The Investigation On The Production Of Anti-neutrophil Cytoplasmic Antibodies In Chronic Bronchitis Rats

Objiective : The purpose of this study was to investigate the pathogenesis of the production of Anti-Neutrophil Cytoplasmic Antibodies(ANCA)in a chronic bronchitis rat model with long-term recurrent infections.Method : The chronic bronchitis model was made by double element of smoking and lipopolysaccharide(LPS)stimulation.The rats were divided into four groups,including LPS and phorbol myristate acetate(PMA)-treated chronic bronchitis rats(Group CB/LPS+PMA),chronic bronchitis rats model(Group CB),PMA-treated healthy rats(Group N/PBS+PMA),heathy rats(Group N).Acute attack stage of chronic bronchitis was induced by the intratracheal instillation of LPS every week.Blood specimens and urine specimens were collected for each week.After repeated stimulation with LPS,immunological markers,which included Citrullinated Histone H3(Cit H3),myeloperoxidase anti-neutrophil cytoplasmic antibodies(MPO-ANCA)and proteinase 3 anti-neutrophil cytoplasmic antibodies(PR3-ANCA)were measured by enzyme-linked immune-sorbent assay(ELISA)at diffrent timepoint.All of the parameters were analyzed.Renal function,urine dipstick and urinary protein at 24 hours were detected.The histopathological lung and kidney tissues were observed by HE staining of paraffin section.Results:(1)The the rat models of chronic bronchitis were established.HE staining of the lung tissue of chronic bronchitis rats showed that there were bronchial mucous glands hyperplasia,inflammatory cells infiltration in the bronchial mucosa,submucosa and muscle layer,and local rupture of smooth muscle tissue in the bronchial wall.(2)Serum levels of Cit H3 in group CB/LPS + PMA at the sixth week were significantly higher than those before the experiment and at the second week(33.97 ± 11.77 ng/ml vs.22.74 ± 2.32 ng/ml,33.97 ± 11.77 ng/ml vs.21.82±2.08 ng/ml)(P<0.05).And compared with those in the N,CB,and N/PBS+PMA groups,serum levels of Cit H3 in group CB/LPS + PMA increased significantly at the sixth week(33.97 ± 11.77 ng/ml vs.24.88 ± 4.98 ng/ml,33.97 ± 11.77 ng/ml vs.21.20 ± 4.35 ng/ml,33.97 ± 11.77 ng/ml vs.23.90 ± 4.66 ng/ml)(P< 0.05).(3)Serum levels of MPO-ANCA in groups N/PBS+PMA and CB/LPS+PMA at the sixth week were significantly higher than those before the experiment and at the second and fourth weeks(54.19 ± 10.61 ng/ml vs.25.67 ± 6.20 ng/ml,54.19 ± 10.61 ng/ml vs.33.56 ± 7.14 ng/ml)(P< 0.01).Serum levels of MPO-ANCA in groups N/PBS+PMA and CB/LPS+PMA were significantly higher than those in groups N and CB at the sixth week(54.19 ± 10.61 ng/ml vs.25.95 ± 2.26 ng/ml,54.19 ± 10.61 ng/ml vs.26.69 ± 4.56 ng/ml)(P< 0.01).(4)The serum PR3-ANCA levels had no statistical significance(P> 0.05).(5)Serum Cit H3 levels were positively correlated with serum MPO-ANCA levels(r = 0.490,P = 0.024).(6)In the lung and kidney tissues of CB/LPS+PMA group MPO and Cit H3 could be detected by confocal immunofluorescence analysis.MPO and Cit H3 were a good colocolization.(7)In the CB/LPS+PMA group,mild renal pathology changes could be observed with inflammatory cells infiltration,while no significant functional changes were detected in lung tissues.Conclusions : Chronic bronchitis rats with the recurrent infections could release large amounts of NETs,in which the exposure of MPO antigen would break the immune tolerance and result in the production of MPO-ANCA.