Lymphocytes upregulate signal sequence-encoding proopiomelanocortin mRNA and beta-endorphin during painful inflammation in vivo
Introduction
Immune cell-derived beta-endorphin1–31 (END) can elicit pain control by activating opioid receptors on sensory nerve terminals within peripheral inflamed tissue (Stein et al., 1990b, Stein et al., 2003). The secretion of END can be triggered by stress, cytokines, catecholamines, corticotropin-releasing factor (CRF) or chemokines (Binder et al., 2004, Cabot et al., 1997, Mousa et al., 2004, Rittner et al., 2006). With prolonged inflammation the number of opioid containing immune cells, the tissue END content, and the efficacy of pain control increase (Machelska et al., 2003, Mousa et al., 2001, Rittner et al., 2001). Immunosuppression reduces stress- or CRF-induced analgesia (Przewlocki et al., 1992, Schäfer et al., 1994, Stein et al., 1990b) and the reconstitution of immunosuppressed rats with lymphocytes reverses this effect (Hermanussen et al., 2004).
END is derived from proopiomelanocortin (POMC) and is processed within the pituitary and various types of non-neuronal cells (Heijnen et al., 1991, Raffin-Sanson et al., 1999, Sharp and Linner, 1993, Slominski et al., 2000, Westly et al., 1986). The first exon of the POMC gene includes promotor binding sites and the mRNA cap region, which are typically untranslated. The second exon encodes the signal peptide sequence necessary for directing the nascent polypeptide to the regulated secretory pathway (Kalies and Hartmann, 1998). The sequences of functionally active peptides such as adrenocorticotrophic hormone, melanocyte-stimulating hormones, and END are contained within the third exon (Drouin et al., 1985).
Previous studies indicate that translation products of POMC transcripts lacking the signal sequence are neither processed to authentic peptides nor secreted (Clark et al., 1990, Rees et al., 2002). Several studies detected truncated POMC transcripts in naïve lymphocytes (Buzzetti et al., 1989, Cabot et al., 1997, DeBold et al., 1988, Lacaze-Masmonteil et al., 1987, Oates et al., 1988, Przewlocki et al., 1992), while findings of full-length POMC mRNA are limited to a single study (Stephanou et al., 1991). However, full-length POMC mRNA may be present under pathological conditions, as demonstrated in a T lymphoma cell line (Buzzetti et al., 1989) and after mitogen treatment of lymphocytes in vitro (Lyons and Blalock, 1997). In the present study we set out to examine the expression of exon 1–3 and exon 2–3 spanning POMC mRNA in lymphocytes from rats with Complete Freund's adjuvant (CFA)-induced paw inflammation. In addition, we quantified exon 2–3 spanning POMC transcripts and END in relation to the development of inflammatory signs and hyperalgesia, we confirmed the functional relevance of END in producing intrinsic pain inhibition in vivo and we verified the specificity of the END antibody by use of END−/− mice.
Section snippets
Experimental animals and induction of inflammation
All experiments were approved by the animal care committee of the Senate of Berlin and strictly followed the guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Male Wistar rats (225–300 g, Charles River Breeding Laboratories) received an intraplantar (i.pl.) injection of 0.15 ml Complete Freund's Adjuvant (CFA; Calbiochem, La Jolla, CA, USA) or 0.15 ml NaCl (controls) into the right hindpaw under brief isoflurane (Rhodia Organic Fine Ldt., Bristol, UK)
Paw inflammation and nociception
Injection of CFA into the right hindpaw of rats resulted in the development of overt signs of inflammation and pain, i.e. increased paw volume and decreased PPT. Paw volume began to increase significantly at 2 h after CFA-inoculation and remained elevated until 96 h compared to contralateral and saline treated hindpaws (P < 0.001, Two-way ANOVA and Bonferroni multiple comparison, Table 2). PPT began to decrease significantly at 2 h and remained reduced until 96 h compared to contralateral and
Discussion
This study demonstrates that: i) rats with hindpaw inflammation show increased hyperalgesia when END is locally neutralized by an antibody or when peripheral opioid receptors are blocked by an antagonist, ii) POMC and END are co-localized in cells of popliteal LN and END content increases with the duration of paw inflammation, iii) exon 1–3 spanning POMC mRNA is detectable in LN beginning at 2 h of inflammation, iv) signal sequence-encoding POMC mRNA is expressed under control conditions and is
Acknowledgement
We thank M. Low for providing the C57BL/6j WT and END−/− mice. This work was supported by the Deutsche Forschungsgemeinschaft (KFO 100/1; GRK 1258). All authors confirm to have no conflicting financial interests.
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2020, Immunology LettersCitation Excerpt :POMC, PENK and PDYN, as well as the related opioid peptides END, ENKs and DYN have been detected in various immune cells accumulating in inflamed tissue in different animal pain models. Hence, POMC mRNA and protein, and END were identified in granulocytes, monocytes/macrophages, T helper (Th; CD4+) lymphocytes and B lymphocytes in the blood and inflamed paws, and in lymph node lymphocytes following CFA-induced hind paw inflammation [23,66,102–110]. POMC mRNA was also found in neutrophils infiltrating tongue in the oral cancer model [111], and END was expressed in mononuclear cells in the bone tumor tissue [112] and in CD4+ T lymphocytes in inflamed colon in the dextran sodium sulphate (DSS)-induced colitis, which models inflammatory bowel syndrome (IBS) [113].
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2013, European Journal of Pharmacology
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Both authors have equally contributed to the present study and share first-authorship.