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Neuroendocrine peptide hormones and their receptors in the immune system
Journal of Neuroimmunology, 10 (1985) 31-40
31
Elsevier JNI 00318
Neuroendocrine Peptide Hormones and their Receptors in the Immune System Production, Processing and Action J. E d w i n Blalock, K e n n e t h L. Bost and Eric M. Smith Department of Microbiology, Unioersityof Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received13 May, 1985) (Revised, received11 June, 1985) (Accepted 11 June, 1985)
Summary Recent findings indicate that the immune and neuroendocrine systems interact and modulate one another functionally. The mechanism for this seems to be that the 2 systems share a set of receptors and ligands (hormones). Cells of the immune system are able to synthesize neuroendocrine peptide hormones which are biologically active and produced in physiologically significant quantities. Furthermore, leukocytes possess functional receptors for these same neuroendocrine hormones which will specifically modulate immune responses. The structural and functional evidence for these interactions is reviewed and discussed in the context of a bidirectional regulatory circuit between the immune and neuroendocrine systems.
Key words:
Corticotropin
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Endorphins
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Irnrnunomodulation
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Leukocytes
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Lymphocytes
Introduction Sufficient evidence has accumulated to leave little doubt that the neuroendocrine system can regulate immunologic functions. The evidence includes the ability to condition immune responses by classical Pavlovian techniques (for review see Ader 1981), positive and negative effects of various brain and pituitary lesions on immunologic functions (Cross et al. 1980; Brooks et al. 1982) as well as the well-known effects of 'stress' on the immune system (for review see Riley 1981). In spite of these and other studies which provide a basis for the phenomena, termed 0165-5728/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
32 psychoimmunology, neuroimmunoendocrinology or neuroimmunomodulation, the biochemical or molecular mechanism by which such control might operate has been lacking. It seems that recent observation of neuroendocrine peptide hormones and their receptors in and on cells of the immune system now provides a molecular basis for the aforementioned control. In this review, we shall discuss the evidence that these peptides and their receptors are in the immune system, and we will suggest how they are controlled by and involved in both immunologic and neuroendocrine functions.
Neuroendocrine Peptide Hormones and their Receptors in the Immune System The first described and perhaps best characterized pituitary peptide hormones in the immune system are corticotropin (ACTH) and endorphins. The leukocyte derived ACTH and endorphins were observed to be identical to their pituitary gland equivalents in terms of bioactivity, antigenicity, molecular weight and retention time on reverse phase high pressure liquiid chromatography (Blalock and Smith 1980, Smith and Blalock 1981: Smith et al. 1982; Lolait et al. 1984; Blalock and Smith 1985; Harbour-McMenamin et al. 1985). Hormone production by the immune system is not limited to the aforementioned peptides but includes vasoactive intestinal peptide (Giachetti et al. 1978; O'Dorisio et al. 1980; Lygren et al. 1984), somatostatin (Lygren et al. 1984) and thyrotropin (TSH) (Smith et al. 1983). While it has yet to be shown for all hormones, there is no doubt that ACTH, TSH and endorphins were synthesized rather than passively acquired by leukocytes since they were intrinsically radiolabeled with amino acids during their induction in vitro (Smith et al. 1983; Blalock and Smith 1985). Preliminary immunologic and biochemical evidence further suggests that human and mouse leukocytes can produce chorionic gonadotropin (CG), growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) (E.M. Smith, D. Harbour-McMenamin, K.L. Bost and J.E, Blalock, unpublished observations). Thus it appears that cells of the immune system have the potential to produce many, if not all, of the known neuroendocrine peptide hormones. In addition to producing peptide hormones, cells of the immune system also seem to possess their receptors. For instance, binding studies suggest that mouse spleen cell populations have 2 types of ACTH receptors, one a high affinity ( K d 0.1 riM) and the other a low affinity (K d 4.8 riM) receptor (Johnson et al. 1982). These seem to roughly correspond with respect to K d to high (K d 0.25 nM) and low (K d 10 nM) affinity receptors on rat adrenal cells (McIlhinney and Schulster 1975). A comparison of the average number of binding sites per cell shows approximately 3000 high and 50000 low affinity sites on splenocytes compared to 3000 high and 30000 low affinity sites on adrenal cells. More recently, we have developed a new technology for the purification of receptors (Bost et al. 1985) and employed it for the isolation and characterization of the adrenal ACTH receptor. This receptor was shown to have a total molecular weight of 225 000 and to be composed of 4 polypeptide chains of 83000, 64000, 52000 and 22000 Da. The ACTH binding site was located on the
33 83 000 Da polypeptide chain (K.L. Bost and J.E. Blalock, submitted for publication). Similar experiments were then done with mouse spleen and human peripheral blood mononuclear cells and a similar if not identical receptor was observed (unpublished observations). Numerous reports exist of specific and high affinity binding sites for neuroendocrine peptide hormones on cells of the immune system, but these to our knowledge are the first data indicating they are biochemically similar, if not identical. Taken together, our conclusion from the above experiments is that there is bidirectional communication between the immune and neuroendocrine systems by virtue of their sharing a common set of structurally identical signal molecules (hormones) and receptors. Binding sites on leukocytes for endogenous opiates and peptide neurotransmitters have also been described. These include but are not necessarily limited to the following. Apparently, the first indirect evidence for opiate receptors was published by Wybran et al. (1979) who showed that morphine and methionine-enkephalin inhibited and enhanced, respectively, T cell rosetting of sheep red blood cells (SRBC). Both the inhibition and enhancement of rosetting could be reversed by the opiate antagonist naloxone, but not by an inactive enantimer, levomoramide. By competitive binding experiments with a radiolabeled ligand, Johnson et al. (1982) directly showed methionine-enkephalin receptors on mouse splenocytes with a single high affinity binding site of approximately 0.6 nM. Lopker et al. (1980) measured stereospecific dihydromorphine binding to human phagocytic leukocytes. Both granulocytes and monocytes had specific receptors with apparent KdS of 10 nM and 8 nM, respectively. Hazum et al. (1979) reported a high affinity fl-endorphin binding site on transformed human lymphocytes with an apparent K d of 3 nM plus a lower affinity site. These, however, were not classic opiate receptors since the binding was not affected by opiate agonists and antagonists. Since binding to opiate receptors classically is through the amino terminal end of the peptide, this nonopiate receptor binding seems to be through the carboxyl end of fl-endorphin (Hazum et al. 1979). This leads to the interesting possibilitS~ that a molecule such as fl-endorphin could bridge 2 different types of lymphocytes by binding through its amino terminus to an opiate receptor on one cell and through its carboxyl terminus to a nonopiate receptor on another lymphocyte. Leukocytes have also been shown to have specific receptors for other neuroendocrine peptides including: neurotensin (Bar-Shavit et al. 1982), substance P (Payan et al. 1984) and vasoactive intestinal peptide (VIP) (Danek et al. 1983; Ottaway et al. 1983). Interestingly, Beed et al. (1983) have shown that the VIP receptor on Molt 4 cells (a continuous line of T lymphocytes) is coupled to the adenyl cyclase system. These data seem to indicate that as is proving the case for peptide hormones, perhaps every neuroendocrine peptide hormone receptor will eventually be found in the immune system.
