WO1996003993A2 - Modulation of thymocyte and t cell functional activity - Google Patents

Abstract

The present invention relates to modulation of cell mediated immune responses, and to modulation or inhibition of thymocyte cancers or thymomas. In a first aspect, the present invention is directed to a method for suppressing an immune response by administering dehydroepiandrosterone (DHEA). In another aspect, the invention relates to a method for enhancing an immune response comprising administering an amount of an inhibitor of calcitonin gene related peptide (CGRP), such as the CGRP antagonist CGRP8-37, or an amount of an inhibitor of DHEA. The invention also provides a method for treating a thymic cancer or thymoma comprising administering an amount of calcitonin gene related peptide (CGRP) effective to induce apoptosis of thymic cancer or thymoma cells. The invention further provides a method for preventing tissue damage comprising administering an amount of an inhibitor of CGRP to inhibit apoptosis of cells in the area of the tissue damage. The invention also relates to a method for identifying an agent capable of inhibiting CGRP-mediated apoptosis.

Description

6/03993

-1- MODULATION OF THYMOCYTE AND T CELL

FUNCTIONAL ACTIVITY

FIELD OF THE INVENTION

The present invention relates to modulation of cell mediated immune responses, and to modulation or inhibition of thymocyte cancers or thymomas.

BACKGROUND OF THE INVENTION

It is likely that neurosteroids and the autonomic nervous system neuropeptides play an important role in the development and homeostasis of the immune system (Bulloch, 1985, Dissert. Abstr. Murofilm Intl. Ann Arbor, 447:727-64; Daynes et al. , 1990, Eur. J. Immunol, 20:793-802; Goetzl et al. , 1990, Adv. Immunol. 48: 161-190; Payan et al., 1986, Adv. Immunol. 39:259-323). One of the peptide/hormone candidates utilized by the nervous and endocrine systems (Sasayama et al., 1991 , General Comparative Endocrinology 83:406-14X) to carry out this function in the higher vertebrate thymus is calcitonin gene related peptide (CGRP). CGRP was first discovered by Rosenfeld et al. (1983, Nature 304: 129-35) and its distribution within the brain (Rethelyi et al. , 1989, Neuroscience 29:225-39; Skofitsch and Jacobowitz, 1985, Peptides 6:721-45) and immune system (Bulloch et al. , 1991 , Prog. Neuroendocrine Immun. 4: 186-194; Bulloch et al. , 1991 , Ann. N. Y. Acad. Sci. 621:218-228; Fink and Weihe, 1988, Neuroscience Letters 90:39; McGillis et al. , 1991 , J. Immunol. 147:3482-89; Weihe et al., 1989, Neuroscience Letters 100:77-82) is unique and intimately tied to the regulation of autonomic neuroendocrine functions (Cavagnaro et al. , 1988, Immunol. 3:228-246). Its binding sites have been well-characterized within the lymphoid tissues of several mammalian species (Bulloch et al. , 1991 , Am. N.Y. Acad. Sci. 621 :218-228; Nillson et al. , 1990, Cell Tissue Res. 262: 125-133; Popper et al. , 1988, Peptides 9:257; Weihe et al. , 1989, Neurosci Lett. 100:77- 82). These studies indicate that the distribution of CGRP innervation and receptor fields varies greatly among these lymphoid tissues (Al-Shawaf et al. , 1990, ABSTR. of 1st Int. Congress Internat. Society Neuroimmune Mod. 216:298; Bulloch et al., 1991 supra; Fink and Weihe, 1988, Neurosci. Lett. 90:39-44, 1988; Henke et al., 1987, Brain Res. 410:404-408; Nillson et al. , 1990, supra; Popper et al. , 1988, supra; Spingall et al. , 1987, J. Autonomic Nervous System 20: 155-166; Weihe et al. , 1989, supra; Wimalawansa et al., 1987, Neuroendocrinol. 46: 131-136) and may influence different compartments of the immune system during critical periods of the development and function of immunocompetent cells. The presence of this peptide within the thymus is of particular interest due to its possible role in the production and selection of functional thymocytes.

Recently Wang et al. (1992, J. Biol. Chem. 267:21052-21057) showed that CGRP inhibits IL-2 secretion in Th-1 CD4⁺ splenocytes, whereas, Daynes et al. (1990, supra) showed that the neurosteroid, dehydroepiandrosterone (DHEA), stimulates IL-2 secretion. Within the periphery and particularly within the thymus gland, this peptide is capable of down regulating the activation of T cells essential to the initiation of a cytotoxic T cell response. This down regulation is dependent on the expression of functional CGRP receptors on the surface of T cells (Bulloch et al., 1995, Am. J. Physiol. 268 (Endocrinol. Metab. 31)E-168-E173) and a rich, discrete distribution of CGRP within the microenvironment where the activation may have occurred (Bulloch et al., 1991 , Prog. Neuroendocrine Immun. 4: 186-194). May et al. (1990, Life Sci. 46: 1627-1631) further demonstrated that DHEA protects the thymus from glucocorticoid induced involution. Both CGRP and DHEA are naturally occurring substances that demonstrate opposing effects in vitro on splenic T cells and thymocytes at physiological doses, so it seems possible that they may operate within thymus in regulating the educative process of subsets of thymocytes.

In the thymus, thymocytes are "selected" by their ability to discriminate between self and non-self and to carry out certain functions that regulate antigen specific cellular and humoral responses in the periphery. Of the Pre T cells that enter and proliferate within the thymus, 95 % are negatively selected and die by a process known as apoptosis (Scollay et al. , 1983, J. Immunol. 132: 1085-1088). Only five percent will be positively selected to emigrate to the periphery as functional T cells. Apoptosis is an active process of gene-directed, non- inflammatory cell death (Kerr and Harmon, 1991, "Definition and Incidence of Apoptosis: An Historical Perspective", Apoptosis: The Molecular Basis of Cell Death, edited by L. David Tomei and Frederick O. Cope. Plainview: Cold Spring Harbor Laboratory Press, p. 5-29; Smith et al., 1994, "Multiple Gene Regulation of Apoptosis", Apoptosis II: The Molecular Basis of Apoptosis in Disease, edited by L. David Tomei and Frederick O. Cope. Plainview: Cold Spring Harbor Laboratory Press, pp. 43-87) which eliminates cells without causing an immune response. This process is not unique to the immune system but also plays an essential role in development and in maintaining homeostasis in generation and regeneration of other tissues. In many tissues, it is induced by stress levels of glucocorticoids, but the mechanisms by which these hormones play a role in apoptosis within the thymus remains unclear primarily due to the amount of circulating corticosterone binding globulin (CBG) (Dhabhar et al., 1993, Brain Research 616:89-98). Thus, it would be of major importance to identify an endogenous thymic neuroendocrine hormone/neurotransmitter that would serve as an internal regulator of development of thymocytes.

The mechanisms that constitute the education of T cells are not fully understood and it is clear that no single signal molecule functions alone to carry out the many complex events involved in this process. The cells that migrate to and develop within the thymus do so in waves and consist of a dynamic heterogeneous population that is subject to the nervous, endocrine, and paracrine microenvironments of the thymus. Accordingly, there is a need in the art to identify factors that regulate these events.

In the thymus, CGRP is found in intrathymic nerves distributed in the corticomedullary boundaries adjacent to the vasculature with branches emanating into the cortical and medullary regions. Some fibers are invested in the arteries but the majority form varicosities among the trabeculae and the cells of the thymus. CGRP is also found in a discrete population of cells located in the medulla, at the cortico-medullary boundary, and in subcapsular and trabecular mast cells. The inventors have shown that this peptide at physiological doses markedly attenuates in vitro mitogen stimulated proliferation. Furthermore, the type I antagonist CGRP₈.₃₇ reverses this effect, and when added to the mitogen stimulated cultures alone it enhances proliferation up to two-fold. This enhancement is not due to a stimulatory effect on cell division but rather an inhibitory effect on endogenous CGRP released in the cultures. This was determined by the fact that when the antagonist was added alone, it did not cause cell division (Bulloch et al., 1991 , Prog. Neuroendocrine Immun. 4: 186-194).

In previous studies (Bulloch et al. , 1993, Abstr. Soc. Neurosci 19:948; Bulloch et al., 1991 , Ann. N.Y. Acad. Sci. 621 :218-228; Bulloch et al., 1991 , Prog. Neuroendocrinol. 4: 186-194; Bulloch et al., 1993, J. Immunol, p.248A), it has been shown that CGRP inhibits Con A activated proliferation of CD4⁺ thymocytes. However, as noted above, CGRP is a potent vasodilator, and thus is impractical for systemic administration to modulate or inhibit T cell responses. Accordingly, there remains a need in the art to identify factors that stimulate local CGRP activity, in order to control cellular immunity while avoiding systemic effects.

Citation of any reference herein should not be construed as an admission that such reference is available as prior art to the instant invention.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to a method for suppressing an immune response. The method comprises administering to a subject suspected of suffering from a disease or disorder that involves an inappropriate immune response an amount of dehydroepiandrosterone (DHEA) effective to induce the activity of calcitonin gene related peptide (CGRP) locally in the area of the inappropriate immune response. More particularly, according to the invention the induction of CGRP activity is sufficient to inhibit a functional activity of a helper (CD4+) T cell.

Thus, a particular advantage of the present invention is that it provides for modulating the magnitude of an immune response via CGRP-mediated regulation of helper T cell activity, without systemic administration of the potent dose of CGRP, which could induce unwanted or adverse side effects.

