WO1991004479A1 - Evaluative means for detecting inflammatory reactivity and for predicting response to stress - Google Patents

Abstract

The present invention relates to a diagnostic test for testing susceptibility of individuals to inflammatory diseases such as rheumatoid arthritis and to means to evaluate potential treatment of arthritis with therapeutic agents directed at the central nervous system (CNS), designed to by-pass the CNS defect. The invention also provides means for evaluating and predicting response to stress in individuals. A counter-regulatory feedback loop exists between the immune and central nervous systems, in which the immune or pro-inflammatory mediators stimulate corticotropin-releasing hormone (CRH) activation of the hypothalamic-pituitary-adrenal (HPA) axis hormonal cascade. The resultant increase in plasma glucocorticoids serves to restrain and limit the intensity of the inflammatory-immune response, through the potent immunosuppressive/anti-inflammatory actions of the glucocorticoids.

Description

EVALUATIVE MEANS FOR DETECTING INFLAMM_AT_ORY REACTIVITY AND FOR PREDICTING RESPON_SE ⁰ STRESS

BACKGROUND OF THE INVENTION The present invention relates to a diagnostic test for testing susceptibility of individuals to inflammatory diseases such as rheumatoid arthritis and to means to evaluate potential treatment of arthritis with therapeutic agents directed at the central nervous system (CNS), designed to by-pass the CNS defect. The invention also provides means for evaluating and predicting response to stress in individuals.

A counter-regulatory feedback loop exists between the immune and central nervous systems, in which the immune or pro-inflammatory mediators stimulate cort- icotropin-releasing hormone (CRH) activation of the hypothalamic-pituitary-adrenal (HPA) axis hormonal cas¬ cade. The resultant increase in plasma glucocorticoids serves to restrain and limit the intensity of the inflam¬ matory-immune response, through the potent immunosuppressive/anti-inflammatory actions of the gluco¬ corticoids.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for testing mammals for susceptibility to inflammatory diseases. In its broadest aspect, the method comprises the steps of administering to a mammal a com¬ pound which is effective in stimulating the hypothalamic- pituitary-adrenal (HPA) axis and measuring the level of hormones secreted by the pituitary and adrenal glands of the mammal. In a more specific aspect, the method com¬ prises the steps of administering to a mammal a compound selected from the group consisting of cytokines, cell growth factors, neuroendocrine hormones such as corti- cotropin releasing hormone (CRH) or arginine vasopressin (AVP), biogenic amines, agonists of biogenic amines, antagonists of biogenic amines, analogues of biogenic amines, monoamine oxidase inhibitors and biogenic amine uptake inhibitors or glucocorticoid receptor antagonists

^"sussTiTUTE SHEET; and measuring the level of glucocorticoids or adrenocorti- cotropic (ACTH) in the blood plasma of the mammal. The substance which is administered should not be the same as the material which is measured. The invention is useful as a model in the study of the mammalian autoimmune diseases. Laboratory animals which may serve as a good model in studying human systems include rats, mice, guinea pigs, rabbits and chickens. However, an ultimate objective of this invention is to provide a method for diagnosing the susceptibility of humans to inflammatory diseases.

The invention is also useful as a means of pre¬ dicting intensity of response to stress and capacity to mount a sustained adaptive response to stress. The use of therapy through CRH activation during an injury or illness can serve the coherently related goals of preventing the immune/inflammatory response from overshooting and of restraining exploratory behavior to diminish exposure to further danger. The hormones to be measured should be hormones which are secreted in increased levels by normal individuals when the compound is administered to the individual but which are not secreted in such high levels after administration of the compound in individuals having an inflammatory disease or susceptibility to an inflam¬ matory disease. Hormones secreted by the pituitary and adrenal glands which can be measured include glucocorti¬ coids such as corticosterone, cortisol, and ACTH. Other hormones which can be measured include CRH, prolactin, arginine vasopressin (AVP), growth hormone (GH) , thyroid stimulating hormone (TSH), and endorphins/enkephalins.

The compound which is used in the test is prefer¬ ably administered intravenously (i.v.), however, other modes of administration such as subcutaneously (s.c.) or orally (p.o.) may be used. The compound is administered together with a suitable non-toxic pharmaceutically acceptable carrier in an amount sufficient to stimulate the hypothalamic-pituitary-adrenal axis. The compound

T should be administered at a time when the hypothalamic- pituitary-adrenal axis is quiescent, i.e., in humans at 8 p.m. However, it could be administered between 8 a.m. - 10 a.m., for example, when giving compounds such as AVP. When an immune/inflammatory mediator such as interleukin-1 is used, the immune inflammatory mediator would probably be administered in a dose of 0.1 μg/kg to 10 g/kg of body weight, preferably 1 μg/kg to 5 g/kg of body weight. When CRH is used, 1 μg ovine CRH per kg body weight is administered i.v. When AVP is used, 0.01 to 2.0 mlU/kg/min is infused i.v. When a biogenic amine or analogue thereof such as quipazine is used, the compound would probably be administered in a dose of 0.01 to lmg of quipazine per kg of body weight, preferably 0.1 to 0.5 mg/kg of body weight. Doses for other biogenic amines or analogues thereof should be determined on a case-by-case basis.

After administration of the compound it is neces¬ sary to wait for a time sufficient to allow the compound to raise the glucocorticoid or ACTH level in the blood plasma of the patient before testing. Generally, it is necessary to wait at least 10 minutes before testing. The glucocorticoid or ACTH level should be measured before the level returns to normal. The glucocorticoid or ACTH level may return to normal within 4 hours after administration of the compound. A preferred waiting period is 15 minutes to 2 hours after administration, more preferably 30 to 60 minutes after administration. If the hormone levels are significantly lower than (such as more than two standard deviations below) the mean established in normal individu¬ als, individuals, then the patient has tested positive for possible susceptibility to inflammatory diseases.

The method is potentially useful for testing for inflammatory diseases including, but not limited to, arthritis, uveoretinitis, pneumonitis, encephalomyelitis, myocarditis, thyroiditis, nephritis, sialoadenitis, adrenalitis, orchitis, multiple sclerosis and hepatic granulomatous diseases. Various immune/inf1-ammatory mediators may be used. Cytokines such as any one of the interleukins (interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-6 (IL-6)), interferons (alpha interferon, beta interferon and gamma interferon) or tumor necrosis factor (TNF) may be used in the test. Other cytokines such as epidermal growth factor (EGF), transforming growth factor- alpha and/or beta (TGF-alpha and/or TGF-beta) may also be used. Biogenic amines such as serotonin, norepinephrine, epinephrine, or dopamine may be used. In addition, analogs and agonists of these biogenic amines such as quipazine may also be used. Additional compounds which may be used include monamine oxidase inhibitors such as tranylcypromine sulfate (30 mg/patient) or isocarboxazid (30 mg/patient) which increase endogenous levels of biogenic amines. Biogenic amine uptake inhibitors such as fluoxetine may also be used.

It is known that inflammatory mediators such as IL-1 cause an increase in plasma corticosterone and ACTH possibly by stimulating the hypothalamic-pituitary-adrenal (HPA) axis. The present invention is based on the finding that susceptibility to arthritis and other inflammatory diseases is related to lack of HPA axis responsiveness to inflammatory mediators and other compounds.

The present invention is also potentially useful as a guide for the treatment of arthritis with agents that may bypass the HPA defect by stimulating the HPA axis centrally or at multiple levels. Such drugs would include the drugs listed below:

Neurotransmitters/monoamines/neuroexcitatory agents: serotonin agonists/releasers/uptake inhibitors: quipazine

1-metachloro-phenyl-piperazine (mCPP) fenfluoramine fluoxetine adrenergic agonists/antagonists/uptake inhibi¬ tors:

SUBSTITUTESHEET idasoxan yohimbine methoxamine desmethy1imipramine ritalin cholinergic agents: arecholine nicotine GABA agonists/antagonists: BCCM-beta carboline

FG 7142 (Sandoz) RO 15788 MAO inhibitors

MAO A:chlorgyline MAO B:phenylzine isocarboxazid trany1cypromine Dopamine uptake inhibitors/releasers buproprion amphetamine

Excitatory amino acids/neuroexcitatory agents: glutamate cocaine Neurohormones: rat/human corticotropin releasing hormone (CRH) corticotropin (ACTH) dexamethasone arginine vasopressin (AVP) thyroxin thyroid stimulating hormone (TSH) estrogen progesterone testosterone

Diapid (Bissendorff, LHRH antagonist) It may also provide a guide for determination of dosage and timing schedule of replacement steroids or other HPA axis hormones such as CRH or ACTH.

