The immune system: our mobile brain?

Immunology Today April 1985

The immune system: our mobile brain? fromJ. E d w i n Blalock and Eric M. Smith In a recent commentary (Nature3 0 9 , 4 0 0 ; 1 9 8 4 ) J o h n M a d d o x suggested that the study of psychological effects on immunologic functions - 'psychoimmunology' - was somewh~tt premature. Specifically, h e argued that while the concept of a link between the central nervous system and the immune system was easily accepted, there was a paucity of data of a mechanistic nature to explain the evidence that the ' mind' can control immune responses. H e doubted, therefore, whether enough is k n o w n to sustain the hope that p s y c h o i m m u n o l o g y was explainable. M u c h evidence to the contrary was apparent in the proceedings of three recent meetings* on the subject which has been variously termed neuroimmunomodulation, p s y c h o i m m u n o l o g y and neuroimmunoendocrinology.

Numerous investigators reviewed past and present data that psychosocial factors such as bereavement can have an adverse effect upon the function of cells of the immune system. Such control can originate in the nervous system: this was elegantly demonstrated by the ability to use classical Pavlovian conditioning to suppress immunologic responses such as antibody production, delayed-type hypersensitivity and to control an experimental model disease of immunologic origin, systemic lupus erythematosus (R. Ader and N. Cohen, Rochester). Pavlovian conditioning can also enhance at least one immunologic function, natural killer cell activity (H. B. Solvason, V. Ghanta and R. Hiramoto, Birmingham; N. H. Spector, Bethesda). Other more direct evidence for a central and peripheral nervous system origin~of immunoregulation came from observations of both positive andnegative effects of electrolytic and chemical lesions of the nervous system on immunologic function and leukocyte cell numbers (R. Cross, J. Jackson, W. Markesbery, W. Brooks and T. Roszman, Lexington; K. Biziere, M. Renoux and G. Renoux, *'Neuroimmunology: exploring parallels ana interactions between the imnmne and nervous systems' October 7-8, 1984, Pasadena; 'Neuromodulation of immunity and hypersensitivity' November 12-14, 1984, Coconut Grove, Florida; the first international workshop on neuroimmunomodulation, November 27-30, 1984, Bethesda.

Tours; B. Arnason, Chicago; B. D. Jankovic, D. L. Jankovic and L. Savoski, Belgrade). At least two nonmutually exclusive pathways for such immunoregulation seem evident. One is the possibility of a 'hard wired' circuit via innervation of the thymus (K. Bullock, Stony Brook), lymph nodes, bone marrow, and spleen (D. Felten, Rochester). This pathway might ulti-

mately act via the effects of neurotransmitters such as norepinephrine and serotonin on leukocytes (T. Roszman andJ. Jackson, Lexington). Indeed, receptors for such substances are present on cells of the immune system. The second immunoregulatory pathway is humoral and acts via pituitary peptide and adrenal steroid hormones. This pathway can be in part evoked by 'stress' and was previously thought to result almost entirely from adrenal steroid hormones. This concept is now in serious doubt because adrenalectomized rats which obviously lack a steroidogenic response are nonetheless immunosuppressed by stress (M. Stein and S. Keller, New York). In addition, at least two types of stress-induced analgesia have been observed, one operating through endogenous opioid peptides (endorphins), the other being nonopioid. Only stress-induced analgesia of the opioid peptide type was associated with immunosuppression, which in turn was correlated with enhanced Continued on p. 116

Complement receptor structure and function from Gordon D. Ross and J o h n P. Atkinson Membrane complement receptors (CRs) are now recognized as important cellbound components of the complement system and research into the structure and function of complement receptors t is one of the main thrusts of current complement research.

CR1 of human erythrocytes was first isolated by Fearon in 1979 and, more recently, CR2, CR3, factor H receptors, DAF, Clq-receptors, and gp45-70 have been isolated and characterized at the protein level. In man, CR1 (C3b/C4b receptor) is an ~ 2 0 0 Kilodalton (M~) polymorphic membrane glycoprotein found on red

tThis is a report of a Complement Receptor Workshop held on October 29-31, 1984, at the Howard Hughes Medical Institute, Coconut Grove, Florida. Abstracts of talks presented at this meeting will be published later this year in the journal Complement.

cells, phagocytes, lymphoc~/tes and in certain tissues such as the kidney glomerulus. Four codominantly expressed allotypic variants of CR1 have been discovered. The mol. wt. and gene frequencies of each type are A, 190 K (0.83); B, 220 K (0.16); C, 160 K(0.01); D, 250 K (<0.01). N-linked sugars account for ~15 K of tool. wt. of each allotype. The molecular basis accounting for this unusual polymorphism in which there is an "~90 K tool. wt. difference in protein mass among alleles is unknown. No functional differences among the four alleles have been Continued on p. 117