In a preferred aspect, the disease or disorder is an autoimmune disease. In particular, the autoimmune disease is selected from the group consisting of multiple sclerosis, Type I diabetes, thyroiditis, myesthemia gravis, and rheumatoid arthritis. The DHEA may be administered systemically or locally to the site of the inappropriate immune response.

In another aspect, the invention relates to a method for enhancing an immune response comprising administering to a subject suspected of suffering from a disease or disorder that involves an ineffective immune response an amount of an inhibitor of calcitonin gene related peptide (CGRP), such as but not limited to the CGRP antagonist CGRP₈.₃ , effective to enhance a functional activity of a helper (CD4+) T cell. Alternatively, the invention provides for administering an amount of an inhibitor of dehydroepiandrosterone (DHEA) effective to enhance a functional activity of a helper (CD4+) T cell.

It is therefore a further advantage of the invention that it provides for independent inhibition and enhancement of different classes of immune response, e.g.. cellular versus humoral immune response.

In yet another aspect, the invention provides a method for treating a thymic cancer or thymoma comprising administering to a subject suspected of suffering from a thymic cancer or a thymoma an amount of calcitonin gene related peptide (CGRP) effective to induce apoptosis of thymic cancer or thymoma cells. Preferably, the CGRP is administered locally to the site of the thymic cancer or thymoma. Alternatively, the CGRP may be administered in a liposome. Liposomal administration of drugs has been found to reduce the systemic, adverse side effects of such drugs.

In still another aspect, the invention provides a method for preventing tissue damage associated with excessive secretion of calcitonin gene related peptide (CGRP) in a hyper-response to an insult, comprising administering an amount of an inhibitor of CGRP effective to inhibit apoptosis of cells in the area of the tissue damage. In a preferred aspect, the inhibitor does not antagonize CGRP-mediated attenuation of helper T cell activity. In a more preferred aspect, an amount of CGRP, or DHEA, or a combination thereof, effective to inhibit a functional activity of a helper T cell is administered with the antagonist of CGRP-induced apoptosis.

Thus, the present invention advantageously provides for regulation of the cellular and physiological events that accompany a serious insult, by simultaneously providing CGRP to inhibit inflammation mediated by infiltrating activated immune cells, and an agent that can inhibit CGRP-mediated apoptosis.

According to the invention, the insult may be severe brain trauma or a stroke leading to brain ischemia, or result from severing a limb, removing an organ for transplantation, myocardial infarct, or pulmonary embolism.

In a specific embodiment, the inhibitor of CGRP-mediated apoptosis is administered in response to head trauma, particularly to avoid neural apoptosis associated with increased CGRP expression that follows trauma and other neural insult. The invention further provides a method for identifying an agent capable of inhibiting CGRP-mediated apoptosis. This method comprises culturing test thymocytes with about 10^"12 M to about 10^~6 M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes for about 8 to about 24 hours; culturing control thymocytes with about 10^"12 M to about 10"⁶ M CGRP for the same time as the test thymocytes in (a); and determining the extent of apoptosis of the thymocytes in each of the cultures. A decrease in the extent of apoptosis of test thymocytes cultured with CGRP and the agent compared to the extent of apoptosis of the control thymocytes is indicative of the ability of the agent to inhibit CGRP-mediated apoptosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. The suppressive effect of CGRP on Con A-stimulated thymocyte proliferation, as determined by tritiated thymidine incorporation, was reversed by CGRPg.₃₇. This effect was found in six independent runs, each with n's of 5-6. One representative experiment using thymocytes from male mice is presented here. CGRP_{g 37} in the presence of Con A enhanced proliferation over Con A alone; the same effect was obtained in seven other experiments besides the ones shown here and in Figure 3. CGRP and CGRP_g_₃₇ in the absence of Con A had no effect on thymocyte proliferation. ^* p < 0.001 compared to Con A alone, using a one way ANOVA followed by Tukey's post hoc comparison. Error bars indicate the standard error of the mean.

FIGURE 2. Dose response effects of CGRP_{g 37} on Con A induced thymocyte proliferation. Con A + CGRP_g.₃₇ enhanced thymocyte proliferation (hatched bars) is statistically greater than proliferation induced by Con A alone at the 2, 4 and 8 microgram/ml dose (open bars). " p < 0.001 , Student's t test. Error bars indicate the standard error of the mean. Male mice were a source of the thymocytes. FIGURE 3. Dose response effect of DHEA on Con A induced thymocyte proliferation. Con A was used at 4 micrograms/ml for all points. Male mice were the source of the thymocytes. One way analysis of variance (ANOVA) indicated a statistically significant treatment effect, p < 0.0001.

FIGURE 4. Effect of CGRP_g_₃₇ on DHEA-induced suppression of Con

A-stimulated thymocyte proliferation. CGRP_g.₃₇ reversed the suppress ive effect of DHEA on Con A, whereas DHEA had no effect on thymocyte proliferation in the absence of Con A (^** p < 0.001 versus DHEA + Con A one way ANOVA followed by Tukey's post hoc comparison). The reversal of DHEA suppression by CGRP_{g 37} was found in 4 separate experiments besides the one shown here, each with n's of 5-6. Male mice were the source of the thymocytes.

FIGURE 5. A. Effects of DHEA, CGRP, and CGRP^ on Con A-induced thymocyte proliferation in male mice. Compared to Con A alone, CGRP (10^~9 M) significantly inhibited suppression of Con A stimulation of thymocyte proliferation (^* p <0.05 using a one way ANOVA followed by Tukey's post hoc comparison). CGRPg.---* (10⁶ M) reversed the effect of CGRP or CGRP plus DHEA f^* p < 0.001). Error bars indicate the standard error of the mean. B. Effects of DHEA, CGRP, and CGRP_g.₃₇ on Con A induced thymocyte proliferation in females. This experiment was carried out in parallel with the stimulation of thymocytes derived from the male mice. The results show that the overall magnitude of responsiveness to Con A was greater in female-derived thymocytes. However, the qualitative responses to CGRP, DHEA, and CGRP_g.₃₇ were the same: i.e. , CGRP (10^~9 M) significantly inhibited suppression of Con A stimulation of thymocyte proliferation and CGRP_{8 37} reversed the effect of CGRP or CGRP plus DHEA (^* p <0.05 using a one way ANOVA followed by Tukey's post hoc comparison). DHEA inhibited Con A induced proliferation (^** p < 0.01). Error bars indicate the standard error of the mean. FIGURE 6. Summary of six separate experiments that demonstrate the specific effect of Con A (4 μg/ml), CGRP (iα⁸M), CGRP₈.₃₇ (10^"6M) in various combinations on apoptosis in thymocyte cultures at (A) 8 and (B) 24 hours. The bars correspond to the following treatments: (a) Con A; (b) CGRP; (c) CGRP₈_₃₇; (d) ConA and CGRP; (e) ConA and CGRP₈.₃₇; (f) CGRP and CGRP₈.₃₇; and (g) ConA, CGRP, and CGRP₈.₃₇. All data points with the exception of CGRP₈₃₇ at 8 hours show significant increase in apoptosis (p < 0.05) when compared to background. The 8-37 antagonist did not block CGRP induced apoptosis either alone or in combination with Con A.

FIGURE 7. Representative experiment (from N=3 experiments) histograms illustrating apoptosis in control (c), corticosterone-treated (a) and CGRP-treated (b) (at physiological doses) thymocyte cultures at (A) 8 and (B) 24 hours. At 24 hours the CGRP apoptotic effect is comparable to corticosterone.

FIGURE 8. Representative experiment (from N=2 experiments) comparing the effect of Con A, CGRP, and

(same concentration as for Figure 6) in various combinations on apoptosis in thymocyte and splenocyte cultures. The first bar represent results for background controls. Bar (a), (b), (e), (f), and (g) correspond to Figure 6; (c) Con A and CGRP; (d) CGRP₈.₃₇.

FIGURE 9. Percent and types (as indicated by CD markers for thymocytes) of total apoptotic cells in (A) control, (B) CGRP (10^" M), and (C) CGRP (10^"8M) + CGRP_{g 37} (10^~6M) treated cultures at 8 hours. The markers evaluated were CD3 (a), CD4/CD8 (b), and CD4 (c). The amount of CD8 apoptotic cells is less than 1 % . The percent of CD- labelled cells was adjusted to reflect the percent of apoptotic cells per group. The experiment is representative of two individual determinations of the type of cells undergoing apoptosis. All apoptotic data points for both experiments fell well within the statistical range represented in Figure 7. FIGURE 10. Representative experiment of N=2 experiments showing the percent of total and CD3 (a), CD4/CD8 (b), and CD4 (c) apoptotic cells at 24 hours. The cell types evaluated and treatments are the same as in Figure 5, so (A) is the control group, (B) received CGRP, and (C) received CGRP-I-CGRP₈.₃₇. The amount of CD8 apoptotic cells is less than 1 % . The percent of CD- labelled cells was adjusted to reflect the percent of apoptotic cells per group.

FIGURE 11. Photomicrograph of CGRP⁺ cells (arrow) within the medulla and at the cortico-medullary junction of the thymus. Schematic insert represents regions of the thymus and the maturational steps that thymocytes undergo as they enter different regions of the thymus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the nascent understanding of the role of calcitonin gene related peptide (CGRP). In particular, the invention relates to the discoveries that a neurosteroid agonizes CGRP-mediated immunomodulation locally; that an antagonist of CGRP can block CGRP's ability to inhibit T cell activation; and that CGRP induces apoptosis in certain cell types.

