Molecular Epidemiological Study On The Association Of ITGAM And FcγRIIIA With Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus is a complex autoimmune disease with multisystem organ involvement. The most prominent manifestations of patients is a large number of multiple autoantibody production and clearance of immune complex disorder leading to complement activation, resulting in multiple system and organ damage. The etiology and pathogenesis of SLE is very complex, probably related to genetic and environmental factors which cause immune dysfunction, resulting in many kinds of immune abnormalities. More than 20 genes which significantly correlated with SLE have been identified through candidate gene-based and family-based linkage approaches and also Genome-wide Association Study (GWAS). Most of these susceptibility genes fall into three key biological pathways implicating immune complexes, host immune signal transduction and interferon pathways in the pathogenesis of SLE. They can induce several abnormity of immune function of human via changing the structure of DNA or activating the immune system. Among these, impairment of clearance of immune complex pathway potentially plays a part in disease initiation and perpetuation. Therefore, study on the relationship between expression of genes which related to the clearance of immune complex and cell function will benefit us with the understanding of the pathogenesis of SLE.ITGAM and FcγRIIIA are two kinds of immune complex clearance and immune-related molecules, whose dysfunction can lead to immune complex processing disorder.ITGAM gene located in 16p11.2, encoding theα-chain ofαMβ2-integrin (also known as Mac-1, CR3, and CD11b/CD18).Recently, many GWAS results that SNP rs1143679 ITGAM-R77H has significant association with the susceptibility of SLE. ITGAM is mainly expressed in neutrophils and monocyte, belongs to the family of surface antigen of leukocyte. It forms special integrin of leukocyte, regulates leukocyte adhesion and emigration from the bloodstream via interactions with a wide range of ligands including ICAM-1, ICAM2, fibrinogen, C3bi, GPIbα, promotes neutrophil adhesion to endothelial cells and participates in phagocytosis of macrophages, neutrophils, monocytes and immune complex process.FcγRIIIA gene located in 1q23 which significantly links with SLE, encoding Receptors for immunoglobulins (Fc receptors) FcγRIIIA, integrates with IgG of Fc segment, the sub-type of integrated IgG displays close relationship with SLE or LN. FcγRIIIA-158V has a higher affinity to the monomer or immune complex of IgG1, IgG3, and IgG4 than FcγRIIIA-158F does. And the difference directly influences the behavior of those cells which express FcγRIIIA. FcγRIIIA is mainly distributed at the surface of T cell and natural killer cell (NK). In immune response, after the integration of Fc receptor with immune complex, it transmits the signal of activation into cell viaγchain of immune receptor tyrosine activated sequence, leads to multiple immunoreactions such as activation of cells, antibody-dependent cytotoxicity, peroxide ions generation, presence of antigen, endocytosis, and cell uptake, plays a very important role in the clearance of immune complex, adjusting the function of immunity of the body. Up to date, there is a lot of case-control studies regarding the association of SLE with FcγRIIIA in several ethnic populations. However, they have yielded apparently inconclusive or contradictory results. These might indicate that heritage could give us some valuable information during the etiology study of complex diseases.In summary, according to the known major pathways of SLE, abnormal immune complex clearance is important for SLE. ITGAM and FcγRIIIA all belong to the immune complex pathway, as the two kinds of immune complex clearance and the important immune related molecules, their dysfunction can lead to immune complex processing disorder.We studied the expression of ITGAM and FcγRIIIA in SLE, for in-depth understanding of the pathogenesis of SLE.And it also provides strong evidence for the early prediction of disease and clinical decision-making as well.Objective1, Compare the difference of FcγRIIIA and ITGAM protein expression in Peripheral blood mononuclear cells (PBMC) of SLE patients and healthy controls, SLE with and without nephritis, active and stable SLE. Analyze the correlation of FcγRIIIA and ITGAM protein expression with SLEDAI, characters of clinical and laboratory. Discuss the correlation between the protein expression of FcγRIIIA and ITGAM in SLE patients.2, Compare the difference of FcγRIIIA and ITGAM mRNA expression in SLE patients and healthy controls, SLE with and without nephritis, active and stable patients. Analyze the correlation of FcγRIIIA and ITGAM protein expression with SLEDAI, characters of clinical and laboratory. Discuss the correlation between the mRNA expression of FcγRIIIA and ITGAM in PBMC of SLE patients.3, Meta analyses the association of ITGAM-R77H and FcγRIIIA-F158V with the susceptibility of SLE, as well as dose-response