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Bmx tyrosine kinase regulates TLR4-induced IL-6 production in human macrophages independently of p38 MAPK and NFκB activity

Christine D. Palmer, Brenda E. Mutch, Sarita Workman, John P. McDaid, Nicole J. Horwood and Brian M. J. Foxwell

Data supplements

  • Supplemental materials for: Palmer et al

    Files in this Data Supplement:

    • Figure S1. Bmx siRNA induces knockdown of Bmx mRNA but not protein (JPG, 49.9 KB) -
      Peripheral blood monocytes were transfected with increasing doses (50, 100 and 200nM) of targeting and control siRNA oligonucleotides and differentiated in the presence of 100ng/ml M-CSF in culture for 4 days. (A) Bmx mRNA levels were assessed by real-time PCR. Mean values (±SEM) for 3 separate donors are shown normalised to buffer control (100%). (B) The expression of Bmx and Lamin positive control (Santa Cruz antibody) were assessed by Western blotting.





    • Figure S2. Bmx kinase dead and kinase deleted mutants do not act as dominant negatives (JPG, 72.7 KB) -
      Macrophages were infected with AdBmx, AdBmx KD (K445E), AdBmx KDel (417-675) or Ad0 as before. Cells were cultured in complete medium and stimulated with LPS (10ng/ml), for 18 hours. LPS-induced TNF and IL-6 production was assessed by ELISA. Mean values (±SEM) for 3 separate donors normalised to LPS-treated controls are shown. Statistical significance was assessed using Student�s t-test.





    • Figure S3. Bmx does not signal upstream of NF-κB (JPG, 44 KB) -
      Mean densitometry units (from Figure 4) pooled for 3 separate donors (±SEM) are shown normalised to 30 min LPS for uninfected controls.

Article Figures & Data

  • Figure 1

    Targeted RNAi knockdown of Btk protein results in decreased TNF, not IL-6, production in human macrophages after LPS stimulation. Peripheral blood monocytes were transfected with increasing doses of targeting and control siRNA oligonucleotides and differentiated in the presence of 100 ng/mL of M-CSF in culture for 4 days. (A) The expression of Btk was assessed by Western blotting. Densitometry units (mean ± SEM) for 4 separate donors are shown normalized to untreated controls. (B) For analysis of cytokine expression, siRNA-transfected M-CSF macrophages were treated with LPS (10 ng/mL) for 18 hours and supernatants assessed for TNF and IL-6 levels by ELISA. Values are shown as mean plus or minus SEM for 4 separate donors normalized to LPS only controls (**P< .01). (C) PBMCs were prepared from XLA and normal male donors (age range, 17–46 years) as described in “Isolation and culture of PBMCs from XLA patients and control donors.” Cells were left undifferentiated (XLA ■, normal ●) or cultured in the presence of M-CSF (100 ng/mL) for 4 days (XLA □, normal ○) and then stimulated with LPS (10 ng/mL) or Pam3C-SK4 (100 ng/mL). Cytokine production was assessed by ELISA 18 hours after stimulation. Each data point shown represents a single donor (n.s., not significant). (D) Bmx and Tec protein expression in matched undifferentiated and M-CSF-treated XLA PBMCs, normal PBMCs, and blood monocytes was assessed by Western blotting. Blots are representative of 4 separate donors for each matched cell population. Statistical significance was assessed using one-way ANOVA and Bonferroni multiple comparison test.

  • Figure 2

    LPS stimulation induces Bmx kinase activity in primary human macrophages. PBMCs were differentiated into macrophages in the presence of 100 ng/mL of M-CSF for 4 days. Cells were infected for 2 hours with adenoviruses overexpressing HA-tagged wild-type Bmx or control adenovirus Ad0 at a multiplicity of infection of 100:1 in serum-free medium. (A) Expression of HA-Bmx at different multiplicities of infection was assessed by Western blotting. (B) For auto-kinase assay, cells were cultured in complete medium and treated with 10 ng/mL of LPS for 0, 5, 10 and 20 minutes. Cells were lysed and HA-Bmx immunoprecipitated from lysates as described in “Immunoprecipitation and kinase assay” and subjected to in vitro kinase assay. (C) Densitometry units (mean ± SEM) pooled for 3 separate donors are shown normalized to untreated controls. Statistical significance was assessed using one-way ANOVA and Bonferroni multiple comparisons test (**P< .01).