CGRP is a 37 amino acid peptide that is well characterized for its vasoactive properties: it is one of the most potent vasodilators known. This peptide has been found to be localized in immune tissues. For example, it is uniquely distributed in cells and nerves of the thymus, and is thought to play a special role in thymus immune function. Previous data have shown that this peptide prevents CD4 (most likely TH1) cells from being activated in the thymus gland and hence may serve as a regional endogenous regulator of the class of the immune response. If CD4+ T cells became activated within the thymus, secretion of lymphokines and cytokines could lead to a cell mediated immune response, with potentially deleterious effects on tissue critical to the education of the immune system. Also, given its vasodilation properties, CGRP may play a role in the exiting of mature lymphocytes from the thymus.

Accordingly, the present invention is directed to regulating immune system function by regulating CGRP activity with agonists and antagonists of CGRP.

Moreover, the present invention is directed in part to the discovery that CGRP mediates its effects through two different receptors, and that these receptors can be independently agonized and antagonized in order to modulate CGRP-mediated responses in vivo. One effect of CGRP is to modulate activation of mature T cells. This effect appears to be modulated through CGRP type 1 receptor (CGRP Rl). A second effect, which operates via a different receptor termed CGRP type 2 receptor (CGRP R2), is to induce apoptosis, e.g. , in a selected subset of T cells. The present invention thus provides for targeting a subset of cells for apoptosis, while preventing suppression of mature T cell activation, for example by providing CGRP (or an analog or derivative thereof, or an agent that stimulates production of endogenous CGRP) with an inhibitor of the CGRP Rl (e.g. , CGRP ₈.₃₇). Alternatively, an agonist of the type 2 receptor that does not affect the type 1 receptor can be provided. In another embodiment, the invention provides for inhibiting CGRP-mediated apoptosis, for example by providing an inhibitor of CGRP R2 when CGRP is administered or its endogenous expression increased; however, this inhibitor will not inhibit CGRP-mediated inhibition of T cell activation. Alternatively, a specific agonist of the type 1 receptor that is inactive at the type 2 receptor may be provided. In this treatment regimen, apoptosis and inflammation can both be avoided, e.g. , following severe brain trauma.

Numerous abbreviations and terms are used herein to simplify the terminology used, and to facilitate a better understanding of the invention.

An "antagonist" is an agent that inhibits or prevents an activity of a molecule. The inhibitory effect of an antagonist can be mediated by competitive inhibition or by non-competitive inhibition. It is contemplated that an antagonist of an activity of CGRP for use according to the invention may intercede at a point upstream or downstream of the events induced by CGRP, i.e. , it may not act directly on CGRP, but nevertheless blocks the effects of CGRP. Such an antagonist may, for example, inhibit expression or cause down-regulation of a CGRP receptor, or activate an opposing cellular activity.

An "agonist" is an agent that promotes an activity of a molecule, i.e. , the opposite of an antagonist. An agonist according to the invention may induce activity or expression of the molecule, or it may demonstrate the same effects as the molecule (i.e. , function analogous to a non-competitive and competitive inhibitor, respectively). As used herein, a CGRP type 1 receptor agonist produces signals associated with CGRP binding with a type 1 receptor, e.g. , thymocyte apoptosis or T cell suppression. Preferably, a CGRP type 1 receptor agonist has no effect on a CGRP type 2 receptor. A CGRP type 2 receptor agonist produces signals associated with CGRP binding to a CGRP type 2 receptor. Preferably, a CGRP type 2 receptor agonist has no effect on a CGRP type 1 receptor. Thus, in a preferred aspect of the invention, CGRP agonists that are highly specific for a single CGRP receptor type are employed. Use of a specific receptor agonist may produce the same effects as administration of CGRP with the antagonist of the other receptor: e.g. , use of a CGRP type 1 receptor agonist may produce the same effects as administration of CGRP with a CGRP type 2 receptor antagonist, and vice versa.

As used herein, the term CGRP refers to calcitonin-gene related peptide. The peptide may be from any animal species, preferably human, and including, but not limited to, other primates, murine, rat, canine, feline, etc. The term further includes the polypeptide that may be obtained from animal sources, such as, but not limited to human, other primate, ovine, porcine, murine, or rat sources. The term also refers to recombinant peptides expressed from genes encoding such peptide. The recombinant peptide may have the same post-translational modifications as the native peptides, or may be differently modified, e.g. , by expression in prokaryotic expression systems, in which the polypeptide will not be glycosylated and may contain an N-terminal methionine residue; expression in yeast expression systems, in which the polypeptide may be decorated with a yeast polysaccharide; or expression in a mammalian expression system, in which native or non-native glycosylation is possible. Similarly, analogs of the naturally occurring peptide, e.g. , that contain conservative amino acid substitutions, or that contain one or more non-peptide bonds, are also contemplated. In addition, fusion polypeptides containing N-terminal (or C-terminal) amino acids that represent cleavage sites, leader sequences, tags (e.g. , a hexa-Histidine tag), etc. are within the scope of the term CGRP. Derivatives of the polypeptide, i.e. , chemically modified forms of the natural peptide, or analogs thereof, such as those prepared by conjugation to a targeting molecule to target the polypeptide across the blood brain barrier (such as transferrin) , conjugation to a hydrophobic peptide or a fatty acid chain to facilitate transport across the blood brain barrier, phosphorylation, carboxymethylation, N-terminal acetylation, pegylation (particularly N-terminal pegylation) or other derivitizations, are also contemplated. Finally, the polypeptide may be a truncated form of the natural peptide, or analog or derivative thereof, provided the truncated form demonstrates a functional activity of CGRP.

As used herein, the term "dehydroepiandrosterone" or "DHEA" (3- hydroxyandrost-5-en-17-one; prasterone; dehydroisoandrosterone; etc.; see The Merck Index, Tenth Edition, index number 7606) refers to a neurosteroid, or analogs or derivatives thereof that demonstrate similar functional activity. DHEA can be obtained from commercial sources, e.g. , Sigma, or can be prepared synthetically (see The Merck Index, supra).

As used herein, the terms "functionally active" and "functional activity" refer to a property of a molecule to modulate target cell phenotype or activity. A composition comprising "A" (where "A" is a single protein, peptide, steroid, etc.) is substantially free of "B" (where "B" comprises one or more contaminating molecule or molecules, not including racemic forms of "A") when at least about 75 % by weight of the proteins, peptide, steroid, etc. (depending on the category of species to which A and B belong) in the composition is "A". Preferably, "A" comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition be substantially free of contamination, and generally that such compositions contain only a single molecular weight species having the activity or characteristic of the species of interest.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al. , Immunology, Second Ed. , 1984, Benjamin/Cummings: Menlo Park, California, p. 384). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Preferably, the adjuvant is pharmaceutically acceptable.

The present invention is based, in part, on the unexpected discovery concerning regulation of thymocyte activity and maturation in the thymus: a natural steroid dehydroepiandrosterone (DHEA) protects the thymic tissue by stimulating the release of CGRP within the thymus gland. Prior studies indicate that DHEA has immunostimulatory activity, e.g. , induces IL-2 secretion by T lymphocytes (Daynes et al. , 1990, Eur. J. Immunol. 20:793-802). This suppressive action is only seen in the thymus, and has not been observed in the spleen. Suppression by DHEA is reversed by the CGRP antagonist CGRP_g_₃₇.

The invention further relates to the unexpected observation that in addition to its modulatory role within the thymus, CGRP causes apoptosis of thymocytes. This effect may be mediated by the type 2 CGRP (CGRP R2) receptor since the antagonist CGRP₈.₃₇ does not prevent or stimulate thymic cell apoptosis. Furthermore, DHEA within the assay time limitations also does not cause apoptosis.

The invention is further based on observations of the role of CGRP after neural injury. The examples, infra, describe that adrenalectomy (ADX) results in death and birth of granule cell neurons in the dentate gyrus (DG) of the hippocampal formation (HF). Cell death is due to the lack of corticosterone occupying the type 1 mineralocorticoid receptor, whereas cell birth may be due to an interference with NMDA receptor activation. Notably, ADX causes a marked elevation of CGRP in the inner third molecular layer (ML) of the DG and in DG hilar and CA3c neurons, termed "the Yellow Brick Road. " CGRP immunoreactivity is primarily found in large dense core vesicles (100 to 200 nm) contained in axons and in small axon terminals which form asymmetrical synapses within the neuropil of the inner third molecular layer and in a few cells within the granule cell layer. The ADX increase of CGRP is not reversed by low doses of corticosterone that occupy mineralocorticoid receptors, nor is the Yellow Brick Road abolished by lesioning input pathways from the hypothalamus. Instead, it appears that the Yellow Brick Road is produced by CGRP elaborated within intrinsic neurons. Estrogen treatment, which stimulates synaptogenesis, causes formation of the Yellow Brick Road without ADX. In ischemic brains (a stroke model), CGRP is expressed in the pyramidal cells of CA1 and a diffuse pattern similar to the cGRP band in the inner third of molecular layer of the DG in the stratum radiatum associated pathway which primarily arise from CA3 and hilar neurons. CGRP is also expressed in hilar neurons. Neurons in colchicine treated rats express dense CGRP-immunoreactivity both along the injection tract and within the target site (the DG). Colchicine has been shown in the brainstem to stimulate CGRP in motor neurons, which indicates that colchicine induces expression of CGRP as well as interferes with spinal formation. These data indicate a protective role of CGRP during reorganization of neural tissue, which could otherwise lead to an inflammatory immune response against apoptic cells and antigens associated with the remodelling process.

