Lymphocytes upregulate signal sequence-encoding proopiomelanocortin mRNA and beta-endorphin during painful inflammation in vivo

https://doi.org/10.1016/j.jneuroim.2006.11.033Get rights and content

Abstract

Proopiomelanocortin (POMC)-derived beta-endorphin1–31 (END) released from immune cells inhibits inflammatory pain. We examined the expression of END and POMC mRNA encoding the signal sequence required for entry of the nascent polypeptide into the regulated secretory pathway in lymphocytes of rats with inflamed hindpaws. Within 12 h of inflammation, END increased in popliteal lymph nodes and at 96 h the intraplantar neutralization of END exacerbated pain. Lymphocytes expressed POMC, END, and full-length POMC mRNA. Semi-nested PCR revealed 8-fold increased exon 2–3 spanning POMC mRNA. Thus, painful inflammation enhances signal sequence-encoding lymphocytic POMC mRNA needed for regulated secretion of functionally active END.

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.

References (49)

  • D.A. Rees et al.

    Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence

    Mol. Cell. Endocrinol.

    (2002)
  • M. Schäfer et al.

    Corticotropin-releasing factor in antinociception and inflammation

    Eur. J. Pharmacol.

    (1997)
  • C. Stein et al.

    Local analgesic effect of endogenous opioid peptides

    Lancet

    (1993)
  • A. Stephanou et al.

    Characteristics and kinetics of proopiomelanocortin mRNA expression by human leucocytes

    Brain Behav. Immun.

    (1991)
  • M. Verma-Gandhu et al.

    CD4(+) T-cell modulation of visceral nociception in mice

    Gastroenterology

    (2006)
  • M. Zimmermann

    Ethical guidelines for investigations of experimental pain in conscious animals

    Pain

    (1983)
  • W. Binder et al.

    Analgesic and antiinflammatory effects of two novel kappa-opioid peptides

    Anesthesiology

    (2001)
  • W. Binder et al.

    Sympathetic activation triggers endogenous opioid release and analgesia within peripheral inflamed tissue

    J. Eur. Neurosci.

    (2004)
  • R. Buzzetti et al.

    Expression of pro-opiomelanocortin gene and quantification of adrenocorticotropic hormone-like immunoreactivity in human normal peripheral mononuclear cells and lymphoid and myeloid malignancies

    J. Clin. Invest.

    (1989)
  • P.J. Cabot et al.

    Immune cell-derived beta-endorphin. Production, release, and control of inflammatory pain in rats

    J. Clin. Invest.

    (1997)
  • H. Cho et al.

    Recombinant guinea pig tumor necrosis factor alpha stimulates the expression of interleukin-12 and the inhibition of Mycobacterium tuberculosis growth in macrophages

    Infect. Immun.

    (2005)
  • A.J. Clark et al.

    In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA

    Mol. Endocrinol.

    (1990)
  • F. de Longueville et al.

    Gene expression profiling of PB and PCN. Correlation between data from DualChip rat hepato and real-time PCR

    (2003)
  • C.R. DeBold et al.

    Proopiomelanocortin gene is expressed in many normal human tissues and in tumors not associated with ectopic adrenocorticotropin syndrome

    Mol. Endocrinol.

    (1988)
  • Cited by (60)

    • Immune cell-mediated opioid analgesia

      2020, Immunology Letters
      Citation 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].

    • Lack of interleukin-17 leads to a modulated micro-environment and amelioration of mechanical hypersensitivity after peripheral nerve injury in mice

      2014, Pain
      Citation Excerpt :

      The evidence presented above clearly suggests that lower expression of endogenous opioid peptides after PSNL would be a result of fewer leukocytes infiltrating the injured site in IL-17−/− mice. Furthermore, previous reports state that the painful stimulus not only mobilizes circulating polymorphonuclear (PMN) cells, monocytes, and lymphocytes and facilitates opioid peptide release, but also increases the expression of opioid peptides, both in vitro and in vivo [20,31,33]. In this regard, it should also be noted that normal and immunocompromised mice receiving an opioid antagonist were found to display augmented hyperalgesia [17], suggesting that opioid peptides are components of an endogenous negative feedback system for control of neuroinflammation after nerve injury.

    • Opioids, sensory systems and chronic pain

      2013, European Journal of Pharmacology
    View all citing articles on Scopus
    1

    Both authors have equally contributed to the present study and share first-authorship.

    View full text