We have found recently that DYN and related opioid peptides can serve as positive regulators of in vitro myelopoiesis. These peptide hormones enhance colony.
Journal
SHORT
of Leukocyte
Biology
42:171-174
(1987)
COMMUNICATION
Dynorphin and Related Opiold Peptides Enhance Tumoricidal Activity Mediated by Murine
Peritoneal
Macrophages
James
and
Department
S. Foster of Microbiology,
Robert
University
N. Moore
of Tennessee,
Knoxville
The influence of dynorphin A (DYN) and related opioid peptides on the tumoricidal function of activated murine peritoneal exudate macrophages (PEM) was investigated. Addition of DYN to macrophage cultures previously activated with mixed a + 3-interferon (IFN-a/fl) and bacterial Iipopolysaccharlde (LPS) significantly enhanced their ability to lyse P815 murine mastocytoma cells in a 16 hr chromium-release assay. The effects of DYN were dependent on prior macrophage activation. Peptide subfragments of DYN were effective in a manner similar to that of the 17-amino-acid parent molecule, indicating that peptide interaction with either ic or &-opioid receptors on the effector cell is effective In potentiating lytic function. The involvement of opiate receptors was confirmed by inhibition of the effects of DYN and leucine enkephalin by the opiold receptor antagonist naloxone. Finally, In addition to IFN-a/i3-primed macrophages, DYN also augmented tumoricidal function in PEM primed for cytotoxicity by either -y-interferon (IFN-’y) or the calcium ionophore A23187, indicating that DYN potentiates function in activated macrophages independent of the specific mode of activation.
Key words:
cytotoxicity,
lipopolysaccharide,
Neuroendocrine mediators influence a broad spectrum of immune functions both in vitro and in vivo [7,11]. Resulting proposals suggest the existence of a neuroimmune regulatory axis whereby intercommunication between the immune and neuroendocnine systems performs homeostatic function [1]. Included in the supporting observations
are
roendocrine nuclear
reported
reports
hormones phagocyte
suggesting
can
system.
that
a variety
influence
cells
Goldman
and
of neu-
of the monoBar-Shavit
[6]
of phagocytic capacity by the neuropeptides substance P and neurotensin. Neurotensin also enhances the tumoricidal activity of ‘y-interferon (IFN‘y)-activated macrophages, whereas stress-related endocnine mediators, specifically adrenocorticotropic hormone
augmentation
and noradrenaline,
inhibit
cytotoxic
capacity
[10].
Opioid peptides also influence macrophage functions in vitro. Methionine-enkephalin enhances antibody-dependent cellular cytotoxicity mediated by munine penitoneal macrophages while inhibiting their phagocytic capacity [5]. In addition, methionine-enkephalin and two other opioid peptides, f3-endorphin and dynorphin A (DYN), stimulate superoxide radical production by macrophages [5 , 17]. We have found recently that DYN and related opioid peptides can serve as positive regulators of in vitro myelopoiesis. These peptide hormones enhance colony formation by marrow-derived precursors in response to macrophage-specific colony-stimulating factor (CSF-1 ; R.N. Moore, manuscript in preparation). To © 1987 Alan
R. Liss, Inc.
mastocytoma
cells
define further the potential regulatory effects of DYN on the mononuclear phagocyte system, we have examined the influence of the peptide hormone on the tumonicidal activity of munine macrophages. We report here the interesting observation that, although DYN and related peptides do not influence the activation of macrophages, these peptide hormones can enhance tumonicidal activity of preactivated macrophage cultures. The data reported are representative, in each case, of multiple experiments with
qualitatively
similar
results
performed
over
a 1 year
period.
Tumonicidal activity represents a “higher-order” macrophage function requiring priming by IFN or other agents and a second signal for activation, provided in vitro by bacterial lipopolysacchanide (LPS) [12,13,161. As is shown in Figure 1 , DYN enhanced the cytotoxicity of
activated
(PEM)
munine
for P815
penitoneal
mastocytoma
exudate
macnophages
cells
as measured in a 16 hr chromium-release assay [13] When added concurrently with tumor target cells to macrophage cultures that had been preincubated overnight with optimally activat.
ing concentrations
Received
February
of IFN-a/fl
24,
1987;
accepted
Reprint requests: Robert N. Moore, Walters Life Sciences Bldg. , University 37996-0845.