Immunologic and Hypothalamic Control of Leukocyte Derived Neuroendocrine Peptide Hormones There seem to be 2 important stimuli which cause leukocyte production of peptide hormones. Immunostimulants were the first described (Blalock and Smith
34 1980) and the particular immunostimulant seems to determine which hormone is elicited (Smith et al. 1983). Viruses (Blalock and Smith 1980; Smith and Blalock 1981), tumor cells (Smith and Blalock 1981) and bacterial lipopolysaccharide (Harbour-McMenamin et al. 1985) caused the de novo synthesis of ACTH and endorphins. A T cell mitogen (staphylococcal enterotoxin A) elicited the production of TSH (Smith et al. 1983) while a mixed lymphocyte reaction resulted in CG production (D. Harbour-McMenamin, E.M. Smith and J.E. Blalock, unpublished observation). Thus the stimulus in part seems to determine the hormone response. Of course an added variable is the cell type which is acted upon by the stimulus. This variable is only now being approached in terms of which cell types and subtypes have the potential to produce a particular hormone. The second type of control is more classically associated with pituitary production of peptide hormones and involves hypothalamic releasing factors. In vitro, corticotropin releasing factor (CRF) was observed to cause the de novo synthesis and release of leukocyte derived A C T H and/~-endorphin. While it occurred at about 10-fold higher concentrations, arginine vasopressin (AVP) alone was also observed to have intrinsic CRF activity. At concentrations that are frequently used on cultured pituitary cells, CRF and AVP together acted in an additive fashion to induce proopiomelanocortin (POMC) derived peptides and such induction was blocked by dexamethasone (Smith et al. 1985). Thus, leukocytes seem quite similar to anterior pituitary cells with respect to control of the POMC gene by a positive hypothalamic signal and feedback inhibition by a synthetic glucocorticoid hormone.
Processing of Proopiomelanocortinby Leukocytes Interestingly, while control of the gene for the A C T H and endorphin precursor (proopiomelanocortin) in leukocytes may be similar to that of anterior pituitary cells, the processing of its products appears somewhat different. For instance, while Newcastle disease virus (NDV) and CRF cause the production of POMC peptides with the molecular weight of A C T H a_ 39 and/~-endorphin (Smith and Blalock 1981; Smith et al. 1985), LPS elicits the production of A C T H and endorphins which correspond to the molecular weight of ACTH1_24 to 26 and a- or y-endorphin (Harbour-McMenamin et al. 1985). Presently, it is not known whether the alternate processing is due to the activation of novel processing enzymes or if a different population of leukocytes is responding to LPS (i.e., compared to NDV or CRF). These findings nonetheless point to alternate proteolytic cleavages of POMC as have been previously observed in the anterior and intermediate lobe of the pituitary as well as the hypothalamus (for review see Krieger 1983). Of course, these results also suggest that cells of the immune system differ from virtually all other extrapituitary tissues where the major proteolytic cleavages are similar to those in the intermediate lobe of the pituitary gland (Krieger 1983). For example, though we detect /~-endorphin, we have yet to observe the' production of an a-melanocyte stimulating hormone (MSH) like peptide. Further, LPS induction of an ACTH with a molecular weight of approximately 2.9 kDa suggests a quite novel processing pathway. Such
35 differential processing points to cells of the immune system having processing pathways which are both unique and in some instances composites of those seen in the anterior and intermediate lobe of the pituitary gland.
