Detection And Clinical Correlation Research Of Serum Gal-9,KL-6 Levels In Patients With Interstitial Pulmonary Disease In Connective Tissue Diseases

Background Interstitial lung disease(ILD)is a kind of acute and chronic nonspecific inflammation of lung stroma.Its etiology is complex and the pathogenesis is unknown.The early stage of interstitial lung disease is alveolitis.With the repeated damage and repair of lung stroma by inflammation,the late stage of pulmonary fibrosis is gradually becoming diffuse interstitial fibrosis.The final outcome of interstitial lung disease as respiratory failure,poor prognosis and high mortality rate.Connective tissue disease is one of the major causes of ILD.CTD belongs to autoimmune disease and belongs to multisystem disease.It is most likely to involve the lung and is manifested as ILD.The pathogenesis of CTD-ILD is not very clear.It is possible that the immune disorder stimulates macrophages and lymphocytes,resulting in the release of cytokines,proteases and oxygen free radicals.These substances can lead to basement membrane damage of alveolar epithelial cells and capillary endothelial cells,and can promote fibroblast proliferation,destruction of alveolar structure,alveolar capillary membrane,which leads to progressive fibrosis,hypoxemia and respiratory failure.Galectin-9(Gal-9)is a chemotactic agent for eosinophils.Gal-9 can mediate the immune response by binding to the CD44 pathway of T cells,immunoglobulin,mucin molecules 3(Tim-3)and T cells.Some studies have found that Gal-9 may inhibit pulmonary fibrosis in patients with CTD.At the same time,examination with immunohistochemistry and lung biopsy in patients with ILD,show that Gal-9 expressed in lung tissue.Gal-9 can induce strong immune response in alveolar type II cells,macrophages and lymphocytes.The level of Gal-9 in the bronchoalveolar lavage fluid inCTD-ILD patients is higher than that of idiopathic ILD and idiopathic nonspecific ILD,.It is indicated that high level of Gal-9 in CTD-ILD patients may be the reason of better prognosis than idiopathic ILD and idiopathic nonspecific ILD.At the same time,the study indicated that Gal-9 mainly derived from lymphocytes.In CTD-ILD,Gal-9 may be combined with Tim-3 to induce apoptosis of Thl/Th17 cells,promote the release of a variety of cytokines,mediate the immune effect,inhibit the proliferation of fibroblasts,and thus inhibit lung fibrosis.Krebs von den Lungen-6(KL-6)is a large molecular sialic acid glycoprotein secreted by alveolar type II epithelial cells.It is less expressed on normal alveolar epithelial cells.The concentration of serum KL-6 in ILD patients is higher than that in patients with other respiratory diseases or normal subjects.When the KL-6 level is greater than 500U/ml,the CTD may be merged with ILD.As far as CTD is concerned,if the level of KL-6 is greater than 500U/ml,it is highly possible to suspect the possibility of combining ILD.In patients with CTD without ILD,serum KL-6 levels are generally lower than 500U/ml.The higher level,of serum KL-6,the more pulmonary exudative lesions,the higher disease activity of ILD,So the serum KL-6 level is related to the rapidly progressing interstitial lung disease(RP-ILD).Objectives Through the detection of levels of KL-6,Gal-9 in patients with CTD-ILD,and comparing with those of CTD-NILD group and normal control group,and analyzing their correlations with laboratory index,pulmonary function,HRCT and pulmonary hypertension,as well as the correlation of Gal-9 and Th17 cells peripheral blood.we preliminary explore the possible role of Gal-9 in the pathogenesis of CTD-ILD,and further verifies the important role of KL-6 for early diagnosis of ILD and assessment of the disease activity.Methods A case control study was conducted to collect 63 patients with CTD-ILD in the department of rheumatology,Guangzhou First People's Hospital.At the same time,60 patients with CTD-NILD and 30 cases of NC which age,gender and sex were matched.The laboratory parameters(ESR,CRP,rheumatoid factor,complement C3,CCP,C4,ANA),lung function(ventilation function,diffusion function),HRCT(dominate with lung exudative lesions,dominate with fibrosis)and pulmonary hypertension(echocardiography)data were examined.The levels of serum Gal-9,KL-6 were detected by enzyme-linked immunosorbent assay(ELISA),Peripheral blood Th17 was detected by flow cytometry in peripheral blood.The serum levels of Gal-9 and KL-6 were compared between group CTD-ILD and group CTD-NILD and group NC respectively.The level of Gal-9,KL-6 and laboratory parameters(ESR,CRP,rheumatoid factor,complement C3,CCP,C4,ANA),lung function,HRCT,and pulmonary hypertension of CTD-ILD patients were analysed.The relationship between the level of serum Gal-9 and expression of Th17 cells in peripheral blood was also analysed.Statistical analysis was conducted by SPSS19.0 software.Results 1?General data There were 63 cases of CTD-ILD,60 cases of CTD-NILD and 30 cases of NC.There was no significant difference in sex and age between the three groups(P>0.05).There was also no statistical difference in the course of disease between the CTD-ILD group and the CTD-NILD group(P >0.05).2?Comparison of serum Gal-9 and KL-6 levels between the three groups(1)Comparison between CTD-ILD group and NC group: The median Gal-9 level in the CTD-ILD group was 102.4(79.2-125.3)ng/L,higher than the median level 76.9(68.3-89.3)ng/L in the NC group(Z=-5.313,P<0.05);The KL-6 mean level in group CTD-ILD(618.76±436.61 U/ml)was significantly higher than that in group NC(278.97±243.33U/ml)(P<0.05).