Thus, the invention is directed to administration of DHEA to induce CGRP activity locally. Local activation of CGRP avoids or reduces deleterious side effects that could accompany systemic administration of CGRP. The invention further relates to administration of CGRP to induce apoptosis of undesired thymocytes, in particular thymic cancers or thymomas. The invention accordingly relates to the identification of specific agonists of the CGRP type-1 receptor, to specific agonists of the type 2 receptor, and to antagonists of the latter activity of CGRP, i.e. , antagonists of CGRP-mediated apoptosis. An antagonist of CGRP-mediated apoptosis would antagonize CGRP activity at the type 2 receptor. Such antagonists are believed to be important to protect tissue from severe trauma or ischemic events. In particular, administration of such an inhibitor of CGRP is particularly desired for brain trauma or stroke, where the injury results in excessive secretion of CGRP in hyper-response to the insult. Thus, while under these circumstances CGRP may have a protective role by preventing deleterious cellular immune responses and inflammation, excessive CGRP may be responsible for the programmed cell death associated with trauma or ischemia.

Accordingly, the invention relates to compositions comprising one or more of CGRP, DHEA, an antagonist of CGRP- or DHEA-mediated helper T cell attenuation, and an antagonist of CGRP-induced apoptosis. Preferably, such compositions are pharmaceutical compositions, suitable for administration to a subject for treatment of a disease or disorder associated with an inappropriate immune response or apoptosis. The methods of the present invention are applicable to treatment of animal subjects, more particularly mammals, and preferably humans. The methods can also be advantageously employed for the treatment ot non-human primate, canine, feline, bovine, ovine, caprine, equine, or non-domesticated animal subjects.

According to the invention, the component or components of a therapeutic composition of the invention may be introduced parenterally, orally, nasally, or rectally. Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. More preferably, where administration of CGRP is indicated to induce apoptosis of a tumor, it may be introduced by injection into the tumor or into tissues surrounding the tumor. In another embodiment, the therapeutic compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid). To reduce its systemic side effects, this may be a preferred method for introducing CGRP.

In yet another embodiment, the therapeutic compound can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al. , N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al. , Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al. , J. Neurosurg. 71 : 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e. , the brain, thus requiring only a fraction of the systemic dose (see, e.g. , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Preferably, a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor.

Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

CD4 (TH1) Modulation

The clinical implications of the discovery of the present inventors have important implications for modulation of helper T cell activity in an immune response. CGRP can suppress certain aspects of the cellular immune response while permitting and possibly even enhancing the humoral immune response. However, since CGRP is a potent vasodilator, and has additional functional activities, systemic administration of this peptide could have undesirable, deleterious side effects.

Thus, it is a significant advantage of the present invention that it provides for administration of the neurosteroid dehydroepiandrosterone (DHEA) to induce the functional activity of CGRP in cells primed to exhibit this activity. For example, in a specific embodiment, infra, thymocytes in in vitro culture suppress T lymphocyte activation by the potent mitogen ConA after DHEA is administered. This DHEA-inhibition is blocked by a CGRP antagonist (CGRP₈.₃₇).

Accordingly, the present invention has important implications for treating diseases or disorders characterized by inappropriate cellular immune activation, particularly where such immune activation occurs in proximity to or in tissues that contain CGRP-producing cells. Since most tissues are likely to include CGRP-containing nerve fibers, the present invention relates to a wide range of therapeutic uses. For example, in cellular (autoimmune) diseases such as rheumatoid arthritis, Type I diabetes, thyroiditis, myesthemia gravis, and, in particular, the neurological disease multiple sclerosis, where the affected tissues include CGRP-containing cells or are innervated with CGRP-producing cells, or both, administration of DHEA can induce local CGRP activity. Local induction of CGRP activity will reduce the frequency and severity of systemic side effects of CGRP, since a local concentration effective to inhibit a functional activity of a T cell can be developed without requiring a systemic concentration of such potency.

In another embodiment, the present invention provides for modulating the class of an immune response by introducing CGRP, or a CGRP type 1 receptor agonist or a molecule that induces CGRP; or an antagonist of CGRP, depending on whether the disease or disorder requires amplification or attenuation of cellular immune response. For example, in diseases such as AIDS where a humoral immune response is inadequate to clear the pathogen, strategies based on the knowledge disclosed herein of the regulatory mechanisms of CGRP (using specific agonists or antagonists to modulate the class of the immune response) can provide a useful therapeutic approach in managing the disease.

An antagonist of CGRP can be a peptide antagonist, such as but not limited to CGRP_{8 37}, an antibody that neutralizes the functional activity of CGRP, a soluble receptor that neutralizes the functional activity of CGRP, or a small molecule analog antagonist. It may also be possible to block CGRP attenuation of cellular immunity by providing an inhibitor of DHEA, such as but not limited to a DHEA antagonist, an antibody to DHEA, or a soluble receptor for DHEA.

Taking the example of treatment of AIDS, the present invention contemplates administration of an antagonist of CGRP that can block CGRP-mediated attenuation of cellular immunity, thus allowing for a more robust cell-mediated immune response.

The immune protection/enhancement mechanisms reported in the literature for DHEA may very well be related to its interaction with CGRP on a regional level and thus offers strategies for regulating the pathophysiology of pathogenic and/or natural insults.

Induction of Apoptosis in Cancers The ability of CGRP to cause apoptosis in thymocytes is an exciting observation, which has significant clinical value as CGRP can be a specific agent for the treatment and management of certain kinds of cancers of the thymus or thymocytes, e.g. , thymomas. Thus, according to the invention, it is possible to induce thymocyte apoptosis by contacting thymocytes or cells of a thymoma with CGRP or a CGRP type 2 receptor agonist. Preferably, the CGRP or type 2 receptor agonist is administered locally to the site of the tumor, in order to avoid excessive systemic exposure and undesirable side effects. (As noted in a specific example, infra, DHEA does not appear to cause apoptosis, and therefore is not believed to be useful for this aspect of the present invention.) Generally, thymomas consist of transformed CD4+CD8+ cells; however, the invention relates to other thymic cancers made up of cells that are CGRP sensitive, particularly cells that bear the CGRP type 2 receptor.

In addition to thymic cancers, it is believed that administration of CGRP can induce apoptosis of other cell types, which may have become transformed as cancer cells. Accordingly, this aspect of the invention extends to the treatment of any cancer or tumor in which the transformed cell has the receptor for CGRP that is involved in apoptosis, i.e. , the type 2 receptor. As used herein, the term "type 2 receptor" refers to a receptor for CGRP that mediates apoptosis, and to which CGRP_{8 37} does not competitively bind or antagonize.

Protection From Tissue Insults Tissue injury from an insult, such as severe trauma or ischemia, is frequently exacerbated by the endogenous responses to these events. For example, traumatic injuries and ischemia induce immune response mechanisms, which frequently escalate to inflammation. Other factors released in response to insults to regulate endogenous responses, e.g. , immune response, may overcompensate, and cause damage themselves. In other words, insults of this sort may lead to disregulation of the endogenous response mechanisms, leading to further tissue injury.

In situations where excessive secretion of CGRP occurs, e.g. , in a hyper-response to insult, administration of an inhibitor of CGRP type 2 receptor activity is desirable to prevent deleterious effects of CGRP to the local milieu. It is believed, based on the present discovery concerning apoptosis induced by CGRP, that CGRP may contribute to the apoptosis of cells in tissues affected by trauma or ischemia. For example, in situations where excessive secretion of CGRP occurs in a hyper- response to severe insult, such as observed in the brain's limbic system, an over- action of the peptide's natural function, inhibition of cellular immune response to avoid a deleterious cellular response may occur. Although not intending to be bound by any particular theory, it is believed that CGRP is released after severe brain trauma or ischemic events to prevent a cellular immune response leading to inflammation, which can itself have devastating effects on local tissues. However, excessive concentrations of CGRP can lead to apoptosis, thus the molecule released to prevent inflammation can itself lead to tissue destruction. Accordingly, the present invention contemplates administering a quantity of an inhibitor or antagonist of CGRP binding or activation via the CGRP receptor that mediates apoptosis, i.e. , the type 2 receptor as defined hereinabove, effective to inhibit or prevent apoptosis, but that does not impair the anti-inflammatory effect of CGRP mediated via the type 1 receptor.

Because it appears that CGRP apoptosis and CGRP immunomodulation activities are mediated through different receptors, the invention contemplates that the type 2 receptor antagonist will not inhibit CGRP binding to the type 1 receptor, which type 1 receptor activity is antagonized by CGRP₈.₃₇. However, in the event that a CGRP antagonist of the receptor involved in apoptosis also antagonizes type 1 receptor activity, an amount of such an antagonist effective to inhibit CGRP- apoptosis receptor activity but ineffective to inhibit type 1 receptor activity may be administered to a subject in need of such treatment.

In addition to inhibition of CGRP-mediated apoptosis, the present invention further provides for attenuating cell-mediated immune responses, i.e. , pathological inflammation, and apoptosis concurrently. Administration of an inhibitor of

CGRP specific for CGRP induction of apoptosis, but that does not inhibit or affect CGRP-mediated helper T cell attenuation, along with CGRP or a CGRP type 1 receptor agonist itself, can provide a potent treatment for conditions characterized by cellular infiltrates, e.g. , during the cellular phase of experimental autoimmune encephalomyelitis (EAE), multiple sclerosis, or in brain cells within the hippocampus after head trauma or ischemia induced by stroke. The CGRP can function to attenuate the cell-mediated immune response, as described above, and the specific type 2 receptor antagonist can prevent CGRP induced apoptosis. Thus, the affected tissue is protected from the effects of CGRP and from inflammation.

