Research ArticleImmunology

T cell–derived soluble glycoprotein GPIbα mediates PGE2 production in human monocytes activated with the vaccine adjuvant MDP

See allHide authors and affiliations

Science Signaling  08 Oct 2019:
Vol. 12, Issue 602, eaat6023
DOI: 10.1126/scisignal.aat6023
  • Fig. 1 Administration of MDP in rabbits induced fever and increased CRP and PGE2 in the blood.

    (A to C) Female NZW rabbits (n = 8) were inoculated with PBS (R1 and R2) or with MDP at 30 or 10 μg/kg (R3, R4, and R5 and R6, R7, and R8, respectively) at time 0. Body temperatures were recorded (A), and CRP (B) and PGE2 (C) were measured in blood samples at time 0 and up to either 72 hours (A and B) or 7 hours (C) after treatment. Data are shown as body temperature measured in each rabbit at indicated time points (A) or as means ± SD calculated for triplicate wells (B and C).

  • Fig. 2 Soluble factor produced by T cells augmented production of PGE2 and proinflammatory cytokines in human monocytes activated with MDP.

    (A) Quantification of PGE2 in cultures of PBMC, monocyte-depleted PBMC (PBMC-Mo), monocytes alone (Mo), and monocytes cocultured with CD3 bead–purified T cells (Mo + Tc) and either not treated (NT) or incubated with MDP overnight. **P ≤ 0.01; ***P ≤ 0.001; and n.s., not significant (P > 0.05), by two-tailed unpaired t test. (B) Quantification of PGE2 in cultures of monocytes not treated (NT) or treated with MDP alone (MDP), with MDP and CD3 bead–purified T cells in separate chambers of transwell plate (MDP/Tc), with MDP and Tc CM (MDP + Tc CM), or with Tc CM alone overnight. *P ≤ 0.05; **P ≤ 0.01; and n.s., not significant (P > 0.05), by two-tailed unpaired t test. (C) Quantification of PGE2 in monocytes incubated alone (Mo) or incubated with Tc CM (Mo + Tc CM) overnight not treated (NT) or treated with MDP or LPS at 50 or 500 ng/ml.**P ≤ 0.01; ***P ≤ 0.001; and n.s., not significant (P > 0.05), by two-tailed unpaired t test. (D to F) Quantification of IL-1β (D), IL-6 (E), and PGE2 (F) in monocytes incubated with MDP or with MDP and Tc CM alone (MDP/Tc CM) or in the presence of 0.01, 0.1, or 1.0 μM indomethacin. Data are means ± SD calculated for triplicate wells in a representative of three experiments performed with cells from individual donors. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and n.s., not significant (P > 0.05), by two-tailed unpaired t test.

  • Fig. 3 Tc CM contributes to increased COX2 gene transcription but not to activation and nuclear translocation of NF-κB.

    (A to C) RT-qPCR for COX2 (A) and mPGES-1 mRNA (B) and quantification of PGE2 (C) in monocytes alone (Mo alone) or in monocytes incubated with Tc CM overnight (Mo + Tc CM) and either not treated (NT) or incubated with MDP in the absence (MDP/No inhibitor) or presence of erlotinib (MDP/Erlotinib). The Ct values for COX2 (A) and mPGES-1 mRNA (B) expression in monocytes were normalized using qPCR reactions with β-actin primers performed in the same samples. Data are mean fold increases in ΔCt values over control (A and B) and means ± SD (C), each calculated from triplicate wells in a representative of three experiments performed with monocytes from individual donors. ***P ≤ 0.001 and n.s., not significant, by two-tailed unpaired t test. (D and E) Western blot (D) and analysis (E) of NF-κB subunit p65 in nuclear extracts (NE) prepared from monocytes activated with MDP and Tc CM in the absence or presence of erlotinib for 3 hours. TATA-binding protein (TBP) was used as loading control for the nuclear extracts. Blot is from one donor of four (others are shown in fig. S7). Data are mean fold increases ± SEM; n = 4. *P ≤ 0.05; **P ≤ 0.01; and n.s., not significant (P > 0.05), by two-tailed unpaired t test.