(2)Comparison between CTD-NILD group and group NC group: The median Gal-9 level in the CTD-NILD group was 133.1(102.8-158.2)ng/L,significantly higher than that in the NC group(Z=-2.563,P<0.05);The level of KL-6 in the CTD-NILD group was(314.54±123.56)U/ml,and therewas no statistical difference between the NC group and the CTD-NILD group(P>0.05).(3)Comparison between CTD-ILD group and CTD-NILD group: The median Gal-9 level in the CTD-ILD group was lower than that in the CTD-NILD group(Z=-3.914,P<0.05);The level of KL-6 in group CTD-ILD was significantly higher than that in group CTD-NILD(P<0.05);3?Correlation of Gal-9 and KL-6 levels with laboratory parameters in CTD-ILD patients:(1)There were no significant differences between serum Gal-9 levels and ESR,CRP,rheumatoid factor,CCP,complement C3,C4 and ANA(P >0.05);(2)There were no significant differences between serum KL-6 levels and ESR,CRP,rheumatoid factor,CCP,complement C3,C4 and ANA(P>0.05)? 4?Correlation of Gal-9,KL-6 levels with lung function in patients with CTD-ILD:(1)Correlation of serum Gal-9 and KL-6 levels with FVC,FEV1,FEV1/FVC and DLCO 1)Serum Gal-9 was positively correlated with DLCO(r=0.650,P=0.022),and it was not significantly correlated with FVC,FEV1,FEV1/FVC(r=0.168,P=0.651,r=0.039,P=0.221,r=-0.348,P=0.138).2)Serum KL-6 was negatively correlated with FEV1(r=-0.301,P=0.019),and it was not significantly correlated with FVC,FEV1/FVC,DLCO(r=-0.191,P=0.155,r=0.328,P=0.092,r=-0.061,P=0.342).(2)Correlation of serum Gal-9 and KL-6 levels with pulmonary ventilation function and diffusion function Grouping: According to the pulmonary ventilation function group,the patients were divided into normal / mild ventilatory dysfunction group and moderate / severe ventilatory dysfunction group.According to the pulmonary diffusion function group,the patients were dividedinto normal / mild diffusion dysfunction group and moderate / severe diffusion dysfunction group.1)Pulmonary ventilation function: the level of serum Gal-9 of Normal/mild ventilation dysfunction group had no significant difference with the level of serum Gal-9 level in severe group/ventilation dysfunction(Z=-0.414,P>0.05);the level of serum KL-6 of Normal/mild ventilation dysfunction group had no significant difference with the level of serum KL-6 in severe group / ventilation dysfunction(P>0.05).2)Pulmonary diffusion function: Normal/mild dispersion of serum level of Gal-9 dysfunction group was higher than that of moderate/severe diffuse dysfunction group(Z=-2.354,P<0.05);there was no significant difference berween the level of serum KL-6 in normal/mild diffuse dysfunction group and that in Medium / severe diffuse dysfunction group(P> 0.05).5?Correlation of serum Gal-9 and KL-6 levels with the imaging findings of high resolution CT(HRCT)in CTD-ILD patients: The images were grouped according to the HRCT findings: ILD group with exudative lesion:The main imaging features of HRCT are grinding glass changes,speckle shadows.ILD group mainly with fibrotic lesions:The main imaging features of HRCT are fibrous,bar like,grid like,honeycomb like.The level of serum Gal-9 in exudative group ILD was higher than that in ILD group mainly with fibrotic lesions(Z=-1.868,P<0.05);The level of serum KL-6 in exudative group ILD was higher than that in ILD group mainly with fibrotic lesions(P<0.05);6?Correlation between Gal-9,KL-6 levels and pulmonary arterial hypertension in patients with CTD-ILD: ILD patients were grouped according to the results of echocardiography: Pulmonary hypertension group(transthoracic Doppler echocardiography and pulmonary artery systolic pressure >35mm Hg)and the group without pulmonary hypertension(transthoracic Doppler echocardiography and pulmonary artery systolic blood pressure less than 35 mm Hg).There was no statistically significant difference in serum Gal-9 concentration between the two groups(Z=-0.093,P>0.05);There were no statistically significant differences in serum KL-6 levels between the two groups(P>0.05)? 7?Correlation analysis of serum Gal-9 level and KL-6 level in patients with CTD-ILD Serum Gal-9 levels were not correlated with KL-6 levels(r=0.110,P=0.391)8?Correlation analysis of Gal-9 concentration and expression of Th17 cells in patients with CTD-ILD Serum Gal-9 concentration was negatively correlated with the expression of Th17 cells in peripherial blood(r=-0.25,P<0.05)?Conclusion 1?The Gal-9 level in the CTD-ILD group was lower than that in the CTD-NILD group.Serum Gal-9 levels were positively correlated with DLCO.The serum Gal-9 levels in the normal/mild diffuse dysfunction group were higher than those in the moderate/severe diffuse dysfunction group.All these indicated that Gal-9 may inhibit the lung interstitial lesion.2?Serum Gal-9 concentration was negatively correlated with the expression of Th17 cells.Whether Gal-9 can inhibit the differentiation of Th17 cells or induce apoptosis of Th17 cells and inhibit the release of various inflammatory cytokines which may play an important role in inhibiting the inflammatory and pulmonary fibrosis of the lung needs further study.3?The level of KL-6 in group CTD-ILD was significantly higher than that in group CTD-NILD.The level of serum KL-6 in exudative group ILD was higher than that in ILD group mainly with fibrotic lesions.These results further confirm that KL-6 can be used as a serological marker for evaluating ILD lesions and disease activity.