© 1985,ElsevierSciencePublishersB.V.,Amsterdam 0167.- 4919/85/$02.00

Immunology Today, vol. 6, No. 4, 1985

116 The immune system--continued from p. 115 tumorigenesis. Both the immunosuppressive and tumor-enhancing effect was blocked by a specific endorphin antagonist, naltrexone (J. Liebeskind and Y. Shavit, Los Angeles). Conversely, administration of met- and leuenkephalins to tumor-bearing mice prolonged the animals' surviv~ and reduced tumor growth (N. Plotnikoff, Tulsa and A. Murgo, Morgantown). These effects might be directly mediated by pituitary peptides, such as endorphins and corticotropin (ACTH), (as opposed to adrenal steroids), an interpretation strongly supported by the observation that A C T H and a-endorplain suppressed in-vitro antibody production, possibly via the opiate and A C T H receptors on lymphocytes (E. Blalock and E. Smith, Galveston; H. Johnson, Gainesville). Thus our concepts of how 'stress' translates into immunosuppression are changing. A 'lymphokine' role for pitmtary and neuro-peptides was not limited to A C T H and endorphins but included a host of other hormones. In a serum substitute system, insulin but not growth hormone (GH) allowed for the generation of a mixed lymphocyte blastogenic response. However, G H addition for the first two days of culture allowed the generation of cytotoxic T cells (C. Snow, Lexington). This in-vitro finding correlates with the known impairment of cellmediated immunity in G H deficient animals, as well as the presence of GH receptors on thymocytes. Arginine, vasopressin and oxytocin replace the IL-2 requirement for interferon production by T cells (Johnson) and thyrotropin enhanced the in-vitro antibody response (Blalock and Smith; Johnson). Substance P (SP) and neurotensin caused degranulation of mast cells and somatostatin (SM) blocked the release of mediators such as leukotrienes from these cells (E. Goetzl, San Francisco). The effect of SM is particularly interesting in light of a recent report of the presence of SM in mononuclear leukocytes. SM will also inhibit a primary antibody response to sheep red blood cells (J. Radosevic-Stasic, Olge Ban). SP also increased T-cell mitogenesis and SP receptors seemed to be restricted to T lymphocytes (D. Payan, San Francisco). That SP may be expressed in a detrimental way was suggested by the high levels of SP which are found in the joints of arthritic animals and the evidence that a substance which is toxic for SP-containing neurons blocks the development of experimental arthritis (J. Levine, San Francisco). These observations link SP activity with faulty immunoregulation in vivo. There might also be a role for a

newly described macrophage chemotactic activity of SP (as well as bombesin, endorphins and enkephalins; M. Ruff and C. Pert, Bethesda) in the attraction of leukocytes into an area of inflammation such as an arthritic joint. Taken together, these various observations seem to provide a vast array of molecules and mechanisms by which the nervous system and pituitary gland might modulate immunity. Neuron-lymphocyte . . . and lymphocyte-neuron communication? Perhaps the most novel aspect of each of the three meetings was the concept that communication between the neuroendocrine and immune systems is reciprocal. For instance, during an immune response, there is an increase in the firing rate of neurons in the hypothalamus as well as an increase in circulating glucocorticoid hormone levels which correlated with increased antibody titers. Supernatant fluids from concanavalin A- or phytohemagglutinin-treated leukocytes raise corticosterone levels after injection into mice, and it was hypothesized that this might ultimately occur through stimulation of the hypothalamus to secrete ACTH-releasing factor (H. Besedovsky, Davos-Platz). The active principle for this reaction was unknown but there are a number of attractive candidates. Synthetic thymosin a-1 increases circulating A C T H and glucocorticoid hormone levels, possibly through an action on the hypothalamus or another portion of the central nervous system (N. Hall, J. McGillis and A. Goldstein, Washington). Thymosin a-1 is present in the supernatant fluids of mitogen-treated leukocytes and so may at least in part be responsible for the effects of these supernatant fluids. A second candidate is IL-2 which was recently shown during clinical testing to raise circulating A C T H and cortisol levels. Thus, classical lymphokines seem to be capable of serving as messengers from the immune to neuroendocrine system. A second, recently discovered communication system involves the production of known neuroendocrine peptide hormones by cells of the immune system. In vitro, human peripheral leukocytes and mouse spleen cells produce A C T H and endorphins in response to virus infection or bacterial lipopolysaccharide (LPS) treatment and have receptors for these hormones (Blalock and Smith). Further, mouse B lymphocytes express messenger R N A for pro-opiomelanocortin (POMC), the common precursor for A C T H and endorphins, after treatment ¢¢ith LPS and dextran sulfate (W. Kuziel, K. Brooks, E. Vitetta, J. Uhr