  • Figure 3

    Bmx regulates TLR-induced TNF and IL-6 production. Macrophages were infected with AdBmx or Ad0 as before. Cells were cultured in complete medium and stimulated with LPS (10 ng/mL), Pam3C-SK4 (100 ng/mL), Malp2 (100 ng/mL), or Flagellin (100 ng/mL) for 18 hours. Cytokine expression in supernatants was assessed by ELISA. (A) LPS-induced TNF, IL-6, IL-8, and IL-10 production was assessed in uninfected, Ad0-infected, and AdBmx-infected macrophages. Values (mean ± SEM) for 4 separate donors normalized to LPS-treated controls are shown. (B) Cytokine production after Pam3C-SK4, Malp2, and Flagellin stimulation was assessed by ELISA. Values (mean ± SEM) for 4 separate donors normalized to Pam3C-SK4- and Malp-2-treated controls are shown. Statistical significance was assessed using Student ttest (*P< .05; **P< .01; ***P< .001).

  • Figure 4

    Bmx does not signal upstream of NF-κB. Macrophages were infected for 2 hours with adenoviruses overexpressing wild-type Bmx or control adenovirus Ad0 in serum-free medium. For luciferase assays, cells were subjected to a second round of infections with NF-κB consensus luciferase adenovirus (AdNFκB-luc) before stimulation. Cells were stimulated with 10 ng/mL LPS for 18 hours for ELISA, 4 hours for luciferase reporter gene assays, and 0.5 and 1 hour for EMSA. (A) Uninfected, Ad0-infected, and AdBmx-infected macrophages were left untreated or preincubated with PSI (1 μM) and TNF and IL-6 production assessed by ELISA. Values (mean ± SEM) are presented as percent compared with LPS-treated uninfected controls (100%; *P< .05; **P< .01). (B) Relative luciferase units (RLU) for uninfected, Ad0-infected, and AdBmx-infected cells in the absence (left hand panel) or presence (right hand panel) of AdNFκB-luc after 4 hours of LPS stimulation are shown as mean (± SEM) for 4 separate donors normalized to LPS only controls. n.s, not significant. (C) Nuclear extracts were collected as described in “Electrophoretic mobility shift assays” and assayed for NF-κB DNA binding by EMSA. Statistical significance was assessed by Student ttest.

  • Figure 5

    Bmx increases TNF and IL-6 mRNA stabilization via 2 distinct downstream pathways. (A) Macrophages were left uninfected or infected with Ad0 and AdBmx as before and left untreated or preincubated with SB203580 (1 μM). TNF and IL-6 production was assessed by ELISA. Values (mean ± SEM) are presented as percent compared with LPS-treated uninfected controls (100%). Statistical significance was assessed using Student ttest. n.s., not significant (*P< .05; ***P< .001). (B) Macrophages were left uninfected or infected with Ad0 and AdBmx as before. Cells were stimulated with LPS (10 ng/mL) for 4 hours before addition of actinomycin D (2 μg/mL). TNF and IL-6 mRNA levels at 0, 15, 30, 60, 90, and 120 minutes after addition of actinomycin D were assessed by real-time PCR. Values (mean ± SEM) for 4 separate donors are shown normalized to time 0 of actinomycin D addition (100%) (**P< .01). (C) Macrophages were left uninfected or infected with Ad0 and AdBmx as before and left untreated or preincubated with SB203580 (1 μM). TNF and IL-6 mRNA levels at 1 and 2 hours after addition of actinomycin D in the presence or absence of SB203580 (1 μM) were assessed by real-time PCR. Values (mean ± SEM) for 4 separate donors are shown normalized to time 0 of actinomycin D addition (100%). Statistical significance was assessed by one-way ANOVA and Bonferroni multiple comparison test (*P< .05; **P< .01).

  • Figure 6

    Bmx targets the 3′UTRs of TNF and IL-6 after LPS stimulation. (A) Schematic representation of the human IL-6 5′ promoter-luciferase-3′UTR (AdIL-6 5′/3′) and IL-6 5′ promoter-luciferase (AdIL-6 5′only) adenoviral constructs. (B) Macrophages were left uninfected or infected with Ad0 and AdBmx before a second round of infections with AdTNF 5′/3′, AdTNF5′, AdIL-6 5′/3′, or AdIL-6 5′ luciferase reporter gene constructs and stimulated with LPS (10 ng/mL) for 4 hours. Relative luciferase units (RLU) for uninfected, Ad0-infected, and AdBmx-infected cells in the absence or presence of TNF reporter viruses (top panel) or IL-6 reporter viruses (bottom panel) after 4 hours of LPS stimulation are shown as mean plus or minus SEM normalized to controls for 4 separate donors. Statistical significance was assessed by Student ttest (*P< .05; **P< .01).

Supplementary Materials

  • Figure S2

    Supplementary PDF file available online.