CSN1S1 alters the morphology of monocytes
After incubation of primary human monocytes with recombinant CSN1S1, changes in cellular morphology were noted. Living cells became adherent to the cell culture dish when incubated for 24 h with 1 μg/ml CSN1S1 and the development of pseudopodia was noted in some of the cells when stimulated with higher concentrations (Figure 1). These effects were not observed in the control cultures without CSN1S1, or in case lower concentrations of CSN1S1 (1 ng/ml or 100 ng/ml) were used (data not shown). For comparison, primary human monocytes were incubated with up to 200 ng/ml LPS, which had no visible effect on cellular morphology (Figure 1).
For a better characterization of cellular morphology, primary cells were cultured in chamber slides and stained. Morphology of living cells suggested a differentiation towards DC or macrophages. Thus, we included primary cells stimulated with GM-CSF or GM-CSF/IL4, and M-CSF or M-CSF/IFNγ for comparison to CSN1S1, as the latter stimulants are known to mediate in vitro differentiation towards the respective cell types[18, 19]. Although we observed rapid morphologic changes in CSN1S1 stimulated cells, in vitro differentiation of monocytes is commonly carried out over 5 days. Thus, cells were incubated with the stimulants for both, 24 h and 120 h. As can be seen in Figure 2, after 24 h, control cells were round with little cytoplasm. In contrast, GM-CSF and GM-CSF/IL-4 induced enlarged cytoplasm. M-CSF stimulated cells displayed a smaller increase in cytoplasm with respect to cells stimulated with GM-CSF or GM-CSF/IL4. Moreover, M-CSF, and especially M-CSF/IFNγ treated cells displayed pseudopodia, which were absent in control cells or cells treated with either GM-CSF or GM-CSF/IL4. Furthermore, cells stimulated with M-CSF/IFNγ formed small aggregates. These modifications observed in M-CSF or M-CSF/IFNγ stimulated cells were similar to changes observed in cells stimulated with CSN1S1, which formed aggregates and developed pseudopodia. After 120 h, many control cells became adherent and showed a greatly enlarged cytoplasm. This was also observed for GM-CSF and GM-CSF/IL-4 treated cells. Stimulation with M-CSF caused the development of pseudopodia besides the occurrence of adherent cells with enlarged cytoplasm. The addition of IFNγ to M-CSF again led to a strong tendency towards cellular aggregation and the development of pseudopodia. This was also true for cells incubated with CSN1S1.
CSN1S1 alters cell-surface marker expression
The cellular morphology of CSN1S1 stimulated cells suggested a differentiation, either into macrophages or into DC. Besides morphological changes, differentiated cells of each type can be distinguished by distinct surface marker expression[18, 19]. Therefore, primary human monocytes were incubated for 24 or 120 h with recombinant CSN1S1 and expression of representative surface markers for differentiation of monocytes towards macrophages or DC were analyzed by flow cytometry after immunolabeling. After 24 h stimulation with 10 μg/ml CSN1S1, upregulations of CD14 (2.5-fold ± 1.0, SD) and CD64 (1.9-fold ± 0.8) were detectable (Figure 3a). Lower concentrations of CSN1S1 had no effect (data not shown). The pattern of surface markers obtained was characteristic for macrophages rather than for DC (CD14high, CD64high, CD83low, CD1alow )[15, 18, 19]. After 120 h of incubation with CSN1S1, CD14 but not CD64 remained significantly upregulated. The pattern of surface markers remained the same (CD14high, CD64high, CD83low, CD1alow) (Figure 3b). Next, we compared the surface markers of monocytes differentiated with CSN1S1 to in vitro differentiated monocytes towards macrophages (M-CSF/IFNγ) or DC (GM-CSF/IL-4). Such differentiation is known to be obtained by stimulation of primary human monocytes for 120 h[20]. As can be seen in Figure 3c, no difference in surface marker expression was observed after 24 h. In contrast, after 120 h, CSN1S1 and M-CSF/IFNγ stimulation resulted in the same phenotype (CD14high, CD64high, CD83low, CD1alow), while GM-CSF/IL-4 caused a significant downregulation of CD14 and CD64 expression with upregulation of CD1a.
CSN1S1 increases phagocytic activity of monocytes
Next, we assessed if incubation of primary human monocytes with CSN1S1 also results in functional changes. Increased phagocytic activity is a characteristic property of monocytes differentiated towards a macrophage-like phenotype[18, 19]. Therefore, the intracellular uptake of labelled zymosan particles into primary human monocytes was assessed in a colorimetric assay after incubation with 10 μg/ml CSN1S1 for 24 h. There was a marked increase in phagocytic activity of cells after 24 h (1.4-fold ± 0.4), which was sustained after 48 h (1.9-fold ± 0.5, Figure 4a).
Influence of CSN1S1 on GM-CSF- and GM-CSF/IL-4-induced DC differentiation
The above data suggested that CSN1S1 skews cellular differentiation of monocytes towards a macrophage-like phenotype. We were therefore interested, if an alternative route of differentiation, i.e. early differentiation of monocytes into DC, could be antagonized by the addition of 10 μg/ml CSN1S1 for 24 h. For this purpose, primary human monocytes were incubated with GM-CSF for 24 h in the presence or absence of CSN1S1 and the expression of cell surface markers was assessed by flow cytometry. As can be seen in Figure 4b, GM-CSF alone induced a characteristic immature DC cell surface marker pattern (CD14low, CD64low, CD83low, CD1ahigh)[15]. The addition of CSN1S1 abolished GM-CSF effects and lead to a macrophage pattern (CD14high, CD64high, CD83low, CD1alow). Besides GM-CSF, the combination of GM-CSF and IL-4 is a strong stimulus for in vitro DC generation[15]. Therefore, we additionally examined the properties of CSN1S1 in influencing GM-CSF/IL-4-induced DC differentiation. GM-CSF/IL-4 similarly caused characteristic immature DC cell surface marker expression (CD14low, CD64low, CD83low, CD1ahigh) after 24 h of incubation, and this effect could not be inhibited by the addition of CSN1S1 (data not shown).