In addition to treatment of trauma and stroke, administration of an inhibiter of CGRP-mediated apoptosis is desired to protect tissues and organs from ischemic and reperfusion injury. Frequently, although ischemia results in some tissue damage, activation of immune response mechanisms and onset of excessive opposing regulatory mechanisms (e.g. , production of profuse amounts of CGRP) during reperfusion of ischemic tissues damages the tissues to a greater extent than the ischemia. Thus, administration of an antagonist of CGRP-mediated apoptosis is desired in the treatment of myocardial tissue after a myocardial infarct, the lung after a pulmonary embolism, the brain after a stroke, and other conditions where the blood supply to an affected organ is limited or temporarily cut off. Similarly, it may be desirable to bathe organs or severed limbs that are to be transplanted or reattached with fluid containing an antagonist of CGRP-mediated apoptosis. More preferably, for treatment of ischemia and maintenance of organs or tissues to be transplanted or reattached, a CGRP type 1 receptor agonist, or alternatively both an antagonist of CGRP-mediated apoptosis (type 2 receptor antagonist) and CGRP are administered. Administration of CGRP will inhibit the immune cell activation that can accompany reperfusion. Moreover, CGRP, as a vasodilator, may facilitate re-opening of the blocked artery causing an ischemic event.

Where CGRP is administered to inhibit cellular immune responses, the invention also contemplates administration of DHEA in place of or concurrently with CGRP. Furthermore, administration of DHEA may be preferred, since this neurosteroid does not appear to induce apoptosis, and thus may obviate or reduce the need for an antagonist of CGRP-induced apoptosis. 6/03993

24

The present invention provides an assay for identifying an antagonist of CGRP- mediated apoptosis. Such an antagonist can be identified by culturing test thymocytes with a concentration of about 10^~12 to about 10^"6 M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes, which agent is present at physiological concentrations, e.g. , about 10^"5 M or less, preferably less than or equal to about 10^"6 M, and more preferably about 10^"7 M. Control thymocytes are cultured with an equal concentration or concentrations of CGRP, without the agent being assayed. The extent of apoptosis of the test and control cultures is determined, e.g. , using a cell viability assay such as trypan blue exclusion or immunocytofluorography. A decrease in the extend of apoptosis in the test culture indicates that the agent antagonizes CGRP-mediated apoptosis.

Such an agent can be further tested to determine whether it antagonizes CGRP type I receptor activity, i.e. , inhibits CGRP attenuation of helper T cell activation. Preferably, the agent will inhibit or antagonize CGRP-induced apoptosis, but not CGRP-mediated attenuation of T cell activation.

The present invention may be better understood by reference to the following non- limiting Examples, which are provided as exemplary of the invention.

EXAMPLE 1: CALCITONIN GENE RELATED PEPTIDE (CGRP) CAUSES APOPTOSIS IN THE MOUSE THYMUS

CGRP has been identified by immunocytochemistry in cell bodies and nerve fibers of the murine thymus. Receptors for CGRP have been characterized within the gland, and their activation by nanomolar levels of CGRP was found to suppress Con A stimulated proliferation of mature virgin CD4 thymocytes. This suppression is blocked by an antagonist for CGRP (CGRP₈.₃₇). CGRP also inhibits the proliferation of Con A and antigen stimulated splenic T cells but to a lesser degree than that observed in the thymus. Given the magnitude of difference between the effects of CGRP on thymocytes versus splenocytes, the present Example was designed in part to determine if some or all of the CGRP induced suppression in the thymus was due to apoptosis.

Thymocytes were plated out with the appropriate reagent and evaluated for apoptosis using FACS analysis and propidium iodide to distinguish apoptotic cells. At 8 and 24 hours Con A did not significantly induce apoptosis in thymocytes whereas CGRP alone (10¹² to 10^"7 M) p <0.04 and in the presence of Con A induced a greater than 2 fold increase in apoptotic cells p < 0.008. The antagonist, CGRP₈.₃₇, did not cause apoptosis alone or in the presence of Con A, nor did it block programmed cell death caused by Con A or CGRP. These data suggest that apoptosis is mediated by a CGRP receptor not sensitive to the antagonist. Since CGRP-mediated inhibition of Con A induced proliferation of thymocytes and splenocytes is blocked by the antagonist, CGRP appears to mediate at least two separate functions on thymocytes via two different CGRP receptors.

These results are explored in greater detail in Example 3, infra.

EXAMPLE 2: RELATIONSHIP BETWEEN

DEHYDROEPIANDROSTERONE (DHEA) AND CALCITONIN GENE RELATED PEPTIDE rCGRP) IN THE MOUSE THYMUS

DHEA and CGRP are naturally occurring substances that are reported to have both opposing and complementary effects on immune functions. The present

Example is directed to determining how they might work together to influence the mitogen-stimulated proliferation of thymocytes. In Con A-induced thymocyte proliferation assays, CGRP and DHEA each inhibited proliferation. When the CGRP antagonist CGRP amino acids 8-37 (CGRP_{8 37}) was added to Con A-stimulated thymocytes, the proliferative response was significantly greater than the Con A response alone, across a range of Con A doses. Moreover, CGRP_{g 37} blocked the inhibitory effect of DHEA. Neither CGRP₈₃₇, CGRP, or DHEA, taken alone or in combination, stimulated thymocyte proliferation in the absence of Con A. CGRP affects the proliferation of CD4⁺ T cells, and thus may be a regional endogenous inhibitor of the proliferation of virgin mature T cells while they remain in the thymus. DHEA appears to act via endogenous CGRP on the thymus CD4⁺ T cell population.

Materials and Methods Animals. BALB/c ity-R male and female mice, 4-8 weeks old, were used in these experiments. The animals were bred and raised pathogen-free in the animal facilities at the VA Medical Center, La Jolla. They were fed commercial food and water ad libitum. Room lighting was controlled in a 12-h cycle. Male mice were used in the experiments except where designated. The mice were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NIH, 1985).

Chemicals. CGRP and CGRP₈.₃₇ were obtained from Peninsula Laboratories, Belmont, California. Con A and DHEA were obtained from Sigma, St. Louis, Missouri. Rabbit anti-CGRP was obtained from Cambridge Research Biochemicals, Wilmington Delaware. Antibody specificity was determined by absorption of the activity with 1 x 10^"5 M CGRP. ABC rabbit staining kits were obtained from Vector Laboratories, Burlingame, California.

Cell Preparation. The assay was performed as described in Mishell and Shiigi (1980, Selected Methods in Immunology, Freeman and Company. New York: Freeman and Company). BALB/c ity-R male and female mice were sacrificed by cervical dislocation, dipped in 70% alcohol, and the thymus was aseptically removed. The tissues were rinsed in several changes of phosphate buffered saline (PBS). Single cell suspensions were made by gently rolling the tissue between the frosted ends of sterile microscope slides. The cells were washed in supplemented RPMI (5 % FCS, 0.03 mg/ml gentamicin, 1 % nonessential amino acids, 1 % sodium pyruvate, 2 mM glutamine and 50 μM 2-mercaptoethanol). Proliferation Assay. Cells were plated in Falcon 96 well flat bottom tissue culture plates at 3xl0⁶ cells per well. The plates were pre-soaked in PBS for an hour to eliminate any plate toxicity. Dose response curves were generated for all reagents to determine optimal doses. Con A was added at concentrations of 2, 4, and 8 μg/ml; CGRP, CGRP₈.₃₇, and DHEA were used at 1 x 10⁶ M to 1 x 10¹³ M. The final total volume per well was 0.25 ml. The plates were incubated for 72 hours at 37°C in a humidified incubator containing 5 % C0₂. Quadruplicate wells were then pulsed with 1 μ Ci per well of ³H-thymidine (Amersham Corp. , specific activity 6.7 Ci/mmol) and incubated for an additional eighteen hours. Cultures were harvested onto glass fiber filter paper, placed in vials with scintillation cocktail, and counted on a scintillation counter. The results were expressed as mean CPM of ³H-thymidine incorporation ± standard error of the means (SEMs). The assays were repeated a minimum of three times. The significance of the results was determined by using a student T test or a one way analysis of the variance (ANOVA) followed by Tukey's post hoc test where indicated.

