In initial experiments, we determined if primary cultures of rat hepatic sinusoidal endothelial cells undergo phenotypic polarization in response to inflammatory mediators known to induce classical and alternative macrophage activation. For comparison purposes we also analyzed the response of rat Kupffer cells. Incubation of Kupffer cells with the classical macrophage activators IFNγ and LPS, alone or in combination, resulted in increased expression of iNOS protein, with no effect on arginase-1 and decreased expression of mannose receptor (). Similar results were observed in hepatic endothelial cells; however the responses to LPS and LPS + IFNγ were reduced, when compared to Kupffer cells. In endothelial cells, IFNγ alone was also less effective in inducing iNOS expression than LPS. To assess alternative activation, cells were treated with IL-10, or the combination of IL-4 + IL-13. In both endothelial cells and Kupffer cells, IL-10 treatment resulted in increased expression of mannose receptor; a small increase in arginase-1 was also observed in endothelial cells. The combination of IL-4 + IL-13 upregulated expression of arginase-1In both cell types, while mannose receptor was only upregulated in endothelial cells. In general, endothelial cells were more sensitive to these cytokines than Kupffer cells. Neither IL-10 nor IL-4 + IL-13 altered iNOS protein expression.
Fig. 1 Classical and alternative activation of Kupffer cells and endothelial cells. Cells, incubated overnight in 12 well dishes (1×106 cells/well), were washed and then incubated with PBS, IFNγ (10 ng/ml), LPS (100 ng/ml), IFNγ + LPS, (more ...)
We also analyzed mRNA expression for iNOS, arginase-1 and mannose receptor in endothelial cells and Kupffer cells treated with inducers of classical and alternative macrophage activation. Consistent with our findings on iNOS protein, iNOS mRNA expression increased in both liver macrophages and endothelial cells in response to IFNγ and/or LPS, with no major effects on arginase-1 or mannose receptor expression ( and not shown). Additionally, iNOS mRNA expression was not altered by incubation of the cells with IL-10 or the combination of IL-4 + IL-13. In endothelial cells, both IL-10 and IL-4 + IL-13 upregulated arginase-1 and mannose receptor mRNA; in Kupffer cells, IL-10 upregulated mRNA for mannose receptor, while IL-4 + IL-13 upregulated arginase mRNA.
Fig. 2 Expression of mRNA for markers of classical and alternative activation in Kupffer cells and endothelial cells. Cells, incubated overnight in 6 well dishes (2×106 cells/well), were washed and then incubated with PBS, IFNγ, IL-10, or IL-4 (more ...)
We next determined if cytokine-induced activation of hepatic endothelial cells and macrophages persisted in the absence of inflammatory stimuli. In these experiments the cells were incubated with IFNγ, IL-10, or IL-4 + IL-13 for 48 h, washed, refed with fresh culture medium without cytokines, and incubated for an additional 24 h prior to analysis. We found that removal of IFNγ from the cultures had no effect on expression of iNOS protein by macrophages or endothelial cells (). Whereas in Kupffer cells, removal of IL-10 had no effect on mannose receptor expression, in endothelial cells, expression of this receptor increased. Similar increases in mannose receptor expression were noted in endothelial cells, but not Kupffer cells, after removal of IL-4 + IL-13.
Fig. 3 Persistence of a polarized phenotype in Kupffer cells and endothelial cells in the absence of inflammatory stimuli. Cells, incubated overnight in 12 well dishes (1×106 cells/well), were washed and incubated with PBS, IFNγ, IL-10, or IL-4 (more ...)
In further experiments, we assessed whether cytokine-induced phenotypic polarization of hepatic endothelial cells and macrophages was reversible. As described above, treatment of both endothelial cells and macrophages with IFNγ for 48 h resulted in increased iNOS expression, while IL-4 + IL-13 treatment upregulated arginase-1 expression (). Subsequent washing and incubation of IFNγ treated cells with IL-4 + IL-13 resulted in increased expression of arginase-1 and decreased expression of iNOS. Conversely, washing and incubation of IL-4 + IL-13 treated cells with IFNγ resulted in increased iNOS expression and decreased arginase-1 expression. In general endothelial cells appeared to be more sensitive to phenotypic switching than Kupffer cells.
