successfully constructed and it will be useful to further study the apoptosis disorders in animal models of SLE.4. AdHAX-1 was administrated to MRL/lpr mice by intraperitoneal injection twice per week for four weeks in series. The results showed that the overexpression of HAX-1 in the spleen lymphocytes resulted in almost complete exacerbation of the SLE-like syndrome in MRL/lpr mice. Almost all autoantibodies were significantly increased and the results had obviously significant difference comparing with the controls (P<0.01 or 0.05), besides circulating immune complexes and anti-histone antibodies. Hypercellularity and increased deposition of IgG in glomeruli were also observed. The levels of interferon gamma were raised. However, when the effective shRNA vector specific for the HAX-1 gene was transfected into the spleen lymphocytes of MRL/lpr mice, the level of interferon gamma was reduced obviously. Our results strongly suggested an anti-apoptotic role for HAX-1 and the involvement of HAX-1 in the pathogenesis of SLE, and that the silence of HAX-1 may be beneficial for the improvement of SLE.Conclusions1. Expression levels of HAX-1 in PBMC of active SLE patients are much higher than in healthy controls and correlated with the active state of SLE.2. HAX-1 can depress cell apoptosis in vitro and in vivo. Overexpression of HAX-1 in Jurkat is anti-apoptotic under the condition ofβ-irradiation, dexamethasone and serum deprivation. AdHAX-1 can decrease the apoptosis of spleen lymphocytes and promote autoantibodies production in MRL/lpr mice.3. HAX-1 is one of the important factors in apoptosis regulation of SLE, but not a decisive one.