The Role Of Different Activated Macrophage Sub-phenotypes In Immune Escape Of Non-small Cell Lung Cancer

Background and ObjectiveMacrophages display remarkable plasticity and can change their physiology in response to environmental factors. These changes can give rise to different sub-populations of the cells with distinct functions. Operationally, two distinct activated states have been defined for macrophages:the M1 macrophages and M2 macrophages, each of them being associated with particular phenotypes and functions. M1 macrophages are generally considered to be potent effector cells that kill microorganisms and tumor cells and produce copious amounts of pro-inflammatory cytokines. In stark contrast, M2 are characterized by:poor antigen-presenting capability, producing factors that suppress T-cell proliferation.We have focused on macrophage heterogeneity in Non-small cell lung cancer. We aimed to determine that whether macrophages were polarized and the possible role of macrophage polarization in Non-small cell lung cancer. Cytometry was used to detect the proportion of M1 macrophages and M2 macrophages in non-small cell lung cancer and to determine whether the proportion is associated with clinicopathological parameters. Immunohistochemical staining for M1 and M2 macrophages symbol molecule was used to investigate whether these two distinct sub-phenotypes states of macrophages in tumour tissue impact the prognosis of patients with non-small cell lung cancer. Immunohistochemical staining for immune cells and cytokine was used to investigate the possible mechanism of macrophages different activation states.In the end, we investigate the mechanism of the differentiation of macrophages into M2 macrophages by cytokines in vitro.Materials and Methods1. The percentages of CD16+CD14+ macrophages and B7-H1+CD14+ macrophages in tumour tissue, pre-tumour tissue and peripheral blood from 22 patients with non-small cell lung cancer, and peripheral blood from 10 healthy control were determined by cytometry. Meanwhile, the relationship between clinicopathological parameters and the percentages of CD16+CD14+ macrophages and B7-H1+CD14+ macrophages was determined.2. Macrophage from tumour tissue and non-tumour tissue were isolated, and were cocultured with red blood cell to investigate the function of phagocytosis.3. Immunohistochemical staining for CD68,TNFα,CD16,CD206,B7-H1 was performed on paraffin embedded tissues from 47 NSCLC case. The results and the relative with clinicopathologic data and prognosis of NSCLC were analyzed.4. Immunohistochemical staining for IL-10,TGF-β1,foxp3,CD56 was performed on paraffin embedded tissues from 47 NSCLC case. The relative with IL-10 expression and macrophages different activation states were analyzed5. We used the cell separate from peripheral blood of healthy people, as the source of macrophages in vitro. We used IFN-γ+LPS,IL-10,Lung cancer cell A549,Lung cancer cell A549+IL-10 antibody to induce macrophages in vitro. Using unstimulated macrophages as control. macrophages stimulated with different method were detected for the levels of membrane proteins CD16 and B7-H1 by FACS, the chemokine production of TNF-αand IL-1ra by ELISA, macrophages engulf red blood cells for the function of phagocytosis, mixed lymphocyte reaction was used to exam stimulatory activity of macrophages.Result1. macrophage phenotypes and function in non-small cell lung cancer①There were increased percentages of B7-H1+CD14+ macrophages in peripheral blood from patients 3.91±1.54% compared with peripheral blood from healthy control 2.13±0.86%; in tumour tissue 42.88±3.52% compared with pre-tumour tissue 17.76±2.74% from patients with non-small lung cancer.②There were decreased percentages of CD16+CD14+ macrophages in tumour tissue 7.76±2.38% compared with pre-tumour tissue 17.76±2.52% from patients with non-small lung cancer, in peripheral blood from patients 20.35±3.54% compared with peripheral blood from healthy control 10.49±1.82%.that The proportion B7-H1+CD14+ macrophages and CD16+CD14+ macrophages is related to TNM stage and tumour size.