Fig. 4 Reversible activation of Kupffer cells and endothelial cells. Cells, incubated overnight in 6 well dishes (2×106 cells/well), were washed and then incubated with PBS, IFNγ or IL-4 + IL-13. After 48 h, the cells were washed and IFNγ (more ...)
In our next series of studies, we assessed the consequences of classical and alternative activation of endothelial cells on hepatocytes. In these experiments, endothelial cells were stimulated with IFNγ and/or LPS, IL-10, or IL-4 + IL-13 for 48 h, washed, and then co-cultured in transwell dishes with hepatocytes. In some experiments hepatocytes were pretreated for 2 h with control or with 5 mM acetaminophen, prior to co-culturing with endothelial cells. Hepatocyte viability was assessed 24 h later. Whereas endothelial cells classically activated with LPS and IFNγ had no effect on control hepatocytes, a 30% decrease in viability was observed in hepatocytes pretreated with acetaminophen (). In contrast, the viability of control hepatocytes was increased in the presence of endothelial cells alternatively activated with IL-4 + IL-13. In acetaminophen pretreated hepatocytes, however, IL-4 + IL-13 activated endothelial cells caused a 20% decrease in hepatocyte viability. Additionally, IL-10 treated endothelial cells suppressed the viability of both control and acetaminophen-injured hepatocytes.
Effects of classically and alternatively activated endothelial cells on hepatocyte viability
Since alternatively activated macrophages are known to promote tissue repair, we next analyzed markers of proliferation (PCNA and β-catenin) and extracellular matrix turnover (MMP-9) in hepatocytes. Consistent with this activity, we found that expression of PCNA, β-catenin and MMP-9 increased in hepatocytes co-cultured with IL-4 + IL-13 activated Kupffer cells (). IL-10 treated Kupffer cells also upregulated hepatocyte MMP-9 expression. Hepatocyte PCNA and β-catenin expression also increased in the presence of control Kupffer cells; IFNγ pretreated Kupffer cells upregulated hepatocyte PCNA. Endothelial cells alternatively activated with IL-4 + IL-13, as well as IL-10, were also found to upregulate markers of tissue repair in hepatocytes. Thus, hepatocyte expression of β-catenin and MMP-9 increased in the presence of endothelial cells activated with IL-4 + IL-13 or IL-10 (). IL-10 treated endothelial cells also upregulated PCNA expression in hepatocytes. In contrast to Kupffer cells, however, endothelial cells classically activated with IFNγ had no effect on hepatocyte expression of these markers. Additionally, while control endothelial cells upregulated hepatocyte MMP-9 expression, KC upregulated hepatocyte PCNA and beta-catenin.
Fig. 5 Effects of NPCs on hepatocyte expression of markers of proliferation and extracellular matrix turnover. Kupffer cells and endothelial cells, cultured overnight on transwell inserts in 24 well dishes, were washed and incubated with PBS, IFNγ, IL-10 (more ...)
In previous studies we demonstrated that injured hepatocytes release factors that induce classical activation of macrophages (Dragomir et al., 2011
; Laskin et al., 1986
). In further experiments, we determined if hepatocytes exert similar effects on sinusoidal endothelial cells. For these studies, endothelial cells were co-cultured in transwell dishes with hepatocytes pretreated for 2 h with control or 5 mM acetaminophen, in the absence or presence of classical and alternative macrophage activators. Endothelial cell expression of iNOS and mannose receptor was assessed 48 h later. Whereas the addition of LPS alone or in combination with IFNγ to co-cultures containing control hepatocytes resulted in increased expression of iNOS in endothelial cells, IL-10 or IL-4 + IL-13 caused an increase in mannose receptor expression (). The effects of these inflammatory stimuli on endothelial cells were markedly enhanced in co-cultures containing acetaminophen injured hepatocytes. Co-culture of endothelial cells and hepatocytes in the absence of inflammatory stimuli had no effect on endothelial cell expression of iNOS or mannose receptor.
Fig. 6 Regulation of endothelial cell activation by hepatocytes. Hepatocytes (HC), cultured overnight on transwell inserts in 24 well dishes, were washed and treated with medium control (Control HC) or medium containing 5 mM APAP (Injured HC). After 2 h, hepatocytes (more ...)