The above noted agents have been selected for possible treatment of inflammatory diseases such as rheumatoid arthritis because they represent a variety of classes of neuroactive agents which would be expected to activate the CRH and/or related arousal systems on a long- term basis (i.e., without inducing tolerance). Such an effect would correct the putative pathophysiological defect in rheumatoid arthritis and, hence, significantly ameliorate inflammatory and/or affective symptoms associated with this illness. Of the above-mentioned compounds, it is expected that 1-metachloro-phenyl-pipera- zine (mCPP) (an antidepressant) , fluoxetine (an antide- pressant), idasoxan (an antidepressant), nicotine, FG 7142 (Sandoz), MAO A:chlorgyline (an antidepressant), and MAO B:phenylzine (an antidepressant) are expected to be preferred compounds for further study. These compounds will probably be administered in the same manner as recommended for their already known indications such antidepressants. Specifically, these compounds should be administered in an amount effective to stimulate the HPA axis which would bypass the defect in the HPA axis. It is also expected that analogues and/or derivatives of the above compounds may be useful.

As indicated in the examples below wherein con¬ trolled stress was introduced into the environment, activity may be used as a means of evaluating the level of hormones secreted by the pituitary or adrenal glands of the mammal being tested.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB show the severity of arthritis post-streptococcal cell wall (SCW) injection in euthymic versus athymic LEW and F344 rats. Severity of arthritis was quantitated by articular index (maximum of 16) for up to 42 days following a sign p. SCW injection. Data repre¬ sent the mean ± S.E.M. for 5 rats per experimental group. Figures 2A through 2D show the plasma corticoste- rone levels induced by SCW, IL-1 alpha, or quipazine in inbred F344/N and LEW/N rats, and in outbred HSD rats. Rats of each strain were injected i.p. with one mediator,

SUBSTITUTESHEET as shown: SCW, (2 mg cell wall rhamnose), 1 μqm recombinant IL-1 alpha, 1 mg quipazine or PBS control. Corticosterone was determined in plasma collected 60 minutes post-injection. Horizontal lines represent means of each group.

Figures 3A through 3F show the time course of plasma ACTH and corticosterone responses to SCW, (panels A and B); human recombinant IL-1 alpha (IL-1) (panels C and D); or quipazine (QUIP) (panels E and F) in F344/N (-^δ-) Versus LEW/N (-0-) rats. Plasma ACTH and corticosterone were quantitated by radioimmunoassay at various time points up to 4 hours following i.p. injection of each agent shown. Data shown are mean ± S.E.M. of a minimum of 5 animals per experimental group. Figures 4A through 4F show the dose responses of plasma ACTH and corticosterone responses to SCW (panels A and B); human recombinant IL-1 alpha (IL-1) (panels C and D); or quipazine (QUIP) (panels E and F) in F344/N ( --- ) versus LEW/N (-0-) rate. Various doses of mediators shown were injected i.p. and plasma ACTH and corticosterone were quantitated by radioimmunoassay 60 minutes post-injection. Data shown are mean ± S.E.M. of a minimum of 5 animals per group.

Figures 5A and 5B show the dose responses of plasma ACTH and corticosterone to various concentrations of human CRH. CRH was injected i.p., and plasma ACTH and corticosterone were measured by radioimmunoassay 60 minutes post injection. Data are mean ± S.E.M. of a minimum of 5 animals per experimental group. Figures 6A through 6D show CRH (A) and enkephalin

(B) transcript levels in the parvocellular neurons of the paraventricular nucleus (PVN) were increased by SCW administration in F344/N rats (≥) but not in LEW/N

(θ)rats. CRH (C) and enkephalin (D) transcript levels in the PVN were not increased by rIL-1 alpha administration in F344/N (3) or LEW/N (O) rats. F344/N and LEW/N rats were injected intraperitoneally with 2 mg cell wall rhamnose/lOOgm rat, or rIL-1 alpha, 1 gm/lOOgm rat, and

*1 i __» tta' t'^v-ⁱ " *^*"«""SET C-WTf^* were sacrificed 0, 2, 4, and 7 hours later. In situ hybridization and determination of the number of copies of probe hybridized per section per PVN were performed as previously described (6, 7). CRH mRNA levels were clearly increased at 4 and 7 hours post-SCW injection in the F344/N rats, both in comparison with baseline and LEW/N levels (e.g., two-tailed T test, p<0.001 at 7 hours between strains) . Although enkephalin mRNA levels rose above baseline in both strains in response to SCW adminis- tration, the F344/N response was greater than the LEW/N response (p.<0.05). A minimum of 6 rats were used for each experimental condition, except the 2 hour time point, for which 4 rats were used. Bars represent S.E.M.

Figures 7A and 7B show the total hypothalamic immuno-reactive CRH (iCRH) content in F344/N (A) and LEW/N (B) rats, measured 4 hours after intraperitoneal injection of various agents. F344/N (A) or LEW/N (B) rats were either untreated or were injected intraperitoneally with PBS, rIL-1 alpha, (1 gm per rat), or SCW (2 mg cell wall rhamnose per rat), and iCRH hypothalamic content was quantitated by radioimmunoassay (10). A minimum of 10 rats per experimental condition was used. Statistical significance was determined by one-way ANOVA followed by Duncan's multiple range test. There was no significant difference between untreated and PBS-treated F344/N rats, nor between any treatment group of LEW/N rats by either criterion. Both rIL-1 alpha and SCW-treated F344/N hypothalami contained significantly more CRH than PBS or untreated F344/N hypothalami, than treated or untreated LEW/N hypothalami (* = ANOVA p<0.0001, Duncan's multiple range p<0.05). Baseline levels of CRH in LEW/N hypothala¬ mi were not significantly different from baseline CRH levels in F344/N hypothalami by Duncan's multiple range test. Figure 8 shows the hypothalamic iCRH secretion from F344/N (a) versus LEW/N (0) rats stimulated in vitro with recombinant IL-1 alpha. Hypothalami from LEW/N or F344/N rats were stimulated for 20 minute periods in vitro with control medium or with IL-1 alpha, at concentrations ranging from 10"¹³M to 10^~6M. iCRH in the culture medium was quantitated by radioimmunoassay (11). Hypothalami from F344/N rats showed an increase of 150% over baseline iCRH secretion, compared to a 10% increase over baseline by LEW/N hypothalami (* = p<0.0001). Statistical signifi¬ cance was determined by Duncan's multiple range test. A minimum of 7 rats per experimental condition was used.

Figures 9A through 9F show the plasma corticosterone (9a) and ACTH (9b) levels in F344/N rats and LEW/N rats exposed to a variety of behavioral stresses versus controls. Rats were exposed to a variety of stressor prior to decapitation for collection of blood. Plasma ACTH and corticosterone were measured by radioimmu- noassay (R.I.A.). Control = rats maintained in a stress free environment while immediately prior to decapitation for collection of blood; stress control = rats housed in the same room as the stressed rats during the period of stress, but not during decapitation; open field = rats exposed to the novel environment of an open field; restraint = rats restrained for 1 hour; swim = animals exposed to swim stress; ether = rats which were exposed to 10,00- ppm ether for 1 hour. Data represent values obtain from two separate experiments. Horizontal lines represent means ± S.E.M.; represents mean ACTH or corticosterone levels which were significantly different from controls (p<0.05 by the Duncan multiple range test); represents mean F344/N and LEW/N plasma ACTH or corticosterone levels which differ significantly from each other in response to the same stress (p<0.05 by the Duncan multiple range test. )

Figure 10 shows the CRH mRNA levels in the paraventricular nucleus of the hypothalamus during restraint stress in F344/N versus LEW/N rats. Rats were restrained for 1,1 or 3 hours prior to decapitation for collection of brains for in situ hybridization for CRH mRNA of the hypothalamus. In situ hybridization was preformed as described by Young (Methods in Enzvmoloσv,

SUBSTITUTESHEET Vol. 168. Hormone Action. Park K. Neuroendocrine

Peptides, (Academic Press, New York, 1989) pp. 702-710.) Briefly, brains were removed and frozen by immersion in isopentane on dry ice. Sections were cut through the PVN of the hypothalamus or through the anterior pituitary using a cryostat and thaw-mounted onto gelatin-coated slides. Sections were fixed in 4% formaldehyde, treated with acetic anhydride, dehydrated and defatted. Oligodeoxynucleotide probes (48 mer), kindly provided by W. Scott Young, III and Michael Brownstein, were labelled using 35S-dATP with terminal deoxytransferase. Approximately 500,000 cpm of probe was applied to each section and hybridization performed overnight at 37°. The sections were washed sequentially in 50% formamide/2X SSC at 40°, exposed to Kodak Ortho M film, and optical density measured with an image analysis system using ³⁵S-standards. CRH mRNA is expressed dpm/mg tissue based on these stand¬ ards. Data represent the mean of 4 to 14 animals- per group = significantly different from LEW/N stress and LEW/N and F344/N controls; P=0,0002 by AVOVA (f-TEST = 4.283, df 72) AND P<0.05 by Fisher PLSD. Control values were not statistically significantly different from each other.

