Interaction Between Hepatic Stellate Cell And Kupffer Cell In Liver Fibrosis

BackgroundA variety of etiologies can lead to liver fibrosis, such as viruses, alcohol, andparasites. Liver fibrotic progression can be partly inhibited by removing orcontrolling the causes, but the fibrosis persists in some exceptions. Betterunderstanding of the fibrosis mechanism will afford opportunities to directly targetthe fibrotic process and develop the therapies of inhibiting or reversing liver diseaseprogression.Liver fibrosis is the result of an imbalance of liver injury and repair. Thisprocess mainly related to two important cells: hepatic stellate cells and Kupffer cells.Hepatic stellate cells mainly produced the predominant extracellular matrix, and itsactivation and apoptosis are closely related to liver fibrosis progression andreversion. Kupffer cells take an important part in the liver innate immune. It alsoplays an important role in the process of liver injury and repair. Besides, both cellslocated in the Disse space of the liver. The hepatic stellate cells and the Kupffer cellsbecome the focus of liver fibrosis research for their functional and spatial effects.Objective1. To observe the inflammatory and fibrotic situation in healthy liver andfibrotic liver, and their activation of hepatic stellate cells and accumulation ofKupffer cells.2. To investigate the interaction of polarizing and polarized macrophage withhepatic stellate cell line LX-2cells.Materials and Methods 1. The cirrhotic liver collected from the liver transplant recipient, while thenormal liver collected from the donor. Due to the limited number of stellate cells andKupffer cells from human liver tissue, we used LX-2cell lines instead of hepaticstellate cells and isolated peripheral blood monocytes polarized macrophages insteadof Kupffer cells. The peripheral blood was bought from Changchun Blood DonorCenter.2. The inflammatory genes (IL-1β, IL-6, IL-10, IL-12, TNF-α) and fibrosisrelated genes (α-SMA, Col1A1, TIMP-1) of liver tissue or perfusate were tested byreal-time quantitative polymerase chain reaction (RT-qPCR). We studied thedifference of hepatic stellate cells and Kupffer cells by immunohistochemistry.α-SMA was used to identify hepatic stellate cells and CD68was used to identifyKupffer cells.3. We used monocyte-macrophage colony stimulating factor and macrophagecolony-stimulating factor to induce M1and M2macrophages, respectively.Cell-surface protein expressions of differentiated macrophages were assessed byflow cytometry. Expression of relevant genes was measured by RT-qPCR.Production levels of tumor necrosis factor-a (TNF-a) and IL-10and were assessedusing enzyme-linked immunosorbent assay (ELISA) method.4. Part of Kupffer cells in the liver derives from the peripheral blood, so theprocess of interaction between LX-2cells and polarizing macrophages was just inthe same stimulation process. Most Kupffer cells in the liver located in the sinusoidand its phenotype changes when the environment changes. The process ofinteraction between LX-2cells and polarized macrophages was just in this process.The macrophages and LX-2cells were co-cultured in a5:1ratio. Phenotype ofdifferentiated macrophages were assessed by flow cytometry. Expression of relevantgenes and production of TNF-a and IL-10was measured by RT-qPCR and ELISA,respectively. Results1. In fibrotic liver, the expression of pro-inflammatory genes (IL-1β, IL-6,IL-12, TNF-α) increased and anti-inflammatory genes (IL-10) decreased. Theexpression of fibrosis-related genes (α-SMA, Col1A1, TIMP-1) also increased.Multi-regression correlation analysis showed that the expression ofpro-inflammatory genes positively correlated with the fibrosis related genes and theanti-inflammatory genes negatively correlated, whereas MMP-2have no significantcorrelation. In addition, the activated hepatic stellate cells and Kupffer cellsaccumulated in the fibrotic liver.2. M1macrophage shaped like fried egg and can express and secret higherpro-inflammatory cytokines IL-6, IL-12, TNF-α and lower anti-inflammatorycytokine IL-10. M2macrophage was elongated, and could express and secret higheranti-inflammatory cytokines IL-10, but less pro-inflammatory cytokines IL-6, IL-12,TNF-α.3. The polarizing macrophages, either M1macrophages or M2macrophages,could promote LX-2cells express more fibrosis-related genes (α-SMA, Col1A1,TIMP-1) and regulative fibrosis genes (MMP-2, MMP-9). There were no significantdifference between the two types of macrophages. LX-2cells could also affectmacrophages polarization process, whether it is to M1or to M2macrophages, theLX-2cell induced macrophages have shown the similar phenotype and function.4. The polarized macrophages, either M1macrophages or M2macrophages,could promote LX-2cells express more fibrosis-related genes (α-SMA, Col1A1,TIMP-1) and regulative fibrosis genes (MMP-2, MMP-9), but the M1macrophagesshowed a stronger pro-fibrotic ability. LX-2cells could also affect macrophagespolarization process, whether M1or M2macrophages, the LX-2cell inducedmacrophages could secrete more IL-10and TNF-α compared with the originalpolarized M1and M2macrophages, which, M1macrophages secreted more TNF-αand M2macrophages secreted more IL-10. Conclusions1. The fibrotic liver had more Kupffer cells and activated hepatic stellate cells,it also had more extracellular matrix and was in pro-inflammation status. Theexpression of fibrosis-related genes α-SMA, Col1A1were positively correlated withthe pro-inflammatory genes, and negatively with the anti-inflammatory genes.2. Different types of macrophages all could activate the LX-2cells and thuspromoting the expression of pro-fibrosis genes. Moreover, the LX-2cells couldimprove the original function of polarized macrophages.