and P. Tucker, Dallas). The same group also showed that the BCL-1 cell line consititutively expressed POMC-related m R N A and raised the possibility that its products, A C T H and fl-endorphin, might alone or in concert represent their previously described low molecular weight B-cell-derived B-cell growth /'actor. Sufficient A C T H is produced in vivo in virus-infected, hypophysectomized (pituitary-less) mice to elicit an increase in circulating corticosterone levels, and spleen cells are the apparent source of this peptide hormone (Blalock and Smith). This same group of investigators also showed that in vitro the P O M C gene products were induced by corticotropin releasing factor and suppressed by dexamethasone. Such data suggested that cells of the immune system can apparently be controlled in a fashion similar to pituitary cells by a positive signal from the hypothalamus (corticotropin releasing factor) and a negative signal from the adrenal gland (a glucocorticoid hormone). Hence, 'stress' as defined by an increase in circulating glucocorticoid hormone levels can, depending on the stimulus, apparently have its ultimate origins not only in the central and peripheral nervous systems but also in the immune system itself. St~ch findings have led to the notion that a primary function of the immune system may be to serve as a sensory organ for stimuli such as bacteria, viruses and tumors cells that are not recognized by the central and peripheral nervous systems (Blalock and Smith), leukocyte information then being transferred to the neuroendocrine system by peptide hormones and lymphokines. To paraphrase one participant (J. Axelrod, Bethesda) at the Washington meeting, certain cells of the immune system may serve as 'free-floating nerve cells'. Perhaps collectively, such cells represent a mobile brain. Supporting this concept, a growing number of neuroendocrine hormone-like peptides and their receptors are being found in cells of the immune system. To date, the list includes the induction ofthryotropin and chorionic gonadotropin in response to a T-cell mitogen (staphylococcal enterotoxin A) and during mixed lymphocyte reactions, respectively (Blalock and Smith); the finding of vasoactive intestinal peptide (VIP) in neutrophils and its receptor on T ceils (S. O'Dorisio, Columbus); a recent report of somatostatin in mononuclear leukocytes; and benzodiazepam receptors on lymphocytes and macrophages (R. Weber and C. Pert, Bethesda). IL-1 induces slowwave sleep (J. Krueger, Chicago) and is endogenous to the brain (A. Fontana,

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Immunology Today, voL 6, No. 4, 1985

efficacy of the opiate antagonist, naloxone, in the treatment of the symptoms of gram negative sepsis and endotoxin shock (J. Holaday, Washington) and the ability of a 'neuropeptide hormone cocktail' to allow for successful allograft transplantation (G. Maestroni and W. Pierpaoli, Ebmatigen)in animal models may be merely the beginning.

Zurich). If more connections between classical lymphokines and the neuroendocrine system are made, these two groups of regulatory molecules may come to be seen as one family of neuroimmunoendocrine substances. Another message from these meetings was that at a molecular level neuroendocrine hormones may act' on cells of the immune system as they do on cells such as neurons. For instance, h u m a n lymphocytes appear to possess voltagegated K ÷ channels as well as Na + and Ca 2+ channels. The K + channels appear to be both modulated by mitogens and involved in the mitogenic response, while the Ca z+ channels may be involved in lymphocyte activation (M. Calahan and S. Gupta, Irvine). In

addition, at least some of the effects that result from VIP binding to its lymphocyte receptor seem to be mediated by an increase in cAMP levels and activation of cAMP-dependent protein kinase (O'Dorisio, Columbus). In conclusion, it appears that not only are we beginning to understand' psychoimmunology' in the whole animal but are making rapid and major advances at the tissue, cellular and molecular level. Such developments portend major changes in the way we view the neuroendocrine and immune systems. These in turn will undoubtedly lead to new strategies for the prophylaxis, diagnosis and treatment of h u m a n diseases of immune, neuroendocrine and psychologic origin. Indeed, the reports of the

The proceedings of the Coconut Grove workshop will be published in The Journal of Immunology. J. Edwin Blalock and Eric M. Smith are at the Department of Microbiology, University of Texas Medical Branch, Galveston, T X 77550, USA.

