Expression Of S100 Protein And Functional Change Of Neutrophils In Acute Kawasaki Disease

Object:KD(Kawasaki Disease) is one of the most common acute vasculitis syndromes in children,which mostly affect arteries,particularly,coronary arteries.Currently,its etiology and etiopathology are still largely unknown.S 100A8,S 100A9 are members of calcium binding protein family,which are mostly secreted by activated neutrophils and monocytes.Several studies have demonstrated that they played vital roles in acute inflammation.However,the expression of these two proteins in KD is rarely studied. This project was to investigate functional change of neutrophils in KD and its relevance to the progress of the disease.Methods:According to the criteria set in 2002 in Japan,34 patients with Kawasaki Disease were enrolled in the study.Meanwhile,20 healthy children with similar age were enrolled as negative controls.Blood samples were collected at the time of diagnosis before the initiation of IVIG(Intravenous Immune Globulin) treatment,and 1 week after IVIG treatment.To evaluate the activation of neutrophils,partial patient underwent DHR assay.0.2 mL of heparin anticoagulated venous blood was collected, 50μL of which was incubated in PBS(phosphate buffer solution),PMA(phorbol myristate acetate) for 15 minutes,fMLP(N-Formyl-Met-Leu-Phe) for 5 minutes at 37℃respectively,then incubated with 25μL of DHR(dihydrorhodamine) for 5 minutes at 37℃,after that erythrocytes were removed by adding 2 mL of hemolysis and cells were washed once with PBS,resuspended in 300-500μL of PBS and then assayed by Flow Cytometry.Neutrophils were extracted from peripheral venous blood and preserved by Trizol reagent,total RNA was extracted from neutrophils.The purity and quantity of RNA were determined according to ultraviolet absorbance at 260nm and 280nm.Gene expression of S100A8 and S100A9 in neutrophils was analyzed by real-time fluorescence quantitative polymerase chain reaction.Results:1,After incubation with PBS and fMLP,the proportions of activated neutrophils in 11 cases before IVIG treatment were higher than those of the control (PBS:P=0.033;fMLP:P=0.000),decreased after IVIG treatment in 6 self control cases (PBS:from 33.5±21.4 to 16.0±11.7,P=0.109;fMLP:from 78.4±15.9 to 30.1±21.1, P=0.001),reached the control level(PBS:P=0.149;fMLP:P=0.867).2,Incubated with PMA,the proportion of activated neutrophils in Kawasaki Disease patients without innate immunodeficiency was more than 90%.3,In KD patients,the expression of S100A8,S100A9mRNA in neutrophils before IVIG treatment was 6.70±0.66 and 6.65±0.57 respectively,which was significantly higher than that of the control(2.55±0.52 and 3.60±0.7 respectively)(P=0.000,0.000).4,Before IVIG treatment in KD patients without CALs,the expression of S100A8,S100A9mRNA in neutrophils was 6.70±0.66 and 6.65±0.57 respectively, decreased to 6.16±0.81 and 6.00±0.77 respectively(P=0.001 and 0.000) after IVIG treatment.5,Before IVIG treatment in KD patients with CALs,the expression of S 100A8,S100A9mRNA in neutrophils was 5.97±0.32 and 6.14±0.45 respectively,increased to 6.66±0.63和6.58±0.45 respectively(P=0.038 and 0.12) after IVIG treatment.6,After IVIG treatment,the expression of S100A8,S100A9mRNA increased in patients refractory to the treatment.Conclusions:In acute Kawasaki Disease,neutrophils not only proliferated but were activated as well,which demonstrates that neutrophils may participate in the onset of KD.After IVIG treatment,the activity of neutrophils was inhibited in some patients, however,in patients with CALs,neutrophils were constantly activated,suggesting that neutrophils inhibition may be one of the mechanisms of IVIG.And neutrophils may play a pivot role in the development of coronary artery lesions in Kawasaki Disease.