  • Fig. 4 Increase in intracellular [Ca2+] is required for production of PGE2 in monocytes.

    (A) PGE2 quantification in monocytes that were not treated (NT), incubated with MDP alone, or incubated with MDP and Tc CM (MDP/Tc CM) either alone (No inhibitor) or in the presence of BAPTA-AM, U73122, or 2-APB. Data are means ± SD from triplicate wells in a representative of three experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by two-tailed unpaired t test. (B) PGE2 quantification in monocytes either unperturbed (Mo alone) or incubated with MDP (Mo/MDP) and either not treated (NT) or treated with 0.1, 0.5, or 1.0 μM ionomycin. Data analysis and N as described in (A). (C) Representative Indo-1 fluorescence time traces in monocytes after treatment with MDP, Tc CM, or MDP and Tc CM. The ratio of mean Indo violet/Indo blue emission values (405 nm:485 nm) indicates changes in free intracellular [Ca2+] over time. Traces are from a representative of three experiments.

  • Fig. 5 T cells isolated using CD3 beads expressed increased levels of GPIbα mRNA and shed GPIbα protein in CM.

    (A) Left: RT-qPCR for GPIbα mRNA in CD3 bead–purified T cells (Tc) and in negatively selected T cells (NSTc) from donors 1 and 2 cultured overnight. The Ct values for GPIbα mRNA expressions in T cells were normalized using qPCR reactions with β-actin primers performed in the same samples. Data are fold increases from triplicate wells. *P ≤ 0.05 and **P ≤ 0.01, by two-tailed unpaired t test. Right: Agarose gels for GPIbα and β-actin transcripts. Representative of two experiments. (B) Western blot of 3.5-fold concentrated CM from CD3 bead–purified T cells (Tc) and from negatively selected T cells (NSTc) from donors 1 and 2 (25 μl of concentrated CM per lane), total cell extracts from platelets (Pla) (0.1 μg of total protein per lane), and rGPIbα (5, 3, and 1 ng of protein per lane). MWM, molecular weight markers; EL, empty lane. Data are representative of three experiments. (C) Western blot of 18-fold concentrated CM from CD3 bead–purified T cells cultured in the absence or in the presence of M8I (7.5 μl of concentrated CM per lane), platelet extracts (0.1 μg of total protein per lane), and an rGPIbα protein (1, 3, and 5 ng of protein per lane). Data are representative of three experiments. (D) Quantification of PGE2 in monocytes not treated (NT) or incubated with MDP alone (MDP), with MDP and Tc CM alone (No inhibitor), or in the presence of M8I or DMSO in control. Data are means ± SD from triplicate wells from two experiments. *P ≤ 0.05; **P ≤ 0.01; and n.s., not significant, by two-tailed unpaired t test.

  • Fig. 6 Antibodies to GPIbα and CD11b reduced COX2, IL1B, and IL6 mRNA expression and PGE2, IL-1β, and IL-6 protein secretion in monocytes activated with MDP and Tc CM.

    (A to F) Quantification of COX2 (A), IL1B (C), and IL6 mRNA (E) expression and quantification of PGE2 (B), IL-1β (D), and IL-6 (F) in monocytes treated with MDP alone or with MDP and Tc CM in the absence or presence of mAbs to GPIbα or Mac-1 (CD11b) or mouse IgG1 in control. The Ct values (A, C, and E) were normalized to that of β-actin performed in the same samples. Data are means ± SD from triplicate wells in a representative of three experiments. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and n.s., not significant, by two-tailed unpaired t test.

  • Fig. 7 Signaling through Mac-1 receptor is required for PGE2 production in MDP-activated monocytes.

