Abstract
BackgroundC5a and its cognate receptor, C5a receptor (C5aR), key elements of complement, are critical modulators of liver immunity and fibrosis. However, the molecular mechanism for the cross talk between complement and liver fibrosis is not well understood. C5a is a potent chemokine regulating migration of cells in the innate immune system. Since activation and migration of hepatic stellate cells (HSC) are hallmarks of liver fibrosis, we hypothesized that C5a contributes to fibrosis by regulating HSC activation and/or migration.ResultsPrimary cultures of mouse HSC increased expression of alpha smooth muscle actin (α-SMA) and collagen 1A (Col1A1) mRNA in response to activation on plastic. Expression of mRNA for C5aR, but not C5L2, a second C5a receptor that acts as a negative regulator, increased in parallel with markers of HSC activation in culture. Increased expression of C5aR on activated HSC was confirmed by immunocytochemistry. Cell surface expression of C5aR was also detected by flow cytometry on activated HSC isolated from mice expressing GFP under the control of the collagen promoter after exposure to chronic carbon tetrachloride. To understand the functional significance of C5aR expression in HSC, we next investigated whether C5a influenced HSC activation and/or migration. Challenge of HSC with C5a during culture had no effect on expression of α-SMA and Col1A1, suggesting that C5a did not influence HSC activation. Another important characteristic of HSC is their migratory capacity; migration of HSC in response to platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) has been well characterized. Challenge of HSC with C5a enhanced HSC migration almost as efficiently as PDGF in a two-dimensional wound healing and Boyden chamber migration assays. C5a also stimulated expression of MCP-1. C5a-induced cell migration was slowed, but not completely inhibited, in presence of 227016, a MCP-1 receptor antagonist, suggesting C5a-induced migration occurs via both MCP-1-dependent and -independent mechanisms.ConclusionsThese data reveal that C5a regulates migration of HSC and suggest a novel mechanism by which complement contributes to hepatic fibrosis. C5a and its receptors are therefore potential therapeutic targets for the prevention and/or treatment of liver fibrosis.
Highlights
Complement component 5a (C5a) and its cognate receptor, C5a receptor (C5aR), key elements of complement, are critical modulators of liver immunity and fibrosis
While expression of C3aR, C5aR, and C5L2 was low in quiescent hepatic stellate cells (HSC), expression of mRNA for both C3aR and C5aR, but not C5L2, increased in parallel with α-smooth muscle actin (α-SMA) and Col1A1 mRNA from day 2 to day 10 in culture (Figure 1C)
Activated HSC isolated from mice treated with Carbon tetrachloride (CCl4) express C5aR and C5L2 While the phenotype of primary HSC activated in culture typically models the phenotype of HSC activated in the liver in response to fibrotic insult, it is critical to validate results of cell culture experiments with data from HSC activated in vivo
Summary
C5a and its cognate receptor, C5a receptor (C5aR), key elements of complement, are critical modulators of liver immunity and fibrosis. C5a is a potent chemokine regulating migration of cells in the innate immune system. Since activation and migration of hepatic stellate cells (HSC) are hallmarks of liver fibrosis, we hypothesized that C5a contributes to fibrosis by regulating HSC activation and/or migration. Hepatic stellate cells (HSC), known as Ito cells or lipocytes, are the major cell type contributing to liver fibrosis. Complement, comprised of a large number of distinct plasma proteins, as well as membrane-bound receptors and regulatory proteins, is a critical component of the innate immune system that provides links to adaptive immunity [9]. C5a, an anaphylatoxin, induces or amplifies multiple innate immune responses. C5a binding to C5aR induces rapid auto-phosphorylation, internalization, and subsequent signalling leading to acute inflammatory responses and chemotaxis [11,12]
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