Results In previous studies (Bulloch et al. , 1991 , Ann. N.Y Acad. Sci. 621 :218-228; Bulloch et al. , 1991 , Prog. Neuroendocrinol. 4: 186-194), we have shown that the thymus contains a rich supply of CGRP expressing nerves and cells and that CGRP inhibits mitogen and antigen driven thymocyte proliferation in BALB/c mice. CGRP-positive immunoreactive cells are concentrated within the medulla and particularly at the cortico-medullary boundaries where mature T cells are found. The ability of CGRP to inhibit IL-2 secretion (Wang et al., 1992, J. Biol. Chem. 267:21052-57) suggested that it might have an inhibitory effect on the proliferative phase of thymocyte maturation. Indeed, previous results indicated the presence of CGRP receptors on thymocytes and that CGRP inhibited thymocyte proliferation at concentrations from 10^"7 to 10^"11'; however, no antagonist for CGRP was available at that time. In order to further investigate the effect of CGRP on thymocyte proliferation, the dose (10~⁶ M) of CGRP₈.₃₇ that totally blocks the exogenous CGRP inhibition of Con A induced thymocyte proliferation was determined (data not shown). This dose was used throughout the remaining experiments. As shown in Figure 1, with thymocytes from the BALB/c ity-R mouse, CGRP significantly inhibited the Con A-induced proliferation, as previously observed with BALB/c and C57B1/6J thymocytes, although the overall incorporation of tritiated thymidine was somewhat lower in the BALB/c ity-R strain. Furthermore, the CGRP antagonist, CGRP₈.₃ , blocked the suppressive effect of CGRP on Con A induced proliferation of thymocytes (Figure 1). Finally, when Con A-stimulated cultures were co-treated with CGRP₈.₃₇, there was a significant enhancement of proliferation over Con A alone. Neither CGRP nor its antagonist caused proliferation in the absence of Con A (Figure 1).

As shown in Figure 2, the enhancement of the Con A response by CGRP_{8 37} using sub-maximal, optimal, and slightly toxic doses of Con A (2, 4, and 8 μg/ml) was examined. In submaximal and optimal doses of Con A, CGRP₈.₃₇ significantly increased the overall proliferative response. Even at a dose of Con A somewhat toxic to the cultures (8 μg/ml), CGRP₈.₃₇ significantly enhanced the proliferative response (Figure 2).

Because DHEA has been reported to stimulated IL-2 production (Daynes et al. , 1990, Eur. J. Immunol. 20:793-802), it was tested in thymocyte proliferation assays, anticipating stimulation. However, the results showed that the addition of DHEA at 10^"6 M to 10^"13 M produced a shallow inhibition of Con A-induced thymocyte proliferation (Figure 3). The addition of CGRP₈.₃₇ to DHEA + Con A -treated cultures restored the response to the level of Con A alone, while DHEA had no effect on thymocyte proliferation in the absence of Con A (Figure 4).

Given the similarity between the magnitude of suppression of DHEA and CGRP on Con A thymocyte proliferation, we then sought to determine if CGRP and DHEA were inhibiting the same or a different population of thymocytes. Also, since DHEA can be converted to androgens and CGRP is present within spinal cord motor neurons (Forger et al. , 1993, Comp. Neurol 330:514-520), which are developmentally sensitive to early androgen exposure, we sought to determine if there was any difference in the sensitivity to DHEA and CGRP in thymocytes derived from males or female mice. As shown in Figure 5, in thymocytes from both male and female mice, the combination of CGRP and DHEA in the presence of Con A resulted in an inhibition of thymocyte proliferation similar to that produced by either CGRP or DHEA alone. In addition, CGRP₈.₃₇ reversed the suppressive effects of the combination of CGRP and DHEA (Figure 5).

In these experiments, in contrast to the data presented in Figure 1 , CGRP₈.₃₇ not only blocked the inhibition by exogenous CGRP of proliferation but also stimulated proliferation above control levels. This discrepancy is a recurrent feature of the data (see Discussion). Nevertheless, CGRP₈.₃₇ was never found to stimulate proliferation in the absence of Con A; instead, it enhanced the effect of Con A alone (Figures 1 and 2).

The only difference noted between the response of thymocytes from males and females was that the magnitude of the response to all reagents was more robust in thymocytes derived from females compared to males (Figure 5).

Discussion The results of this Example indicate that exogenous CGRP inhibits Con A-stimulated proliferation of immature thymocytes by a mechanism that is inhibited by 10^"6 M CGRP₈.₃₇. Moreover, and contrary to expectations, DHEA inhibited Con A-stimulated thymocyte proliferation; this DHEA-mediated inhibition was also reversed by CGRP₈.₃₇. Furthermore, CGRP_{8 37} enhanced the Con A-stimulated proliferation. Taken together with the presence of CGRP-immunoreactivity in cells within the cortico-medullary boundaries and within medulla of the thymus, these results suggest that one of the roles of endogenous CGRP may be making a contribution to the containment of proliferation. Given the location of CGRP-immunoreactive nerves and cells within the thymus and our previous characterization of CGRP receptors and function on thymic and splenic T cells, it is possible that endogenous CGRP might be involved in preventing newly evolved virgin mature CD4⁺ T cells from being inappropriately activated within the thymus.

A possible role for endogenous CGRP in thymus. Although the CGRP secreting cells have not yet been identified in the thymus, it is clear that the CGRP is not derived from the serum in the medium since the level of CGRP in fetal calf serum is below the femtomolar range. It is possible that CD4⁺ Th-1 cells produce CGRP and release it as their own autocrine feed-back mechanism to shut down IL-2 synthesis. Conceivably, DHEA could also induce CGRP secretion in Th-1 cells in conjunction with its ability to activate IL-2 synthesis as part of a feedback loop within the thymus (Daynes et al. , 1990, supra).

Although the tritiated thymidine responses are somewhat lower in the BALB/c ity-R mouse than were observed in other strains, the results showing inhibition of Con A stimulation by exogenous CGRP are qualitatively similar to those observed in mice of other strains. In addition, the present results indicate that not only does CGRP_{8 37} block the effects of exogenous CGRP on Con A stimulated thymocyte proliferation, but it also appears to blocks the effect of endogenous CGRP. This observation is based on the fact that Con A-stimulated cultures treated with CGRP₈.₃₇ incorporate approximately twice the amount of thymidine compared to cultures treated with Con A alone. It is highly unlikely that, in addition to blocking the CGRP effect, CGRP₈.₃₇ initiates proliferation in other cell types by binding to an unknown receptor, because in cultures without Con A. CGRP_{8 37} did not induce proliferation.

In the experiments summarized in Figure 5, in contrast to the data presented in Figure 1 , CGRP₈.₃₇ not only blocked the inhibition by exogenous CGRP of proliferation but also stimulated proliferation above control levels, much as it did when CGRP₈.₃₇ was added in the presence of Con A alone. Experimental data in addition to that presented in Results, indicates the following: 1) that CGRP_8.37 reversed the inhibition by exogenous CGRP to the baseline of Con A alone in 4 experiments (e.g., Figure 1) and to above baseline in 2 experiments; 2) for DHEA inhibition of the Con A stimulation, CGRP_g.₃₇ restored proliferation to baseline in 2 experiments (e.g. , Figure 4) and above baseline in 2 experiments; 3) for the effect of Con A alone (e.g. , Figure 3), CGRP₈.₃₇ elevated proliferation in 8 out of 8 independent experiments; 4) when CGRP and DHEA were added together to inhibit proliferation, as shown in Figure 5, CGRP_{g 37} restored proliferation to baseline in 1 experiment and elevated it above baseline in 3 experiments. Given the fact that these assays were conducted over a 4 day interval, there was no control over the amount of endogenous CGRP that may have been present and released.

Parallel experiments to the ones reported in this paper were carried out on splenocytes and it was found that Con A induces proliferation and that exogenous CGRP inhibits Con A-induced stimulation and the antagonist, CGRP₈.₃₇, reverses this effect. However, in spleen, in contrast to the results in thymus, CGRP₈.₃₇ was never found to potentiate the Con A effect. This fits with the postulated role of endogenous CGRP in thymus, since unlike thymus, the spleen contains very sparse innervation by CGRP fibers and very few CGRP positive cellular profiles.

Possible mechanism of DHEA action. Daynes et al. (1990, supra) reported that DHEA induced IL-2 secretion in vitro, whereas, Wang et al. (1992, supra) reported that CGRP inhibited IL-2 secretion in Th-1 CD4⁺ cell lines. Furthermore, Risdon et al. (1991 , Exper. Hematol. 19: 128-131 ) showed that dietary DHEA reduced thymic cellularity but had no proliferative effect on mature T cells. In addition, DHEA protected the thymus against involution by glucocorticoids (Daynes et al. , 1990, supra). This led to consideration of the possibility tnat DHEA may protect or interfere with the CGRP suppression of thymocyte proliferation in Con A treated cultures. However, the results showed that proliferation was inhibited by DHEA to a degree similar to that caused by CGRP. Moreover, combining both DHEA and CGRP did not consistently increase the level of inhibition, nor did this combination block inhibition. Furthermore, neither DHEA and CGRP_g.₃₇, separately or together, caused proliferation of thymocytes in the absence of Con A, so the stimulation of IL-2 by DHEA observed by Daynes (1990, supra) in cloned cell lines or in T cells derived from DHEA-treated mice must be minimal in fresh or Con A stimulated thymocytes.

Since DHEA is primarily synthesized by the testes in mice, an assay to determine if there was a difference between the proliferation of male and female thymocytes stimulated by Con A in the presence of DHEA was performed. The results show a quantitative but not qualitative difference. However, the quantitative differences between the male and female responses are not surprising due to the well-characterized differences between the immune responses of males and females (Ahmed et al. , 1985, J. Immunol. 134:204-209).

