Effect And Mechanisms Of CsA On Bleomycin-induced Pulmonary Interstitial Fibrosis

BackgrandInterstitial lung disease, also called diffuse interstitial lung disease, is agroup of respiratory disorders characterized by the presence of activebreathing difficulties, diffuse infltration on chest radiographs, restrictiveventilatory disorder, low diffusing capacity of lung for carbon monoxide, andhypoxemia. The main pathologic changes are diffuse inflammation ofpulmonary parenchyma, interstitium and interstitial pulmonary fibrosis. It iscommon clinically that in part of patients with ILD are secondary torheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus,polymyositis/dermatomyositis, ankylosing spondylitis and mixed connectivetissue disease. Once formed, interstitial fibrosis will irreversibly cause seriousand even life-threatening respiratory and circulatory dysfunction. Accordingly,in order to lucubrate our understanding of the mechanisms of pulmonaryfibrosis, especially recognize the course of early stage, pathogenesis and treatment of the disease will be solved.CD147, also known as the extracellular matrix metalloproteinase inducer,is a member of immunoglobulin superfamily, espressing all over the body.CyPA, targeted by cyclosporin A (CsA), is present in a wide variety of cellswithin the cytosolic protein. CyPA is the ligand of CD147. CsA is a potentimmunosuppressive agent, specially acts upon lymphocytes, is used for theprevention and treatment of allogeneic organ transplant, bone marrowtransplant rejection or graft versus host reaction clinically, also forautoimmune diseases such as lupus nephritis insensitived to otherimmunosuppressive therapy, refractory nephrotic syndrome and so on.This institute reported in previous studies, that CyPA which was highlyexpressed in inflammatory lesions, was chemotactic in aggregation ofinflammatory cells in articular lesions. In the pathologic process of RA joint,CyPA plays an important role in the pathological changes and developmentthrough its interaction with CD147. And CsA can inhibits this effect. In orderto ascertain whether CyPA-CD147in interstitial lung disease has the sameeffect and mechanism, on the basis of previous work, we establish a murinemodel of pulmonary interstitial fibrosis to study the early course ofinflammatory cell and molecular basis, and use CsA for therapeuticintervention, preliminary discuss CD147-CyPA in the pathogenesis pulmonaryfibrosis.AimTo establish a murine model of pulmonary interstitial fibrosis, observeinflammatory cell infiltration and collagen deposition in the pathologicalprocess and its cellular and molecular basis. Explore the effects andmechanisms of CsA on the inflammation and fibrosis of the model mice. Methods and resultsInduction of murine models of pulmonary interstitial fibrosis and itsresearch systemInduction of murine models of pulmonary interstitial fibrosisSeventy-two C57BL/6female mice were assigned randomly to theBLM-induced group, the saline-induced group and negative control group,with24mice in each group. BLM-induced mice were administrated BLM5.0mg/kg（concentration:2.0mg/ml） intratracheally to induce pulmonaryinterstitial fibrosis, saline-induced mice were administrated with equal volumeof saline instead, while there is no handling on negative control group.Testing and researchOn the4th,7th and14th day after administration,8mice of each groupwere sacrificed,500μ L peripheral blood was obtained and combined withheparin sodium for anticoagulation. Leukocytes were counted by countingchamber. The proportion of CD4~+T cells, CD14~+monocytes and CD19~+Bcells were quantified by flow cytometry. The top-left lung was surgicallyremoved, stabilize with4%0.1PBS paraformaldenyde for24hours. Afterroutine dehydration and paraffin denvelopment, the3μ m laraffin slices werecut from the paraffin-embedded lung samples for HE and Masson staining.Ashcroft standard was used for evaluation of pulmonary interstitial fibrosis.Total BALF cell was counted by counting chamber, with its smears stainedwith Giemsa.ResultsPeripheral blood and BALF cell analyzationThe quantity of total leucocyte was higher in BLM-induced mice than those in saline-induced, with its peak on the7th day (10.96±0.35)×10~9/L. Theproportion of CD14~+monocytes in BLM-induced mice were5.94%(4d)、5.85%(7d)、4.76%(14d) respectively which were increased markedly thanthose in saline-induced at the same time. While CD19~+B cells inBLM-induced mice were higher than that of saline-induced on the7th and14th day. There is no significant difference statistically in the number ofleucocyte, the proportion of CD14~+monocytes and CD19~+B cells betweensaline-induced group and negative control group. With the prolonged time ofadministration, the total cell counts of BALF in BLM-induced mice were(35.2±10.10)×10~6/lung,(23.1±15.52)×10~6/lung and (14.89±1.33)×10~6/lung, much higher than saline-induced group at the same time.Lymphocytes played an important role in BALF cell augmentation (7d：104.90/HP), and remained generous until the14th day. Mononuclearmacrophages changed similar to lymphocytes, while neutrophils wereincreased at the early stage (4d,7d). There is no significant differencestatistically in the number of total cells, mononuclear macrophages,lymphocytes and neutrophils between saline-induced group and negativecontrol group.Histologic examination of lung tissuesWith