Immunologic Functions of Neuroendocrine Peptide Hormones Besides the classic role of inducing adrenal steroidogenesis (Mcllhinney and Schulster 1975), Johnson et al. (1982) found that ACTH itself is also a potent inhibitor of antibody production. In a plaque forming cell (PFC) assay in vitro which measures antibody secreting cells, ACTH 1_39 suppressed the response to both a T lymphocyte dependent antigen, sheep red blood cells (SRBC) and T independent antigen, dinitrophenol-Ficoll. Inhibition of antibody to SRBC required only one fourth the amount of ACTH necessary for equal inhibition of the DNP-Ficoll response. This suggests that T cell function may be more sensitive to ACTH than B lymphocyte function. Interestingly, while highly purified and synthetic ACTHl_39 is suppressive, ACTHl_24 is not inhibitory (Johnson et al. 1984). This is in contrast to ACTH's steroidogenic activity in which ACTHl_39 or ACTHx_ 24 are equally active (Mcllhinney and Schulster 1975). It was necessary for ACTH to be present at the time of addition of antigen for maximum inhibition and thiol reducing agents blocked ACTH suppression of the antibody response. Since these characteristics are also associated with suppression of antibody by interferon (IFN), it seems that the mechanisms through which ACTH inhibits the antibody response may be quite similar to that of a lymphokine, IFN (Johnson et al. 1982). Since ACTH and endorphins are products of the same polyprotein precursor and are both elevated during stress, endorphins were evaluated for immunomodulatory activity. Related peptides, the enkephalins also were tested for modulation of antibody synthesis, t~-Endorphin, the shortest of the 3 endorphin species was the only one to affect PFC formation and it was suppressive (Johnson et al. 1982). Both leucine and methionine-enkephalin suppressed the antibody response in vitro to SRBC though to a lesser degree than t~-endorphin. Since the endorphins and enkephalins share common amino terminal sequences and all bind to opiate receptors, the extra amino acid(s) on the carboxyl terminus of ~,- and fl-endorphin may be responsible for their inactivity in this system. Although fl-endorphin had no effect of its own, it as well as naloxone would compete with and block a-endorphin's suppressive effect. Thus it seems that binding of certain endogenous opioid peptides to an opiate-like receptor on lymphocytes will suppress antibody production. Interestingly, TSH caused an effect that was opposite to that of ACTH and ~-endorphin. TSH enhanced the antibody response in vitro to SRBC and this effect required the addition of the hormone during the early phase of the response (1-2 days) (Blalock et al. 1985). It is intriguing that 2 different stimuli can elicit the leukocyte production of 2 different hormones (TSH and ACTH) and these in turn have opposite actions on the immune system. Like B lymphocyte function, T cell function is also modulated by peptide hormones. Gilman et al. (1982) found that fl-endorphin enhanced the proliferative
36 response of rat splenic lymphocytes to the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). The enhancement was dose dependent but was not blocked by the opiate antagonist naloxone. The inability of naloxone to block the enhancement suggests that ~-endorphin does not act through a classic opiate receptor in this system (Simon and Miller 1981). In contrast, McCain et al. (1982) found that human T cell mitogenesis was effectively suppressed by /~-endorphin. While these results seem conflicting, they might be explained as being due to different species and relative concentrations. McCain et al. (1982) utilized higher /~-endorphin concentrations. Opiates frequently display opposite activities between high and low concentrations (Faith et al. 1983) which could account for these discrepancies. Plotnikoff and Miller (1983) subsequently found that methionine and leucine enkephalins enhanced PHA induced human lymphocyte mitogenesis. Endogenous opiates have also been implicated in the regulation of natural killer (NK) cell activity. In vitro, Mathews et al. (1983) and Kay et al. (1984) have found that B-endorphin in a dose dependent fashion enhances N K activity 20-55%. Again, the enhancement was observed with very low doses (1 × 10 14 M) and increased up to 1 x 10 - 6 M. Interestingly, y-endorphin also enhanced N K activity but was less potent and a-endorphin had very little effect at all (Kay et al. 1984). Thus, the endorphin enhancement of N K activity was opposite of that seen with antibody synthesis where the shorter c~-endorphin was more suppressive than ~,- and /3-endorphin. N K enhancement seemed to be mediated through opiate receptor(s) since it was blocked by naloxone in both cases. Mononuclear cell chemotaxis is also stimulated by both ,8-endorphin and methionine-enkephalin (Van Epps and Saland 1984). The response to both neuropeptides was bimodal and active at very low concentrations (1 × 10 ~4 M). This effect also seems to be mediated through opiate receptors as evidenced by naloxone blockage of migration. Besides the endorphins and enkephalins, ACTH also modulates the cell mediated immune response. ACTH was found to suppress lymphokine, IFN-'f, production in vitro (Johnson et al. 1984). Full length, ACTH~ 39 (both natural and synthetic) suppressed the induction of IFN-y. Shorter fragments of ACTH, ACTHl_24 and c~-MSH (ACTHI_13) did not suppress. Also, CLIP (corticotropin-like intermediate lobe peptide) which corresponds to ACTH~8 39, did not affect IFNq, production (Johnson et al, 1984). Once again, there seems to be no correlation between ACTH's steroidogenic and immunomodulatory activities since ACTH~_24 has full steroidogenic capability and is not immunomodulatory. Furthermore, this varied immunomodulatory activity associated with different sizes of endorphins and ACTH suggests that immunoregulation could be a function of differential processing of these neuropeptides as was discussed above.
