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, 289 (51), 35582-92

Dendritic Cell IL-1α and IL-1β Are Polyubiquitinated and Degraded by the Proteasome

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Dendritic Cell IL-1α and IL-1β Are Polyubiquitinated and Degraded by the Proteasome

Joseph S Ainscough et al. J Biol Chem.

Abstract

IL-1α and β are key players in the innate immune system. The secretion of these cytokines by dendritic cells (DC) is integral to the development of proinflammatory responses. These cytokines are not secreted via the classical secretory pathway. Instead, 2 independent processes are required; an initial signal to induce up-regulation of the precursor pro-IL-1α and -β, and a second signal to drive cleavage and consequent secretion. Pro-IL-1α and -β are both cytosolic and thus, are potentially subject to post-translational modifications. These modifications may, in turn, have a functional outcome in the context of IL-1α and -β secretion and hence inflammation. We report here that IL-1α and -β were degraded intracellularly in murine bone marrow-derived DC and that this degradation was dependent on active cellular processes. In addition, we demonstrate that degradation was ablated when the proteasome was inhibited, whereas autophagy did not appear to play a major role. Furthermore, inhibition of the proteasome led to an accumulation of polyubiquitinated IL-1α and -β, indicating that IL-1α and -β were polyubiquitinated prior to proteasomal degradation. Finally, our investigations suggest that polyubiquitination and proteasomal degradation are not continuous processes but instead are up-regulated following DC activation. Overall, these data highlight that IL-1α and -β polyubiquitination and proteasomal degradation are central mechanisms in the regulation of intracellular IL-1 levels in DC.

Keywords: Dendritic Cell; IL-1α IL-1β; Inflammation; Interleukin 1 (IL-1); Proteasome; Ubiquitylation (Ubiquitination).