and
LPS,
April
significant
(p
5 x 108 U/mg protein) at a concentration of 400 U/mI, LPS (Escherlchla coil 0111 :84; SIgma) at 10 ng/ml, or both. DYN (113; Sigma) was added at the indicated concentrations, and tumoricidal activity was assessed in a 16 hr chromium-release assay [101. The P815 murine mastocytoma cell line (kindly provided by Drs. J.L. Pace and S.W. Russell, University of Florida, Gainesville) served as the tumor target. P815 cells were labeled with 200 Ci/mI of Na51CrO4 (Amersham Corp, Arlington Heights, IL) and added to wells with activated PEM. After 16 hr, half of the supernatant (100 I) was removed from each well and the radioactivity for each sample determined in a gamma spectrometer. Percent specific chromium release as an index of tumor killing was calculated after subtrating spontaneous isotope release from P815 incubated In RPMI alone and relative to total detergent-releasable counts. All samples were assayed at least in triplicate. Results are presented as mean ± i SE and are representative of data from multiple qualitatively similar experiments. Statistical significance was evaluated by Student’s t test.
0.05) enhancement of tumonicidal activity was evident at DYN (fragment 1-13) concentrations ranging from i0 to l0 10 M. The observed enhancement of cytolysis, though variable in magnitude, was typically 50 % of the control value and was consistently
1-17
from
30%
demonstra-
to
were
activated
overnight
with IFN-a//3
and LPS before
addition
ofpeptides and P815 targets. Assay conditions were as given in Figure 1 . The data presented are representative of three separate experiments yielding similar results. bDYN subfragments (all from Sigma Chemical Co.) were added at a concentration of l0 M. CMean specific chromium release ± 1 SE for quadruplicate determinations. dSignificantly different from control at p < 0.05.
ble in multiple
experiments.
Little
or no cytolytic
activity
was evident when macrophages were preincubated with either IFN-a//3 or LPS alone, irrespective of added DYN, nor did even prolonged incubation with DYN alone induce lytic activity (not shown). The augmentative effect on cytolysis mediated by DYN seems to reflect an influence on the effector cell population. elicit chromium release from labeled treatment of P815 with DYN influence
DYN
P815 their
itself ,
did
not
nor did presusceptibility
to lysis by activated macnophages (not shown). Thus DYN appeared to act on preactivated macrophages, enhancing lytic function, but did not act as an additional priming or second signal. Subfragments of the DYN molecule (Fig. 2) [4] show differential selectivity for opiate receptors. The 17-aminoacid parent DYN molecule and amino-terminal fragments larger than nine amino acids exhibit selective affinity for the K-opioid receptor [2,3]. Leucine-enkephalin, represents the first five amino-terminal amino
which acids of
DYN, binds selectively to the #{244}-opioidreceptor [3] DYN subfragments were tested for their ability to enhance tumonicidal function. Results are given in Table 1 PEM were activated with IFN-a/fl and LPS, and DYN .
.
Dynorphin-Enhanced TABLE 2. Augmentation and Leucine-Enkephalin
of Tumoricidal Function by DYN Is Abrogated by Naloxonea
Macrophage
173
Cytotoxicity
TABLE 3. Effect of DYN (1-17) on Tumoncidal Activity of Murine PEM Primed With lFN-a/3, IFN-y, or lonophore A23187
Specific
chromium
release
( %)b
Naloxone (10_6 M)c
+
Peptide
added
Control
None DYN (l0 M) DYN(10’#{176}M) Leucine-enkephalin (1O M) Leucine-enkephalin (10’#{176}M) PEM
were
activated
35.0 48.8 50.8 47.6 44.0
± 1.4 ± 21d ± 1.7” ± O.8 ±
30.6 48.8 36.2 46.8
± 0.5 ± 21d ± 1.6 ± 0#{149}5d
35.4 ± 1.7
2.0”
PEM
with
IFP-a/$
and
LPS
overnight
before addition of DYN (1-17) and naloxone as noted and P815 targets. Assay conditions were as given for Figure 1 . The data presented are representative of three separate experiments yielding similar results. ‘Mean specific chromium release ± 1 SE for triplicate determinations. CNaloxone was acquired from Pitman-Moore, Inc. (Washington Crossing, NJ). dSignificantly different from control at p < 0.05.
DYN
with”
IFN-a/3 + LPS IFN-a/i3 + LPS IFN--y” + LPS IFN-y’ + LPS A23l87 + LPS A23187C + LPS PEM
as before
activated
were
(1-17)”
Specific chromium release (%)C
-
34.0
±
1.9
+
53.7
±
3.3
-
33.6
± 3.2
+
55.1
± 2.9
-
42.4 ± 0.9 60.3 ± 391
+
activated
with
the agents
listed
for 4 hr at 37#{176}C before
addition of DYN (1-17) and P815 targets. Concentrations of priming agents were IFN-a/f3 at 200 U/mI, recombinant murine IFN-y at 10 U/ml, and ionophore A23 187 at 2 x l06 M. LPS was added at 10 ng/ml. Assay conditions were as given for Figure 1. The data presented are representative of two separate experiments yielding similar results. bDYN (1-17) was added at 10 ‘#{176}M. CMean specific chromium release ± 1 SE for triplicate determinations. dRecombinant murine IFN-y was kindly provided by Dr. Albert Zlotnik (DNAX cThe ionophore 1Significantly
Research Institute, Palo Alto, CA). A23l87 was acquired from Sigma. different
at p < 0.05.