Neuroendocrine Functions of Leukocyte Derived ACTH and Endorphins The observation that cells of the immune system are a source of secreted A C T H suggested that in certain circumstances the pituitary gland should not be required for an ACTH mediated steroidogenic response. This was borne out by the observa-
37 tion that Newcastle disease virus (an inducer of leukocyte ACTH in vitro) infection of hypophysectomized mice caused a time dependent increase in corticosterone production which was inhibited by dexamethasone. Unless the mice were pretreated with dexamethasone, their spleens were positive for ACTH (Smith et al. 1982). Based on these observations, we have suggested a lymphoid-adrenal axis exists which may account for the increases in corticosteroid levels which are generally observed during infections. Further, such an axis might also explain the earlier observation of bacterial polysaccharide (Piromen) induced cortisol responses in 7 out of 8 patients who underwent pituitary stalk sectioning (Van Wyk et al. 1960). More recently, a patient presented with the clinical and laboratory features of the ectopic ACTH syndrome. While the absence of a gradient in ACTH concentration between the inferior petrosal sinuses and the periphery argued against pituitary overproduction, no ACTH producing tumor was found and bilateral adrenalectomy was performed to correct the hypercortisolism. Six months later, a pseudotumor containing only normal fat and inflammatory :issue was observed in the patient. Upon removal of this inflammatory tissue, basal plasma ACTH levels immediately returned to normal and leukocytes within the inflammatory tissue stained positively for ACTH by an immunoperoxidase procedure (Dupont et al. 1984). Collectively, these studies seem to leave little doubt that cells of the immune system are able to influence directly adrenal functiol~ via ACTH production and consequently may also indirectly affect hypothalamic and pituitary function. Gram-negative sepsis and endotoxin induced shock may represent another situation in which leukocyte production of proopiomelanocortin derived peptides play pivotal roles. For instance, endorphins have been implicated in the pathophysiology associated with these trauma since the opiate antagonist naloxone improved survival rates and blocked a number of cardiopulmonary changes associated with these conditions (Reynolds et al. 1980). Further, 2 separate pools of endorphins have been observed following endotoxin administration and it was suggested that one might originate in the immune system (Carr et al. 1982). Considering the potent immunologic effects of endotoxin as well as its ability to induce leukocyte production of endorphins in vitro (Harbour-McMenamin et al. 1985), cells of the immune system seem the most likely source of endogenous opiates that are observed during Gram-negative sepsis and endotoxin shock. Consistent with this idea is the observation that lymphocyte depletion, like naloxone treatment, blocked a number of endotoxin induced cardiopulmonary changes (Bohs et al. 1979). Our interpretation of these results is that lymphocyte depletion removes the source of the endorphins while naloxone blocks their effector function. This may represent a situation where immune system derived endorphins are involved in the manifestations of disease states. Conclusions
It seems clear from the number of examples by various laboratories as cited above, that cells of the immune system can synthesize neuroendocrine hormones. Furthermore, these molecules are biologically active. Besides endocrine functions
38 they may also bind to receptors on lymphocytes and regulate immune responses. For what purpose do leukocytes synthesize and respond to neuroendocrine hormones'? Our bias is that it is a means by which the immune system can serve a sensory role (Blalock 1984a, b). Instead of classic sensory stimuli (physical, chemical, visual, etc.), the immune system recognizes immune type stimuli such as viruses, bacteria and tumors. Production of neuroendocrine hormones by the stimulated leukocytes could then alter homeostasis in a fashion similar to the pituitary gland during classic sensory stimuli recognition. Presumably, the hormones will also modulate host defense mechanisms. In the case of A C T H and endorphins, negative feedback through other endocrine hormones such as corticosteroids will shut off lymphocyte A C T H and endorphin synthesis. The ability of C R F to induce lymphocyte A C T H suggests that classic sensory stimuli can also induce the lymphocyte arm of the circuit. The implications for this system are numerous. One is the possibility for differential regulation. Since various immune inducers stimulate different hormones from leukocytes, there would be a variety of alterations in homeostasis. The particular responses elicited will correspond to the lymphocyte population being induced or hormone being produced. Also, differential processing of the same hormone such as A C T H could cause alternate immunoregulation since the effects of A C T H on the immune system vary with the length of the molecule. An understanding of these interactions may help explain the pathophysiology of diseases which have both immune and endocrine components. Furthermore, it may provide a rationale for new clinical therapies. As an example, it may be possible to partially reconstitute pituitary hormone deficiencies with the appropriate immune stimulus or hypothalamic releasing factor which in turn would act on the immune system. Indications that this may be possible come from the data showing the induction of corticosterone in hypophysectomized mice by a viral antigen.
Acknowledgements Supported in part by Office of Naval Research G r a n t N0014-84-K-0486 and N I H G r a n t R01 AM 338-39-01A1. K.L.B. was supported by a McLaughlin Postdoctoral Fellowship. We thank Diane Weigent and Rhonda Peake for their skilful typing of this manuscript.