A plausible model to explain the relationship between DHEA and CGRP has CGRP inhibiting proliferation of CD4⁺ cells, and DHEA acting by causing release of endogenous CGRP from a population of thymic cells. As to the mechanism of the inhibitory effect of CGRP on mitogen-stimulated proliferation, it may be a direct inhibition of IL-2 production as suggested by the findings of Wang et al. (1992). However, it might also involve a mediation by prolactin, since T cells have been shown to respond to prolactin during proliferation (Sabharwal et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7713-7716). Furthermore, IL-2 driven proliferation of T cells requires prolactin, and CGRP is known to inhibit prolactin release (Elie et al., 1990, Neuropeptides 16: 109-113, Fahim et al., 1990, Neuroendocrinol. 51:688-693; Netti et al. , 1989, Neuroendocrinol. 49:242-247). Physiological significance for thymus function. An in vivo role for these in vitro observations seems quite plausible given the function of the thymus and the distribution of CGRP nerves and cells. The thymus is a primary lymphoid organ. Its role is to produce a population of T cells that can recognize foreign antigens and protect the body from infection both through cellular and humoral immunity. T cells are necessary for the activation of both classes of the immune response in the periphery. Of the immunocompetent cells that leave the thymus, only a fraction of those meet with a specific antigen, clonally expand and are capable of carrying out their effector function. Most die without being activated or are negatively selected and undergo programmed cell death (apoptosis). Although most education and production of T cells takes place during prepubescence, new antigen sensitive T cells are produced throughout life within a thymic environment that is subject to the nervous, endocrine, and paracrine factors.

The thymus, which is not designed as an effector organ, cannot have mature T cells activated within its boundaries, particularly cytotoxic T cells. Wang et al. (1992, supra) have presented data that indicates that CGRP affects CD4⁺ Th-1 cells. The activated peripheral CD4⁺ Th-1 cell helps in the generation of antigen specific CD8⁺ cells through its production of IL-2. However, this normally takes place in secondary lymphoid tissue. Thus, there must be some mechanism to protect mature CD4⁺ Th-1 cells and CD8⁺ cells that are about to leave the thymus from premature activation. The distribution of CGRP-immunoreactive nerves and cells in areas within the thymus that are rich in mature T cells offers such a mechanism. CGRP containing cells within the medulla and the cortico-medullary boundary may suppress such accidental activation and proliferation of functional, educated T cells.

CGRP is one of the most potent vasodilator known (Brain et al., 1984, Nature 313:54-55). Its presence in nerves may slow cells in the perivascular space from premature activation while simultaneously permitting them to leave the thymus. The in vitro suppression of T cell proliferation by DHEA through what appears to be a CGRP-related mechanism, may represent an in vivo neurosteroid/hormonal regulation within the thymus of inappropriate activation of mature T cells.

EXAMPLE 3: CALCITONIN GENE RELATED PEPTIDE (CGRP)

CAUSES APOPTOSIS IN MOUSE THYMUS

In the present Example, the neuroendocrine role of CGRP within the thymus is examined to determine if its attenuation of thymocyte proliferation is simply due to a quiescence of the CD4 population and/or a reduction in the available pool of cells via apoptosis that were destined to be CD4 antigen sensitive cells.

Materials and methods Animals. Four to six week old BALB/C male and female mice were obtained and maintained as described in Example 2.

Chemicals. CGRP, CGRP₈.₃₇, and Con A were obtained as described in Example 2. Corticosterone was obtained from Steraloids, Wilton, NH.

Cell Preparation. Thymus and spleen cells were obtained as described in Example 2. The trabeculae and non-suspended clumps were allowed to sediment out.

Suspended cells were removed, centrifuged, resuspended and counted for viability using trypan blue exclusion.

Proliferation Assay. Cells were plated in Falcon 96 well flat bottom tissue culture plates at 3x106 viable cells/well for thymocytes and 2x105 viable cells/well for splenocytes. The plates were incubated for 72 hours at 37oC in a humidified incubator containing 5 % C02. Wells were then pulsed with 1 μCi/well of ³H-thymidine (Amersham Corp., specific activity 6.7 Ci/mmol) and incubated for an additional eighteen hours. Cultures were harvested onto glass fiber filter paper, placed in vials with scintillation cocktail, and counted on a scintillation counter. The results were expressed as mean CPM of ³H-Thymidine incorporation +/- standard error of the means (SEMs). The significance of the results was determined by using a student T test or a one way analysis of the variance (ANOVA) followed by Tukey's post hoc test where applicable.

Apoptosis. Cells were prepared as described above and harvested at 8 and 24 hours. Cultures treated with corticosterone and CGRP were subjected to dose response evaluations to determine the optimal dose for inducing apoptosis within the physiological range (10^"8 M for CGRP, and 10^"7 M for corticosterone) (data not shown). At the end of the incubation period, the cells were washed twice and resuspended in PBS. The cells were then fixed in 50% EtOH/PBS.

Samples were analyzed for apoptosis after staining the ethanol fixed cells with propidium iodide (PI) using a modification of the method of Telford, et al. (1992, Cytometry 13: 137-43). The fixed cells were washed once in PBS and then resuspended in a 50 μg/ml solution of PI containing 10 μg/ml RNase. After a 20 minute incubation at room temperature, 20,000 cells per sample were analyzed on a Coulter Elite Flow Cytometer (Coulter Corp. , Hialeah, FL) using a doublet discrimination protocol. The percent of apoptotic cells was calculated using the Multicycle software from Phoenix Flow Systems.

Detection of T cell specific markers (CD antigens). Thymocytes were prepared as described above and incubated for 8 and 24, hours. After the incubation period, cells were harvested, washed twice and resuspended in PBS, and counted using trypan blue exclusion to test for viability. Cells (10⁶) were then labelled with antibodies to CD3, CD4, or CD8, using a 1/100 dilution of antibody. FITC labelled hamster anti-mouse CD3, rat anti-mouse CD4 and CD8, and PE labelled rat anti-mouse CD8 were used for double staining with the FITC labelled CD4 (Pharmingen San Diego, CA). The cells were incubated on ice for 45 minutes, washed, and resuspended in 0.5 ml 2% formaldehyde in PBS.

Single and double stained thymocytes were analyzed on a Becton Dickinson FACStar PLUS flow cytometer (Becton Dickinson, Mt. View, CA). Ten million cells were analyzed per sample. Viable cells and non viable cells, as determined by their forward versus 90 degree scatter patterns, were examined separately for surface antigens. Data were evaluated using Multiplus Software from Phoenix Flow Systems.

The percent of CD-labelled cells was adjusted to reflect the percent of apoptotic cells per group. For example, if a culture condition had 10% total apoptotic cells, of which 50% were double positive, then the adjusted percentage of 5 % would reflect the actual number of apoptotic CD double positive cells.

Results In the present Example and in previous studies (Bulloch et al. , 1990, FASEB 4:a2044 (Abstract); Bulloch et al., 1991 , Prog. Neuroendocrine Immun. 4: 186-194; Bulloch et al. , 1991 , Ann. N. Y. Acad. Sci. 621:218-228) it is demonstrated that CGRP is distributed in discrete regions within the thymus that define the borders between negatively and positively-selected thymocytes (mature and immature thymocytes, respectively). Example 2 also shows that the ability of mature, virgin thymocytes to respond to the mitogen Con A is inhibited by CGRP and that this inhibition is mediated by a type 1 CGRP receptor that is sensitive to the antagonist CGRP_{8 37}(Bulloch et al., 1995, supra). Furthermore, there is an increase in proliferation in cultures treated with the antagonist alone, which indicates that endogenous CGRP plays a role in suppressing activation and proliferation of the mature thymocytes within the gland.

In order to determine if CGRP suppression of proliferation was due to a reduction in the number of mature thymocytes available for stimulation or a reduction in the pool of thymocytes that was converted into mature thymocytes, the effects of CGRP on apoptosis were examined. The data in Figure 6 show that CGRP either alone or in the presence of Con A induces apoptosis in thymocytes with a significance of p < 0.05 when compared to cultures containing medium alone. The induction of apoptosis by CGRP is not blocked by the antagonist CGRP_8.37. In fact, the antagonist, which has no significant effect by itself on apoptosis at 8 hours seems to enhance the effects CGRP at both 8 and 24 hours. It also induces a significant rise in apoptotic cells by itself at 24 hours. These data indicate that the effect of CGRP on thymocyte apoptosis is mediated through a CGRP receptor other than the type one receptor (Figure 6).

In order to evaluate the potency of this endogenous ligand of apoptosis, the ability to produce apoptosis was compared to that of another naturally occurring inducer of apoptosis, the glucocorticoid corticosterone. Figure 7 shows that at physiological doses, CGRP is as lethal an inducer of apoptosis as corticosterone at 24 hours. Furthermore, it was determined that CGRP at this dose does not block the corticosterone induced apoptosis when co-incubated with this steroid (data not shown).

In order to test the specificity of CGRP induced apoptosis, its ability to induce apoptosis in mitogen stimulated splenocyte cultures was evaluated. Neither CGRP nor its antagonist induces apoptosis in splenocytes either at 8 or 24 hours, whereas, they induce marked apoptosis in parallel thymic cultures.

To determine if CGRP-induced apoptosis targeted any particular functional class of thymocytes, flow cytometry was used to examine cultures at 8 and 24 hours. The results indicate that CGRP induced apoptosis in some CD3, CD4/CD8 and CD4 positive thymocytes but not CD8 positive thymocytes (Figures 9 and 10). However, since these data show that CD4/CD8 double positive cells are approximately equal in number to the CD3 positive cells it is plausible, given that most double positive thymocytes are also positive for CD3, that the apototic CD4 positive cells in these cultures are the CD3 negative/CD4 positive immature thymocytes that occupy the outer cortical epithelial region of the gland (Figure 11).