To determine whether DYN would enhance tumoniciwere added at i0 M. All the DYN fragwhether ic- or 5-receptor-specific, augdal function when PEM were primed with agents other than IFN-a/13, PEM were primed with optimal concenmented cytolysis at nanomolar concentrations, indicating trations of IFN-a//3, recombinant IFN-’y (14), or the that enhancement of cytolysis may be mediated through calcium ionophore A23187 [9], along with LPS and DYN either receptor. DYN fragment 1-8, which reacts with added as indicated. Results are given in Table 3. Enboth ic- and #{244}-receptors [3] was also active. Dose-rehancement of tumonicidal function by DYN was indepensponse curves for DYN and DYN subfragments were optimal at 108_10 10 M; however, no real difference dent of the nature of the priming stimulus, an increase in was noted in the capacity of the subfragments to augment cytolysis in the presence of DYN being evident when lytic function, independent of receptor specificity (not PEM were primed with IFN-”y or A23187 as was observed previously with IFN-a/fl. DYN was also found to shown). augment lysis when the amounts of IFN-a//3, IFN-y, or To confirm that DYN-related peptides are indeed funcLPS were increased to levels tenfold above optimal (not tioning via opiate receptors, the effect of the general shown). The results indicate that DYN enhances the opioid receptor-antagonist naloxone on DYN and leufunction of maximally activated cells irrespective of the cine-enkephalin-enhanced tumoricidal function was investigated. As can be seen in Table 2, naloxone at a nature or quantity of activating agents. The results demonstrate that DYN and related opioid concentration of 106M abrogated the effects of both DYN and leucine-enkephalin when these peptides were peptides can enhance the tumonicidal function of munine macrophages. This effect of DYN is dependent on prior added to activated PEM at 10 M. Naloxone had no activation, DYN alone being unable to elicit effect, however, when the peptides were present at a macrophage an activated state. Maximal augmentation of lytic funcconcentration of i07 M. Lower concentrations of naloxone were unable to block the effects of DYN or leucinetion typically occurred at DYN concentrations of l0 8_ 10 M, concentrations within the physiological realm enkaphalin at 10 M (not shown). These results are consistent with the relatively poor affinity of naloxone [8]. Subfragments of the DYN molecule were also effecfor K- and b-receptors [ 15] The slight depression by tive, indicating that this effect may be mediated by the Knaloxone of activity in macrophages not treated with and/on #{244}-opioidreceptors. This is supported by the demopioid was consistently observed; however, the depresonstration that the effects of DYN and leucine-enkephalin sion was never statistically significant. The results sugare inhibitable by the opioid antagonist naloxone. gest that DYN and leucin-enkephalin mediate their effects Regarding the mechanism by which DYN potentiates on activated PEM through classical opioid mechanisms, cytolytic function, little is known. Mediation of the effect of activated macrophages to DYN for likely interacting with the effector cell at K- and/or #{244}-is rapid, exposure 4 hr, followed by removal of the peptide, being sufficient opioid receptors. subfragments ments tested,
.
174
Foster
and Moore
to give full augmentation (not shown). An increase in overall capacity for killing could be related to enhanced production of oxidative metabolites [17] or to potentiation of other stages in the actual lytic cycle. Investigations of the biochemical events in DYN-induced analgesia have focused on the capacity of DYN to modulate calcium conductivity and thus cellular response [18]. With respect to this, it is of interest that DYN maintains effectiveness when macrophages are primed with the calcium ionophore A23187, although the significance of this observation is not clear. Modulation of calcium fluxes may represent one mechanism by which DYN influences responsiveness of the activated, cytolytic cell although other regulatory modes doubtless participate. Our results demonstrate that DYN and related peptide hormones can positively influence tumonicidal effector function in activated macrophages. Taken together with the augmentative influence of DYN on myelopoiesis and other reported functional effects [17], the observations reported here suggest a potential role for DYN and related opioid mediators in modulation of macrophage growth and effector function. Framed in terms of a neuroimmune regulatory axis [1], DYN may represent an important neuroendocrine influence on the mononuclear phagocyte system.
ACKNOWLEDGMENTS This work was supported in part by National Institutes Health grant AI-18960. R.N.M. is the recipient of National Institutes of Health Research Career Development Award AI-00657. of
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