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40 Mcllhinney, R.A.J. and D. Schulster, Studies on the binding of 125I-labelled corticotropin to isolated rat adrenocortical cells, J. Endocrinol., 64 (1975) 175-184. O'Dorisio, M.S., T.M. O'Dorisio, S. Cataland and S.P. Balcerzak, Vasoactive intestinal peptide as a biochemical marker for polymorphonuclear leukocytes, J. Lab. Clin. Med., 96 (1980) 666-670. Ottaway, C.A., C. Bernaerts, B. Chart and G.R. Greenberg, Specific binding of vasoactive intestinal peptide to human circulating mononuclear cells, Can. 3. Physiol. Pharmacol., 61 (1983) 664-671. Payan, D.G., D.R. Brewster and E.J. Goetzl, Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts, J. Immunol., 133 (1984) 3260-3265. Plotnikoff, N. and G.C. Miller, Enkephalins as immunomodulators, Int. J. Immunopharmacol., 5 (1983) 437-441. Reynolds, D.G., N.V. Guill, T. Vargish, R.B. Hechner, A.I. Fader and J.W. Holaday, Blockade of opiate receptors with naloxone improves survival and cardiac performance in canine endotoxic shock, Circ. Shock, 7 (1980) 39-48. Riley, V., Psychoneuroendocrine influences on immunocompetence and neoplasia, Science, 212 (1981) 1100-1109. Simon, E.J. and J.M. Miller, Opioid peptides and opiate receptors. In: G.J. Seigel, R.W. Albers, B.W. Agaranoff and R. Katzman (Eds.), Basic Neurochemistry, Little, Brown and Co., Boston, MA, 1981, p. 255. Smith, E.M. and J.E. Blalock, Human lymphocyte production of ACTH and endorphin-like substances: Association with leukocyte interferon, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7530-7534. Smith, E.M., W.J. Meyer and J.E. Blalock, Virus-induced increases in corticosterone in hypophysectomized mice: A possible lymphoid-adrenal axis, Science, 218 (1982) 1311-1312. Smith, E.M., M. Phan, D. Coppenhaver, T.E. Kruger and J.E. Blalock, Human lymphocyte production of immunoreactive thyrotropin, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6010-6013. Smith, E.M., A.C. Morrill, W.J. Meyer and J.E. Blalock, Corticotropin releasing factor induction of leukocyte derived immunoreactive ACTH and endorphins, Nature (London) (1985) in press. Van Epps, D. and L. Saland, /~-Endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis, J. lmmunol., 132 (1984) 3046-3053. Van Wyk, J.J., G.S. Dugger, J.F. Newsome and P.Z. Thomas, The effect of pituitary stalk section in the adrenal function of women with cancer of the breast, J. Clin. Endocrinol. Metab., 20 (1960) 157-172. Wybran, J., T. Appelbroom, J.-P. Famaey and A. Govaerts, Suggestive evidence for receptors for morphine and methionine-enkephalin on normal blood T lymphocytes, J. Immunol., 123 (1979) 1O68-1070.
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Elsevier JNI 00318
Neuroendocrine Peptide Hormones and their Receptors in the Immune System Production, Processing and Action J. E d w i n Blalock, K e n n e t h L. Bost and Eric M. Smith Department of Microbiology, Unioersityof Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received13 May, 1985) (Revised, received11 June, 1985) (Accepted 11 June, 1985)
Summary Recent findings indicate that the immune and neuroendocrine systems interact and modulate one another functionally. The mechanism for this seems to be that the 2 systems share a set of receptors and ligands (hormones). Cells of the immune system are able to synthesize neuroendocrine peptide hormones which are biologically active and produced in physiologically significant quantities. Furthermore, leukocytes possess functional receptors for these same neuroendocrine hormones which will specifically modulate immune responses. The structural and functional evidence for these interactions is reviewed and discussed in the context of a bidirectional regulatory circuit between the immune and neuroendocrine systems.
Key words:
Corticotropin
-
Endorphins
-
Irnrnunomodulation
-
Leukocytes
-
Lymphocytes
Introduction Sufficient evidence has accumulated to leave little doubt that the neuroendocrine system can regulate immunologic functions. The evidence includes the ability to condition immune responses by classical Pavlovian techniques (for review see Ader 1981), positive and negative effects of various brain and pituitary lesions on immunologic functions (Cross et al. 1980; Brooks et al. 1982) as well as the well-known effects of 'stress' on the immune system (for review see Riley 1981). In spite of these and other studies which provide a basis for the phenomena, termed 0165-5728/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
32 psychoimmunology, neuroimmunoendocrinology or neuroimmunomodulation, the biochemical or molecular mechanism by which such control might operate has been lacking. It seems that recent observation of neuroendocrine peptide hormones and their receptors in and on cells of the immune system now provides a molecular basis for the aforementioned control. In this review, we shall discuss the evidence that these peptides and their receptors are in the immune system, and we will suggest how they are controlled by and involved in both immunologic and neuroendocrine functions.
Neuroendocrine Peptide Hormones and their Receptors in the Immune System The first described and perhaps best characterized pituitary peptide hormones in the immune system are corticotropin (ACTH) and endorphins. The leukocyte derived ACTH and endorphins were observed to be identical to their pituitary gland equivalents in terms of bioactivity, antigenicity, molecular weight and retention time on reverse phase high pressure liquiid chromatography (Blalock and Smith 1980, Smith and Blalock 1981: Smith et al. 1982; Lolait et al. 1984; Blalock and Smith 1985; Harbour-McMenamin et al. 1985). Hormone production by the immune system is not limited to the aforementioned peptides but includes vasoactive intestinal peptide (Giachetti et al. 1978; O'Dorisio et al. 1980; Lygren et al. 1984), somatostatin (Lygren et al. 1984) and thyrotropin (TSH) (Smith et al. 1983). While it has yet to be shown for all hormones, there is no doubt that ACTH, TSH and endorphins were synthesized rather than passively acquired by leukocytes since they were intrinsically radiolabeled with amino acids during their induction in vitro (Smith et al. 1983; Blalock and Smith 1985). Preliminary immunologic and biochemical evidence further suggests that human and mouse leukocytes can produce chorionic gonadotropin (CG), growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) (E.M. Smith, D. Harbour-McMenamin, K.L. Bost and J.E, Blalock, unpublished observations). Thus it appears that cells of the immune system have the potential to produce many, if not all, of the known neuroendocrine peptide hormones. In addition to producing peptide hormones, cells of the immune system also seem to possess their receptors. For instance, binding studies suggest that mouse spleen cell populations have 2 types of ACTH receptors, one a high affinity ( K d 0.1 riM) and the other a low affinity (K d 4.8 riM) receptor (Johnson et al. 1982). These seem to roughly correspond with respect to K d to high (K d 0.25 nM) and low (K d 10 nM) affinity receptors on rat adrenal cells (McIlhinney and Schulster 1975). A comparison of the average number of binding sites per cell shows approximately 3000 high and 50000 low affinity sites on splenocytes compared to 3000 high and 30000 low affinity sites on adrenal cells. More recently, we have developed a new technology for the purification of receptors (Bost et al. 1985) and employed it for the isolation and characterization of the adrenal ACTH receptor. This receptor was shown to have a total molecular weight of 225 000 and to be composed of 4 polypeptide chains of 83000, 64000, 52000 and 22000 Da. The ACTH binding site was located on the
33 83 000 Da polypeptide chain (K.L. Bost and J.E. Blalock, submitted for publication). Similar experiments were then done with mouse spleen and human peripheral blood mononuclear cells and a similar if not identical receptor was observed (unpublished observations). Numerous reports exist of specific and high affinity binding sites for neuroendocrine peptide hormones on cells of the immune system, but these to our knowledge are the first data indicating they are biochemically similar, if not identical. Taken together, our conclusion from the above experiments is that there is bidirectional communication between the immune and neuroendocrine systems by virtue of their sharing a common set of structurally identical signal molecules (hormones) and receptors. Binding sites on leukocytes for endogenous opiates and peptide neurotransmitters have also been described. These include but are not necessarily limited to the following. Apparently, the first indirect evidence for opiate receptors was published by Wybran et al. (1979) who showed that morphine and methionine-enkephalin inhibited and enhanced, respectively, T cell rosetting of sheep red blood cells (SRBC). Both the inhibition and enhancement of rosetting could be reversed by the opiate antagonist naloxone, but not by an inactive enantimer, levomoramide. By competitive binding experiments with a radiolabeled ligand, Johnson et al. (1982) directly showed methionine-enkephalin receptors on mouse splenocytes with a single high affinity binding site of approximately 0.6 nM. Lopker et al. (1980) measured stereospecific dihydromorphine binding to human phagocytic leukocytes. Both granulocytes and monocytes had specific receptors with apparent KdS of 10 nM and 8 nM, respectively. Hazum et al. (1979) reported a high affinity fl-endorphin binding site on transformed human lymphocytes with an apparent K d of 3 nM plus a lower affinity site. These, however, were not classic opiate receptors since the binding was not affected by opiate agonists and antagonists. Since binding to opiate receptors classically is through the amino terminal end of the peptide, this nonopiate receptor binding seems to be through the carboxyl end of fl-endorphin (Hazum et al. 1979). This leads to the interesting possibilitS~ that a molecule such as fl-endorphin could bridge 2 different types of lymphocytes by binding through its amino terminus to an opiate receptor on one cell and through its carboxyl terminus to a nonopiate receptor on another lymphocyte. Leukocytes have also been shown to have specific receptors for other neuroendocrine peptides including: neurotensin (Bar-Shavit et al. 1982), substance P (Payan et al. 1984) and vasoactive intestinal peptide (VIP) (Danek et al. 1983; Ottaway et al. 1983). Interestingly, Beed et al. (1983) have shown that the VIP receptor on Molt 4 cells (a continuous line of T lymphocytes) is coupled to the adenyl cyclase system. These data seem to indicate that as is proving the case for peptide hormones, perhaps every neuroendocrine peptide hormone receptor will eventually be found in the immune system.
Immunologic and Hypothalamic Control of Leukocyte Derived Neuroendocrine Peptide Hormones There seem to be 2 important stimuli which cause leukocyte production of peptide hormones. Immunostimulants were the first described (Blalock and Smith
34 1980) and the particular immunostimulant seems to determine which hormone is elicited (Smith et al. 1983). Viruses (Blalock and Smith 1980; Smith and Blalock 1981), tumor cells (Smith and Blalock 1981) and bacterial lipopolysaccharide (Harbour-McMenamin et al. 1985) caused the de novo synthesis of ACTH and endorphins. A T cell mitogen (staphylococcal enterotoxin A) elicited the production of TSH (Smith et al. 1983) while a mixed lymphocyte reaction resulted in CG production (D. Harbour-McMenamin, E.M. Smith and J.E. Blalock, unpublished observation). Thus the stimulus in part seems to determine the hormone response. Of course an added variable is the cell type which is acted upon by the stimulus. This variable is only now being approached in terms of which cell types and subtypes have the potential to produce a particular hormone. The second type of control is more classically associated with pituitary production of peptide hormones and involves hypothalamic releasing factors. In vitro, corticotropin releasing factor (CRF) was observed to cause the de novo synthesis and release of leukocyte derived A C T H and/~-endorphin. While it occurred at about 10-fold higher concentrations, arginine vasopressin (AVP) alone was also observed to have intrinsic CRF activity. At concentrations that are frequently used on cultured pituitary cells, CRF and AVP together acted in an additive fashion to induce proopiomelanocortin (POMC) derived peptides and such induction was blocked by dexamethasone (Smith et al. 1985). Thus, leukocytes seem quite similar to anterior pituitary cells with respect to control of the POMC gene by a positive hypothalamic signal and feedback inhibition by a synthetic glucocorticoid hormone.
