Volume 54, Issue 1, Pages 71-79 (July 1998) Interleukin (IL)-1 and IL-4 synergistically stimulate NF-IL6 activity and IL-6 production in human mesangial cells  Yuichi Nakazato, Tomoko Hayashida, Yoshihiko Kanno, Hiroyuki Sasamura, Hirokazu Okada, Hiromichi Suzuki, Takao Saruta  Kidney International  Volume 54, Issue 1, Pages 71-79 (July 1998) DOI: 10.1046/j.1523-1755.1998.00967.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Effects of interleukin (IL)-1 and IL-4 on IL-6 production by human mesangial cells (MC). (A) Quiescent MC were incubated in serum-free medium containing IL-1α (10ng/ml) and/or IL-4 (0.01 to 10ng/ml) for 24hours. At the end of the incubation period, supernatants were collected and IL-6 concentrations were quantitated by ELISA. IL-4 alone had no statistically significant effect on IL-6 release, but potentiated the effect of IL-1α. Data shown represent one experiment. Two other experiments gave similar results. N = 3, Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Effects of IL-1 and IL-4 on expression of IL-6 mRNA in human mesangial cells (MC). (A) Quiescent MC were treated with vehicle, IL-1α (10ng/ml), IL-4 (10ng/ml), or IL-1+IL-4 for four hours. Total RNA was isolated and analyzed by Northern blotting. The blot was re-probed for G3PDH mRNA to ensure equivalent RNA loading between lanes. The autoradiograph is representative of four independent experiments. (B) Densitometric data in each experiment are expressed relative to the value of IL-1 treated cells, and are shown as means ±SD of four experiments. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Effect of IL-4 on degradation of IL-6 mRNA in IL-1-activated human mesangial cells (MC). Quiescent MC were incubated with IL-1α (•; 10ng/ml), or IL-1α plus IL-4 (▪; 10ng/ml) for four hours followed by addition of actinomycin D (5 μg/ml, final). Total RNA was extracted after 0, 1, 2, and 4hours of actinomycin D treatment and subjected to Northern blot analysis. Autoradiographic signals of IL-6 mRNA and 18S rRNA were quantitated by densitometry. IL-6 mRNA levels were normalized for 18S rRNA levels in each sample and expressed as percentages of time 0 values. The plotted data represent the average values of two separate experiments. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Effects of IL-1 and IL-4 on nuclear factor-κB (NF-κB) activity in mesangial cell (MC) nuclear extracts. Quiescent MC were treated with IL-1α (10ng/ml) or IL-4 (10ng/ml) for the final 0.5, 2, 6, or 12hours of a 36hour-incubation in serum-free medium. After preparation of nuclear extracts, binding activities were assessed by EMSA using labeled-NF-κB consensus oligonucleotides. Arrows indicate specific DNA-protein complexes, which were specifically competed by excess unlabeled probe Figure 5c. Representative data are from three independent experiments. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Effects of combined treatment with IL-1 and IL-4 on nuclear factor-κB (NF-κB) activity. Mesangial cells (MC) were treated with vehicle (control), IL-1 (10ng/ml), IL-4 (10ng/ml), or IL-1+IL-4 for 30minutes (A) or two hours (B). NF-κB activity was analyzed as in Figure 4. The specificity of the complexes formed with an IL-1-treated human MC nuclear extract (HMC NE) was examined by including 100-fold excess unlabeled NF-κB probe, anti-p65 antiserum, or anti-p50 antiserum in the binding mixtures (C). Antiserum to p65 caused a supershifted band (arrowhead). NF-κB activity in HeLa cell nuclear extract (HeLa NE) was analyzed in parallel as a positive control. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Effects of IL-1 and IL-4 on the nuclear localization of nuclear factor-κB (NF-κB) p65. Nuclear extracts were prepared from MC treated with vehicle, IL-1α (10ng/ml), IL-4 (10ng/ml), or IL-1+IL-4 for 30minutes or one hour. An equal amount of protein was loaded onto each lane, and analyzed by Western blot using affinity purified anti-p65 antiserum. Representative data are from three independent experiments. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 7 Effects of IL-1 and IL-4 on the nuclear localization of nuclear factor-κB (NF-κB) p50. Nuclear extracts prepared from MC treated with cytokines for 30minutes were used for Western analysis using anti-p50 antiserum. Representative data are from two independent experiments. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 8 Effects of IL-1 and IL-4 on nuclear factor (NF)-IL6 binding activity. Nuclear extracts were prepared from quiescent MC that were treated with IL-1α (10ng/ml) or IL-4 (10ng/ml) for 0.5, 2, 6, and 12hours. The binding activities to the NF-IL6 motif were analyzed by EMSA using a 32P-labeled 27bp synthetic oligonucleotide. Arrows indicate specific oligonucleotide-protein complexes. Two other experiments gave similar results. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 9 Effects of combined treatment with IL-1 and IL-4 on binding activities to NF-IL6 motif. Quiescent MC were treated with vehicle (control), IL-1 (10ng/ml), IL-4 (10ng/ml), or IL-1+IL-4 for two hours (A) or five hours (B), and nuclear extracts were analyzed by EMSA. Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 10 Specificity of binding activities to NF-IL6 motif in nuclear extract from IL-1+IL-4 stimulated cells. Nuclear extract from human MC treated with IL-1+IL-4 for five hours (HMC NE) was incubated for 30minutes at 4°C in the presence of 100-fold excess unlabeled 27bp NF-IL6 probes (NF-IL6 oligo), 14bp NF-IL6 probe (NF-IL6 oligo, 14bp), CREB, or AP2 oligonucleotides, or 1 μl of anti-NF-IL6 antiserum or anti-p65 antiserum together with 32P-labeled 27bp NF-IL6 probe. The two oligonucleotide-protein complexes indicated by the arrows were markedly displaced by cold 27bp probe and a shorter 14bp oligonucleotide having a core NF-IL6 binding sequence. Anti-NF-IL6 antibody partially displaced them and formed a weak supershifted band (arrowhead). Kidney International 1998 54, 71-79DOI: (10.1046/j.1523-1755.1998.00967.x) Copyright © 1998 International Society of Nephrology Terms and Conditions