SVIBOR - Project code: 1-08-216


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Project code: 1-08-216

Modulation of NK-activity by Leu- and Met-enkephalin

Main researcher: GABRILOVAC, JELKA (12736)

Type of research: basic
Duration from: 01/01/91. to 12/31/93.

Papers on project (total): 34
Papers on project quoted in Current Contents: 10
Institution name: Institut "Ruđer Bošković", Zagreb (98)
Department/Institute: Department of Experimental Biology and Medicine
Address: Bijenička c. 54, p/p/ 1016, 10000 Zagreb
City: 10000 - Zagreb, Croatia
Fax: 385 (0)1 425-497
Phone: 385 (0)1 4561-111
Fax: 385 (01) 425-497
E-mail: gabrilovac

Summary: The ability of endogenous opioid peptides Leu- and Met-enkephalin (LENK, MENK) to modulate cytotoxic activity of NK-cells in vivo was tested 2 hours after intraperitoneal (i/p) injection of enkephalin (7.5 or 10 mg/kg body weight) into CBA mice, or in vitro, after 18-hour incubation of human peripheral blood lymphocytes (PBL) with enkephalins of the broad range of concentrations (10-7 M to 10-14 M), respectively. Both enkephalins bidirectionally modulated ( i.e. stimulated or suppressed) NK-activity both after in vivo and in vitro treatment. Bidirectional modulation of NK-activity by LENK/MENK was associated with bidirectional changes of the ability of PBL to secrete interferon (IFN) in vitro, as well as with bidirectional changes in intracellular cAMP level, suggesting their causal relationship. In vivo, beside NK-cytotoxicity LENK also altered the number of NK-cells in mouse spleen. In addition to the effects on NK-cells, LENK also exerted bidirectional effects on the migratory ability of mouse spleen cells, both after in vivo and in vitro treatment. The direction of this effect varied with the level of intracellular cAMP supporting the evidence for the role of cAMP in signal transduction of enkephalins. The observed effects of enkephalins on NK-activity, NK-cell number, cell motility could only partly be reversed by opioid receptor antagonist naloxone, suggesting beside opioid, involvement of nonopioid mechanism(s) in their action. 1994/95 Extending the investigation of endogenous opioid peptides as immunomodulators, we tested whether altered immunoreactivity induced by enkephalins in vivo, may reflect redusribution of the lymphocyte populations in peirpheral organs, due to activation of HPA axis and increase of glucocorticoids. Additionally, the effect od Leu-enkephalin (LENK) on the expression of MHC-I antigens was also examined, as similar interaction was reported for another opioid peptide, b-endorphin. The data obtained have shown that LENK does not alter the cellular composition in the spleens of CBA mice, 2 h after injection (10 mg/kg): the frequency of CD3+, CD4+, CD8+, and sIg+ cells, as well as the density of these markers remained unchanged. However, LENK modulated the expression of MHC-I on the spleen cells: it was down-regulated in nonstimulated, and up-regulated in Con-A-stimulated cells. The in vitro treatment of spleen cells with LENK (10-8 M to 10-12 M) confirmed the data obtained in vivo: LENK did not alter the expression of CD3, CD4, CD8, and sIg, but did alter the expression of MHC-I, by down-regulating it on nonstimulated, and by up-regulating it on the Con-A-stimulated spleen cells. These changes were mainly due to T (CD3+) ymphocytes, and within T lymphocytes to CD4+. However, an opposite effect of LENK on MHC-I expression was observed on CD8+ cells. Both down- and up-regulation of the MHC-I expression were dose-dependent. The peak of LENK activity varied with the cell population and the state of cell activity. Generally, a higher concentration of LENK (10-10 M) was needed to affect naive cells, and lower (10-12 M) to affect Con-A-stimulated splenocytes. The in vitro results suggest a direct effect of LENK on lymphocytes, confirming our previous data. Altered expression of MHC-I antigens in the presence of LENK suggests an association between the binding sites for LENK and these antigens. The observed interaction of LENK and MHC-I may be the mechanism underlying the LENK-induced functional alterations reported earlier.

Keywords: NK-cells, NK-activity, Leu-enkephalin, Met-enkephalin, naloxone, opioid receptors, cAMP, interferon, cell migration, mechanism of immunomodulation, HPA axis, lymphocyte redistribution, MHC-I antigen expression

Research goals: The aim of the project was to test the ability of endogenous opioids Leu- and Met-enkephalin (LENK, MENK) to modulate cytolitic activity of NK-cells. Whereas the in vivo experiments can not distinguish between direct and indirect (via central nervous system) effects of enkephalins, the in vitro experiments suggest their direct effects on cells of the immune system. Involvement of opioid receptors in NK modulation was tested by using the nonselective opioid receptor antagonist, naloxone. Reversion of the effects induced by enkephalins after blocking of opioid receptors by naloxone was expected, if their action is mediated through opioid mechanism. In order to elucidate the mechanism of signal transduction by enkephalins the level of intracellular cAMP was determined after in vitro tretament of lymphocytes with MENK. Alteration in level of intracellular cAMP was expected if it is involved in signal transduction. Direct effects of LENK, as well as the role of cAMP in them, was also tested by measuring migratory ability of mouse splenocytes in vitro. To that purpose agents that intefere with cAMP metabolism, both at its afferent and at efferent arc, were used. Further, it was tested whether modulation of NK-cytotoxicity was associated with alteration of the NK-cell number. To that purpose the phenotype of NK-cells was determined. Finally, it was tested whether the modulation of NK-activity by enkephalins involves secretion of interferon (IFN), one of the key regulators of NK-cells. 1994/95 We have previously reported that endogenous opioid peptides -enkephalins - modulate immune reactivity of mouse spleen cells after in vivo (i/p) application. The immunomodulatory activity could partly be ascribed to the activation of HPA axis, judging from its abrogation by adrenalectomy. Increased glucocorticoid level, observed in enkephalin-treated mice, may cause redistribution of lymphocytes in periphery, which may result in altered immunoreactivity. This possibility was tested by analysing the cellular composition in the spleens of mice 2 h after i/p injection of Leu-enkephalin (LENK), by using the CD3, CD4, CD8 and sIg markers. In addition, the effects of LENK on the expression of MHC-I antigens was tested, as similar interaction of another opioid peptide, b-endorphin, had been reported. In order to examine the direct effect of LENK on lymphocyte marker expression, in vitro experiments have been performed by incubating the spleen cells with LENK (10-8 M to 10-12 M). In order to assess the influence of the state of cell activity on the LENK effect (its direction, and the active LENK concentration), Con-A was used as a stimulator.


  1. Name of project: >095 Haematologische und immunologische Untersuchungen von Leukaemie-Erkrankungen verschiedener Genese (Leukaemia, immunotherapeutic Model)
    Name of institution: GSF-Forschungszentrum fuer Umwelt und Gesundheit, Institut fuer Immunologie
    City: 70 - Muenchen, SR Njemačke


  1. Name of institution: GSF'Forschungzentrum fuer Umwelt und Gesundheit GmbH Institut fuer Immunologie
    Type of institution: State institute
    Type of cooperation: Occasional exchange of experts
    City: 70 - Muenchen, SR Njemačke


  1. Name: .
    Type of achievement: Other

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Last update: 10/11/95