Cooperative induction of cytokines by AP-1 and NF-κB
To further characterize the involvement of NF-κB in cytokine regulation, we treated cells with an NF-κB activation inhibitor (NAI). The results showed that NAI selectively down-regulated NF-κB activation (figures 4a and 4b) and did not alter AP-1 activity (figures 4c and 4d). Exposure of Jurkat T-cells to NAI resulted in a modest reduction of CXCL8 following PMA exposure, while it did not alter the CXCL8 release following HK E. coli exposure (figure 5a). NAI did not affect TNF expression (figure 5b) indicating that NF-κB is not the main regulator of CXCL8 or TNF following either PMA or HK E. coli exposure in Jurkat T-cells. In contrast, NAI resulted in a complete inhibition of IL-6 following PMA exposure and a 45% inhibition following HK E. coli exposure (figure 5c), suggesting an involvement of NF-κB in IL-6 regulation.
Figure 4  Inhibition of NF-κB activity by NAI. Jurkat T-cells were transfected with luciferase reporter plasmids containing either NF-κB or AP-1 cis-acting elements. The cells were incubated with NF-κB activation inhibitor (NAI) for 1 h followed by stimulation with PMA (162 nM, A and C) or HK E. coli (5 × 107 CFU/ml, B and D) for 24 h. (A, B) NF-κB, but not (C, D) AP-1 was inhibited. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 4). Controls were set to 1.
Figure 5  Involvement of NF-κB in cytokine regulation. Cytokine/chemokine levels were determined using ELISA following incubation of Jurkat T-cells with NAI (1 h) and stimulation (24 h). (A) CXCL8 expression was partially inhibited following PMA stimulation (grey bars), whereas the levels were not altered following stimulation with HK E. coli (black bars), this indicates an induction mainly regulated by AP-1. (B) TNF expression was not affected by NAI. (C) IL-6 release was completely inhibited by NAI following PMA exposure, indicating regulation through NF-κB since IL-6 expression was significantly increased in response to HK E. coli than PMA. Statistical significance from the positive control (PMA/HK E. coli) was determined using Student's t-test. (n = 3).   Ca2+ was observed to increase AP-1 activity (figure 1c) and reduce NF-κB activity (figure 2e); therefore, we exposed T-cells to a PKC inhibitor together with PMA to determine its effect on cytokine expression. Inhibition of PKC reduced CXCL8 release from 7 ng/ml to 3 ng/ml while it had a modest effect on IL-6 and TNF (figure 6a-c). This prompted us to test the effect of JNK inhibition on PMA-induced cytokine expression. JNK is involved in the regulation of a multitude of different transcription factors, including the phosphorylation and activation of c-Jun, c-Fos and p53, leading to cellular apoptosis [26]. Inhibition of the JNK pathways resulted in a down-regulation of both CXCL8 and IL-6, while no clear effect was observed on TNF expression (figure 6a-c). Analysis of mRNA levels using RT-qPCR (table 2) showed that PMA induced both il-6 and cxcl8 mRNA (5.1-fold and 111.8 fold respectively). Addition of the NF-κB inhibitor NAI and the JNK inhibitor reduced the il-6 expression below basal levels. In contrast, while the cxcl8 levels were suppressed by the same treatments the levels remained elevated above basal level.
Figure 6  Involvement of PKC and JNK in cytokine expression. Jurkat T-cells were incubated with PKC- and JNK inhibitors for 1 h followed by stimulation with PMA (162 nM) for 24 h. (A) CXCL8 expression was inhibited in a dose-dependent manner by both inhibitors. (B) JNK-I revealed a stronger inhibitory effect on IL-6 expression than the PKC-I. (C) TNF expression was not affected by either inhibitor. Statistical significance from the positive control (PMA) was determined using Student's t-test. (n = 3).
Table 2  Jurkat T-cells were treated with 10 nM NAI, 10 μM JNK-I or10 nM PKC-I for 2 h followed by induction with 162 nM PMA for 24 h.  Gene expression was determined using RT-qPCR. The Ct values were normalized against 18S. Statistical significance from the positive control (PMA) was determined using Student's t-test. Concentrations are given as fg/ml, values are presented as mean ± SEM, n = 3.