br Materials and methods br Results

Materials and methods
Results
Discussion Taken together with our previous report, our current data demonstrate induction of Chk in monocytes by IL-3, IL-4, IL-13, and GM-CSF ((Musso et al., 1994) and Fig. 1, Fig. 2, Fig. 3, Fig. 4). Chk GS-7340 receptor is also inducible by stem cell factor or phorbol 12-myristate 13-acetate (PMA) in megakaryblasts (Grgurevich et al., 1997, Bennett et al., 1994). These findings and the fact that Chk can negatively regulate Src-family proteins (Avraham et al., 1995, Chow et al., 1994b, Price et al., 1997, Price et al., 1999, Zrihan-Licht et al., 1998, Hirao et al., 1997), which are critical for monocyte differentiation, have led us to propose that Chk may play an important role in monocyte differentiation. The fact that induction of Chk is prevented and/or reversed by treatment of PBMs with IFN-γ (Fig. 4, Fig. 5, Fig. 6) is particularly intriguing and raises the question of how IFN-γ inhibits GM-CSF mediated increases in Chk expression. We can suggest several possible explanations of how this might occur. The first possibility is that IFN-γ decreases the GM-CSF receptor expression on the cell surface. This explanation seems unlikely because IFN-γ actually increases the expression of the gene encoding the beta subunit of the GM-CSF receptor (Hallek et al., 1992) and because continuous stimulation with GM-CSF is not necessary for continued expression of Chk (Fig. 4A). Secondly, IFN-γ might induce a suppressor of cytokine signaling (SOCS) molecule to inhibit signaling by binding to the signaling complex of IL-4 family members. This hypothesis is consistent with the requirement for protein synthesis for IFN-γ-mediated suppression. However, this explanation would not explain why continuous stimulation with IL-4 family members is not necessary for continued expression of Chk (Fig. 4A) since it is believed that SOCS proteins inhibit cytokine signaling by binding directly to signaling complexes at the cytoplasmic tail of the receptors (Krebs and Hilton, 2000). A third explanation is that IFN-γ stimulation causes expression of cellular proteins that bind to sequences in the Chk promoter, and serve as dominant repressors of the promoter. This possibility can only be examined in more detail once the Chk promoter has been characterized. However, promoter regulation alone would not explain how IFN-γ is able to apparently reverse GM-CSF mediated increases in Chk expression within 6–8h (Fig. 6) since the Chk message appears to be stable for approximately 48h ((Musso et al., 1994) and Fig. 2). Preliminary data from our laboratory suggests a fourth explanation, namely that IFN-γ stimulation somehow decreases the stability of Chk mRNA and/or protein in addition to repressing the Chk promoter. Cyclohexamide treatment of GM-CSF stimulated monocytes results in the loss of detectable amounts of Chk protein within 5h which suggests a relatively short half-life (1–2h) for Chk compared to Csk (data not shown). Additionally, in order to detect Chk in human monocytes, they needed to first be treated with the cell permeable protease inhibitor DFP (see Section 2) which suggests that Chk is highly labile in these cells. Chk protein stability and half-life suggest that the nature of this protein is one that necessitates quick elimination, and IFN-γ induced blockage of the Chk promoter and degradation of the Chk message/protein can explain both the blockage and rapid reversal of Chk protein expression in human monocytes. Since IFN-γ and IL-4 are prototypic Th1 and Th2 cytokines respectively, our results imply that Chk may have a role in the differentiation of monocytes during a humoral immune response verses a delayed type hypersensitivity response. Our results also suggest that monocytes present during a humoral response might be capable of “converting” to macrophages involved in a DTH response. However, macrophages involved in a DTH response (in the presence of high concentrations of IFN-γ) are incapable of reverting to the pre-DTH stage in terms of their inability to induce Chk following exposure to IFN-γ. These possibilities are currently being tested in Chk−/− mice.