Following liver injury, hepatic stellate cells (HSC) undergo proliferation and migrate into damaged areas in response to chemotactic factors. HSC have been shown to regulate leukocyte trafficking by secreting monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes and lymphocytes. In this study, we explored whether MCP-1 exerts biological actions on HSC. HSC were isolated from normal human livers, cultured on plastic, and studied in their myofibroblast-like phenotype, and three different cells lines were used. Chemotaxis was measured in modified Boyden chambers. Phosphatidylinositol 3-kinase (PI 3-K) was assayed on phosphotyrosine immunoprecipitates. Exposure of HSC to MCP-1 stimulated migration of HSC in a dose-dependent fashion. Maximal stimulation was obtained with 250 ng/mL MCP-1, which resulted in a 3-to 4-fold stimulation of cell migration. Checkerboard analysis showed that the increase in cell migration was almost completely a result of chemotaxis rather than chemokinesis. In contrast, in quiescent HSC, MCP-1 did not exert any effect on cell migration. In leukocytes, MCP-1 activates the pertussis toxin-sensitive CCR2 receptor. However, transcripts for CCR2 could not be shown in HSC, and pertussis toxin only modestly inhibited MCP-1-induced migration. Exposure of HSC to MCP-1 was associated with an increase in cytosolic calcium concentration, PI 3-K activity, protein tyrosine phosphorylation. Blocking calcium influx or pretreatment of HSC with the PI 3-K inhibitor wortmannin markedly reduced cell migration. This study shows, for the first time, a potential direct profibrogenic action of MCP-1 via HSC chemotaxis. MCP-1-dependent signals in these cells are not transduced by CCR2 and may be mediated by alternative chemokine receptors. (HEPATOLOGY 1999;29:140-148.)
The tissue response to injury involves the coordinated recruitment and activation of a number of cells in the attempt to repair the damage provoked by toxic, infectious, or immunological mechanisms. Inflammatory cells recruited at sites of damage are responsible for the scavenging of the necrotic cells, whereas myofibroblasts secrete extracellular matrix components and restore the integrity of the tissue. Within the liver, hepatic stellate cells (HSC) are responsible for this second part of the wound-healing response. 1,2 In normal liver, HSC fulfill the role of retinoid storage and metabolism, and their phenotype is referred to as quiescent. However, following injury, HSC undergo differentiation toward an activated phenotype characterized by proliferation and increased secretion of extracellular matrix components. This process is associated with enhanced or de novo expression of receptors for several soluble mediators, such as platelet-derived growth factor (PDGF), transforming growth factor-β€, or thrombin, which mediate the increase in cell proliferation and extracellular matrix production. [3][4][5] Therefore, HSC are the main cell type involved in the deposition of matrix that leads to fibrosis and cirrhosis. Another feature of cells involved in tissue repair is their ability to migrate into the damaged areas according to concentration gradients of chemotactic factors. 6 We have recently shown that HSC share this ability to respond to chemotactic factors such as PDGF. 7 Recent investigation has pointed out additional characteristics of the HSC that are relevant for the hepatic wound healing response. 2 HSC have been shown to express several molecules that are capable of regulating leukocyte trafficking, including chemokines. [8][9][10][11][12] These latter are a group of cytokines that exhibit chemoattractant properties for relatively specific groups of leukocytes. Four classes of chemokines have been recognized according to the position of conserved cysteine residues and differences in the spectrum of target cells. 13 The group of CXC chemokines includes a variety of factors, such as interleukin-8, which are mainly, but not exclusively, chemotactic for neutrophils. 13 Lymphotactin, a cytokine that specifically attracts lymphocytes, is the only known member of the C class of chemokines. 13 A novel cell-associated chemokine characterized by a CX3C motif and higher molecular weight has been recently identified. 14 Chemokines of the CC groups include molecules where two conserved cysteine residues are adjacent, and are generally chemotactic for monocytes and lymphocytes. Members of this group include monocyte chemotactic protein (MCP)-1, -2, -3, -4, and -5, macrophage inflammatory protein-1β£ and 1β€, and RANTES. 13 Studies conducted by our group and by other investigators have indicated that HSC express and secrete different members of the CC chemokine subfamily, including MCP-1 and macrophage inflammatory protein-1. 9,15,16 Several lines of evidence indicate that MCP-1 may Abbreviations: HSC, hepatic stellate cells; PDGF, platelet-derived growth factor; MCP-1, monocyte chemotactic protein-1; ERK, extracellular signal-regulated kinase; PBMC, peripheral blood mononuclear cells; cDNA, complementary DNA; PCR, polymerase chain reaction; PI 3-K, phosphatidylinositol 3-kinase; PTX, pertussis toxin; EGTA, ethylene glycol-bis(β€-aminoethyl ether) N,N,NΠ,NΠ-tetraacetic acid.
From the Istituto di Medicina Interna and