Processing of Proopiomelanocortinby Leukocytes Interestingly, while control of the gene for the A C T H and endorphin precursor (proopiomelanocortin) in leukocytes may be similar to that of anterior pituitary cells, the processing of its products appears somewhat different. For instance, while Newcastle disease virus (NDV) and CRF cause the production of POMC peptides with the molecular weight of A C T H a_ 39 and/~-endorphin (Smith and Blalock 1981; Smith et al. 1985), LPS elicits the production of A C T H and endorphins which correspond to the molecular weight of ACTH1_24 to 26 and a- or y-endorphin (Harbour-McMenamin et al. 1985). Presently, it is not known whether the alternate processing is due to the activation of novel processing enzymes or if a different population of leukocytes is responding to LPS (i.e., compared to NDV or CRF). These findings nonetheless point to alternate proteolytic cleavages of POMC as have been previously observed in the anterior and intermediate lobe of the pituitary as well as the hypothalamus (for review see Krieger 1983). Of course, these results also suggest that cells of the immune system differ from virtually all other extrapituitary tissues where the major proteolytic cleavages are similar to those in the intermediate lobe of the pituitary gland (Krieger 1983). For example, though we detect /~-endorphin, we have yet to observe the' production of an a-melanocyte stimulating hormone (MSH) like peptide. Further, LPS induction of an ACTH with a molecular weight of approximately 2.9 kDa suggests a quite novel processing pathway. Such
35 differential processing points to cells of the immune system having processing pathways which are both unique and in some instances composites of those seen in the anterior and intermediate lobe of the pituitary gland.
Immunologic Functions of Neuroendocrine Peptide Hormones Besides the classic role of inducing adrenal steroidogenesis (Mcllhinney and Schulster 1975), Johnson et al. (1982) found that ACTH itself is also a potent inhibitor of antibody production. In a plaque forming cell (PFC) assay in vitro which measures antibody secreting cells, ACTH 1_39 suppressed the response to both a T lymphocyte dependent antigen, sheep red blood cells (SRBC) and T independent antigen, dinitrophenol-Ficoll. Inhibition of antibody to SRBC required only one fourth the amount of ACTH necessary for equal inhibition of the DNP-Ficoll response. This suggests that T cell function may be more sensitive to ACTH than B lymphocyte function. Interestingly, while highly purified and synthetic ACTHl_39 is suppressive, ACTHl_24 is not inhibitory (Johnson et al. 1984). This is in contrast to ACTH's steroidogenic activity in which ACTHl_39 or ACTHx_ 24 are equally active (Mcllhinney and Schulster 1975). It was necessary for ACTH to be present at the time of addition of antigen for maximum inhibition and thiol reducing agents blocked ACTH suppression of the antibody response. Since these characteristics are also associated with suppression of antibody by interferon (IFN), it seems that the mechanisms through which ACTH inhibits the antibody response may be quite similar to that of a lymphokine, IFN (Johnson et al. 1982). Since ACTH and endorphins are products of the same polyprotein precursor and are both elevated during stress, endorphins were evaluated for immunomodulatory activity. Related peptides, the enkephalins also were tested for modulation of antibody synthesis, t~-Endorphin, the shortest of the 3 endorphin species was the only one to affect PFC formation and it was suppressive (Johnson et al. 1982). Both leucine and methionine-enkephalin suppressed the antibody response in vitro to SRBC though to a lesser degree than t~-endorphin. Since the endorphins and enkephalins share common amino terminal sequences and all bind to opiate receptors, the extra amino acid(s) on the carboxyl terminus of ~,- and fl-endorphin may be responsible for their inactivity in this system. Although fl-endorphin had no effect of its own, it as well as naloxone would compete with and block a-endorphin's suppressive effect. Thus it seems that binding of certain endogenous opioid peptides to an opiate-like receptor on lymphocytes will suppress antibody production. Interestingly, TSH caused an effect that was opposite to that of ACTH and ~-endorphin. TSH enhanced the antibody response in vitro to SRBC and this effect required the addition of the hormone during the early phase of the response (1-2 days) (Blalock et al. 1985). It is intriguing that 2 different stimuli can elicit the leukocyte production of 2 different hormones (TSH and ACTH) and these in turn have opposite actions on the immune system. Like B lymphocyte function, T cell function is also modulated by peptide hormones. Gilman et al. (1982) found that fl-endorphin enhanced the proliferative
36 response of rat splenic lymphocytes to the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). The enhancement was dose dependent but was not blocked by the opiate antagonist naloxone. The inability of naloxone to block the enhancement suggests that ~-endorphin does not act through a classic opiate receptor in this system (Simon and Miller 1981). In contrast, McCain et al. (1982) found that human T cell mitogenesis was effectively suppressed by /~-endorphin. While these results seem conflicting, they might be explained as being due to different species and relative concentrations. McCain et al. (1982) utilized higher /~-endorphin concentrations. Opiates frequently display opposite activities between high and low concentrations (Faith et al. 1983) which could account for these discrepancies. Plotnikoff and Miller (1983) subsequently found that methionine and leucine enkephalins enhanced PHA induced human lymphocyte mitogenesis. Endogenous opiates have also been implicated in the regulation of natural killer (NK) cell activity. In vitro, Mathews et al. (1983) and Kay et al. (1984) have found that B-endorphin in a dose dependent fashion enhances N K activity 20-55%. Again, the enhancement was observed with very low doses (1 × 10 14 M) and increased up to 1 x 10 - 6 M. Interestingly, y-endorphin also enhanced N K activity but was less potent and a-endorphin had very little effect at all (Kay et al. 1984). Thus, the endorphin enhancement of N K activity was opposite of that seen with antibody synthesis where the shorter c~-endorphin was more suppressive than ~,- and /3-endorphin. N K enhancement seemed to be mediated through opiate receptor(s) since it was blocked by naloxone in both cases. Mononuclear cell chemotaxis is also stimulated by both ,8-endorphin and methionine-enkephalin (Van Epps and Saland 1984). The response to both neuropeptides was bimodal and active at very low concentrations (1 × 10 ~4 M). This effect also seems to be mediated through opiate receptors as evidenced by naloxone blockage of migration. Besides the endorphins and enkephalins, ACTH also modulates the cell mediated immune response. ACTH was found to suppress lymphokine, IFN-'f, production in vitro (Johnson et al. 1984). Full length, ACTH~ 39 (both natural and synthetic) suppressed the induction of IFN-y. Shorter fragments of ACTH, ACTHl_24 and c~-MSH (ACTHI_13) did not suppress. Also, CLIP (corticotropin-like intermediate lobe peptide) which corresponds to ACTH~8 39, did not affect IFNq, production (Johnson et al, 1984). Once again, there seems to be no correlation between ACTH's steroidogenic and immunomodulatory activities since ACTH~_24 has full steroidogenic capability and is not immunomodulatory. Furthermore, this varied immunomodulatory activity associated with different sizes of endorphins and ACTH suggests that immunoregulation could be a function of differential processing of these neuropeptides as was discussed above.