the prolonged time of BLM administration, it showed wideralveolar septum and more infiltrating inflammatory cells which consisted ofgenerous lymphocyte, mononuclear macrophages and neutrophils than thosein saline-induced mice. With the prolonged time of BLM administration, therewas much more collagen deposition and fibroblasts which accumulatedgenerously within the interstitium and around the bronchiole, in addition toarchitectural destruction. Collagen score is1.5at the4th of the experiment, and increased to6.92at the14th day, fibrosis obviously. There was fewerinflammation and fibrosis in saline-induced group and negative control group.Effect and mechanisms of CsA on bleomycin-induced murinepulmonary interstitial fibrosisTreatment of murine pulmonary interstitial fibrosisNinety-six C57BL/6female mice were assigned randomly into fourgroups: CsA30mg treatment group, CsA50mg treatment group, treatmentcontrol group and BLM model group, with24mice in each group. All groupswere administrated BLM5.0mg/kg (concentration:2.0mg/ml) intratracheallyto induce pulmonary interstitial fibrosis. The day of BLM injection wasassumed as day0of the experiment. At the beginning of day1, mice intreatment groups were intraperitoneal injected with CsA once daily (30mg/kg,50mg/kg), control treatment group were injected with equal volume of normalsaline instead, while no therapy was treated on BLM model group.Testing and researchOn the4th,7th and14th day after administration,8mice of each groupwere sacrificed,500μ L peripheral blood was obtained and combined withheparin sodium for anticoagulation. Leukocytes were counted by countingchamber. The proportion of CD4~+T cells, CD14~+monocytes and CD19~+Bcells were quantified by flow cytometry. The top-left lung was surgicallyremoved, stabilize with4%0.1PBS paraformaldenyde for24hours. Afterroutine dehydration and paraffin denvelopment, the3μ m laraffin slices werecut from the paraffin-embedded lung samples for HE and Masson staining,and for immunohistostaining to detect expression of CD147. Ashcroftstandard was used for evaluation of pulmonary interstitial fibrosis. Total BALF cell was counted by counting chamber, with its smears stained withGiemsa.ResultsPeripheral blood and BALF cell analyzationThe quantity of total leucocytes in BLM-induced mice treated with CsAwere (5.11±0.25)×10~9/L and (5.63±0.34)×10~9/L, much lower than those inBLM-induced mice (7.07±0.51)×10~9/L (P＜0.01). The proportion of CD14~+monocytes in BLM-induced mice treated with CsA was decreased markedly,especially on the4th day(＜3.2%), While CD19~+B cells were cut down on the7th and14th day. There was no significant difference statistically in thenumber of leucocytes, the proportion of CD14~+monocytes and CD19~+B cellsbetween BLM model group and treatment control group. The total cell countsof BALF in BLM-induced mice treated with CsA were also reduced at thebeginning of the treatment, with (4.47±0.88)×10~5/lung in30mg groupand(14.67±0.26)×10~5/lung in50mg group which were much lower thanBLM-induced mice (35.20±1.10)×10~5/lung (P＜0.01). Many types of cellswere decreased in BLM-induced mice treated with CsA, with lymphocytesreduced markedly, which was (16.56±6.44)/HP and (25.29±14.16)/HP. Andkeepping on this low level until14th day. Neutrophils was differentsignificantly at the early stage(7d、14d). There was no significant differencestatistically in the number of BALF total cells, lymphocytes, mononuclearmacrophage and netrophils between BLM model group and treatment controlgroup.Histologic examination of lung tissuesThe inflammation of pulmonary interstitial fibrosis in BLM-inducedmice treated with CsA was relieved markedly, with therapeutic effect of50mg group better than that of30mg treatment. There was lesser fibroblastsaggregation and collagen deposition within the interstitium and around thebronchiole, while the two treatment groups had no significant differencestatistically. With CsA treatment, collagen scores all less than2.0, indicatingimprovement of interstitial fibrosis in the lung. There was no significantdifference statistically of inflammation and fibrosis between BLM modelgroup and treatment control group.Expression of CD147in the murine lung by SP staining.CD147expressing cells such as mononuclear macrophages, neutrophilsand lymphocytes infiltrating lung tissue of BLM-induced mice, were reducedsharply in CsA treatment groups, even if there was no significant differencebetween the two groups statistically.ConclusionThis report meets with success in inducing pulmonary interstitial fibrosiswith BLM and establishing its research system, as well as treating with CsA.It was proved that the early manifestation of BLM-induced pulmonaryinterstitial fibrosis is generous aggregation of inflammatory cells to the lungtissue and spread all over the body, the inflammatory response was theninitiated and will contribute to lung injury and fibrosis at the later stage alongwith the disease development. Treatment with CsA earlier will not only inhibitthe inflammation, but also restrain disease development, suggesting that CsAmay inhibitted BLM-induced pulmonary interstitial fibrosis by blocking theinteraction of CD147-CyPA to reduce infiltration of lymphocytes, neutrophilsand mononuclear macrophages to the lung tissue. The research system and theresults of the experiment will lay the foundation of further clarification.