We found that macrophages, rather than cholangiocytes, produce chemoattractants in response to RRV infection. Using an in vitro infection system of a cholangiocyte cell line previously shown to be susceptible to RRV and to express an array of cytokines and chemokines (6
), we did not find support for a direct role of these cells in the promotion of chemotaxis of inflammatory cells. Searching for other cells with potential role in the regulation of the inflammatory response in experimental biliary atresia, we detected RRV in hepatic mononuclear cells 3–14 days after infection, and identified the virus in macrophages. Using the macrophage cell line Raw 264.7 to examine the role of these cells in production of chemoattractants to inflammatory cells, we found that they secrete pro-inflammatory cytokines previously linked to pathogenesis of biliary atresia [examples: IFNγ and TNFα, ref (6
)] and high levels of the chemokine Mip2/Cxcl2. Most notably, conditioned media from RRV-infected macrophages induced prominent migration of neutrophils, which was dependent on Mip2/Cxcl2. These data identify macrophages as a new cellular target of RRV in experimental biliary atresia and point to its role as a source of soluble mediators that amplify the inflammatory population of the hepatobiliary environment.
Cholangiocytes are key epithelial targets of RRV and undergo injury by hepatic NK and CD8+ cells during pathogenesis of experimental biliary atresia (6
); hepatocytes are also susceptible to RRV, but do so at a lower multiplicity of infection. The injury by inflammatory cells might relate to the recognition of viral epitopes in infected cells and/or to aberrant expression of MHC-associated molecules. However, recent work by another laboratory showed that RRV-exposed cholangiocytes do not appear to function as antigen-presenting cells (12
). Based on the findings of increased expression of chemokines by cholangiocytes, investigators have suggested that infected cells may play a role in immunomodulation (11
). Addressing this scenario, we first found an increase in the mRNA expression for cytokines and chemokines, but the expression at the protein level was either below the detectable levels by ELISA (for TNFα, IFNα, INFβ, INFγ, and IL1β) or still very low when compared to the levels produced by the macrophage cell line (for Mip2). The reasons for the discrepancy between the levels of mRNA expression detected by real-time PCR and the protein levels are not obvious, but may include the variable rates of translation of mRNA transcripts. Regardless of the cause for the discrepancies, we did not find support for the release of biologically sufficient amounts of chemoattractants to inflammatory cells by cholangiocytes. It is possible that the ongoing production of chemokines by cholangiocytes for expanded periods of time (beyond the 24 hours investigated in our studies) could generate a higher concentration of chemoattractants. However, we chose to stay within the constraints of 24 hours of infection in order to simulate early biological events (i.e., periductal inflammation and cholangiocyte injury) induced in the model of experimental biliary atresia (6
). The lack of support for a role of cholangiocytes in antigen presentation or as a source of chemoattractants does not negate the possibility that cholangiocytes are critical elements of the pathogenesis of biliary atresia. For example, RRV-harboring cholangiocytes are cellular targets of NK and CD8+ cells. Further, they must maintain mucosal continuity along the intra- and extra-hepatic biliary tracts. When this continuity is disrupted by NK cell-mediated injury, duct damage occurs and the phenotype of experimental biliary atresia emerges over time. If NK cells are depleted in vivo, cholangiocyte injury is negligible and the atresia phenotype is prevented (22
The detection of RRV proteins in hepatic macrophages expands the types of cells that are initially targeted by the virus in the neonatal liver, and formally implicates the innate immune system in pathogenesis of experimental biliary atresia. It is possible that the RRV signal observed in hepatic macrophages represents viral antigens that have been processed and are being presented on the cell surface. If this is correct, macrophages may serve as antigen-presenting cells to induce an adaptive response targeted to bile ducts. Our data adds an additional role for macrophages as producers of inflammatory mediators that recruit neutrophils to the site of infection. One of these mediators is Mip2/Cxcl2, as supported by the induction of Mip2/Cxcl2-dependent chemotaxis of neutrophils by conditioned media from RRV-infected Raw 264.7 cells and by the increased expression of Mip2/Cxcl2 mRNA in livers as early as 3 days after RRV infection. We do not know whether Mip2/Cxcl2-dependent chemoattraction of neutrophils is essential to the pathogenesis of bile duct injury in vivo, but the data reported herein form the basis for this line of future investigation.
Patient-based studies have implicated macrophages in pathogenesis of biliary atresia. The enriched expression of the lipopolisaccharide receptor CD14 in Kupffer cells and the population of the livers of children with biliary atresia by activated macrophages suggest that these cells contribute to the creation of a pro-inflammatory environment at diagnosis and/or after portoenterostomy (24
). At least in one report, a greater population of macrophages in the affected liver was associated with poor outcome (25
). Despite the lack of evidence of active viral infection at the time of diagnosis in most patients with biliary atresia, a previous exposure to viral proteins may be sufficient for the induction of pro-inflammatory signals. For example, the non-structural protein 4 (NSP4) of RRV has been shown to trigger the expression of inducible-nitric oxide synthase in ileal macrophages after RRV infection (19
). While the use of cell culture systems and the experimental model of rotavirus-induced biliary atresia in newborn mice are powerful tools to understand components of operative biological processes, it is important to recognize that findings from either system require validation in tissues of affected children. Further dissection of the role of macrophages in pathogenesis of disease, either as an antigen-presenting cell or as one of the cellular targets of a viral insult, will also require the use of in vitro systems using human cells. This line of studies are likely to decipher how macrophages or macrophage-derived signals directly regulate mechanisms of disease and can be potential therapeutic targets to block progression of disease to end-stage cirrhosis.