relationship. Method60 SLE patients were recruited from the Department of Rheumatology at Anhui Provincial Hospital and the First Affiliated Hospital of Anhui Medical University. All the patients fulfilled the classified criterion emendated by the American Rheumatology Association in 1977, and other systematic diseases were excluded. According to age and sex matched, control group included 60 healthy voluntary people from The First Affiliated Hospital and The Affiliated Provincial Hospital of Anhui Medical University, and postgraduate students. Clinical and laboratory data of SLE patients were collected, disease activity was measured using the SLEDAI, and survey questionnaire was designed according to the SLE diagnosis and classification. After obtaining the informed consent, blood samples were collected.Flow cytometry staining with FITC, PE, and PE-Cy5 was used to analyze the expression of ITGAM and FcγRIIIA in SLE patients and normal controls. The correlation of expression levels of ITGAM and FcγRIIIA with the development of SLE was also discussed. Real-time quantitive Polymerase chain reaction (Q-PCR) was used to detect the expression level of ITGAM and FcγRIIIA mRNA in SLE and controls. The correlation of mRNA levels of ITGAM and FcγRIIIA with SLE was also discussed. We surveyed studies on ITGAM and FcγRIIIA and SLE/LN using Medline, Blackwell, and EMBASE databases up to January 2009. Two investigators independently assessed the data quality and extracted the data. A Meta-analysis was peformed to assess the risk of ITGAM and FcγRIIIA polymorphsim for SLE.SPSS 10.01 software was applied for statistical analysis.T-test or Mann-Whitney test was used to compare the difference of the expression levels of ITGAM and FcγRIIIA. Relationship of expression levels of the two genes with SLEDAI and clinical and laboratory features was also analyzed. Relationship of mRNA level of FcγRIIIA and ITGAM in PBMC of SLE patients was also analyzed.Results1. The expression difference and clinical significance of ITGAM in PBMC of SLE patients 1.1 Difference of the expression of ITGAM in PBMC of SLE patients and controls, SLE with and without nephritis, active and stable SLE.Compared with controls, expression of protein ITGAM in T cell, neutral granule cell, and monocyte of SLE patients is significantly lower(29.47±12.07 vs. 48.77±17.81,P<0.001 ; 258.81±120.02 vs. 415.00±254.78 , P=0.013 ; 380.54±100.26 vs. 548.22±272.07,P=0.003). Compared with SLE without nephritis, the expression of ITGAM in neutrophil of SLE with nephritis is significantly lower(242.90±119.91 vs. 322.45±106.47,P=0.043). But the expression of ITGAM in PBMC of SLE patients in active and stable SLE did not show significant difference(P>0.05).1.2 Correlation of SLEDAI with expression of ITGAM.The expression level of ITGAM in SLE patients does not show significant association with SLEDAI(P>0.05). SLEDAI shows positive correlation with the expression of ITGAM in neutrophil and monocyte, negative correlation with the expression of ITGAM in T cell of SLE patients. However, the difference does not show statistical significance(P>0.05).1.3 Correlation of ITGAM expression with clinical and laboratory features.SLE patients with serositis or neuropsychiatric involvement show increased expression level of ITGAM (P =0.049, 0.047 respectively). SLE patients with lower C3 also have decreased expression of ITGAM(P = 0.033). Those SLE patients with anti-RNP positive, higher ESR also have a higher expression of ITGAM (P =0.049, 0.003 respectively).1.4 Expression of ITGAM mRNA in PBMC of SLE patientsCompared with controls, SLE patients have lower expression of ITGAM mRNA in PBMC (P = 0.007). The expression of ITGAM mRNA between active and stable SLE patients does not show statistical difference(P > 0.05).1.5 Correlation of SLEDAI with mRNA expression of ITGAM.Expression of ITGAM mRNA in PBMC of SLE patients has significant positive correlation with SLEDAI. (r = 0.272,P < 0.035).1.6 Correlation of ITGAM mRNA expression with clinical and laboratory features. Expression of ITGAM mRNA does not show significant correlation with the laboratory feature of SLE(P > 0.05).2. Expression and clinical significance of FcγRIIIA in PBMC of SLE patients2.1 Difference of FcγRIIIA expression in PBMC between SLE patients and controls, SLE with and without nephritis, active and stable SLE.Compared with SLE without nephritis, the expression of FcγRIIIA in NK cell of SLE patients with nephritis is significantly higher(P=0.011). Compared with stable SLE patients, the expression of FcγRIIIA in NK cell of active SLE is obviously higher(P=0.022). Nevertheless, compared with controls, the expression of FcγRIIIA in NK cell, monocyte in SLE patients did not show statistical difference(P > 0.05).2.2 Correlation of SLEDAI with expression of FcγRIIIA in PBMC of SLE patients.Expression of FcγRIIIA mRNA has negative correlation with SLEDAI, but not obvious (r = 0.246,P>0.05).2.3 Correlation of expression of FcγRIIIA in PBMC of SLE patients with their clinical and laboratory features.SLE patients with arthritis