③The percentage of macrophages engulf red blood cells 30.21±1.73% in tumor tissue is lower than the percentage of macrophages engulf red blood cells 38.11±2.35% in pre-tumor tissue.the phagocytosis index of macrophages engulf red blood cells 0.86±0.09 in tumor tissue is lower than The percentage of macrophages engulf red blood cells 0.97±0.15 in pre-tumor tissue.2. Clinical significance of macrophage phenotypes in non-small cell lung cancerTNF-a positive expression was detected on macrophage, and positive rate on macrophage in the cancer nests was 21.28% lower than the cancer margin 48.93 %and pre-tumor 42.10%tissue(P<0.05). CD 16 positive expression was detected on macrophage, and positive rate on macrophage in the cancer nests was 34.04% lower than the cancer margin 51.16% and pre-tumor tissue 78.94%(P<0.05). CD206 positive expression was detected on macrophage, and positive rate on macrophage in the cancer nests was 65.95% and in the cancer margin was 74.41 % higher than the pre-tumor tissue 36.84%(P<0.05). B7-H1 positive rate in the cancer nests was 48.93% and in the cancer margin was 74.41% higher than and pre-tumor tissue 31.57%(P<0.05). TNF-a and CD16 expression on macrophage in the cancer nests was negatively correlated with the tumor stage(P<0.05), positively with postoperative prognosis(P<0.05). B7-H1 and CD206 expression on macrophage in the cancer nests was positively correlated with the tumor stage(P<0.05), B7-H1 negatively with postoperative prognosis(P<0.05).3. the relationship between the non-small cell lung cancer microenviroment and macrophage phenotypes①the numbers of FOXP3+ cells and PD-1+ cells from tumour tissue were significantly higher than non—tumour tissue. the numbers of CD56+ NK cells from tumour tissue were significantly lower than non—tumour tissue. The tumour which expression of CD206 and B7-H1 have the higher numbers of FOXP3+ cells.②IL-10 positive expression were detected on cancer cells of non-small cell lung cancer, and positive rate was 70.21% higher than the pre-tumor lung tisse 26.31 %. The expression was positively correlated with the tumor stage(P<0.05), negatively with postoperative prognosis(P<0.05). The expression of IL-10 was negatively correlated with the expression of TNF-a and CD 16 (P<0.05), positively with the expression of CD206 and B7-H1 in tumor(P<0.05).4. The function of IL-10 on macrophage phenotypes①There was IL-10 expression on Lung cancer cell A549.②It was shown that macrophages stimulated with IFN-γ+LPS expressed higher level of CD 16 and TNF-a than the other four groups; while macrophages stimulated with IL-10 and Lung cancer cell A549 supernatants expressed higher level of B7-H1 and IL-1ra than the other three groups. The macrophages stimulated with IL-10 and Lung cancer cell A549 supernatants decrease the function of phagocytosis than the macrophages stimulated with IFN-γ+LPS. The stimulatory activity of macrophages in mixed lymphocyte reaction was decrease in IL-10 group and Lung cancer cell A549 supernatants group than the IFN-γ+LPS group.Conclusion1. There are sub-phenotypes of Ml macrophages and M2 macrophages in the non-small cell lung cancer. The number of these two type of macrophage in different site of tumour was different. There were more M2 macrophages in inside of tumor while there were more M1 macrophages in pre-tumour tissue. The expression of proteins on these two type of macrophages were correlated with the tumor stage and postoperative prognosis. The above results suggested that most of the macrophages were M1 macrophages at the early stage of the tumors, while most of the macrophages were M2 macrophages at the later stage of the tumors.2. IL-10 positive expression was detected on non-small cell lung cancer cells, was positively correlated with the tumor stage, and negatively with postoperative prognosis.There was negative relationship between the expression of IL-10 and the number of M1 macrophage, and positive relationship between the expression of IL-10 and the number of M2 macrophage. The results suggested that IL-10 may play an important roll in M2 macrophages differentiation.3. There was positive relationship between the number of Trcg T cell and M2 macrophage, the results imply that Trcg T cell might play an important role in immune escape of the non-small cell lung cancer.4. There was IL-10 expression on Lung cancer cell A549. In vitro, The molecules of M1 macrophage, like CD 16 and TNF-α,could be induced by IFN-y+LPS,while the molecules of M2 macrophage, like B7-H1 and IL-1ra could be induced by IL-10 and A549 supernatants. The phagocytic function of macrophage could be decreased when the co-culture of macrophage with IL-10 and Lung cancer cell A549 supernatants, and could be increased when the co-culture of macrophage with IFN-y+LPS. The results indicated that IL-10 induce macrophages to differentiate into M2 macrophages in vitro.