Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice

Abstract

BackgroundT cell-derived opioids play a key role in the control of inflammatory pain. However, the nature of opioids produced by T cells is still matter of debate in mice. Whereas β-endorphin has been found in T lymphocytes by using antibody-based methods, messenger RNA (mRNA) quantification shows mainly mRNA encoding for enkephalins. The objective of the study is to elucidate the nature of T cell-derived opioids responsible for analgesia and clarify discrepancy of the results at the protein and genetic levels.MethodsCD4+ T lymphocytes were isolated from wild-type and enkephalin-deficient mice. mRNA encoding for β-endorphin and enkephalin was quantified by RT-qPCR. The binding of commercially available polyclonal anti-endorphin antibodies to lymphocytes from wild-type or enkephalin knockout mice was assessed by cytofluorometry. Opioid-mediated analgesic properties of T lymphocytes from wild-type and enkephalin-deficient mice were compared in a model of inflammation-induced somatic pain by measuring sensitivity to mechanical stimuli using calibrated von Frey filaments.ResultsCD4+ T lymphocytes expressed high level of mRNA encoding for enkephalins but not for β-endorphin in mice. Anti-β-endorphin polyclonal IgG antibodies are specific for β-endorphin but cross-react with enkephalins. Anti-β-endorphin polyclonal antibodies bound to wild-type but not enkephalin-deficient CD4+ T lymphocytes. Endogenous regulation of inflammatory pain by wild-type T lymphocytes was completely abolished when T lymphocytes were deficient in enkephalins. Pain behavior of immune-deficient (i.e., without B and T lymphocytes) mice was superimposable to that of mice transferred with enkephalin-deficient lymphocytes.ConclusionsRabbit polyclonal anti-β-endorphin serum IgG bind to CD4+ T lymphocytes because of their cross-reactivity towards enkephalins. Thus, staining of T lymphocytes by anti-β-endorphin polyclonal IgG reported in most of studies in mice is because of their binding to enkephalins. In mice, CD4+ T lymphocytes completely lose their analgesic opioid-mediated activity when lacking enkephalins.

Mu and delta opioid receptor knockout mice show increased colonic sensitivity

“…Transcripts for POMC were undetectable in the colon, whereas previous studies reported positive immunolabeling for beta‐endorphin in human or mouse colonic tissue (Verma‐Gandhu et al., ; Hughes et al., ). This may be caused by differences in experimental conditions including mouse strain and the use of immunohistochemistry rather than RT‐PCR (Basso et al., ).…”

Endogenous control of inflammatory visceral pain by T cell‐derived opioids in IL‐10‐deficient mice

“…23 Data were acquired for each individual sample on 100,000 cells by forward and side scatter intensity on F I G U R E 1 Piroxicam treatment induces (accelerates) colitis in IL-10 −/− mice. Cells were incubated with antibodies in FACS buffer (PBS 1% FCS, 5 mmol/L EDTA, 0.1% NaN 3 ) for 30 minutes on ice.…”

“…After washing, cells were fixed, permeabilized, and then stained with either rabbit anti-Met-enkephalin polyclonal IgG antibodies (Merck-Millipore-Chemicon International) or control rabbit non-immune serum IgG (Jackson Immunoresearch Laboratory, Baltimore, PA) as previously described. 23 Data were acquired for each individual sample on 100,000 cells by forward and side scatter intensity on F I G U R E 1 Piroxicam treatment induces (accelerates) colitis in IL-10 −/− mice. IL-10 −/− mice were fed with standard chow diet without (white symbols) or with piroxicam (black symbols) for 10 d (n = 26 for each group of mice).…”

The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

“…through the arachidonic acid cascade, they also play a role in mediating endogenous peripheral analgesia, as reflected by the increased expression of opioid receptors on the peripheral terminals of sensory neurons and heightened local production of opioid peptides at the site of inflammation (10,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This suggests an integral role of the immune system in mediating analgesia in response to noxious stimuli.…”

“…Deletion of genes coding for proenkephalin (PENK) in the brain and immunocytes results in down-regulation of Met-ENK release, suggesting that ENK is synthesised from the same precursor protein molecule in both nervous and immune systems (137). In addition, the production of peripheral opioid peptides involves both circulatory and resident inflammatory cells such as T-and B-lymphocytes, granulocytes (during early inflammation) and monocytes/macrophages (during late inflammation) (16,18,(138)(139)(140)(141).…”