    (A) Western blot for CD11b in THP-1 cells transfected with control or with siRNA targeting CD11b. Representative of two experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B and C) RT-qPCR for COX2 (B) and IL1B (C) expression in THP-1 cells transfected with control siRNA or CD11b siRNA and either not treated (NT) or treated with MDP and Tc CM (MDP/Tc CM). Data are mean fold increases ± SD over control (without MDP and Tc CM treatment) in triplicate wells in a representative of three experiments. *P ≤ 0.05 and n.s., not significant, by two-tailed unpaired t test. (D) Ca2+ transients in response to Tc CM were recorded in monocytes untreated or pretreated with antibodies to CD11b or with mouse IgG1 (mIgG1). The Indo-1 ratio of emission intensities (405 nm:485 nm) over time is presented. Representative of three experiments. (E) Quantification of mouse Cox2 mRNA in the liver and spleen of WT (w/t), CD18 KO, and αM KO mice at 2 hours after inoculation of MDP. The data show fold increases in Cox2 mRNA ΔCt values in mice that received MDP (six mice per group) over average Cox2 ΔCt values in mice that received PBS (four mice per group); fold increase for each mouse and means calculated for the groups of mice are shown. *P ≤ 0.05 and **P ≤ 0.01, by two-tailed unpaired t test. (F) Quantification of PGE2 in monocytes (Donor 1, Donor 2, and Donor 3) not treated (rGPIbα/0) or treated with rGPIbα at 1, 10, or 20 μg/ml or with H19 peptide (20 μg/ml) alone (MDP 0) or in the presence of MDP at 50 ng/ml. Data are means ± SD from triplicate wells. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by two-tailed unpaired t test. (G) PGE2 quantification in monocytes incubated with rGPIbα (10 and 20 μg/ml) alone [not treated (NT)], with rGPIbα in the presence of MDP alone (No inhibitor), or with rGPIbα and MDP in the presence of U73122 or 2-APB. Data are means ± SD from two independent experiments. *P ≤ 0.05 and **P ≤ 0.01, by two-tailed unpaired t test.

  • Fig. 8 Model of PGE2 production in monocytes activated with MDP and Tc CM.

    MDP alone induced nuclear NF-κB and low levels of COX2 transcription and PGE2 production. CD3 bead–activated T cells shed GPIbα protein by the activity of metalloprotease. Triggering of Mac-1 in monocytes by T cell–derived GPIbα activates PLC/InsP3/InsP3R pathway and induces release of calcium from the endoplasmic reticulum. Increase in cytosolic calcium provides a second signal essential for increased COX2 transcription and subsequent production of PGE2 in MDP-activated monocytes.

  • Table 1 PGE2-inducing activity of serum-free Tc CM and NSTc CM used for MS.

    Quantification of PGE2 in monocytes treated with MDP in the presence of conditioned medium (CM) prepared from CD3 bead–isolated T cells (Tc CM) or from negatively selected T cells (NSTc CM) cultured overnight in serum-free Expi293 medium.

    CM*PGE2 ± SD (pg/ml)Fold increase
    Tc CMNSTc CM
    Neat2,309.0 ± 4.4250.0 ± 8.39.2
    Concentrated66,769.8 ± 14,258.85,128.0 ± 1,784.013.0

    *Tc CM and NSTc CM were left untreated (Neat) or were concentrated using a 50-kDa MWCO centrifugal filter (Concentrated).

    †Monocytes were treated with MDP in the presence of Tc CM Neat or Concentrated or with MDP in the presence of NSTc CM Neat or Concentrated overnight. Monocyte cell culture supernatants were assayed for PGE2 production. Data are shown as means ± SD from triplicates.

    ‡Fold increase was calculated for PGE2 production in monocytes incubated with MDP and Tc CM versus MDP and NSTc CM. Representative of three experiments.

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/12/602/eaat6023/DC1

      Fig. S1. PGE2 dose response in monocytes treated with MDP alone or with MDP in the presence of Tc CM.

      Fig. S2. CM from CD3 bead–treated, negatively selected T cells increased PGE2 production in monocytes activated with MDP.

      Fig. S3. Tc CM strongly augmented production of IL-8 and only minimally increased that of RANTES and IL-12 p40 in monocytes activated with MDP.

      Fig. S4. Production of PGE2 in monocytes activated with MDP and Tc CM is IL-1β independent.

