PI3K limits TNF-alpha production in CD16-activated monocytes

Abstract

IgG complexes bind to Fc receptor family members FcgammaRI (CD64), FcgammaRII (CD32) and FcgammaRIII (CD16), activating cell MAPK and PI3K resulting in increased cytokine production from particular leukocytes. The signaling molecules involved in cytokine production after cross-linking CD16 have not been determined in monocytes. To address this question, TNF-alpha, IL-1beta and IL-6 were measured in activated monocytes after inhibiting MEK1/2, PI3K and glycogen synthase kinase-beta (GSK-3beta). The roles of GSK-3beta and NF-kappaB were then determined using reporter assays and siRNA treatment. The data suggested that an MAPK pathway stimulated TNF-alpha release but that active PI3K limited TNF-alpha, IL-1beta and IL-6 cytokine production after cross-linking CD16. PI3K was also shown to limit nuclear translocation of NF-kappaB. The limiting effect of PI3K on TNF-alpha production from activated monocytes depended on the decrease of GSK-3beta activity, which significantly reduced the transactivation of NF-kappaB. Moreover, the TNF-alpha production induced by CD16 cross-linking was reduced in monocytes after treatment with siRNA against NF-kappaB, implying that this transcription factor functioned in TNF-alpha production. The results suggest that CD16 cross-linking activated PI3K and that active PI3K limited TNF-alpha production by inhibiting GSK-3beta activity, that blocked the action of NF-kappaB.

Figure 1. A MAPK pathway regulated TNF-α release from monocytes after CD16 stimulation

Primary human monocytes were pretreated with kinase inhibitors PD98059 (A) and U0126 (B) at two concentrations for 1 hour prior to the addition of anti-CD16 antibody (clone 3G8) or an IgG isotype control. Supernatants were collected 48 hours later and the concentration of TNF-α was measured by ELISA. Total protein from the cells was then measured. Y-axis shows the concentration of TNF-α expressed as pg of cytokine per mg of total cellular protein. *p<0.05, as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 2. PI3K limits cytokine release from monocytes after CD16 stimulation

Cultured human primary monocytes were pretreated with the PI3K inhibitor LY294002 one hour prior to the addition of anti-CD16 antibody (clone 3G8) or an IgG isotype control antibody. Supernatants were collected 48 hours after the addition of the antibodies. Secreted TNF-α (A), IL-1β (B) and IL-6 (C) were measured by ELISA. Total protein from the cells was then quantitated. Y-axis shows the concentration of cytokines as pg of cytokine per mg of total cellular protein. *p<0.05, as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 3. PI3K inhibitor wortmannin increased TNF- α from activated monocytes

Cultured human primary monocytes were pretreated with the PI3K inhibitor wortmannin one hour prior to the addition of anti-CD16 antibody (clone 3G8) or an IgG isotype control. Supernatants were collected 48 hours after the addition of the antibodies. Secreted TNF-α was measured by ELISA and the concentration of TNF-α was expressed as pg of cytokine per mg of total tissue protein. *p<0.05, as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 4. PI3K and MAPK pathway interactions

Cultured human primary monocytes were pretreated with the MEK1/2 inhibitor PD98059 (A) or U0126 (B) and the PI3K inhibitor LY294002 (A) or wortmannin (B) one hour prior to the addition of anti-CD16 antibody (3G8) or an IgG isotype control antibody. Supernatants were collected 48 hours after the addition of the antibodies and secreted TNF-α was measured by ELISA. TNF-α concentration is expressed as pg of cytokine per mg of total tissue protein. *p<0.05, as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 5. PI3K and GSK-3β function to inhibit TNF-α release

(A) Cultured human primary monocytes were pretreated with PI3K kinase inhibitor LY294002 or GSK-3β kinase inhibitor LiCl one hour prior to the addition of anti-CD16 antibody (clone 3G8) or vehicle. Monocytes were collected 0, 15 or 60 minutes after the addition of the CD16 antibody or vehicle, and 10 µg of total protein was loaded in each lane for western blot analysis. The western blot was probed with an antibody against Ser388 phosphorylated c-Raf antibody (73 kDa) or a Ser9 phosphorylated GSK-3β (46KDa). (B) Cultured human primary monocytes were pretreated with kinase inhibitors one hour prior to the addition of anti-CD16 antibody (clone 3G8) or an IgG isotype control. Supernatants were collected 48 hours after the addition of the antibodies. TNF-α concentration was determined by ELISA and expressed as pg of cytokine per mg of total tissue protein. *p<0.05 as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 6. PI3K effects nuclear localization of p65 and phosphorylated p65

Human primary monocytes were pretreated with vehicle or PI3K kinase inhibitor LY294002 one hour prior to the addition of anti-CD16 antibody (3G8). Monocytes were collected 0, 15 or 60 minutes after the addition of the CD16 antibody. Nuclear extracts were prepared, and the amounts of p65 (A) and p65 phosphorylated at Ser536 (B) were determined by ELISA. *p<0.05 as determined by two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 7. NF-κβ transactivation was regulated by GSK-3β

Human monocytes were transfected with a luciferase construct containing five 5’ NF-κβ sites (GGGGACTTTCC). A SV40 Renilla luciferase construct was co-transfected to control for variability in transfection efficiencies. 24 h after transfection, monocytes were treated with the PI3K kinase inhibitor LY294002 or wortmannin or the GSK-3β kinase inhibitor LiCl. One hour after the addition of the kinase inhibitors, monocytes were treated with an anti-CD16 antibody (3G8) or an IgG isotype control antibody. Luciferase activity was measured 5 hours after the addition of the antibody and reported as relative light units (RLU), which is the luminescent signal from the firefly luciferase normalized to an internal control Renilla luciferase construct. *p<0.05, as determined by a two-way ANOVA and a Duncan’s post-hoc test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.

Figure 8. Reducing NF-κB expression decreased NF-κβ transactivation and CD16-induced TNF-α production in activated monocytes

Human primary monocytes were transfected with siRNA bearing homology to the NF-κB gene or a control siRNA construct. (A) Monocytes were isolated 48 hours following transfection, and the total amount of cellular NF-κB was measured by ELISA. (B) 24 h after transfection, monocytes were again transfected with a luciferase construct containing five 5’ NF-κB sites and a SV40 Renilla luciferase construct. 24 h following the second transfection the monocytes were treated with an anti-CD16 antibody (clone 3G8) or an IgG isotype control antibody. Luciferase activity was measured 5 hours after the addition of the antibody and reported as relative light units (RLU), which is the luminescent signal from the firefly luciferase normalized to an internal control Renilla luciferase construct. (C) Monocytes were treated with an anti-CD16 antibody (clone 3G8) or an IgG isotype control antibody 48 hours after transfection. Supernatants were collected 48 hours after the addition of the antibodies and TNF-α concentration was measured by ELISA and expressed as pg of cytokine per mg of total tissue protein. *p<0.05, as determined by the Student’s t-test. Each experiment was repeated three times with monocytes from different donors. Graphs show the mean + SEM.