DETAILED DESCRIPTION OF THE INVENTION A single intraperitoneal injection of an aqueous suspension of Group A Streptococcal cell wall fragments (peptidoglycan-group specific polysaccharide) into LEW/N female rates induces severe, rapid onset, acute thymic- independent arthritis, followed by a chronic proliferative and erosive thymic-dependent arthritis. However, histo¬ compatible F344/N female rats, along with several other histocompatible strains, develop only minimal, transient swelling of the hind paws (1, 2) . Strain dependent differences in response to the cell walls are also noted in the degree of splenic hypertrophy, blood leukocytosis, and the development of hepatic granulomata. The develop¬ ment of severe inflammatory pathology in the LEW/N rat is not related to differences in the quantities, the sites of

_SUBSTITUTE SHEΓT_^ localization, or the duration of persistence of the cell wall fragments. In both LEW/N and F344/N rats, the cell walls localize to the spleen, liver, bone marrow and synovial blood vessels of the peripheral joints (1, 3, 4). LEW/N rats develop a more persistent inflammatory reaction at the sites of cell wall localization than do F344/N rats (5). In vitro analyses of various mononuclear cell responses to cell walls have not provided fundamental in¬ sights into the mechanisms underlying the marked suscept- ibility to streptococcal cell wall-induced disease in the LEW/N rats and resistance in F344/N rats, although small differences between strains have been noted (6-8, and Wilder et al, unpublished data).

Histologically, the earliest changes in the acute phase arthritis in LEW/N rats are synovial microvascular injury associated with increased endothelial cell la expression. This is rapidly followed by infiltration of la positive macrophages in the synovium (1). At all time points F344/N rats exhibit little or no inflammation compared to LEW/N rats. This difference in the degree of synovial macrophage and endothelial cell la expression, that parallels the development of arthritis, is the most striking immunohistological difference between the two strains. This difference is even observed in athymic LEW.rnu/rnu and F344.rnu/rnu rats. This suggests that there may be a factor or factors regulating both la expression and the acute thymic-independent phase of SCW arthritis, and that the difference in arthritis suscepti¬ bility between the two strains may be related to the presence of a down-regulator of la operative soon after injection of SCW in F344/N but not in LEW/N rats.

Corticosteroids are both potent endogenous down- regulators of la expression, and potent endogenous immuno- suppressive and anti-inflammatory agents (9-13). Corti- costerone is released early in the course of inflammation through stimulation of the HPA axis by inflammatory media¬ tors such as endotoxin and interleukin-1 (IL-1) (14-23). Since SCW activate macrophages and stimulate release of IL-1, and are chemically related to endotoxin (bacterial lipopolysaccharide, LPS) (24), and since IL-1 is critical in maintaining the normal feedback loop between the immune system and central nervous system (CNS) (11-23, 25-26), the early ACTH and corticosterone responses to SCW and IL- 1 alpha in inbred F344/N and LEW/N rats and outbred HSD rats were compared. Since serotonin (5-HT) is also released from platelets during inflammation, and down- regulates la expression (27, 28), and since 5-HT pathways represent another route of hypothalamic-pituitary stimula¬ tion (29-31), the effect of the serotonin agonist, quipa¬ zine, on acute ACTH and corticosterone responses in F344/N, LEW/N and HSD rats was also compared. Further¬ more, to evaluate the direct involvement of glucocorticoids in the observed SCW susceptibility of LEW/N rats and SCW resistance of F344/N rats, the ability of replacement doses of glucocorticoids to suppress the SCW susceptibility of the former and the ability of a potent glucocorticoid antagonist RU 486 to reverse the SCW resistance of the latter was examined.

Animals: One hundred gram, virus antibody free, female, inbred F344/N and LEW/N rats, and outbred Harlan- Sprague-Dawley (HSD) rats, purchased from Harlan Sprague Dawley (Indianapolis, IN), were acclimatized to 12 hour on-12 hour off light cycles, prior to intraperitoneal injection of various inflammatory mediators.

Drugs and inflammatory mediators: Group A Strep- tococcal cell wall peptidoglycan-group specific carbohy¬ drate (SCW) was prepared in phosphate buffered saline (PBS), as previously described (1). It was injected at a concentration of 0.02 to 2 mg of cell wall rhamnose per rat. Recombinant human interleukin-1 alpha (IL-1 alpha): IL-1 alpha (32) was a generous gift from Drs. P. Kilian and P. Lomedico, (Hoffman-LaRoche, Nutley, N.J.). It was injected at doses ranging from 0.1 to 5 gm per rat. Specific activity ranged from 3 x 10⁸ to 2.5 x 10⁹ Units/μgm. 1 unit of IL-1 activity was defined in the D10 cell bioassay, as previously described (32). Endotoxin levels in final concentrations injected were less than 0.0013 EU/100 μl . Quipazine was purchased from Sigma Chemical Company (St. Louis, MO). It was injected at doses ranging from 0.1 to 5 mg per rat. Dexamethasone for cell culture was purchased from Sigma Chemical Company (St. Louis, MO), and used in doses ranging from 0.01 μg to 100 μgva. per rat. RU 486: The glucocorticoid receptor antagonist, RU 486, (33, Philibert, D., Deraedt, R. , & Teutsch, G. (1981) Proc. VIII International Congress of Pharmacology, p. 668.) was a generous gift from Roussel- UCLAF (Paris, France). It was suspended in sterile normal saline for intraperitoneal (i.p.) injection, at doses ranging from 0.03 mg to 3 mg per rat. LY53857: The serotonin (5-hydroxytryptamine, 5-HT₂) antagonist, LY53857 (6-methyl-l-[l-methylethyl] ergoline-8-carboxylic acid, 2- hydroxy-1-methylpropyl ester [Z]-2-butenedioate) (34), was a generous gift from Dr. M. Cohen, Lilly Research Labora¬ tories, Eli Lilly and Co. (Indianapolis, IN). Rat/human corticotropin releasing hormone (CRH) was purchased from Peninsula Laboratories (Belmont, CA) , and was used at doses ranging from 0.01 to 8 μgm per rat.

Hormone assays: Plasma corticosterone was quantitated by radioimmunoassay (35) kit purchased from Radioassay Systems Laboratories, Inc., Immunochem Corpora- tion (Carson, CA) . Adrenocorticotrophic hormone (ACTH) levels were determined by radioimmunoassay, as previously described (36). Rats were injected i.p. between 10 and 11 AM, and blood was collected from 30 minutes to 4 hours post-injection, for plasma ACTH and corticosterone mea- surements. Inter- and intra-assay control variability for corticosterone was 1.2% and 3.4% respectively; inter- and intra-assay control variability for ACTH was 8.0% and 2.8% respectively.

Severity of arthritis: Severity of arthritis was quantitated by articular index, performed by a single blinded observer, as previously described (2). Briefly, articular index is the sum of the severity of arthritis

(scale 0-4, 4 - most severe arthritis) of each of the limbs. Maximum articular index is 16.

Data analysis: A minimum of 5 rats per experi¬ mental group were studied, and experiments were repeated a minimum of 3 times. Data shown are mean ± standard error of the mean (S.E.M.) of each group. Experimental groups were compared to vehicle treated controls and to each other, and statistical significance between groups were determined by unpaired Student t test. Total ACTH and corticosterone released were calculated by integrating the areas under the curves, using the trapezoid rule.

