Opioid peptides: do they have immunological significance?

TIPS - September 1985

368

Opioid peptides: do they have immunological significance? Hansj6rg Teschemacher and Lothar Schweigerer Almost a decade after the discovery of the endogenous opioids, their physiological role is still unclear. Hansj6rg Teschemacher and Lothar Schweigerer describe recent studies which indicate that opioid peptides might have immunological significance. These peptides can modulate several immune functions and appear to be constituents of the immune system themselves. It is possible that opioid peptides are involved in the immune response to stress or even in the immune defence against neoplastic disease. A series of endogenous opioids have been discovered since the enkephalins were first described in 1975. However, although an impressive amount of information about their structure, biogenesis and location in mammals and lower animal classes has been collected, their physiological significance remains elusive 1. However, there is n o w evidence that opioid peptides, rather than playing one unique role, have multiple physiological functions - at least in mammals; it is also quite clear that their functional importance is in no way restricted to the brain which stood sponsor for the name of the first detected endogenous opioids - the 'enkephalins'. While the opioid puzzle is far from being solved, recent findings indicate that opioid peptides may play a physiological role in the immune system. A n u m b e r of reports have described modulation of i m m u n e competence by administration of opioids or opiold antagonists to animals or humans, (e.g. opiate addicts2). Initially, it was unclear whether or not these 'pharmacological' effects reflected physiological functions of endogenous peptides, but a growing line of evidence n o w suggests that this is indeed the case 3,4.

Opioid peptides interact with cellular and humoral components of the immune system Opioid peptides

can interact

Hansj~rg Teschemacher is Professor of Pharmacology, at the Rudolf-Buchheim-Institut fiir Pharmakologie der Justus-Liebig-Universitiit Gie~en, Frankfurter Str. 107, D-6300 Gieflen, FRG. Lothar Schweigerer is a Research Fellow at the University of California, School of Medicine, Cancer Research Institute, 1282-M, San Francisco, CA 94143, USA. 1985, Elsevier Science Publishers B.V., A m s t e r d a m

with specific binding sites on both immunocompetent macromolecules and cells, and can elicit effects on immunocompetent cells, e.g influence antibody response, T cell rosette text, T cell proliferation etc. The mechanisms underlying these effects are not clear; both opioid or non-opioid receptors may be involved. Moreover, cells and opioid peptides used in the reported studies should have been derived from the same species. This is not clear for most of these studies and, thus, some of the respective resuits might be artifacts of heterologous conditions.

Complement system The 31-residue opioid peptide, ~-endorphin, appears to be one of the most prominent candidates for a functional interaction with the immune system, and its C-terminal fragment appears to be of major importance in its proposed immune functions. Specific binding of h u m a n ~endorphin to the terminal complexes of h u m a n complement SC5b-9 and C5b-9 (m) has been recently demonstratedS; interestingly, binding is not via the opioid-active N-terminus, but via a C-terminal fragment, i.e. ~endorphin binds to non-opioid sites on terminal complement complexes. This interaction might have physiological significance in endotoxemia. Endotoxin is k n o w n to cause secretion of ~-endorphin immunoreactive materials into the blood 6, but has also been shown to induce the formation of the SC5b-9 complex in serum 7. Thus, both ligand and binding site are provoked by the same stimulus, possibly representing links in a specific regulatory process.

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Lymphocytes H u m a n ~-endorphin has also been demonstrated to bind to cultured h u m a n lymphocytes via a C-terminal fragment 8 and can apparently modulate T cell mitogen-induced proliferation of T lymphocytes 9,1°. This modulation was shown variously to be stimulatory or inhibitory, presumably due to different types or stages of differentiation of the T cells tested; also ceils used in these studies were from different species. The proliferative effects could not be blocked by the specific opioid antagonist naloxone, suggesting that they are elicited via non-opioid receptors; moreover, o~-endorphin 1° (containing the opioid-active N-terminus of ~-endorphin) was ineffective. Whereas ~-endorphin-induced T cell proliferation appears to be mediated via non-opioid receptors, T cell functions seem to be modulated by opioid peptides via opioid receptors. [Met]enkephalin (a pentapeptide, whose amino acid sequence is identical to the opioid-active N-terminus of ~endorphin and which has 6 agonist activity 1) was found to increase the percentage of active T cell rosettes in tests performed with mononuclear cells from blood of normal volunteers 11 or lymphoma patients 12. As far as tested 11, this effect was inhibited by naloxone. A ~-type opioid agonist, morphine, was inhibitory in this test; this inhibition could also be blocked by naloxone 11. A so far u n k n o w n type of opioid receptor, or non-opioid receptors may be involved here. In-vitro antibody production of mouse spleen cells can apparently be modulated by opioid peptides via opioid receptors. [Met]enkephalin, ~-endorphin and ~endorphin were shown to inhibit the antibody response to sheep erythrocytes, i.e. to a T celldependent antigen in a naloxonereversible manner 13. The order of potency was: o~-endorphin > [Met]enkephalin > ~-endorphin. The reason for this different behaviour is unclear. Monocytes ~-endorphin and [Met]enkephalin stimulate chemotaxis of h u m a n blood mononuclear cells, again via opioid receptors 14 ; the stimulated cells had the morphological appearance of monocytes