The role of M-CSF in CSN1S1-mediated cellular differentiation
We previously reported that monocytic cells secrete GM-CSF in response to CSN1S1[13]. GM-CSF is known to influence the differentiation of monocytes towards a DC phenotype[21]. According to the present results, autocrine stimulation with CSN1S1 induced GM-CSF must therefore be overcome by alternative stimuli to allow for a differentiation towards the observed macrophage-like phenotype. We speculated that autocrine stimulation with M-CSF secreted upon CSN1S1 induction, upregulation of the M-CSF receptor CD115, or downregulation of the GM-CSF receptor CD116 could be responsible for the observed effects. First, primary human monocytes were stimulated with 10 μg/ml CSN1S1 for 24 h and M-CSF secretion into supernatants was quantified by ELISA. As can be seen in Figure 4c, CSN1S1 increased the secretion of M-CSF into culture supernatants 29-fold. As a control, an M-CSF antibody was added to the experiments in order to demonstrate its capacity to bind all secreted M-CSF after stimulation (Figure 4c). In the next step, differentiation of primary human monocytes was induced by 24 h incubation with 10 μg/ml CSN1S1 and the expression of CD14 and the M-CSF and GM-CSF receptors (CD115 and CD116, respectively) were determined by flow cytometry and immunolabeling (Figure 4d). CSN1S1 lead to the expected upregulation of CD14, while the expression of CD115 and CD116 remained unchanged. The addition of an M-CSF antibody to CSN1S1 stimulated primary human monocytes in the same concentration that was demonstrated to bind the secreted M-CSF protein (Figure 4c) did not alter the expression of CD14 or the receptors CD115 and CD116. Thus, neither changes in the expression of M-CSF, nor up- or downregulation of M-CSF receptor (CD115) or GM-CSF receptor (CD116) respectively, explained the preferential shift of monocyte differentiation towards macrophages in culture conditions that contain both M- and GM-CSF.
CSN1S1 induced differentiation and cytokine expression may partially be mediated via MAPK
We previously reported that CSN1S1 upregulates the expression and secretion of GM-CSF in monocytes in a p38 MAPK-dependent fashion[13]. We were therefore interested to analyze if cellular differentiation and the expression of other proinflammatory cytokines is also dependent upon MAPK pathways. In a first step, we analysed if addition of inhibitors of the MAPK pathways, i.e. JNK, p38, and ERK, influenced overall survival of primary human monocytes. As can be seen in Figure 5a, there was no significant effect on cellular vitality of monocytes by addition of inhibitors for 24 h in the concentrations used in subsequent experiments. Next, we assessed if the addition of these inhibitors was biologically effective in suppressing MAPK mediated signalling. LPS-signalling is known to be mediated via all three MAPK: JNK, p38, and ERK, and results in IL-1b expression[22]. Therefore, primary human monocytes were stimulated with LPS for 24 h in the presence and absence of MAPK inhibitors and IL-1b-mRNA expression was measured as a control experiment. As can be seen in Figure 5b, all inhibitors significantly suppressed IL-1b-mRNA expression to a similar degree. In order to identify a putative signal transduction mechanism responsible for CSN1S1-induced cellular differentiation, we then tested the ability of MAPK inhibitors to impede the generation of a macrophage-like phenotype. Primary human monocytes were therefore incubated with MAPK inhibitors before stimulation with 10 μg/ml CSN1S1. Cell surface markers CD14 and CD64, upregulated during CSN1S1-induced differentiation as described above, were assessed by flow cytometry and immunolabeling. As depicted in Figure 5c, CSN1S1-mediated upregulation of CD14 was significantly decreased by inhibition of ERK, but not p38 and JNK. Next, we were interested to see if this effect was specific for CSN1S1 stimulated cells or if ERK-inhibition generally reduces CD14 expression in monocytes treated with M-CSF or GM-CSF. To this end, cells were treated with MAPK inhibitors prior to stimulation with M-CSF. As shown in Figure 5d, upregulations of CD14 in M-CSF treated cells were not influenced by inhibition of ERK as in CSN1S1 treated cells, but by inhibition of JNK. In contrast, MAPK inhibition did not influence surface marker expression in cells stimulated with GM-CSF for 24 h (Figure 5e). This suggests that signal transduction of CSN1S1-mediated differentiation is at least in part distinct from pathways used by M-CSF.
In order to evaluate whether CSN1S1 induces the expression of proinflammatory cytokines via the same route, primary human monocytes were stimulated with CSN1S1 and IL-1b and IL-6 protein-secretion into culture supernatants was measured in the presence and absence of MAPK inhibitors. As can be seen in Figure 5f-g, a significant reduction in the upregulation of IL-1b was noted with inhibition of p38 and JNK while IL-6 was decreased with inhibition of JNK only. Of note, inhibition of ERK did not reduce increased cytokine secretion in a significant manner.
The above results suggested that CSN1S1 engages all 3 MAPK molecules to either exert effects on cellular differentiation or proinflammatory cytokine expression. To confirm this notion, we additionally assessed activation, i.e. phosphorylation, of MAPK after incubation of primary human monocytes with 10 μg/ml CSN1S1 for 24 h by Western blot. As can be seen in Figure 5h, p38, JNK, and ERK were all phosphorylated when stimulated with CSN1S1, but not in control experiments without CSN1S1.