RESULTS Thymic-Independent and Thvmic-Dependent Phases of LEW/N SCW Induced Arthritis Figures 1A and IB show the repeat of an earlier experiment (1) in which the articular index (Al) was assessed in euthymic versus athymic LEW and F344 rats injected with SCW. Five animals in each group were treated with a single intra-peritoneal dose of SCW at day 0, and observed from 6 weeks. The arthritis induced in euthymic LEW/N rats is diphasic, with a rapid-onset acute inflammatory component developing as early as 24 hours after injection of SCW, and a later chronic component developing at 3 to 6 weeks post-injection. Athymic LEW.rnu/rnu rats do not develop the late phase arthritis, but do develop the early inflammatory component and a continue low grade chronic synovitis. The early phase of SCW arthritis in LEW rats is, therefore, thymic-indepen- dent, and the late phase is thymic-dependent. The very small percentage of euthymic and athymic F344 rats that develop mild arthritis develop only the early thymic- independent component, which rapidly resolves. The pre¬ sence of a strain difference in the acute, thymic-indepen- dent phase of SCW arthritis in athymic LEW.rnu/rnu versus F34 .rnu/rnu- rats indicates that the thymic-independent phase of the arthritis is genetically regulated, and that the regulating factor or factors are operative very early in the disease. Corticosterone Responses to SCW, IL-1 Alpha and Quipazine in Outbred HSD Rats Versus Inbred F344/N and LEW/N Rats Since, as discussed above, corticosterone is a potent down-regulator of la expression which is released early in the course of inflammation through stimulation of the HPA axis by inflammato y mediators (14-23) , the early ACTH and corticosterone responses to SCW, IL-1 alpha and the serotonin (5-HT) agonist, quipazine, in inbred F344/N and LEW/N rats and outbred HSD rats were compared. Intraperitoneal SCW, IL-1 alpha, and the serotonin

(5-HT) agonist quipazine all induced marked plasma corti¬ costerone responses in F344/N rats at one hour post i.p. injection (Figures 2A through 2D and Table 1) . In con¬ trast, these agents induced only minimal (SCW, quipazine) or absent (IL-1 alpha) plasma corticosterone responses in LEW/N rats (p<0.01). Outbred HSD rats exhibited mean corticosterone responses intermediate between the low LEW/N and high F344/N responses. Corticosterone responses of HSD rats showed a wide spread, and fell into two groups: one overlapping the low LEW/N responses, and the other overlapping the high F344/N responses.

Table 1. Plasma corticosterone in PBS, SCW, IL-1 alpha or quipazine treated HSD, F344/N or LEW/N rats.

PLASMA CORTICOSTERONE, ng/ l (mean ± S.E.M.)

Data represents mean ± S.E.M. of plasma corticosterone shown in Figures 2A through 2D. Plasma corticosterone was determined by radioimmunoassay of plasma collected 60 minutes post-i.p. injection of PBS, SCW (2 mg cell wall rhamnose/rat) , IL-1 alpha (1 μg /rat) or quipazine (1 mg/rat) in HSD, F344/N or LEW/N rats. The one hour time point of corticosterone mea¬ surement and doses of mediators used were those found to be associated with maximal corticosterone responses in time course and dose response experiments (Figures 3A through 3F and 4A through 4F).

Time Course Kinetics of Plasma ACTH and

Corticosterone Versus Responses to SCW, IL-1 Alpha and

Quipazine in LEW/N Versus F344/N Rats

Figures 3A through 3F show that while plasma ACTH peaked at 30 to 60 minutes post-injection in both

F344/N and LEW/N rats, the LEW/N plasma ACTH response to

SCW, IL-1 alpha and quipazine was consistently lower than the F344/N response at all time points. Similarly, the

LEW/N plasma corticosterone response was lower than the F344/N response at all time points. Total ACTH and corticosterone secreted over the entire time course in response to SCW, IL-1 alpha, or quipazine was significantly less in LEW/N rats than in F344/N rats

(Table 2). In F344/N rats, compared to LEW/N rats, plasma ACTH increased more than 3 fold as much in response to IL-

1 alpha; more than 2 fold as much in response to SCW, and more than 1.6 fold in response to quipazine. F344/N rats increased plasma corticosterone more than 2 fold in response to SCW and IL-1 alpha, and 1.4 fold in response to quipazine when compared to LEW/N rats.

Table 2. Total plasma ACTH and corticosterone secreted over 4 years in response to SCW, IL-1 alpha or quipazine in LEW/N versus F344/N rats.

TOTAL ACTH (ng/mlx240 min) (mean ± S.E.M.)

F344/N LEW/N n p *F/L

SCW 94.5±4.7 IL-1 64.9±5.4 Quipazine 95.5±12.0

S_UBSTITUTESHEET TOTAL CORTICOSTERONE, (/zg/mlx240 min)

(mean ± S.E.M. )

SCW 184.913.9 IL-1 104.5+3.7

Quipazine 119.6±5.4

*F/L = ratio of total ACTH or corticosterone secreted by F344/N (F) rats versus LEW/N (L) rats. Data represent mean ± S.E.M. of total plasma ACTH and corticosterone secreted by F344/N versus LEW/N rats in response to i.p. SCW (2 mg cell wall rhamnose/rat) , IL-1 alpha (1 g/rat), or quipazine (1 mg/rat) . Data were derived, using the trapezoid rule, by calculation of the area under time course curves shown in Figures 3A through 3F.

Dose-Responses of Plasma ACTH and Corticosterone to SCW, IL-1 Alpha and Quipazine in LEW/N Versus F344/N Rats

Figure 4A through 4F show the plasma ACTH and corticosterone responses of LEW/N versus F344/N rats treated with varying doses of SCW, IL-1 alpha or quipazine. At all mediator doses tested, LEW/N rats had lower plasma ACTH and corticosterone levels than F344/N rats. Plasma ACTH and Corticosterone Responses of LEW/N

Versus F344/N Rats to Rat/Human CRH LEW/N ACTH and corticosterone responses to various doses of i.p. rat/human CRH were lower than F344/N responses (Figures 5A and 5B) . Pituitary, Adrenal and Thymus Weights in F344/N Versus LEW/N Rats Pituitary weights, although not significantly different, were greater in F344/N compared to LEW/N rats (Table 3). F344/N adrenal gland weights were slightly but significantly greater than adrenal gland weights from age- matched LEW/N rats (p<0.01). LEW/N thymus weights were significantly higher than F344/N thymus weights (p<0.01), in age-matched rats.

SUBSTITUTESHEET Table 3. Pituitary, adrenal and thymus weights in age- matched LEW/N and F344/N rats.

Strain (n) Pituitary (n) Adrenal (n) Thymus Weight Weight

Weight

(mg) (mg) (mg)

(27) 15.6+0.5 (10)

(26) 13.3±0.7 (10)

<0.01 <0.01

Inhibition of SCW Arthritis By Dexamethasone Treatment of LEW/N Rats Since corticosterone responses to SCW were clearly blunted in LEW/N rats, the effect of the corticosteroid, dexamethasone, on severity of arthritis in SCW treated LEW/N rats (Table 4) was evaluated. Five animals per group were injected i.p. with SCW (2 mg cell wall rham- nose/rat) on day 0, together with various doses of dexa¬ methasone. Dexamethasone treatment was continued for 72 hours at doses ranging from the physiologic replacement range of 0.5 μq twice daily (b.i.d.) or 1 μq once daily (QD) to doses in the pharmacologic range, (10 - 100 μq QD) . Not only did dexamethasone doses in the pharmacologic range totally suppress the arthritis induced by SCW, but doses in the physiologic range (1 μq QU or 0.5 μq b.i.d.) also significantly suppressed the severity of arthritis as determined by arthritis index (A.I.) compared to SCW plus saline treated controls (p<0.05). Table 4■ Dexamethasone suppression of SCW arthritis in LEW/N rats (72 hrs).

*A.I. significantly less than A.I. of control animals (dexamethasone dose 0, saline only) (p<0.05). LEW/N rats were injected with a single dose of SCW (2 mg cell wall rhamnose/rat) , followed by dexamethasone, at doses indicated, or saline controls. Dexamethasone injections were given once (QD) or twice (b.i.d.) daily for a total of 72 hours, and severity of arthritis (A.I., articular index) was quantitated at 72 hours post-SCW injection by a single blinded observer.

Effect of the Corticosterone Receptor Antagonist, RU 486. or the 5-HT, Antagonist, LY53857, on F344/N Rats Treated with SCW Table 5 shows the effect or treatment of F344/N rats with SCW plus the corticosterone receptor antagonist RU 486, or SCW plus the 5-HT₂ antagonist LY53857, compared to either agent alone. Five animals per group were injected i.p. with SCW (2 mg cell wall rhamnose per rat) on day 0. Various doses of RU 486 were injected i.p. sim¬ ultaneously with SCW, and the RU 486 was readministered once daily for up to 72 hours post-SCW injection LY53857 treatment was begun simultaneously with SCW and continued twice daily for 72 hours. Minimal mortality was observed in F344/N rats treated with SCW alone, and no mortality was observed in F344/N rats treated with RU 486 or LY53857 alone. Doses of RU 486 which had no effect alone (3) mg QD), were highly toxic when administered i.p. together with SCW, resulting in 100% mortality. Doses of RU 486 as

H ET low as 0.03 mg QD, when administered to SCW-treated rats, were still associated with significant inflammatory morbidity and mortality compared to controls. RU 486 has previously been shown to exacerbate carrageenin-induced inflammation, without significant mortality (33). Increased mortality in the SCW-arthritis model was probably related to the severe peritonitis which developed in association with the combined i.p. administration of the two agents. At doses of RU 486 low enough to permit survival, surviving rats developed acute arthritis, in some cases of moderate severity, e.g., mean A.I. = 4.5 at 0.3 mg RU 486 dose. Concurrent treatment of F344/N rats with SCW and the 5-HT₂ antagonist LY53857 was not associ¬ ated with significant mortality, but was associated with development of mild to moderate arthritis compared to control rats treated with either agent alone (p<0.05). Although not all dosing variables were explored, the data clearly show that blocking the effects of corticosterone or 5-HT in SCW treated F344/N rats results in severe or even fatal systemic inflammatory disease.

