Novel orphanin FQ/nociceptin transcripts are expressed in human immune cells
Introduction
Orphanin FQ or Nociceptin (OFQ/N) is a neuropeptide that has been implicated in the mediation of pain sensation and auditory perception, as well as complex behaviors such as lordosis, locomotion, learning and anxiety (reviewed by Harrison and Grandy, 2000). The 17-amino acid peptide is derived from a larger precursor encoded by a transcript derived from four exons (Mollereau et al., 1996). OFQ/N mediates its actions via a G protein coupled receptor termed the NOP receptor, also known as ORL-1 (Mollereau et al., 1994). The NOP receptor has striking similarity to the classical opioid receptors (mu, delta and kappa) both in primary sequence, as well as in secondary messenger activation. NOP receptor activation of Go/i subunits leads to the inhibition of adenylate cyclase, activation of MAPK, inhibition of Ca2+ conductance and potentiation of K+ currents Connor et al., 1996a, Connor et al., 1996b, Hawes et al., 1998, Matthes et al., 1996, Meunier et al., 1995, Reinscheid et al., 1995.
The NOP receptor expression in immune cells has been well documented. Halford et al. (1995) demonstrated that mouse peripheral blood lymphocytes (PBLs) express NOP receptor transcripts following mitogen activation. Other studies demonstrated NOP expression in human immune tissues, PBLs and numerous cell lines Miotto and Evans, 1996, Wick et al., 1995 and that the RNA levels for the NOP receptor in human mixed lymphocytes or selected cell lines were comparable to those of the human central nervous system (Peluso et al., 1998). NOP expression in immune cells was also demonstrated with ligand binding studies Hom et al., 1999, Serhan et al., 2001, as well as agonist-stimulated GTPγS binding experiments (Peluso et al., 2001). Furthermore, NOP receptor expression has been implicated in the regulation of immune function such as antibody production (Halford et al., 1995), modulation of cAMP in human B lymphocyte cell line (Raji) (Hom et al., 1999) and neutrophil chemotaxis (Serhan et al., 2001).
Due to the previous findings that established the expression of NOP receptor in immune cells, it was of interest to determine if human immune cells were also capable of synthesizing the NOP receptor ligand OFQ/N. We have previously demonstrated that human neuronal OFQ/N transcripts can be alternatively spliced to generate a total of four possible messages (Arjomand and Evans, 2001). Of these, the two longer transcripts were demonstrated to be conserved in both mouse and rat, whereas the shorter transcripts were novel in human and lacked exon 2 which contains the putative translational start site and signal peptide. Based on these findings, we sought to determine whether human PBLs expressed OFQ/N transcripts, and if so, identify the various splice variants. Here we report that upon activation, human PBLs express OFQ/N messages, while resting PBLs express novel OFQ-like messages that utilize a novel 5′ exon, and presumably, an alternative transcriptional start site and promoter. Further analyses of sorted PBLs revealed that expression of these novel transcripts were limited to CD19+B lymphocytes.
Section snippets
Cell culture
Human peripheral blood lymphocytes (PBLs) were isolated by Ficoll gradient centrifugation and cultured in RPMI containing 10% human serum, 2 mM l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 200 U/ml of recombinant human IL-2 at 37 °C and 5% CO2. PBL activation was induced with 1 μg/ml of phytohemagglutinin (PHA). Leukocyte subsets were isolated by immunomagnetic positive selection for CD3+, CD4+, CD8+, CD14+, CD19+, CD56+ cells (Miltenyi Biotec, Auburn, CA).
CEM, THP, Jurkat and
Human spleen expresses OFQ/N transcripts lacking exon 2
We have previously described the existence of low abundant, alternatively spliced OFQ/N messages in human brain whereby exon 2, which encodes the translational start site, was deleted (Arjomand and Evans, 2001). The RPA probe was designed to distinguish the two alternatively spliced forms of OFQ/N transcripts by spanning portions of exons 2 and 3 (Fig. 1A). A protected fragment of 457 nt corresponds to transcripts containing exon 2, whereas a 326-nt protected fragment corresponds to OFQ/N
Discussion
The opioid system has been implicated to play a role in regulation and maintenance of the immune system by modulating factors such as antibody and cytokine production, phagocytosis, chemotaxis and cellular proliferation and differentiation (reviewed by Salzet et al., 2000, McCarthy et al., 2001). In addition, immune-derived opioid peptides have been shown to induce analgesia at the site of inflammation Mousa et al., 2001, Stein, 1995. Hence, the opioid system may not only function as an immune
Acknowledgements
This work was supported by the National Institute on Drug Abuse Grant DA-05010. JA is a Hatos scholar and supported in parts by the Norman Cousins Center for Psychoneuroimmunology and Training Grant T32-MH17140. The authors are grateful to Bojana Rnjak and Dr. Jim Boulter for technical support and to Duane Keith for the critical reading of the manuscript.
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