Elsevier

Biochemical Pharmacology

Volume 63, Issue 10, 15 May 2002, Pages 1901-1910
Biochemical Pharmacology

Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid

https://doi.org/10.1016/S0006-2952(02)00982-6Get rights and content

Abstract

Interleukin (IL)-12 plays a pivotal role in the development of T helper type 1 (Th1)-immune response, which may have therapeutic effects on diseases associated with pathologic Th2 responses such as allergic disorders and asthma. In this study, we investigated the effects of berberine, a benzodioxoloquinolizine alkaloid with anti-microbial and anti-tumor activities, on the production of IL-12 p40, an inducible subunit of IL-12, in mouse macrophages. Berberine-induced IL-12 p40 production and activation of p38 mitogen-activated protein kinase (MAPK) in dose-dependent manners, which were significantly inhibited by p38 MAPK inhibitors and yohimbine, indicating that p38 MAPK and α2-adrenergic receptor were involved in the induction of IL-12 p40 production in mouse macrophages by berberine. Furthermore, berberine significantly enhanced IL-12 p40 production in mouse macrophages when combined with lipopolysaccharide, a well-known inducer of IL-12 production. These findings may explain some of the known biological effects of berberine and suggests berberine as an immunotherapeutic compound for induction of IL-12, which is potentially applicable for tumors, infectious disease, and airway inflammation.

Introduction

Interleukin (IL)-12 is a heterodimeric cytokine comprised of two disulfide-linked subunits of 35 (p35) and 40 (p40) kDa encoded by two separate genes. Secretion of p40 or p70 is limited to cells of the macrophage/monocyte lineage and occurs only after activation of these cells [1]. In contrast, the IL-12 p35 chain is produced by a number of cell types and constitutively expressed. Thus, p40 expression governs the production of the bioactive p70, which induces the production of IFN-γ and, in turn, drives the production of a number of inflammatory cytokines. Additionally, IL-12-induced IFN-γ can direct activated CD4+ T lymphocytes to differentiate into T helper type 1 (Th1) cells [2]. Adequate production of IL-12 is essential for the maintenance of normal host defense mechanisms and this key role has raised considerable interest in the mechanisms involved in the regulation of IL-12 biosynthesis [3], [4]. Inducible expression of IL-12 has been documented in macrophages and dendritic cells after stimulation by microbial antigens or via CD40–CD40L interaction [5], [6]. In lipopolysaccharide (LPS)- and IFN-γ-treated monocytes, the expression of IL-12 p40 has been shown to be primarily regulated at the transcriptional level, which involves functional synergy with transcription factors, C/EBP or Ets families with NF-κB [7], [8], [9], [10].

Berberine is a benzodioxoloquinolizine alkaloid that has been isolated from Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Berberis aristata (tree turmeric). Its extracts and decoctions have demonstrated significant anti-microbial activity against a variety of organisms including bacteria, viruses, fungi, protozoans, helminths, and chlamydia [11]. Furthermore, berberine has shown a number of beneficial effects, including immunostimulation via increased blood flow to the spleen, macrophage activation, elevation of platelet counts in cases of primary and secondary thrombocytopenia. In addition, berberine may possess anti-tumor promoting properties as evidenced by inhibition of cyclooxygenase-2 transcription and N-acetyltransferase activity in colon and bladder cancer cell lines, and transient, but marked, inhibitory action on the growth of mouse sarcoma cells in culture [12].

p38 mitogen-activated protein kinase (MAPK) pathway is activated by environmental perturbation (e.g. osmotic changes, heat shock) and by inflammatory cytokines including TNF-α and IL-1 [13]. This pathway has been proposed to function in the regulation of cytokine production [14], [15], B cell and T cell proliferation and differentiation [16], [17], [18], the innate immune response [19], cell cycle control [20], [21] and apoptosis [22], [23]. Recent studies have shown that the p38 MAPK, activated through MAPK kinase 3 (MKK3), may be involved in the production of proinflammatory cytokines by both antigen-presenting cells and CD4+ T cells [24], [25], [26].

In this report, we have demonstrated that berberine significantly induced IL-12 p40 production in mouse macrophages and, furthermore, enhanced IL-12 p40 production in macrophages when combined with LPS. Berberine-activated p38 MAPK in macrophages and p38 MAPK inhibitors significantly suppressed this berberine-induced IL-12 p40 production, indicating that p38 MAPK pathway may be involved in the berberine-mediated induction of IL-12 p40 production from mouse macrophages.