Table 5. Effects RU 486 or LY53857 on mortality and arthritis in F344/N rats treated with SCW

Agent(s) Mortality Incidence of Severity of Injected Arthritis in Arthritis in

Surviving Rats Surviving

Rats

(A.I.)

SCW + saline 2/15 1/13 0.2

saline + RU 486 3.0 mg 0/6 0/6 0

SCW + RU 486 0.03 mg 1/5* 2/4 0.5±0.3

SCW + RU 486 0.1 mg 2/5* 1/3 0.3±0.3 SCW + RU 486 0.3 mg 3/5* 1/2 4.5±4.5**

SCW + RU 486 1.0 mg 4/5* 0/1 0

SCW + RU 486 3.0 mg 5/5

saline - LY53857 0.3 mg 0/7 0/7 0 SCW + LY53857 0.03 mg 1/5 2/4 2±1.2**

SCW + LY53857 0.3 mg 0/5 2/5 2±1.5

♦Surviving rats receiving RU 486 plus SCW showed ruffled fur and peritoneal inflammation at necropsy. ** p<0.05 compared to articular index (A.I.) of SCW plus saline treated F344/N rats were treated with a single injection of SCW (mg cell wall rhamnose/rat) , followed by daily i.p. injections of RU 486, or twice daily i.p. injections of LY53857, at doses indicated. Control animals were treated with SCW plus saline, RU 486 plus saline, or LY53857 plus saline. Articular index was quantitated by a blinded observer at 72 hours post-SCW injection. Maximum A.I. is 16. F344/N rats' and LEW/N rats' levels of CRH, ACTH and corticosterone responses to behavior stress were compared as indicated in Figures 9A through 9F. The differential pituitary-adrenal responses were measured throughout the time course of exposure to restraint as indicated in tables 6 and 7.

SUBSTITUTE SHEET Table 6. Total hypothalamic CRH content, and pituitary ACTH content in F344/N and LEW/N rats exposed to restraint

F344/N LEW/N p

total CRH content/hypothalamus (pg/hypothalamus)

control 1706+153 2352+109 <0.05

3 hour restraint 2159176 2436166 <0.05 p <0.05N.S.

total ACTH content/pituitary (mg/pituitary)

Legend: Total hypothalamic CRH content and pituitary ACTH content was measured in hypothalami and pituitaries from LEW/N or F344/N rats restrained for three hours compared to controls. Hypothalami were excised and CRH and ACTH quantitated after extraction, as previously described (3). Data represent mean 1 S.E.M. of 10 animals per condition. Statistical significance was determined by ANOVA followed by Duncan multiple range.

Table 7. Plasma ACTH and corticosterone and behavioral responses to restraint and open field in F344/N, LEW/N and HSD rats

F344/N LEW/N HSD

Restraint

Plasma ACTH (pg/ml) control 135.719.6 180.5114.2 170.5113.9

3 hour restraint 231.5121.5 170.4110.6*

157.6125.4*

Plasma corticosterone (ng/ml) control 20+8

3 hour restraint 3321104

Fecal boli

3 hour restraint 6.411.1

Open Field

Outer squares crossed Inner squares crossed Rears Fecal boli (open field) Grooming

Legend: Plasma corticosterone and behavioral responses to restraint or open field testing. F344/N, LEW/N or HSD rats were either restrained for 3 hours, or exposed to an open field (5). Behavior in the open field was character¬ ized by quantitation of numbers of outer and inner squares crossed, number of fecal boli produced, and number of grooming behaviors. Data represent mean 1 S.E.M. of 10 animals per group. Experiments were performed in tripli¬ cate.* = significantly different from F344/N, p,0.05, ANOVA followed by Duncan Multiple Range test.

SUBSTITUTE SHEET DISCUSSION One of the earliest events that occurs in streptococcal cell wall injected LEW/N rats, and even in athymic nude LEW.rnu/rnu rats, is enhanced la expression on synovial endothelial cells. This develops concomitantly with the inflammatory process, and the intensity of expression parallels the severity of the arthritis. In marked contrast, insignificant enhancement of la antigen expression develops in SCW-injected euthymic and athymic F344 rats (1). Corticosteroids are both potent endogenous down-regulators of la expression and potent endogenous immunosuppressive and anti-inflammatory agents (9-13). In the experiments reported here, it has been shown that acute corticosterone responses to SCW IL-1 alpha and quipazine are severely depressed in arthritis susceptible, high la-expressing LEW/N rats, compared to arthritis resistant low-la expressing F344/N rats. Outbred HSD rats, which exhibit an intermediate mean susceptibility to SCW induced arthritis with wide vari- ability (2), also showed an intermediate mean and wide variability of corticosterone responses to these media¬ tors. Furthermore, replacement of corticosterone with physiologic doses of dexamethasone significantly sup¬ pressed the severity of SCW arthritis in LEW/N rats. Conversely, antagonism of corticosterone in F344/N rats, with the corticosterone receptor antagonist RU 486, was associated with increased mortality, exacerbation of inflammation, and development of mild to moderate acute arthritis in this otherwise resistant strain. It is clear from these studies that, whether present on a genetic basis, as in LEW/N rats, or on a pharmacological basis, as in RU 486 treated F344/N rats, a deficiency in the corti¬ costerone response to SCW is associated with development of susceptibility to SCW-induced inflammatory disease. Elevation of corticosterone during inflammation results from stimulation of the hypothalamic-pituitary- adrenal (HPA) axis by inflammatory and immune mediators, such as endotoxin (bacterial lipopolysaccharide, LPS) and

^IJSSTTΓUT SHIFΓT interleukin-1 (IL-1) (14-23). Since SCW (peptidoglycan- group specific polysaccharide) are chemically related to endotoxin, and stimulate the release of IL-1 from activated macrophages (24) , SCW could increase corti- costerone via IL-1 stimulation of the HPA axis. The studies presented here show that, in addition to depressed corticosterone responses to SCW, LEW/N rats have absent corticosterone responses to IL-1. Recent studies have shown that IL-1 stimulates the HPA axis at primarily the hypothalamic level by inducing CRH release (15-23). LEW/N rats have depressed ACTH responses which parallel the low corticosterone responses to SCW and IL-1, in contrast to F344/N rats which have higher ACTH and corticosterone responses. These initial findings suggest that the HPA axis defect in LEW/N rats is at either the hypothalamic and/or the pituitary level.

The smaller adrenal glands and the larger thymuses of LEW/N compared to F344/N rats, are also consistent with deficient HPA axis responses and chronic mild hyposecre- tion of corticosterone. LEW/N rats also have depressed ACTH and corticosterone responses to exogenous CRH. This could be secondary to inadequate priming of the anterior pituitary corticotroph by endogenous CRH or other ACTH secretagogues, or to some inherent defect of this cell. It is impossible from the data presented here to precisely define the site of the defect, whether hypothalamic or pituitary, particularly since chronic over- or understimu- lation of the HPA axis, from whatever cause, could result in secondary changes in baseline and stimulated levels of CRH, ACTH and corticosterone production, as well as secondary changes in adrenal and thymus sizes (13, 37). Further studies are required to determine the precise anatomical and molecular site of the lesion in LEW/N rats. The observation that LEW/N rats are deficient in ACTH and corticosterone responses to the 5-HT agonist quipazine, as well as to IL-1 and SCW, suggests that the defect in these rats is not solely at the level of IL-1 stimulation of the HPA axis. The greater LEW/N

SUBSTITUTESHEET corticosterone response to quipazine compared to IL-1 alpha may be related to the multiple pathways through which 5-HT and 5-HT agonists stimulate the HPA axis (31, 38-40), perhaps allowing quipazine to partially bypass the LEW/N defect in IL-1 - hypothalamic-pituitary pathways. This hypothesis is supported by existing data indicating that serotonin represents both a major CRH and a potent ACTH secretagogue (31, 41, 42). The importance of 5-HT pathways in intactness of the inflammatory mediator - HPA axis loop and arthritis resistance is also suggested by the association of arthritis with LY53857 treatment of SCW-injected rats.

