Background Oxidative stress i.e. accumulation of reactive oxidative species has been found to be implicated pathogenesis of many autoimmune diseases including systemic lupus erythematosus. Although our body has natural process of scavenging reactive oxidative species but whenever balance inclines towards accumulation, oxidative stress begins to build in. These accumulated ions lead to damage at cellular level and at molecular levels also. In our NK cell specific study we evaluated oxidative stress and expression level of killer immunoglobulin receptors. Killer cell immunoglobulin like receptors binds to mhc class 1 receptors. They work in antagonistic manner they are either activating for NK cell activity or inhibiting. So the balance between two categories is critical for self tolerance. We have evaluated expression level of kir2dl4 which binds to HLA-G ligand and activating in nature and KIR3DL1 on other hand interacts with HLA-Bw4 and prevent NK cell killing of healthy cells.
Methods Lupus Patient and healthy subjects Lupus patients are enrolled from outpatient department (OPD) of rheumatology clinic, PGIMER, Chandigarh.
Flowcytometric analysis PBMC isolated were incubated with antibodies conjugated to APC,PE, PerCP/cy5.5 for surface staining of antigens cd56, kir2dl4 and kir3dl1. DCFDA dye based analysis was al done for estimation cellular ROS levels.
Quantitative real-time polymerase chain reaction: NK cells were sorted from using isolation kit from stemcellTM(cat no.17955). cDNA was synthesized from RNA separated from isolated nk cells. Gene primers were run on the StepOnePlus RealTime PCR Systems and analyzed with StepOne Software V2.1 (Applied Biosystems New York, USA).
Results Fraction of cd3-cd56 in pbmc (%) were reduced in SLE patients (5.395%± 0.6900 N=27) as compared to healthy control (10.32 ± 1.729 N=16), P Value0.0037
ROS increased in cd3–cd56+ cells of SLE patients as compared to healthy control.
Cd56 dim and cd56 bright cells of SLE have significantly higher fraction expressing kir2dl4.
Lower Cd56 dim cells of SLE patients express kir3dl1 on surface.
Mean mRNA level in SLE patients of HMOX–1 is 2.56 times higher as compared to healthy control. (p–value=0.0532)
Mean mRNA Expression level of kir3dl1 are not significantly different in SLE patients and healthy control
Mean mRNA expression level of kir2dl4 is 20 times higher in SLE patients as compared to healthy control(p–value=0.0260)
Conclusion In our study NK cell of patients have been found more oxidatively stressed as compared to healthy and Increased mRNA expression of HMOX-1 also indicate same. Significantly increased expression of KIR2dl4 at mRNA and surface expression may be responsible for killing HLA-G bearing self cells. Surface expression of Kir3dl1 has been found to be significantly decreased in CD 56 dim cells, probable consequence is reduced tolerance. Although we could find any significant correlation of mRNA expression kir2dl4 and kir3dl1 with that of hmox-1, so cannot conclude any relationship between these receptor and oxidative stress.
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