Complementreceptors-continuedJromp. 115 detected. Moreover, the glomerular C3 receptor is functionally, immunochemically, and structurally identical to CR1 of peripheral blood cells. The human promyelocyte HL-60 line is a convenient cell for the study of the biosynthesis of CR1 since induction of differentiation in these cells results in the expression of large amounts of this receptor. Evidence was provided for a new gene cluster of functionally related complement proteins in man that included CR1, C4-binding protein (C4-bp), and factor H (H). These proteins have important regulatory functions in the control of complement activation. Each binds to C3b and/or C4b, disassembles the C3/C5 convertase enzymes bound to C3b or C4b, and serves as a cofactor for the proteolytic inactivation of C3b and C4b by factor I (1). Although no structural or antigenic similarities among these proteins had been noted in the past, analysis of polymorphic variants indicates that the genes for these three proteins are linked in man. It is particularly interesting that CR1, an integral membrane protein, is genetically linked to two plasma proteins with similar function. This newly identified complement regulatory locus is not linked to the H L A complex which is the site for the genes encoding the C3-convertase forming complement proteins, C4, C2, and factor B (B). It is interesting that genes coding for many of the complement proteins are arranged in clusters and appear to be evolutionarily preserved. Further knowledge of the genetics and function of this newly identified cluster may provide important insights into the mechanisms for control of putative immune complexmediated illnesses. The C3d receptor (CR2) of h u m a n B

lymphocytes is a 140 K tool. wt. membrane glycoprotein. A previously reported 72 K tool. wt. C3d receptor of Raji cells was shown to be a fragment of the 140 K tool. wt. molecule. The biosynthesis of this receptor was studied in a h u m a n B lymphoblastoid cell line, HBS. A slightly smaller precursor was identified and N-linked sugars were shown not to be required for the C3dbinding function or for transport to the plasma membrane. Although the function of this molecule on B cells remains an enigma, several groups substantiated the exciting observation that this molecule serves as a receptor for EpsteinBarr (EB) virus (see below). CR3, the iC3b receptor, is a member of a family of three structurally related leukocyte cell surface molecules known as LFA-1, Mac-1/OKM1, and p150,95. These molecules each contain two noncovalently associated glycoprotein subunits known as a and/3, that are linked together in an 0q//1 structure. The achain subunits are distinct but show sequence homology, while the 95 K tool. wt. //-chains are identical. These molecules are involved in cell adhesion (see below) and the Mac-1/OKM1 molecule is the CR3 receptor specific for ftxed iC3b. A series of patients with recurrent bacterial infections has been reported who are deficient in this entire membrane antigen family. Cloning of the structural genes is in progress. A fourth type of receptor for f~xed C3 has been proposed, but nothing is known of its structure or function. CR4 is present on neutrophils and monocyte/ macrophages and has a specificity similar to CR2. Neutrophils formed rosettes with EC3dg bearing large amounts of fixed C3, and these rosettes were not blocked by a mixture of antiCR1, anti-CR2, and anti-CR3. Also,

neutrophil binding of [ J25I]C3dg-dimers was inhibited by fluid-phase C3dg, iC3b, and C3d, but not by C3b, nor by antibodies to CR1, CR2 and CR3. Until now there has been little previous knowledge of the structure of the C5a receptor. Despite its extraordinarily high binding affinity on intact cells, no C5a receptor activity was detectable in soluble fractions of granulocytes generated with several different detergents. Experiments to identify the C5a receptor of granulocytes by photoaffinty cross-linkage were reported at the meeting. A covalent complex containing [125I]C5a and a membrane component was found to have a tool. wt. of 6070 K. A new group of C3b binding m e m brane proteins (gp45-70) of h u m a n peripheral blood cells was described whose function is unknown. These molecules are similar to CR1 in binding specificity in that C3b or C4b, but not C3d, are ligands. They are distinct in cell distribution (in most cases a reciprocal relationship is present) and do not cross-react with polyclonal antibodies to CR1, CR2, CR3, or DAF (decay accelerating factor). However, a C3b-binding protein of similar tool. wt. ( ~ 65 K) is present on rabbit alveolar macrophages. Interestingly, a murine C3b-binding protein of "-"65 K rnol. wt. was found to be immunoprecipitated by a polyclonal rabbit anti-human CR1 antibody. The binding sites for bovine conglutinin and CR2 within the C3 mole~cule were localized. The approach '~employed should be applicable to other functional domains in C3 as well. Using purified C3 fragments and synthetic peptides (the latter based on eDNA data), the binding site for conglutinin (but not for CR3) was shown to be the carbohydrate moiety of the 27 K tool. wt.