The greater LEW/N corticosterone and ACTH response to SCW compared to IL-1 may also be related to stimulation of the HPA axis at multiple levels by the many inflammatory mediators released by SCW, including IL-1, interleukin-2 (IL-2), tumor necrosis factor (TNF) and 5- HT. Although 5-HT does not cross the blood brain barrier, 5-HT released during inflammation could hypothetically directly stimulate pituitary release of ACTH (31, 41, 42). The physiologic relevance of such a potential route of 5- HT stimulation of the HPA axis is, however, not clear, since it would be dependent on adequate systemic concen¬ trations of 5-HT reaching central sites. Taken together, the experiments reported here show that LEW/N rats represent a strain of rats genetically deficient in ACTH and corticosterone responses to several inflammatory or stress mediators, including SCW and IL-1, and 5-HT agonist, quipazine, and exogenous CRH. In contrast, F344/N rats represent a histocompatible, relatively SCW arthritis-resistent strain with intact, potent ACTH and corticosterone responses to the same inflammatory or stress mediators. Responses of HSD rats represent those of an outbred population, covering a wide range of both inflammatory mediator-HPA axis ACTH and corticosterone responses and SCW-arthritis susceptibility.

LEW/N and F344/N rats, therefore, represent a unique animal model for a genetically determined defect in

SUBSTΪTU \ _S .S-._ the CNS - inflammatory/immune system feedback loop. Whether of hypothalamic or pituitary origin, this defect is associated with increased susceptibility to arthritis in response to SCW, and could also contribute to the increased susceptibility to other experimentally-induced inflammatory diseases observed in LEW/N rats (43-50). The data, coupled with the markedly enhanced inflammatory disease in SCW-injected F344/N rats following pharmacologic interruption of the HPA axis, and suppres- sion of arthritis severity in SCW-injected LEW/N rats following replacement doses of dexamethasone, provide strong evidence that arthritis susceptibility in the LEW/N rat, and resistance in the F344/N rat is regulated, at least partially, through corticosterone production and HPA axis responsiveness to inflammatory and possibly other stress mediators.

The data may also have implications for susceptibility to rheumatoid arthritis in humans. Rheuma¬ toid arthritis is associated with a class II major histo- compatibility complex (MHC) epitope that is shared amongst several different haplotypes. Studies examining the contribution of class II MHC (la) type to rheumatoid arthritis susceptibility have suggested that MHC type and sequence are only partially responsible for susceptibility to rheumatoid arthritis (51). Another, as yet undefined factor, perhaps one controlling regulation of la expres¬ sion, may therefore contribute to susceptibility to rheumatoid arthritis. The data presented here are consis¬ tent with the concept that the additional factors regulat- ing both la expression and susceptibility to arthritis are corticosteroids and responsiveness of the HPA axis to inflammatory and possibly other stress mediators. Several other lines of evidence support this concept. Rheumatoid patients are exquisitely sensitive to the disease-sup- pressing effects of low doses of corticosteroids; rheuma¬ toid arthritis frequently remits during hypercortisolemic states, such as pregnancy, and exacerbates during hypo- cortisolemic states such as the post-partum period. These observations, coupled with our data, suggest that evalua¬ tion of hypothalamic-pituitary-adrenal axis responsiveness to inflammatory and possibly other stress mediators, in patients with rheumatoid arthritis, may provide new insights into the disease process.

Additional evidence indicates that susceptibility to streptococcal cell wall (SCW)-induced arthritis in the Lewis (LEW/N) rat, a model for human rheumatoid arthritis, is due, in part, to defective inflammatory and stress mediator-induced activation of the hypothalamic-pituitary- adrenal axis. To explore the mechanism of this defect, the functional integrity of the hypothalamic corticotropin releasing hormone (CRH) neuron in LEW/N rats was examined and compared to histocompatible, arthritis-resistant F344/N rats. In response to SCW or recombinant inter- leukin-1 alpha (rIL-1 alpha), LEW/N rats showed profoundly deficient paraventricular nucleus CRH mRNA levels, hypo¬ thalamic CRH content and CRH release from explanted hypothalami in organ culture. These data provide strong evidence that the defective LEW/N ACTH and corticosterone responses to inflammatory and other stress mediators, and LEW/N susceptibility to experimental arthritis, are due in part to a hypothalamic defect in the synthesis and secre¬ tion of CRH. The additional finding of deficient expres- sion in LEW/N rats of the hypothalamic enkephalin gene, which is coordinately regulated with the CRH gene in response to stress, suggests that the primary defect is not in the CRH gene, but is rather related to its inappro¬ priate regulation. We have found that, compared to F344/N rats, LEW/N rats' CRH, ACTH and corticosterone responses to behavioral stresses were profoundly blunted, consistent with their severely defective CRH, ACTH and corticosterone responses to inflammatory mediators. Hence, LEW/N rats secreted significantly less ACTH and corticosterone than did F344/N rats in response to all stressors, i.e., novel environ¬ ment, swim, restraint and ether (Figures 1A and IB). These differential pituitary-adrenal responses were

SU _S ITUTE SHEET maintained throughout the time course of exposure to restraint (3 hour plasma ACTH: LEW/N = 170 1 10 pg/ml; F344/N = 232 1 20 pg/ml; p<0.05; 3 hour plasma corti¬ costerone: LEW/N = 19 1 7 ng/ml; F344/N=332 1 100 ng/ml, p<0.05). However, by three hours in response to ether, the differences in plasma ACTH and corticosterone levels in the two strains tended to narrow (3 hour plasma ACTH: LEW/N = 269 1 8 pg/ml; F344/N = 377 1 46 pg/ml p<0.05; 3 hour plasma corticosterone: LEW/N = 414 1 12 ng/ml; F344/N = 522 1 36 ng/ml, p = N.S. The relatively greater corticosterone response to swim stress and ether in LEW/N rats probably reflects the greater relative potency of these stressful stimuli compared to open field and re¬ straint stresses. Furthermore, ether is known to activate the HPA axis through both central pathways and direct stimulation of pituitary corticotroph cells as well.

Consistent with the differential plasma ACTH and corticosterone responses to physical restraint, LEW/N rats failed to show any increase in CRH mRNA expression in the PVN of the hypothalamus in contrast to F344/N rats in which hypothalamic CRH mRNA expression increased signi¬ ficantly during restraint. These findings are consistent with our previous data showing deficient LEW/N plasma ACTH and corticosterone responses and PVN CRH mRNA expression in response to inflammatory mediators such as SCW, IL-1 alpha or quipazine, and suggest that hypothalamic CRH biosynthesis in LEW/N rats, compared to F344/N rats, is blunted in response to a wide range of stressful stimuli, from chemical inflammatory mediators to behavioral stress stimuli. Also consistent with their plasma ACTH and corticosterone responses to ether, LEW/N CRH mRNA levels in the PVN were increased over basal levels. These findings demonstrate that during exposure to a powerful stress stimulus, such as ether, the defect in CRH biosyn- thesis in LEW/N rats can be enhanced, but still not to the level observed in F344/N rats.

In response to intraperitoneal injections of Group A streptococcal cell wall peptidoglycan-polysaccharide

_UBSTITUTESHEET (SCW), inbred Lewis (LEW/N) female rats develop severe proliferative and erosive arthritis which mimics human rheumatoid arthritis. Histocompatible Fischer (F344/N) rats, on the other hand, do not develop arthritis in response to the same SCW stimulus (1-8) . In light of recent postulated evidence that inflammatory mediator- activation of glucocorticoid secretion is one mechanism by which the immune response is appropriately regulated and restrained pituitary-adrenal responsiveness to SCW and other inflammatory stimuli in LEW/N and F344/N rats was previously explored (23, 52, 53). It was found that LEW/N rats have defective HPA axis responses to inflammatory and other stress mediators and that the response of F344/N rats to the same stimuli is intact, or above normal. Specifically, LEW/N rats, in contrast to F344/N rats, have markedly impaired plasma ACTH and corticosterone responses to intraperitoneally-injected SCW, to recombinant human interleukin-1 alpha (rIL-1 alpha), to the serotonin agonist, quipazine, and to synthetic rat/human corti- cotropin releasing hormine (CRH) . In addition, LEW/N rats, compared to F344/N rats, have smaller adrenal glands and larger thymuses, consistent with chronic lack of stimulation by ACTH and corticosterone, respectively. Furthermore, arthritis and severe inflammation can be induced in otherwise SCW arthritis - resistant F344/N rats, by interruption of the HPA axis at its effector end- point, with the glucocorticoid receptor antagonist, RU 486. Taken together, these data indicate that LEW/N rats' pituitary and adrenal hyporesponsiveness to inflammatory and other stress mediators is a major factor contributing to their susceptibility to SCW arthritis and other experi¬ mental inflammatory diseases (43-50).

