T helper 2 cytokines induce preproenkephalin mRNA expression and proenkephalin A in human peripheral blood mononuclear cells

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Abstract

We investigated the regulatory influence of several cytokines on the expression of preproenkephalin (PPE) mRNA in human peripheral blood mononuclear cells (PBMC). By use of a quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), we demonstrate that the T helper 2 cytokines IL-4 and IL-10 are more potent in upregulating PPE mRNA expression in human PBMC than the T helper 1 cytokines IL-2 and γ-IFN. In addition, TGF-β is also an effective inducer of PPE mRNA. TGF-β, IL-4 and IL-10 increase the cytoplasmatic concentration of met-enkephalin in PBMC. Secretion of met-enkephalin in the culture supernatant of IL-4- or IL-10-stimulated PBMC could not be observed, but proenkephalin A-derived met-enkephalin containing peptides could be demonstrated. IL-4 and IL-10 do not induce PPE mRNA via the same pathways. We could observe that PKA is involved in IL-4 mediated PPE mRNA induction, whereas IL-10 apparently uses another route.

Introduction

Opioid peptides represent a group of regulatory mediators that mediate interactions between the nervous system and the immune system (Weigent and Blalock, 1987). These peptides are encoded by three different genes, the pro-opiomelanocortin (POMC) gene, the prodynorphin gene and the preproenkephalin (PPE) gene. The precursor proteins derived from these three different genes are tissue-specifically processed into biologically active peptides (Udenfriend and Kilpatrick, 1984; Lynch and Snyder, 1986). One of these peptides is met-enkephalin, a five amino acid opioid peptide, which is derived from the propeptide proenkephalin A (PEA). Besides neuronal cells (Eiden, 1987), cells of the immune system are also capable of expressing PPE mRNA and producing met-enkephalin. This has first been observed in a murine T helper cell clone (Zurawski et al., 1986), and has also been demonstrated in activated murine B cells from spleen, bone marrow, lymph node, and in murine macrophages (Martin et al., 1987; Rosen et al., 1989). We have shown earlier that mitogen-activated human peripheral blood mononuclear cells (PBMC) and articular chondrocytes both express the mRNA for PPE (Kuis et al., 1991; Villiger and Lotz, 1992).

Enkephalins also modulate immune function. They stimulate T-cell migration (Heagy et al., 1990), inhibit primary and secondary antibody synthesis (Johnson et al., 1982; Munn and Lum, 1989), and modulate lymphocyte proliferation (Hucklebridge et al., 1990; Linner et al., 1995). Moreover, enkephalins were shown to stimulate non-specific effector functions such as chemotaxis (van Epps and Saland, 1984), superoxide production (Foris et al., 1984), antibody dependent cellular cytotoxicity (ADCC) (Foris et al., 1984) and NK cell function (Faith et al., 1984).

Cytokines are important mediators in inflammation. They influence the inflammatory response, but also signal the brain which leads to, among other things, increases in CRH expression in the hypothalamus and activation of the hypothalamus–pituitary–adrenal (HPA) axis (Berkenbosch et al., 1987; Weigent and Blalock, 1987; Blalock, 1994; Weigent and Blalock, 1995). This paper focusses on another property of cytokines: the capacity to induce the expression of the opioid peptide met-enkephalin in PBMC.

The present study investigates by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) which cytokines can influence the PPE mRNA expression. Moreover, we could demonstrate that PPE mRNA is translated into PEA-derived intermediate peptides present in culture supernatant.

Section snippets

Cell isolation

Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by Ficoll–Hypaque density gradient centrifugation of heparinized blood.

Culture conditions

Cells were cultured in 24-well tissue culture plates (Costar Europe) at a density of 106 cells/ml per well at 37°C in a humidified atmosphere of 5% CO2 in air. The culture medium consisted of RPMI-1640 (Gibco, Grand Island, NY) supplemented with 5% heat inactivated fetal calf serum (Gibco, Grand Island, NY), 2 mM l-glutamine, 100 U/ml penicillin and

Quantitative RT-PCR for PPE mRNA expression

To analyze the expression of PPE mRNA, we developed a quantitative RT-PCR method. Subsequently, we performed Southern blot hybridization to confirm the specificity of the product. For the quantitative RT-PCR a synthetic variant of PPE RNA was constructed consisting of 568 bp. Cellular RNA results in a fragment of 930 bp. As is shown in Fig. 1A, addition of increasing amounts of the synthetic RNA fragment to the RT-PCR reaction leads to a linear increase in product. Addition of two different

Discussion

This study describes that cytokines can induce PPE gene expression and met-enkephalin production in human PBMC. PPE mRNA is detected at low levels in resting cells, but increases four-fold after activation with the cytokine TGF-β and the TH2 cytokines IL-4 and IL-10. The TH1 cytokines IL-2 and γ-IFN have only a marginal effect on PPE gene expression. In addition, the predominantly monocyte-derived cytokines IL-1β and IL-6 are also weak inducers of PPE mRNA in PBMC.

TH2 cytokines represent

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