      Fig. S5. CM from CD3 bead–treated, negatively selected T cells increased COX2 mRNA expression in monocytes activated with MDP.

      Fig. S6. Production of IL-1β and IL-6 in monocytes activated with MDP and Tc CM is sensitive to RIP2 kinase inhibitor erlotinib.

      Fig. S7. Western blot of NF-κB p65 in nuclear extracts of monocytes activated with MDP and Tc CM in the absence or presence of erlotinib.

      Fig. S8. Tc CM but not NSTc CM induced calcium flux in monocytes.

      Fig. S9. Flow cytometry of bead-purified T cells with antibodies to CD41a.

      Fig. S10. Gating strategy to exclude presence of CD41a+ platelets in subsets of sorted CD4+ and CD8+ T cells.

      Fig. S11. CM from sort-purified T cells incubated with CD3 beads overnight induced COX2 mRNA and PGE2 in monocytes activated with MDP.

      Fig. S12. Platelets did not induce PGE2 in MDP-treated monocytes.

      Fig. S13. Cox2 mRNA Ct values in the spleen and liver of WT, CD18 KO, and αM KO mice injected with MDP or PBS.

      Fig. S14. rGPIbα increased PGE2, IL-1β, and IL-6 in MDP-treated monocytes.

      Fig. S15. Western blot of NF-κB p65 in nuclear extracts prepared from monocytes activated with MDP and rGPIbα.

      Table S1. Production of PGE2 in monocytes activated with MDP and Tc CM but not with MDP and NSTc CM.

      Table S2. Increased PGE2-inducing activity in concentrated Tc CM.

      Table S3. List of proteins increased twofold or higher in Tc CM compared with NSTc CM.

    • This PDF file includes:

      • Fig. S1. PGE2 dose response in monocytes treated with MDP alone or with MDP in the presence of Tc CM.
      • Fig. S2. CM from CD3 bead–treated, negatively selected T cells increased PGE2 production in monocytes activated with MDP.
      • Fig. S3. Tc CM strongly augmented production of IL-8 and only minimally increased that of RANTES and IL-12 p40 in monocytes activated with MDP.
      • Fig. S4. Production of PGE2 in monocytes activated with MDP and Tc CM is IL-1β independent.
      • Fig. S5. CM from CD3 bead–treated, negatively selected T cells increased COX2 mRNA expression in monocytes activated with MDP.
      • Fig. S6. Production of IL-1β and IL-6 in monocytes activated with MDP and Tc CM is sensitive to RIP2 kinase inhibitor erlotinib.
      • Fig. S7. Western blot of NF-κB p65 in nuclear extracts of monocytes activated with MDP and Tc CM in the absence or presence of erlotinib.
      • Fig. S8. Tc CM but not NSTc CM induced calcium flux in monocytes.
      • Fig. S9. Flow cytometry of bead-purified T cells with antibodies to CD41a.
      • Fig. S10. Gating strategy to exclude presence of CD41a+ platelets in subsets of sorted CD4+ and CD8+ T cells.
      • Fig. S11. CM from sort-purified T cells incubated with CD3 beads overnight induced COX2 mRNA and PGE2 in monocytes activated with MDP.
      • Fig. S12. Platelets did not induce PGE2 in MDP-treated monocytes.
      • Fig. S13. Cox2 mRNA Ct values in the spleen and liver of WT, CD18 KO, and αM KO mice injected with MDP or PBS.
      • Fig. S14. rGPIbα increased PGE2, IL-1β, and IL-6 in MDP-treated monocytes.
      • Fig. S15. Western blot of NF-κB p65 in nuclear extracts prepared from monocytes activated with MDP and rGPIbα.
      • Table S1. Production of PGE2 in monocytes activated with MDP and Tc CM but not with MDP and NSTc CM.
      • Table S2. Increased PGE2-inducing activity in concentrated Tc CM.
      • Table S3. List of proteins increased twofold or higher in Tc CM compared with NSTc CM.

      [Download PDF]

    Navigate This Article