Pituitary ACTH hyporesponsiveness to stimuli can be primary, or secondary to lack of hypothalamic stimulation (36). In order to determine whether the impaired ACTH and corticosterone responses of LEW/N rats were hypothalamic in origin,the ability of streptococcal cells walls (SCW) or rIL-1 alpha to affect in vivo hypo-

SUBSTITUTE SHEET thalamic CRH secretion in LEW/N and F344/N rats was compared. Results of these studies show that the LEW/N HPA axis defect involves the hypothalamus. In contrast to F344/N rats, neuronal synthesis and secretion of CRH within the PVN was markedly impaired.

The influence of inflammatory mediators on hypo¬ thalamic expression of CRH mRNA was quantitated by .in situ hybridization with a CRH probe in PVN sections from F344/n and LEW/N rats injected intraperitoneally with either SCW or rIL-1 alpha. 80-100 g virus antibody-free, F344/n and LEW/N female rates (Harlan Sprague Dawley, Indianapolis, IN) were injected intraperitoneally with SCW (2 mg cell- wall rhamnose per 100 gm rat), or recombinant interleukin- 1 alpha (rIL-1 alpha, 1 μgm/100 gm rat). Recombinant IL-1 alpha was a kind gift from Drs. P. Kilian and P. Lomedico, Hoffman-LaRoche, Nutley, NJ. Blood was immediately collected and used to determine plasma corticosterone and ACTH levels (35, 36). The brains were removed, and 4 mm coronal slices containing the PVN were frozen and stored at -80°C until 12 μm. frozen sections were cut and thaw-mounted onto twice gelatin-coated slides. They were then processed for jLn situ hybridiza¬ tion with CRH or enkephalin probes and analyzed as previ¬ ously described (54, 55, 56). The hybridizations were performed at 37°C for 20-24 hours in 600 mM Tris-HCl (pH 7.5), 50% formamide, 4 mM EDTA, 0.1% sodium pyrophosphate, 0.2% SDS, 0.2 mg/ml heparin sulfate, and 10% dextran sulfate. The probes had specific activities of 10-15,000 Ci/mmol. Between 4 to 7 hours after injection of SCW, CRH mRNA levels increased significantly in F344/N PVN, but did not increase in LEW/N PVN (Figure 6A) . This lack of a CRH biosynthetic response to SCW in LEW/N rats could be secondary to a defect in the CRH gene or in steps leading to its activation. Coordinate regulation of the CRH and enkephalin genes in the PVN after application of two different stresses has been previously described (57, 58). In order to determine whether induction of the enkephalin gene by SCW was also defective in LEW/N rats, adjacent

SUBSTITUTE SHEET sections from the animals treated with SCW were examined for enkephalin expression by .in situ hybridization. The response was much greater in F344/N PVN than in the LEW/N PVN (Figure 6B) . These results suggest that a common pathway that activates the CRH and enkephalin genes in the PVN in F344/N and other normal rats (57, 58) is defective in LEW/N rats.

Bacterial endotoxin lipopolysaccharide, (com¬ ponents of SCW) and IL-1 stimulate the HPA axis (14-22, 59). Since SCW induce IL-1 release (24), IL-1 is one probable mediator of SCW HPA axis stimulation. Indeed, rll-l alpha did induce significant (P<0.01) increases in plasma corticosterone and ACTH in F344/N rats, compared to LEW/N rats (F344/N ACTH = 480 pg/ml, LEW/N ACTH = 70pg/ml; F344/N corticosterone = 488 ng/ml, LEW/N corticosterone = 78 ng/ml, at one hour post-injection) (See preceding discussion). A similar single intraperitoneal injection of rIL-1 alpha (1 microgram per rat) however, did not significantly increase CRH mRNA or enkephalin mRNA in the PVN over baseline in either strain (Figures 6C and 6D) . This discrepancy between the ability of rIL-1 alpha to augment plasma ACTH and corticosterone, and its inability to augment CRH mRNA levels in the PVN in F344/N rats, suggests that rIL-1 alpha, at the dose used, stimulates secretions, but not transcription of CRH. Alternatively, any increase in transcript levels induced by rIL-1 alpha may be below the level of sensitivity of the ii situ hybridization assay. The increase of CRH mRNA levels induced by SCW, in contrast to the lack of CRH mRNA response to rIL-1 alpha, could be related to two mechanisms; the more sustained nature of the SCW stimulus compared to rIL-1 alpha, or the multiple factors released by SCW, which should stimulate the CRH neuron via multiple pathways. Total hypothalamic CRH content reflects the balance between CRH synthesis and secretion. Four hours after intraperitoneal injection of LEW/N or F344/N rats with SCW (2 mg cell wall rhamnose per rat) , recombinant

^USSTITUTE SHEE ; IL-1 alpha (1 μq per rat), or phosphate buffered saline (PBS, sterile, endotoxin free, GIBCO, Grand Island, NY), rats were decapitated, and hypothalami were rapidly removed, quick-frozen on dry ice and extracted. Total immunoreactive CRH (iCRH) was quantitated by radioimmuno¬ assay, as previously described (60). Hypothalamic immunoreactive CRH (iCRH) content, measured 4 hours after intraperitoneal injection of SCW, rIL-1 alpha, or phos¬ phate buffered saline (PBS) is shown in Figures 7A and 7B. In F344/N rats, hypothalamic content of ICRH increased more than two fold over controls in response to either intraperitoneal SCW or rIL-1 alpha. iCRH content in PBS- injected and untreated animals was not significantly different. In contrast, iCRH content did not change in LEW/N rats injected with PBS, SCW or rIL-1 alpha, compared to untreated LEW/N rats. The lack of change in hypotha¬ lamic iCRH content of LEW/N rats in response to in vivo administration of SCW or rIL-1 alpha is consistent with the LEW/N rats' defective response of CRH mRNA to these mediators. The SCW-induced increase in hypothalamic iCRH in F344/N rats is consistent with their ability to in¬ crease CRH mRNA levels in response to SCW. rIL-1 alpha's capacity to increase CRH mRNA levels in response to SCW. rll-l alpha's capacity to increase hypothalamic iCRH content in F344N rats, but not CRH mRNA levels in the PVN of these rats, suggests that IL-1 alpha may increase the rate or efficiency of CRH mRNA translation and/or post- transnational processing without causing detectable increases in CRH transcript levels. In order to determine whether IL-1 stimulation of

CRH secretion is also defective in LEW/N rats, the ability of rIL-1 alpha to induce iCRH release jLn vitro from LEW/N versus F344/N hypothalamic explants was compared. Hypotha¬ lamic explants, obtained from untreated age-matched F344/N or LEW/N rats, were cultured in the presence of various concentrations of rIL-1 alpha and release of iCRH into the culture supernate was quantitated by radioimmunoassay. Hypothalamic explants were rapidly removed from untreated

SUBSTITUTESHEE rats, as previously described (61, 62). The explants were incubated overnight at 37°C, 5% C0₂, in medium 199, (M199, GIBCO, Grand Island, NY), with 0.1% bovine serum albumin (BSA, grade V, Sigma Chemicals, St. Louis, MO). The hypothalami, in 48 well tissue culture plates, were then serially transferred every 20 minutes through a series of six wells containing one of the following additives, in order: control M199 (3 wells, total 60 minutes); M199 plus recombinant IL-1 alpha (10"¹³M to 10"⁶M; 2 wells, total 40 minutes); or 60 mM potassium chloride (1 well, 20 minutes). Immunoreactive CRH (iCRH) was assayed directly in the media, by a sensitive radioimmunoassay, as previ¬ ously described (63). Only results from viable hypothala¬ mi, represented by those with a ≥ 90% iCRH response to 60 mM KC1 over basal values, were included in the analyses. Figure 8 shows that rIL-1 alpha (10"¹³M to 10"⁶M induced a 150% increase in iCRH secretion over baseline from F344/N hypothalami, and no increase in iCRH secretion over baseline from LEW/N hypothalami. As discussed previously, the LEW/N rats' defec¬ tive ACTH and corticosterone responsiveness to inflam¬ matory and other stress mediators is one critical factor in their susceptibility to SCW-induced arthritis. Our current findings suggest that the deficient LEW/N ACTH and corticosterone responses and associated susceptibility to arthritis are related to a lack of hypothalamic synthesis and secretion of CRH, and perhaps other stress hormones, in response to inflammatory and other stress mediators. The coordinate defect in LEW/N enkephalin mRNA synthesis in response to SCW provides evidence at the LEW/N rats' CRH biosynthetic defect is not specific to the CRH gene, but may result from a defect in its regulation. The present findings suggest a unique model for a mammalian autoimmune disease, in which a central nervous system defect results in an illness characterized by inadequate immune/inflammatory counter-regulation. Such a mechanism may also be relevant to human illnesses such as rheumatoid arthritis.