Figures

FIGURE 1.
FIGURE 1.
Intracellular expression and secretion of IL-1 by BMDC: impact of LPS and ATP. BMDC (106 cells/ml) were cultured for 24 h in the presence of medium alone or increasing doses of LPS (A and D), or for 4 h with medium alone or LPS (0.1 μg/ml) and then challenged with ATP (0–10 mm) for 30 min (B, C, E, and F). Supernatants and cell lysates were harvested and analyzed for the presence of IL-1α or IL-1β using cytokine-specific ELISA. IL-1 content of cell lysates is displayed in A (■, IL-1α; □, IL-1β) and supernatants (secreted IL-1) in B (IL-1α) and C (IL-1β) as mean ± S.E. (n = 3). Supernatants (SN) and lysates (Lys) were also analyzed by Western blotting using either anti-IL-1α (D and E) or anti-IL-1β (D and F) antibodies. A protein marker lane on each gel was used to determine molecular weight. Representative blots are shown in each case.
FIGURE 2.
FIGURE 2.
IL-1 is degraded intracellularly in BMDC by a process up-regulated during DC activation. BMDC (106 cells/ml) were incubated with LPS (0.1 μg/ml) for various time periods (0 to 48 h). Supernatants (○) and lysates (●) were analyzed for the presence of IL-1α (A), IL-1β (B), and IL-6 (C) using specific ELISA (single experiment). In some experiments, cells were incubated with LPS for 4 or 48 h, with the final 44 h at 37 or 4 °C, and IL-1α (■) and IL-1β (□) content was measured in lysates by ELISA (D; n = 3). In other experiments, cells were incubated with LPS for various periods of time (1 to 5 h), with the final 1 h of culture in the presence of CHX (10 μg/ml)(E–H). Lysates were prepared and analyzed for the presence of IL-1α (E and F) and IL-1β (G and H) by ELISA. Data are displayed from one representative experiment with respect to actual cytokine levels for each time period before and after a 1-h CHX treatment (●, 1–2 h; ■, 2–3 h; ▴, 3–4 h; ♦, 4–5 h) (E, IL-1α; and G, IL-1β). The rate of IL-1 degradation for each time period (ng/106 cells/h) was calculated by deducting cytokine levels after the CHX incubation from baseline levels prior to CHX addition (F, IL-1α; and H, IL-1β). Data shown are mean ± S.E. (n = 3). Statistical significance of differences between 4 (D) or 1–2 h-treated samples versus other treatment groups (F and H) was determined by one-way ANOVA, *, p < 0.05; **, p < 0.01.
FIGURE 3.
FIGURE 3.
IL-1 degradation in BMDC is dependent upon the proteasome. BMDC (106 cells/ml) were incubated with medium alone (M; A and B) or LPS (0.1 μg/ml; C and D) or poly(I:C) (100 μg/ml; G) for 8 or 12 h with the final 4 h in the presence of 10 μm of the proteasome inhibitor MG132 (A–D and G) or with an equivalent volume of solvent (DMSO) alone. Supernatants and lysates were prepared and analyzed for the presence of IL-1α (A and C) and IL-1β (B and D) using cytokine-specific ELISA. For both IL-1α and IL-1β, secreted (supernatant) cytokine levels were below the limit of detection (data not shown). Lysates prepared in parallel (■, medium; □, LPS) were also analyzed for IL-1β mRNA using RT-PCR and the ΔΔCt method. Results were normalized against naive BMDC and the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) (E). These lysates (and recombinant (Rm) IL-1β control) were also analyzed by Western blotting using an anti-IL-1β (F) antibody. A representative blot is shown. A protein marker lane on the gel was used to determine molecular mass. Data shown are mean ± S.E. (n = 4). A one-way ANOVA was used to determine statistical significance of differences between treatment groups. *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
IL-1 degradation is dependent on the proteasome in macrophages. J774 (A–C and E–G) or BMDM (D and H) (106 cells/ml) were incubated with LPS (1 μg/ml) for 16 h with a final 4 h in the presence of CHX (10 μg/ml) and DMSO, MG262 (A and E), ALLN (B and F), or β lactone (C and G) (all 0–50 μm for J775 cells). For BM-derived macrophages a single concentration of each proteasome inhibitor was used (50 μm for ALLN; 30 μm for both MG262 and β lactone). Supernatants and lysates were prepared and analyzed for the presence of IL-1α (A–D) and IL-1β (E–H) using cytokine-specific ELISA. For both IL-1α and IL-1β, secreted cytokine levels were below the limit of detection (data not shown). Data shown are mean ± S.E. (n = 3). A one-way ANOVA was used to determine statistical significance of differences between the DMSO-treated samples versus the other treatment groups. *, p < 0.05; **, p < 0.01.
FIGURE 5.
FIGURE 5.
IL-1 degradation in BMDC is not dependent upon autophagy. BMDC (106 cells/ml) were incubated with medium alone (M; A and B) or LPS (0.1 μg/ml; C, D, and F) for 8 h or for 12 h with the final 4 h in the presence of 10 μm of the autophagy inhibitor wortmanin (Wort) (A-D) or with an equivalent volume of solvent (DMSO) alone, or both wortmanin and MG132 in various combinations of concentrations (F; 0.1, 1, or 10 μm). Supernatants and lysates were prepared and analyzed for the presence of IL-1α (A and C) and IL-1β (B, D, and F) using cytokine-specific ELISA. For both IL-1α and IL-1β, secreted (supernatant) cytokine levels were below the limit of detection (data not shown). In addition, BMDC (106 cells/ml) were incubated with LPS (0.1 μg/ml) for 8 h, or 12 h with the final 4 h in the presence or absence of wortmanin (0.1, 1, or 10 μm). Lysates were prepared and analyzed by Western blotting using an anti-LC3 antibody (E). A protein marker lane on the gel was used to determine molecular weight. Data shown are mean ± S.E. (n = 4). A one-way ANOVA was used to determine statistical significance of differences between treatment groups. *, p < 0.05; **, p < 0.01.
FIGURE 6.
FIGURE 6.
IL-1 is polyubiquitinated in DC. 107 BMDC (106 cells/ml) were incubated with medium or LPS (0.1 μg/ml) for 12 h with the final 4 h in the presence or absence of 10 μm of the proteasome inhibitor MG132 (A–D). In addition, 107 BMDC (106 cells/ml) were incubated with medium or LPS (0.1 μg/ml) for 4, 8, or 12 h with the final 4 h in the presence of 10 μm of the proteasome inhibitor MG132 (E and F). Cells were lysed, an aliquot of lysate was retained as whole cell lysate (WCL) and the remainder was immunoprecipitated with anti-IL-1α (A, C, and E) or anti-IL-1β antibody (B, D, and F). The same volume of lysis buffer alone was immunoprecipitated (IP) with anti-IL-1α or anti-IL-1β antibody (immunoprecipitation negative control; IP-ve control). The samples were analyzed by Western blotting using an anti-IL-1α antibody (A), an anti-IL-1β antibody (B), or an anti-ubiquitin antibody (C–F). For Fig. 4D, results are from 2 separate gels, run and developed concurrently. A protein marker lane on each gel was used to determine the molecular weight. Representative blots are shown in each case.
FIGURE 7.
FIGURE 7.
Ubiquitinated IL-1 expression following LPS priming and ATP challenge. 107 BMDC (106 cells/ml) were incubated with medium or LPS (0.1 μg/ml) for 12 h with the final 4 h in the presence or absence of 10 μm of the proteasome inhibitor MG132. Cells were then challenged with 10 mm ATP for 30 min or left untreated. Cells were lysed, an aliquot of lysate was retained as whole cell lysate (WCL) and the remainder was immunoprecipitated (IP) with anti-IL-1α (A and C) or anti-IL-1β antibody (B and D). The samples were analyzed by Western blotting using an anti-IL-1α antibody (A), an anti-IL-1β antibody (B), or an anti-ubiquitin antibody (C and D). A protein marker lane on each gel was used to determine molecular weight. Representative blots are shown in each case.

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References

    1. Steinman R. M. (2003) The control of immunity and tolerance by dendritic cells. Pathol. Biol. 51, 59–60 - PubMed
    1. Banchereau J., Steinman R. M. (1998) Dendritic cells and the control of immunity. Nature 392, 245–252 - PubMed
    1. Mellman I., Steinman R. M. (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106, 255–258 - PubMed
    1. Medzhitov R. (2010) Inflammation 2010: New adventures of an old flame. Cell 140, 771–776 - PubMed
    1. Netea M. G., Nold-Petry C. A., Nold M. F., Joosten L. A., Opitz B., van der Meer J. H., van de Veerdonk F. L., Ferwerda G., Heinhuis B., Devesa I., Funk C. J., Mason R. J., Kullberg B. J., Rubartelli A., van der Meer J. W., Dinarello C. A. (2009) Differential requirement for the activation of the inflammasome for processing and release of IL-1β in monocytes and macrophages. Blood 113, 2324–2335 - PMC - PubMed

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