Section snippets

Mice, monoclonal antibodies, cytokines, and reagents

Female DBA/2 mice were obtained from the Japan SLC, Inc. and used at 6–10 weeks of age. Anti-IL-12 p40 mAbs C17.8 and C15.6 were purified from ascitic fluid by ammonium sulfate precipitation followed by DEAE–Sephagel chromatography (Sigma Chemical Co). Anti-p38 MAPK mAb and anti-phosphotyrosine mAb were purchased from Santa Cruz Biotechnology, Inc. Recombinant murine IL-12 was generously provided by Dr. Stanley Wolf (Genetics Institute, Cambridge, MA). LPS (from Escherichia coli 0111:B4),

Berberine induces IL-12 p40 production from splenic macrophages

To assess the effect of berberine on IL-12 production, splenic macrophages were treated with berberine (0.1–1 μg/mL) for 48 hr and the levels of IL-12 p40 protein in the culture supernatants were determined by a sandwich ELISA for IL-12 p40. An IL-12 p40 subunit was known as the highly inducible and tightly regulated component of IL-12 [30]. Berberine strongly induced IL-12 p40 production in a dose-dependent manner (Fig. 1). Treatment of macrophages with 1 μg/mL berberine induced approximately

Discussion

In this report we have demonstrated that berberine can significantly induce IL-12 p40 production through p38 MAPK activation and can synergize to induce IL-12 p40 production when combined with LPS. IL-12 plays an essential role in the optimal generation of IFN-γ-secreting Th1 cell under many experimental conditions. Through this activation, and through its ability to induce directly IFN-γ secretion from both T and NK cells, IL-12 plays a central role in both innate and adaptive immunity

Acknowledgments

The authors thank S. Wolf, Y.K. Choe and J. Cheong for their generous gift of valuable reagents, and J.W. Lee and Y.C. Lee for helpful discussions. This work was supported by grants from the HMP (01-PJ1-PG3-21200-0021).

References (58)

  • X. Gao et al.

    Immunomodulatory activity of resveratol: suppression of lymphocyte proliferation, development of cell-mediated cytotoxicity, and cytokine production

    Biochem. Pharmacol.

    (2001)
  • M. Suzuki et al.

    The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-α- or IL-1β-stimulated rheumatoid synvial fibroblasts

    FEBS Lett.

    (2000)
  • Z.H. Nemeth et al.

    Calcium channel blockers and dantrolene differentially regulate the production of interleukin-12 and interferon-gamma in endotoxemic mice

    Brain Res. Bull.

    (1998)
  • M.B. Faries et al.

    Calcium signaling inhibits interleukin-12 production and activates CD83(+) dendritic cells that induce Th2 cell development

    Blood

    (2001)
  • K. Kang et al.

    IL-12 synthesis by human Langerhans cells

    J. Immunol.

    (1996)
  • M.K. Gately et al.

    The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses

    Annu. Rev. Immunol.

    (1998)
  • F. Mattner et al.

    Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response

    Eur. J. Immunol.

    (1996)
  • F. Koch et al.

    High level IL-12 production by murine dendritic cells: up-regulation via MHC class II and CD40 molecules and down-regulation by IL-4 and IL-10

    J. Exp. Med.

    (1996)
  • M. Cella et al.

    Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T–T help via APC activation

    J. Exp. Med.

    (1996)
  • X. Ma et al.

    The interleukin-12 p40 gene promoter is primed by interferon-γ in monocytic cells

    J. Exp. Med.

    (1996)
  • T.L. Murphy et al.

    Regulation of interleukin-12 p40 expression through an NF-κB half site

    Mol. Cell Biol.

    (1995)
  • S.E. Plevy et al.

    Multiple control elements mediate activation of the murine and human interleukin-12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins

    Mol. Cell Biol.

    (1997)
  • J. Han et al.

    An MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells

    Science

    (1994)
  • J.C. Lee et al.

    A protein kinase involved in the regulation of inflammatory cytokine biosynthesis

    Nature

    (1994)
  • R. Beyaert et al.

    The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis in response to tumor necrosis factor

    EMBO J.

    (1996)
  • M. Rincon et al.

    Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway

    EMBO J.

    (1998)
  • A. Craxton et al.

    p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes

    J. Immunol.

    (1998)
  • Z.S. Han et al.

    A conserved p38 mitogen-activated protein kinase pathway regulates Drosophila immunity gene expression

    Mol. Cell Biol.

    (1998)
  • K. Takenaka et al.

    Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest

    Science

    (1998)
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