Modulation of NK-activity by Leu- and Met-enkephalin
Main researcher
: GABRILOVAC, JELKA (12736) Assistants
MARTIN-KLEINER, IRENA (56662)
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
Communication
Fax: 385 (0)1 425-497
Phone: 385 (0)1 4561-111
Fax: 385 (01) 425-497
E-mail: gabrilovac olimp.irb.hr
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.
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.
COOPERATION - PROJECTS
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
COOPERATION - INSTITUTIONS
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