SUBSTITUTE SHEET Arginine Vasopressin Stimulation Test:

There are currently two methods available to test the integrity of HPA axis responses in humans: the CRH stimulation test, and the arginine vasopressin (AVP) stimulation test (Meller, W.H. et al, J. of Psychiatric Research, 21, No. 3, 269-277 (1987). Further details of this test can be found in NIH Clinical Project no. 87-M- 85a which is hereby incorporated by reference. Both these methods have been developed at the NIH Clinical Center, and have been extensively studied in diseases such as depression and Cushings' syndrome. The CRH stimulation test determines sensitivity of ACTH and cortisol responses to exogenous CRH, and therefore tests the integrity of pituitary corticotroph cells' responses to stress. Since defective pituitary responsiveness to stress could be primary, or could be secondary to long-term hypothalamic defects as well, the CRH test also indirectly tests the integrity of hypothalamic responses to stress. A more direct measure of the integrity of hypothalamic CRH responses to stress is the AVP stimulation test. AVP synergizes with CRH in stimulating ACTH release. Decreased endogenous CRH would therefore result in de¬ creased ACTH and cortisol secretion in response to exoge¬ nous AVP. Maximal ACTH and cortisol responses to AVP stimulation occur at 9:00 a.m., when CRH should be at its peak. Maximal ACTH and cortisol responses to CRH stimula¬ tion occur when CRH is at its nadir, at 4:00 p.m. An intravenous infusion of arginine vasopressin of 1.0 mlU/kg/min administered over a one hour period between 9:00 and 10:00 a.m. has been shown to have minimal side effects, and to maximally synergize with endogenous CRH in stimulating ACTH release. However, the infusion could be given at 8:00 to 9:00 p.m., depending on results of preliminary testing. AVP Stimulation Test:

Arginine vasopressin will be administered by an intravenous infusion of 1.0 mlU/kg/min of arginine vasopressin over a one hour period between 9:00 a.m. and

«M«»S~5TUTE SHESr. f 10:00 a.m. The infusions will be by means of a digitally controlled Extracorporeal Constant Infusion Pump, Model 2100. If any given dosage side effects such as nausea or gastrointestinal cramping occur, the infusion will be stopped immediately. Upon cessation of side effects, the infusion may again commence at a dose established to be free of side effects from a given individual. Side effects should be rare even at the maximal doses proposed for this study. In addition to the standard intracath placed for infusion of arginine vasopressin, blood will be drawn through a scalp vein needle in the contralateral arm, every 15 minutes before, during, and for two hours after the infusion. Eight cc's will be taken for every sample, and a total of 104 cc's will be taken for each infusion. Blood will be assayed for ACTH and cortisol, and in some cases also for other hormones such as beta- endorphin, growth hormone, prolactin and oxytocin.

Synthetic arginine vasopressin will be obtained from the Parke-Davis Company. Park-Davis markets a highly purified synthetic peptide with a sequence of naturally occurring arginine vasopressin and the commercial designa¬ tion Aqueous Pitressin.

The components for use by the means described herein can be assembled as kits for use in accord with the teachings of the application. Such kits may contain the hypothalamic-pituitary-adrenal (HPA) axis stimulating drug, tracers such as 1125 or an ELISA tracer, reagents specific for use in the assay chosen, antibodies to HPA axis hormone, and, our use as controls, standardized plasma. Any of the HPA axis hormones such as GABA agonists/antagonists, MAO inhibitors, Dopamine uptake inhibitors/releasers, Cholinergic agents, serotonin agonists/releasers/uptake inhibitors, adrenergic agonists/antagonists/uptake inhibitors named at page 5 are appropriate for use in kits in accord with the teachings of this disclosure. 1-metachloro-phenyl-piperazine (mCPP) is a particularly preferred stimulant.

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Claims

WHAT IS CLAIMED IS:

1. A method for testing the susceptibility of a mammal to inflammatory diseases which comprises the steps of: Administering to a mammal a compound which is effec- tive in stimulating the hypothalamic-pituitary-adrenal axis; and measuring the level of hormones secreted by the pituitary or adrenal glands of said mammal.

2. The method of claim 1, which comprises the steps of administering to a mammal a compound selected from the group consisting of cytokines, growth hormones, neuroendocrine hormones such as corticotropin releasing hormone or arginine vasopressin (AVP), biogenic amines, agonists of biogenic amines, analogs of biogenic amines, monoamine oxidase inhibitors and biogenic amine uptake inhibitors; and measuring the level of glucocorticoids or adrenocorticotropic hormone in the blood plasma of said mammal.

3. The method of claim 1, wherein said inflam¬ matory disease is arthritis, uveoretinitis, pneumonitis, encephalomyelitis, multiple sclerosis and hepatic granulo- matas.

4. The method of claim 1, wherein said compound is interleukin-1, serotonin, corticotropin releasing hormone, AVP or quipazine and said hormone is cortisol, corticosterone or adrenocorticotropic hormone.

5. The method of claim 1, wherein the level of said hormones secreted by the pituitary and adrenal glands are measured 10 minutes to 4 hours after administration said compound.

6. A method for testing the susceptibility of the mammal to arthritis which comprises the steps of: Admin¬ istering to a mammal an amount of interleukin-1 or quipazine effective to stimulate the hypothalamic- pituitary-adrenal axis; and measuring the level of adreno- corticotropic hormone or corticosterone in the plasma of said mammal between 10 minutes and 4 hours after administration of said interleukin-1 or quipazine.

7. The method of claim 6, wherein said mammal is a laboratory animal.

8. The method of claim 6, wherein said mammal is a human.

9. A method for treatment of inflammatory diseases which comprises the step of: administering to a patient suffering from an inflammatory a compound which stimulates the hypothalamic-pituitary-adrenal axis in an amount effective to stimulate said axis.

10. The method of claim 9, wherein said compound is administered to a patient suffering from rheumatoid arthritis.

11. A kit comprising: (1) a HPA axis stimulating agent; and (2) a means of measuring the HPA axis hormone response in the patients's plasma.

12. A kit of claim 11 having, additionally, a tracer.

13. A kit of claim 11 containing antibodies to the HPA axis hormone.

14. A kit of claim 11 wherein the means of measuring the HPA axis hormone response is an ELISA test.

15. A kit of claim 14 wherein the kit contains an ELISA tracer.

16. A kit of claim 11 wherein the stimulating agent is a GABA agonist/antagonist.

17. A kit of claim 11 wherein the stimulating agent is 1-metachloro-phenyl-piperazine.

18. A kit of claim 11 containing therein a HPA axis stimulating agent; an ELISA tracer, antibodies to HPA axis hormone, and standardized plasma as a control.

19. A kit of claim 11 containing ^x125 as a trac¬ er.

20. The use of a compound to test the suscepti¬ bility of a mammal to inflammatory diseases which comprises the steps of: Administering to a mammal an effective amount of a compound which stimulates the hypo¬ thalamic-pituitary-adrenal axis; and measuring the level of hormones secreted by the pituitary or adrenal glands of said mammal.

21. The use of claim 20, which comprises the steps of administering to a mammal an effective amount of a compound selected from the group consisting of cyto¬ kines, growth hormones, neuroendocrine hormones such as corticotropin releasing hormone or arginine vasopressin (AVP), biogenic amines, agonists of biogenic amines, analogs of biogenic amines, monoamine oxidase inhibitors and biogenic amine uptake inhibitors; and measuring the level of glucocorticoids or adrenocorticotropic hormone in the blood plasma of said mammal.

22. The use of claim 20, wherein said inflam¬ matory disease is arthritis, uveoretinitis, pneumonitis, encephalomyelitis, multiple sclerosis and hepatic granulo- matas.

23. The use of claim 20, wherein said compound is interleukin-1, serotonin, corticotropin releasing hormone, AVP or quipazine and said hormone is cortisol, corticoste¬ rone or adrenocorticotropic hormone.

24. The use of claim 20, wherein the level of said hormones secreted by the pituitary and adrenal glands are measured 10 minutes to 4 hours after administration said compound.

25. The use of a compound to test for the sus¬ ceptibility of a mammal to arthritis which comprises the steps of: Administering to a mammal an effective amount of interleukin-1 or quipazine which stimulates the hypothalamic-pituitary-adrenal axis; and measuring the level of adrenocorticotropic hormone or corticosterone in the plasma of said mammal between 10 minutes and 4 hours after administration of said interleukin-1 or quipazine.

26. The use of claim 25, wherein said mammal is a laboratory animal.

27. The use of claim 25, wherein said mammal is a human.

28. The use of a compound to treat inflammatory diseases which comprises the step of: administering to a patient suffering from an inflammatory disease, a compound which stimulates the hypothalamic-pituitary-adrenal axis in an amount effective to stimulate said axis.