Study On The Role Of Macrophage Subsets And Lunx MRNA In The Pleural Effusion

Background: Lung cancer and tuberculosis infection are the main factors for the formationof pleural effusion. Macrophages are infiltrating components in the pleural fluid. Macrophagescan phagocytize pathogen, scavenge the foreign sunstance and dead cells, and as a type ofantigen-presenting cells in the innate immune system, can efficiently present antigen to T cells intissue, which results in T cell activation. Macrophages acquire distinct functional phenotypes,depending on the microenvironment, and macrophages can be classically activated as M1oralternatively activated as M2. M1macrophages exert the anti-inflammatory and anti-tumor effect,while M2macrophages repair the inflammation and induce the growth of tumor. The differentactivation states of macrophages determine their role in different diseases. Macrophages are thefirst line to defend the mycobacterium tuberculosis (M.tb) infection. Whether the M. tb cansurvive or reproduce is closely related to the functional status of macrophages;on the other hand,tumor-associated macrophages (TAMs) promote tumor growth and angiogenesis via releasingmany chemokines, and induce the degradation of the matrix, and further promote tumor invasionand metastasis. The role of macrophages in patients with tuberculous (TPE) or malignant pleuraleffusion (MPE) in patients remains to be further studied. In addition, in recent years, althoughdiagnosis technology of cancer has made considerable progress, there are still some difficultiesfor the identification of benign and malignant pleural effusion. Cytology is the most importantmethod to distinguish between benign and malignant pleural effusion. However, hyperplasticmesothelial cells, rhagiocrine cells, and degenerative mesothelial cells often display specialmorphological characteristics in the pleural effusion, which makes it difficult to identify thetumor cells. Lunx is distinctively expressed in pulmonary carcinoma, with little or no expressionin other human tumors and normal tissues. The detection of Lunx mRNA maybe helpful for theidentification between benign and malignant effusion. Therefore, this study was to determine thechanges of macrophage subsets in TPEs and MPEs, and provide a theoretical basis for furtherresearch. In addition, we assess the clinical significance of Lunx mRNA detection in diagnosingMPEs caused by pulmonary carcinoma.Aim：The study is aimed to exam the role of different subsets of macrophages in pleural fluid (PF) and peripheral blood (PB) from patients with new onset TPEs and MPEs andinvestigating the role of macrophages in the pleural effusions, and further to assessing theclinical significance of Lunx mRNA detection in diagnosing MPEs caused by pulmonarycarcinoma.Methods：The numbers of PB and PF macrophage subsets in patients with new onset TPEand MPE were determined by flow cytometry. The concentrations of serum and PF cytokineswere determined by cytometric bead array (CBA) and enzyme-linked immunosorbentassay(ELISA). The potential association between the cytokines and the values of clinical measureswere analyzed. The levels of Lunx mRNA in the PF were determined by real-time PCR. Theclinical parameters including blood counts, erythrocyte sedimentation rate (ESR), T.SPOT.TB,PF Potential of hydrogen (PH), lactate dehydrogenase (LDH), glucose, albumin,carcino-embryonic antigen (CEA), cast-off and et al. were completed by the trained specialist.Results：1. The numbers of PB CD14+CD163-and CD14+CD163-IL-12+M1macrophagesin the patients with onset TPE were significantly greater than those in the HC, but less thanthose in the PF of the patients. The concentrations of serum IL-12were correlated positivelywith the numbers of PB CD14+CD163-IL-12+M1in the patients2. The numbers of CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CD115+,CD14+CD163+CD206+CD115+M2in the PB were significantly greater than those in the PF ofonset TPE patients.3. The numbers of PB CD14+CD163-MAC387+and CD14+CD163-MAC387-M1macrophages in the patients with onset TPE were significantly greater than those in the HC, butless than those in the PF of the patients.4. The concentrations of serum IL-1, IL-6, IL-8, IL-12, TNF, and TGF-in the onsetTPE patients were significantly higher than that in the HC, but lower than that in the PF of thepatients. In contrast, there was no significant difference in the levels of serum IL-10between theHC and patients, but the levels of serum IL-10were significantly lower than that in the PF ofpatients.5. The levels of serum TNF-and IL-12was correlated positively with the values of ESRand the numbers of ESAT-6-and CFP-10-specific IFN-γ secreting T cells in the onset TPEpatients. In addition, the levels of PF TNF-were correlated positively with the concentrationsof ADA and LDH in the patients.6. The numbers of PB CD14+CD163+and CD14+CD163+IL-10+M2macrophages in thepatients with MPE were significantly greater than those in the HC, but less than those in the PF of the patients. The numbers of PB CD14+CD163-IL-12+M1macrophages in the patients weresignificantly less than those in the HC, but greater than those in the PF of the patients.7. The numbers of CD14+CD163+, CD14+CD163+CD206+, CD14+CD163+CD115+,CD14+CD163+CD206+CD115+M2in the PB of MPE patients were significantly greater thanthose in the HC, but less than those in the PF of the patients.8. The numbers of PB CD14+CD163+MAC387+M2and CD14+CD163+MAC387-M2macrophages in the patients with MPE were significantly greater than those in the HC, but lessthan those in the PF of the patients.9. The concentrations of serum IL-1, IL-6, IL-8, IL-10, TNF, and TGF-in the MPEpatients were significantly higher than that in the HC, but lower than that in the PF of thepatients. However, there was no significant difference in the levels of serum IL-12between theHC and patients.10. The levels of PF TNF-and IL-12was correlated positively with the values of CEAand LDH in the MPE patients, and the levels of PF IL-10were correlated positively with theconcentrations of CEA in the patients11.The specificity and sensitivity of Lunx mRNA for the diagnosis of MPE caused bypulmonary carcinoma.were95.9%and84.9%. The area under the ROC curve was0.922, andhigher than0.821for cast-off cells or0.798for CEA.12. Lunx mRNA is distinctively expressed in pulmonary carcinoma. All of theLunx-positive patients with MPEs were diagnosed with pulmonary carcinoma, and allextrapulmonary carcinoma patients were Lunx-negative. The positive predictive value of LunxmRNA for the source of tumor cells was100%.13. Lunx mRNA expression for MPE patients caused by pulmonary carcinoma decreasedafter the first session of chemotherapy in the CR and PR groups, increased in the PD group,there was no change in the NC group14. The median overall survival for MPE patients caused by pulmonary carcinoma was53weeks in the decreased Lunx mRNA expression group, and it was25weeks in the increasedLunx mRNA expression group. The patients in the decreased Lunx mRNA expression grouphad longer overall survival times than those in the increased Lunx mRNA expression groupConclusions:1. M. tb infection induced a polarized pro-inflammatory M1response in thepatients with onset TPE, especially at the pathological site. The data indicate that the increasedM1responses contribute to the early pathogenesis of TPE in humans. 2. M2macrophages are predominant in TAMs in the PF of MPE patients, which furtherindicate the important role of M2macrophages in the pathogenesis of MPE.3.Lunx mRNA is an effective marker of pulmonary carcinoma, and should berecommended to use in the clinic. To monitor the Lunx mRNA expression levels may help thedoctor predict the response of patients to the chemotherapy, understand the stage of the diseases,and further assess the prognosis.