TIPS - September 1985

369 agonist w i t h some ~ activity), ~e n d o r p h i n (mixed 8-/~-agonist) and m o r p h i n e (~-agonist) antagonize a P G E l - i n d u c e d i n h i b i t i o n of IgE-mediated serotonin release from rat mast cells. The naloxonereversible nature of these effects indicates that they are m e d i a t e d b y o p i o i d receptors 16 (of the ~- or h-type). Natural killer cells The most interesting of the in-vitro findings on opioid-immune system interactions is the enhancement of natural cytotoxicity by opioid p e p t i d e s and the implications of this for i m m u n e defence of neoplastic disease. [3-endorphin (10-14 M) and [Met]enkephalin (10-9M) have been shown to enhance the activity of natural killer (NK) cells

and this classification was supp o r t e d b y o b s e r v e d reduction of chemotactic effects after removal of glass w o o l - a d h e r e n t cells. The chemotactic response was blocked b y naloxone and d i s p l a y e d peak activities at l ( k 8 and 10-12 M, suggesting the presence of multiple receptors p o s s i b l y located on various s u b p o p u l a t i o n s of cells. The chemotactic effect elicited b y the frequently used chemotactic p e p t i d e , f M e t - L e u - P h e , proved naloxone-reversible indicating that such effects might be m e d i ated via e n d o g e n o u s opioids. Granulocytes A m o d e r a t e chemotactic response to [Met]enkephalin and [~-endorphin has also been shown for neutrophils 14. Interference of o p i o i d p e p t i d e and granulocyte effects on the vascular e n d o t h e l i u m seems to be based on an o p i o i d p e p t i d e interaction with the prostaglandin system 1~. H u m a n umbilical vein endothelial cells increase prostacyclin production in response to stimulation b y arachidonic acid or thrombin. This response is enhanced by [Met]enkephalin in a naloxonereversible manner. Since prostacyclin inhibits adherence of granulocytes to the e n d o t h e l i u m and, thus, g r a n u l o c y t e - m e d i a t e d endothelial damage, o p i o i d p e p t i d e s might u n d e r certain conditions p r o v i d e protection against granul o c y t e - m e d i a t e d vascular injury. Interestingly, the described effects were elicited at p e p t i d e concentrations as low as 10-12 M. Mast

cells

[Met]enkephalin

(h-

w h e t h e r this material represents authentic o¢-endorphin, an c¢e n d o r p h i n precursor or some other p e p t i d e with no sequence homology. Macrophages ]~-endorphin immunoreactive materials, p r o b a b l y [~-endorphin, [3-1ipotropin and a precursor thereof, have been detected in a s u b p o p u l a t i o n of mouse spleen macrophages 19, but have not been identified so far in peripheral blood leukocytes. It might be possible that these o p i o i d p e p t i d e s play a local imm u n e regulatory role in the spleen microenvironment. Interferon H u m a n leukocyte interferon has been reported to display opioid activity in o p i o i d receptor b i n d i n g assays and other test systems 2°. There are obviously no structural homologies between o p i o i d p e p t i d e s and the h u m a n leukocyte interferons so far identified20,21; this p h e n o m e n o n requires further elucidation.

Opioid peptides: links between neuroendocrine and immune systems? O p i o i d p e p t i d e s have been regarded as ' n e u r o p e p t i d e s ' (neurotransmitters or neuromodulators) which also play roles in mammalian endocrine functions 1. This picture is fragmentary and it is n o w k n o w n that in a d d i t i o n o p i o i d p e p t i d e s modulate a n u m ber of i m m u n e functions. However, although they are themselves constituents of some comfrom peripheral h u m a n blood 17. Although the effect was shown to be blocked b y naloxone indicating o p i o i d receptor involvement, no e n h a n c e m e n t of NK activity was found for o¢-endorphin or morphine; this requires further elucidation.