have significantly lower expression of FcγRIIIA(P = 0.025). On the contrary, SLE patients with myositic have higher expression(P = 0.043). SLE patients with alopecie or nephritis have higher expression of FcγRIIIA (P =0.013, 0.017 respectively). With the decrease of C3 and leucocyte, the SLE patients have obviously increased expression of FcγRIIIA(P = 0.007 and 0.008 respectively). SLE patients with higher IgA have lower expression of FcγRIIIA (P = 0.043).2.4 Expression of FcγRIIIA mRNA in PBMC of SLE patientsCompared with controls, SLE patients have lower expression of FcγRIIIA mRNA in PBMC (P =0.001). Compared with SLE without nephritis, expression of FcγRIIIA mRNA of SLE with nephritis is obviously lower(P = 0.015). The expression of FcγRIIIA mRNA between active and stable SLE patients does not show statistical difference(P > 0.05).2.5 Correlation of SLEDAI with mRNA expression of FcγRIIIA. Expression of FcγRIIIA mRNA has negative correlation with SLEDAI , but it does not show statistical difference (r = -0.246,P > 0.05).2.6 Correlation of FcγRIIIA mRNA expression with clinical and laboratory features.SLE patients with arthritis have higher FcγRIIIA mRNA level (P = 0.025). SLE patients with anti-SSB positive, anti-RNP positive, decease of C3, and increase of IgG have significantly lower expression of FcγRIIIA mRNA(P values are 0.041, 0.003,0.029, and 0.003 respectively). Expression of FcγRIIIA mRNA does not show obvious correlation with clinical feature(P > 0.05).2.7 Correlation of expression of FcγRIIIA with ITGAM proteinExpression of ITGAM in neutrophil shows significant positive correlation with that in monocyte of SLE patients.(r = 0.661,P<0.001). Expression of ITGAM in monocyte of SLE patients has positive correlation with its expression of FcγRIIIA, but negative correlation with the expression of FcγRIIIA in NK cell. The expression of ITGAM in neutrophil has negative correlation with its expression in NK cell and monocyte. Expression of ITGAM in T cell has negative correlation with the expression of FcγRIIIA in NK cell, positive correlation with the expression of FcγRIIIA in monocyte on the contrary. However, they all do not show statistical difference(P > 0.05).2.8 Correlation of expression of FcγRIIIA mRNA with ITGAM mRNAExpression of FcγRIIIA mRNA has negative correlation with ITGAM mRNA of SLE patients. But it does not show statistical difference (r = -0.019,P > 0.05).3. Meta analysis: FcγRIIIA-F158V and ITGAM-R77H polymorphism in susceptibility to SLE. 3.1 Meta analysis of FcγRIIIA-F158V in susceptibility to SLE.17 studies were selected for the Meta analysis of FcγRIIIA and SLE, including 3,493 SLE patients and 2,426 controls. Among those SLE patients, 1,711 with nephritis, 1,782 without nephritis. 1,855 SLE patients were European, 409 patients were African, and 1,229 patients were Asian. In the meta-analysis, we performed three different comparisons: SLE vs. disease-free control, SLE with nephritis vs. SLE without nephritis, and SLE without nephritis vs. disease-free control. The results indicated that 158F allele presented overall consistent association evidence for SLE [OR1.27,95% confidence interval (CI) 1.13–1.43]. Moreover, FF homozygosity shows dose-effect relationship for SLE with maximal OR 1.68 (95% CI 1.26–2.23). Furthermore, 158F allele has significant correlation with European and Asian SLE(OR1.30,95%CI 1.07–1.58;OR1.24, 95%CI 1.04–1.48). In addition, FF homozygosity only shows dose-effect correlation in Asian SLE. In particular, 158F allele shows obvious correlation with Asian SLE with nephritis(OR1.26, 95%CI 1.06–1.50). FF homozygosity has more significant correlation with European and Asian SLE than does with African SLE(OR1.61,95%CI 1.03–2.53;OR1.70,95%CI 1.12–2.58).3.2 Meta analysis of ITGAM - R77H in susceptibility to SLE.5 GWAS studies were selected for meta analysis, including 12,123 SLE patients and 17,106 control subjects. Meta analysis indicated that risk allele A has significant correlation in susceptibility to SLE (OR1.795,95%CI1.676-1.921), homozygosity of AA vs. GG (OR3.540, 95%CI 2.771-4.522). AG vs GG (OR 1.750;95%CI 1.617-1.895), dominant model (OR 1.857;95%CI 1.719-2.005), recessive model (OR 3.041;95%CI 2.384-3.878) of ITGAM rs1143679 were significantly increased in SLE.Conclusions1. Protein and mRNA expression of ITGAM and FcγRIIIA in PBMC has close association with SLE.Different FcγRIIIA expression of SLE patients with different clinical features.2. ITGAM-R77H and FcγRIIIA-F158V Gene polymorphism strongly influences susceptibility to SLE. Moreover, FcγRIIIA-158F shows dose-effect relationship. 158F allele shows significant correlation with Asian SLE with nephritis.Taken together, we may confirm that FcγRIIIA and ITGAM correlated with the development of SLE through gene expression and genetic susceptibility. As important factors in pathway of immune complex, FcγRIIIA and ITGAM might play an important role in the development of SLE, providing more effective diagnostic and prognosis and target clinical management tools for this complex autoimmune disease.