Neuroendocrine Functions of Leukocyte Derived ACTH and Endorphins The observation that cells of the immune system are a source of secreted A C T H suggested that in certain circumstances the pituitary gland should not be required for an ACTH mediated steroidogenic response. This was borne out by the observa-
37 tion that Newcastle disease virus (an inducer of leukocyte ACTH in vitro) infection of hypophysectomized mice caused a time dependent increase in corticosterone production which was inhibited by dexamethasone. Unless the mice were pretreated with dexamethasone, their spleens were positive for ACTH (Smith et al. 1982). Based on these observations, we have suggested a lymphoid-adrenal axis exists which may account for the increases in corticosteroid levels which are generally observed during infections. Further, such an axis might also explain the earlier observation of bacterial polysaccharide (Piromen) induced cortisol responses in 7 out of 8 patients who underwent pituitary stalk sectioning (Van Wyk et al. 1960). More recently, a patient presented with the clinical and laboratory features of the ectopic ACTH syndrome. While the absence of a gradient in ACTH concentration between the inferior petrosal sinuses and the periphery argued against pituitary overproduction, no ACTH producing tumor was found and bilateral adrenalectomy was performed to correct the hypercortisolism. Six months later, a pseudotumor containing only normal fat and inflammatory :issue was observed in the patient. Upon removal of this inflammatory tissue, basal plasma ACTH levels immediately returned to normal and leukocytes within the inflammatory tissue stained positively for ACTH by an immunoperoxidase procedure (Dupont et al. 1984). Collectively, these studies seem to leave little doubt that cells of the immune system are able to influence directly adrenal functiol~ via ACTH production and consequently may also indirectly affect hypothalamic and pituitary function. Gram-negative sepsis and endotoxin induced shock may represent another situation in which leukocyte production of proopiomelanocortin derived peptides play pivotal roles. For instance, endorphins have been implicated in the pathophysiology associated with these trauma since the opiate antagonist naloxone improved survival rates and blocked a number of cardiopulmonary changes associated with these conditions (Reynolds et al. 1980). Further, 2 separate pools of endorphins have been observed following endotoxin administration and it was suggested that one might originate in the immune system (Carr et al. 1982). Considering the potent immunologic effects of endotoxin as well as its ability to induce leukocyte production of endorphins in vitro (Harbour-McMenamin et al. 1985), cells of the immune system seem the most likely source of endogenous opiates that are observed during Gram-negative sepsis and endotoxin shock. Consistent with this idea is the observation that lymphocyte depletion, like naloxone treatment, blocked a number of endotoxin induced cardiopulmonary changes (Bohs et al. 1979). Our interpretation of these results is that lymphocyte depletion removes the source of the endorphins while naloxone blocks their effector function. This may represent a situation where immune system derived endorphins are involved in the manifestations of disease states. Conclusions
It seems clear from the number of examples by various laboratories as cited above, that cells of the immune system can synthesize neuroendocrine hormones. Furthermore, these molecules are biologically active. Besides endocrine functions
38 they may also bind to receptors on lymphocytes and regulate immune responses. For what purpose do leukocytes synthesize and respond to neuroendocrine hormones'? Our bias is that it is a means by which the immune system can serve a sensory role (Blalock 1984a, b). Instead of classic sensory stimuli (physical, chemical, visual, etc.), the immune system recognizes immune type stimuli such as viruses, bacteria and tumors. Production of neuroendocrine hormones by the stimulated leukocytes could then alter homeostasis in a fashion similar to the pituitary gland during classic sensory stimuli recognition. Presumably, the hormones will also modulate host defense mechanisms. In the case of A C T H and endorphins, negative feedback through other endocrine hormones such as corticosteroids will shut off lymphocyte A C T H and endorphin synthesis. The ability of C R F to induce lymphocyte A C T H suggests that classic sensory stimuli can also induce the lymphocyte arm of the circuit. The implications for this system are numerous. One is the possibility for differential regulation. Since various immune inducers stimulate different hormones from leukocytes, there would be a variety of alterations in homeostasis. The particular responses elicited will correspond to the lymphocyte population being induced or hormone being produced. Also, differential processing of the same hormone such as A C T H could cause alternate immunoregulation since the effects of A C T H on the immune system vary with the length of the molecule. An understanding of these interactions may help explain the pathophysiology of diseases which have both immune and endocrine components. Furthermore, it may provide a rationale for new clinical therapies. As an example, it may be possible to partially reconstitute pituitary hormone deficiencies with the appropriate immune stimulus or hypothalamic releasing factor which in turn would act on the immune system. Indications that this may be possible come from the data showing the induction of corticosterone in hypophysectomized mice by a viral antigen.
Acknowledgements Supported in part by Office of Naval Research G r a n t N0014-84-K-0486 and N I H G r a n t R01 AM 338-39-01A1. K.L.B. was supported by a McLaughlin Postdoctoral Fellowship. We thank Diane Weigent and Rhonda Peake for their skilful typing of this manuscript.
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