Discussion In previous studies (Bulloch et al. , 1990, FASEB 4:a2044 (Abstract); Bulloch et al. , 1991 , Prog. Neuroendocrine Immun. 4: 186-194; Bulloch et al. , 1991, Ann. N. Y. Acad. Sci. 621 :218-228), CGRP immunopositive nerves and cells were observed to occupy discrete regions within the thymus where immature and mature virgin thymocytes are located (Figure 11) and can suppress mitogen and antigen stimulated thymocyte and splenocyte proliferation (Bulloch et al. , 1991 , Prog. Neuroendocrine Immun. 4: 186-194; Bulloch et al., 1995, supra; Umeda et al., 1988, Biochem. Biophys. Res. Commun. 154:227-35). These studies have provided invaluable information as to the putative function of this peptide within the gland and have laid the ground work for exploring nervous, neuroendocrine and immunological interactions.

The present Example shows that CGRP has another unique immune function in that it is capable of inducing apoptosis in a manner as potent as the endogenous glucocorticoid, corticosterone.

The initial question posed in this study, which asks whether apoptosis can account for the poor response of CGRP-treated thymocytes to Con A, can only be partially answered by these experiments. The marked decrease of CD4/CD8 positive and CD4⁺CD8^" thymocytes due to apoptosis during the first twenty four hours of incubation may in part explain the poor proliferative response of the CGRP treated Con A cultures. It does not, however, explain the effect fully because Con A treated splenic cells are suppressed by CGRP but they are refractory to CGRP induced apoptosis. Furthermore, the type one receptor antagonist, CGRP₈.₃₇, reverses the effect of CGRP on proliferation in the thymus but has no effect on blocking apoptosis. The data of the present Example would then indicate that CGRP affects mitogen induced proliferation and apoptosis by two different pathways involving two different receptors, a type one and a non-type one receptor. It is also interesting to note that the CD4 subset of thymocytes, whether CD3^4" or not, are driven into apoptosis up to three-fold over control cultures. This phenomenon is also indicative of the relationship of CGRP to the CD4 subset of thymocytes/T cells and suggests that its function in immune system is intimately linked to the expression of this molecule.

The distribution of CGRP+ nerves and cells in areas within the thymus that are relatively rich in mature T cells suggests the endogenous role of this peptide within the thymic parenchyma is to contain accidental proliferation and activation of T cell responses that would be deleterious to the survival and function of the gland. Likewise, CGRP is a potent vasodilator (Brain et al., 1984, Nature 313:54-55), and its presence in nerves may protect the perivascular space from premature activation of immunocytes while simultaneously permitting the cells to exit the thymus. It now seems to have an additional function, in that it can induce apoptosis in immature thymocytes and may serve as a primary agent of endogenous apoptosis or as a last line of defense against immature or auto-reactive thymocytes accidentally exiting the thymus. In this respect CGRP can be viewed as an anti-autoimmune agent for the thymus.

Recently, Schmeid et al. (1993, Am. J. Pathol. 143:446-52) have shown that apoptosis also serves as a natural way to eliminate inflammatory T cells in critical sites of the central and peripheral nervous system. Given the select distribution and release of this neuropeptide throughout the peripheral and central nervous system, it is postulated that CGRP may serve to regulate the type of immune response by restraining the T-cell cytotoxic response as well as by inducing apoptosis in other immune cells in different stages of their activation within environments crucial to the survival of the species. The type of CGRP receptor involved in apoptosis still needs to be identified. It is also important to determine if this receptor is expressed in other T and accessory immune cells during different phases of their life cycle. For example, Duke (1991 , "Apoptosis in Cell Mediated Immunity" , Apoptosis: The Molecular Basis of Cell Death, edited by L. D. Tomei and F. O. Cope, Plainview: Cold Spring Harbor Laboratory Press, pp. 209-226) states that IL-2 prevents induction of apoptosis in the peripheral CTLL response. Since Wang et al. (1992, J. Biol Chem 267:21052-57) showed that CGRP inhibits 11-2 production in a cloned TH-1 cell, it is possible that clearance of cytotoxic T cells, after pathogens have been eliminated, may be linked in the later phases of this response to down regulation of IL-2 by CGRP in CGRP enriched regions and/or to the expression of the apoptotic CGRP receptor on these cells.

EXAMPLE 4: (CGRP) RELEASE IN THE HIPPOCAMPAL

FORMATION MAY SUPPRESS INFLAMMATORY IMMUNE RESPONSES

ADX results in cell death in granule cells of the dentate gyrus (DG). Cell death occurs via apoptosis, thereby preventing the hippocampal formulation (HF) from undergoing massive neuronal damage due to cytokine release and a consequent inflammatory immune response. CGRP is increased in the HF after several types of insults, including ischemia and colchicine injection, in addition to ADX. As shown in Examples 2 and 3, CGRP also suppresses cellular immune responses in specific organs such as the thymus, by inducing apoptosis in thymocytes, suppresses cell-mediated immunity and blocks macrophages from presenting antigen. Given the immunological and behavioral effects of this peptide, the distribution of CGRP in the HF of ADX rats has been evaluated. Light microscopic analysis shows CGRP-immunoreactivity (CGRP-ir) is distributed in the inner third molecular layer (ITML) of the DG and in DG hilar and in CA3c neurons. In ADX rats, CGRP-ir is quite intense. Electron microscopic analysis of the DG shows that CGRP-ir is primarily found in large, dense core vesicles (100-120 nm) contained in axons and in small axon terminals which form asymmetrical synapses with dendritic spines in the ITML and a few cells within the granule cell layer. Fornix lesions did not reduce CGRP-ir in the HF of ADX rats, indicating that the CGRP is mostly derived from intrinsic neurons rather than the subcortical pathway. These studies suggest that CGRP plays a role in regulating immune responses in the HF, and leads to a clearer understanding of pathological and psychological effects of CNS trauma.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for suppressing an immune response comprising administering to a subject suspected of suffering from a disease or disorder that involves an inappropriate immune response an amount of dehydroepiandrosterone (DHEA) effective to induce the activity of calcitonin gene related peptide (CGRP) locally in the area of the inappropriate immune response.

2. The method according to Claim 1 , wherein the induction of CGRP activity is sufficient to inhibit a functional activity of a helper (CD4+) T cell.

3. The method according to Claim 1 , wherein the disease or disorder is an autoimmune disease.

4. The method according to Claim 3, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, Type I diabetes, thyroiditis, myesthemia gravis, and rheumatoid arthritis.

5. The method according to Claim 1 , wherein the DHEA is administered via an intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, oral, nasal, or rectal route.

6. The method according to Claim 1 , wherein the DHEA is administered locally to the site of the inappropriate immune response.

7. A method for enhancing an immune response comprising administering to a subject suspected of suffering from a disease or disorder that involves an ineffective immune response an amount of an inhibitor of calcitonin gene related peptide (CGRP) effective to enhance a functional activity of a helper (CD4+) T cell.

43 8. The method according to Claim 7, wherein the inhibitor is a peptide corresponding to CGRP₈.₃₇.

9. A method for enhancing an immune response comprising administering to a subject suspected of suffering from a disease or disorder that involves an ineffective immune response an amount of an inhibitor of dehydroepiandrosterone (DHEA) effective to enhance a functional activity of a helper (CD4+) T cell.

10. A method for treating a thymic cancer or thymoma comprising administering to a subject suspected of suffering from a thymic cancer or a thymoma an amount of calcitonin gene related peptide (CGRP) effective to induce apoptosis of thymic cancer or thymoma cells.

11. The method according to Claim 10, wherein the CGRP is administered locally to the site of the thymic cancer or thymoma.

12. The method according to Claim 10, wherein the CGRP is administered in a liposome.

13. A method for preventing tissue damage associated with excessive secretion of calcitonin gene related peptide (CGRP) in a hyper-response to an insult comprising administering an amount of an inhibitor of CGRP effective to inhibit apoptosis of cells in the area of the tissue damage.

14. The method according to Claim 13, wherein the inhibitor does not antagonize CGRP-mediated attenuation of helper T cell activity.

15. The method according to Claim 14, further comprising administering an amount of CGRP, or DHEA, or a combination thereof, effective to inhibit a functional activity of a helper T cell.

16. The method according to Claim 13, wherein the insult is severe brain trauma or a stroke leading to brain ischemia.

17. The method according to Claim 13, wherein the insult results from severing a limb, removing an organ for transplantation, myocardial infarct, or pulmonary embolism.

18. The method according to Claim 13, wherein the inhibitor of CGRP is identified by the method comprising: a) culturing test thymocytes with about \Qrⁿ M to about 10^"6 M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes for about 8 to about 24 hours; b) culturing control thymocytes with about 10^"12 M to about 10^~6 M CGRP for the same time as the test thymocytes in (a); and c) determining the extent of apoptosis of the thymocytes in each of the cultures; wherein a decrease in the extent of apoptosis of test thymocytes cultured with CGRP and the agent compared to the extent of apoptosis of the control thymocytes is indicative of the ability of the agent to inhibit CGRP-mediated apoptosis.

19. A method for identifying an agent capable of inhibiting CGRP-mediated apoptosis comprising: a) culturing test thymocytes with about 10^"12 M to about 10^~* M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes for about 8 to about 24 hours; b) culturing control thymocytes with about 10^"12 M to about 10^"6 M CGRP for the same time as the test thymocytes in (a); and c) determining the extent of apoptosis of the thymocytes in each of the cultures; wherein a decrease in the extent of apoptosis of test thymocytes cultured with CGRP and the agent compared to the extent of apoptosis of the control thymocytes is indicative of the ability of the agent to inhibit CGRP-mediated apoptosis.