Opioid peptides occur in cells or macromolecules with immune competence Some c o m p o n e n t s of the imm u n e system m a y produce their own o p i o i d peptides. Plasma cells Using i m m u n o c y t o chemical techniques, an o~-endorp h i n immunoreactive material was found in a s u b p o p u l a t i o n of plasma cells of the canine colonic mucosa TM. It is not k n o w n as yet

370 p o n e n t s of the i m m u n e system, there is no evidence, so far, to suggest that they are produced b y the i m m u n e system in order to exert those m o d u l a t i o n s as 'imm u n o p e p t i d e s ' . The available data w o u l d rather suggest their classification as ' n e u r o i m m u n o p e p tides'; opioid peptides m i g h t link n e u r o e n d o c r i n e and i m m u n e systems m e d i a t i n g tonic or temporary control of the i m m u n e system b y superior n e u r o e n d o c r i n e regulatory centers, e.g. in the CNS. Such a hypothesis w o u l d be compatible with the broad spectrum of opioid ligands on the n e u r o e n d o c r i n e side a n d opioid receptors m e d i a t i n g a whole series of opioid effects on the i m m u n e side. Certain i n - v i v o data support this concept of a ' n e u r o i m m u n o p e p t i d e ' function. For example, there are data 22 which indicate that in rats subjected to inescapable footshock stress (intermittently applied), analgesia as well as a s u p p r e s s i o n of natural killer (NK) cell cytotoxicity occurs; both of these stress effects are blocked b y the opioid antagonist naltrexone, i n d i c a t i n g that they are mediated b y release of e n d o g e n ous opioids. Presumably, stress reactions such as the observed NK s u p p r e s s i o n are results of a process starting with a central nervous reaction to the stressor which then triggers a n u m b e r of regulatory m e c h a n i s m s along a neuronendocrine pathway ending u p with a final step such as NK suppression. According to this model, release of e n d o g e n o u s opioids could have occurred in the CNS or at a n y stage ' d o w n s t r e a m ' , along the n e u r o e n d o c r i n e pathway. It is unlikely that the opioid could be released at the final stage to directly influence the NK system, since opioids either are inactive in v i t r o or actually e n h a n c e NK cytotoxicity17. Thus, although opioid peptides may not necessarily link n e u r o e n d o c r i n e a n d i m m u n e systems directly, they may represent i m p o r t a n t links of n e u r o e n d o c r i n e - - i m m u n o r e g u latory processes.

T I P S - S e p t e m b e r 1985

A decreased survival time in comparison with controls was observed in rats with transplanted m a m m a r y ascites tumors, w h e n the animals had b e e n earlier exposed to the same stress paradigm23; interestingly, this stress effect, i.e. survival time reduction, again proved to be mediated b y e n d o g e n o u s opioids as s h o w n b y its blockade b y naltrexone. It is tempting, of course, to interpret these findings in terms of an e n d o g e n o u s opioid release triggering NK cytotoxicity suppression responsible for e n h a n c e d t u m o r development and, thus, for survival time reduction. However, it is not conclusively k n o w n whether this opioid release-dep e n d e n t survival time reduction is caused b y e n h a n c e d tumor development, with or w i t h o u t participation of NK cytotoxicity, or solely b y m e c h a n i s m s restricted to the n e u r o e n d o c r i n e system. It is also u n k n o w n whether NK cytotoxicity suppression and survival time reduction are mediated via identical opioid release m e c h a n i s m s and where such release mechanisms may be located. However, recent evidence has s h o w n that injection of ~ - e n d o r p h i n into the nucleus of the raphe m a g n u s of rats led to an increased n u m b e r of artificial p u l m o n a r y metastases from a Walker 256 t u m o r 24, suggesting that e n d o g e n o u s opioids released in the CNS u n d e r physiological conditions such as stress may, in fact, induce i m m u n e m o d u l a t i o n relevant to tumor development. The 'neuroimmunopeptide' concept gains 'psychoneuroimmunological' significance in cancer patients who are forced to cope with the knowledge of a disease which is d e p e n d e n t in its progress on the status of their i m m u n e defence, in particular, NK activity. In animals the "opioid stress' paradigm as described above induces a 'learned helplessness' p h e n o m e n o n . This p h e n o m e n o n may occur in cancer patients in the form of apathetic psychological responses to their situation; the result could be i m m u n o s u p -

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23 Lewis,J. W., Shavit, Y., Terman,G. W., Nelson, L.R., Gale, R. P. and Liebeskind, J. C. (1983)Peptides 4, 635--638 24 Simon, R. H., Arbo, T. E. and Lundy, J. (1984) Brain Res. Bull. 12, 487-491