GB2289218A - Inhibition of TNFalpha production with agonists of the A2b subtype of the adenosine receptor - Google Patents

Abstract

TNF alpha production is inhibited by contacting the A2b subtype of the adenosine receptor with an adenosine receptor agonist, especially in monocytes in which cAMP accumulation is increased due to activation of adenylate cyclase. The agonist is preferably adenosine 5'-(N-cyclopropyl)carboxamidoadenosine, 5'-(N-ethyl)carboxamideadenosine, (R)-N<6>-phenyl-2-propyladenosine or cyclohexyladenosine. The agonists may be used in the therapy of autoimmune states. A process for the identification of A2b adenosine receptor agonist, or selective, compounds is described, involving treating monocytes with the compound to determine the degree of TNF alpha inhibitor, and selecting those compounds which either bind specifically to the A2b adenosine receptor or which include cAMP increase in a cell line expressing the receptor.

Description

TITLE OF THE INVENTION INHIBITION OF TNFa PRODUCTION BY A2b ADENOSINE RECEPTOR AGONISTS AND ENHANCERS BACKGROUND OF THE INVENTION I. FIELD OF THE INVENTION: The present invention concerns the use of compounds identified as specific modulators of adenosine's physiological actions. The pharmacology of these compounds is characterized through the use of cloned human adenosine Al, A2a, A2b and A3 receptor subtypes. This invention discloses that compounds identified as agonists of the A2b adenosine receptor subtype are useful in inhibiting the production of tumor necrosis factor (TNFa) by monocytes and/or macrophages.

Therefore this invention comprises a method of treatment or prevention of disease states induced by production of TNFa. These conditions include, but are not limited to autoimmune diseases including rheumatoid arthritis, rheumatoid spondylitis, inflammatory bowl disease (ulcerative colitis and Crohns disease), intestinal pathology associated with graft vs. host disease, organ transplant reactions, septic shock, fever and myalgia due to infection and cachexia associated with chronic infections, malignancy and aquired immune deficiency syndrome, pulmonary diseases such as pulmonary sarcoidosis, silicosis, chronic pulmonary inflammatory disease, adult respiratory distress syndrome.

2. BACKGROUND: Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular, nervous, pulmonary, renal and immune systems. Adenosine has been demonstrated to terminate superventricular tachycardia through blockage of atrioventricular nodal conduction (J.P. DiMarco, et al., (1985) J. Am. Col. Cardiol. 6:417-425, A. Munoz, et al., (1984) Eur.

Heart J. 5:735-738). Adenosine is a potent vasodilator except in the kidney and placenta (R.A. Olsson, (1981) Ann. Rev. Physiol. 43:385 395). Adenosine produces bronchoconstriction in asthmatics but not in nonasthmatics (Cushly et al., 1984, Am. Rev. Respir. Dis. 129:380384). Adenosine has been implicated as a preventative agent and in treatment of ventricular dysfunction following episodes of regional or global ischemia (M.B. Forman and C.E. Velasco (1991) Cardiovasc.

Drugs and Therapy 5:901-908) and in cerebral ischemia(M.C. Evans, et al., (1987) Neurosci. Lett. 83:287, D.K.J.E.,Von Lubitz, et al., (1988) Stroke 19:1133).

Dog Al and A2a adenosine receptors were the first adenosine receptors to be cloned. See F. Libert, et al., (1989) Science 244:569-572, C. Maennant, et al., Biochem. Biophys. Res. Comm., (1990) 173:1169-1178, and F. Libert, et al. (1991) EMBO J. 10:16771682. The rat Al adenosine receptor was cloned by L.C. Mahan, et al., (1991) Mol. Pharm. 40:1-7 and S.M. Reppert, et al., (1991) Mol.

Endocrin. 5:1037-1048, the rat A2a by Fink et. al., (1992) Mol. Brain Res. 14:186-195, and the rat A2b by Stehle et al. (1992) Mol.

Endocrinol. 6:384-393. Cloning of the rat A3 adenosine receptor was reported by Meyerhof et al., (1991) FEBS Lett. 284:155-160 and Zhou et al., (1992) PNAS USA 89:7432-7436. Cloning of the sheep A3 adenosine receptor has been reported by Linden et al., (1993) Mol.

Pharm. 44:524-532. Cloning of the human Al, A2a, A2b and A3 receptors were reported in GB 2264948-A (9/15/93). The human Al adenosine receptor differs by 18 amino acids from the dog Al sequence and 16 amino acids from the rat Al sequence. The human A2a adenosine receptor differs by 28 and 71 amino acids, respectively from the dog and rat A2a sequences. The amino acid sequence for the human A3 receptor is 72% identical with the rat A3 receptor and 85% identical with the sheep A3 receptor sequences.

The actions of adenosine are mediated through G-protein coupled receptors, the Al, A2a, A2b and A3 adenosine receptors. The adenosine receptors were initially classified into Al and A2 subtypes on the basis of pharmacological criteria and coupling to adenylate cyclase (Van Caulker, D., Muller, M. and Hamprecht, B. (1979) J. Neurochem.

33, 999-1003.). Further pharmacological classification of adenosine receptors prompted subdivision of the A2 class into A2a and A2b subtypes on the basis of high and low affinity, respectively, for adenosine and the agonists NECA and CGS-21680 (Bruns, R.F., Lu, G.H. and Pugsley, T.A. (1986) Mol. Pharmacol. 29, 331-346; Wan, W., Sutherland, G.R. and Geiger, J.D. (1990) J. Neurochem. 55, 17631771). The existence of Al, A2a and A2b subtypes has been confirmed by cloning and functional characterization of expressed bovine, canine, rat and human receptors. A fourth subtype, A3, had remained pharmacologically undetected until its recent identification by molecular cloning. The rat A3 sequence, tgpcrl, was first cloned from rat testis by Meyerhoff et al. (see above). Subsequently, a cDNA encoding the identical receptor was cloned from striatum and functionally expressed by Zhou et al. (see above). When compared to the other members of the G-protein coupled receptor family, the rat sequence had the highest homology with the adenosine receptors ( > 40% overall identity, 58% within the transmembrane regions). When stably expressed in CHO cells, the receptor was found to bind the radioligand 125I-APNEA (N6 2-(4-amino-3-iodophenyl)ethyladenosine) and when transfected cells were treated with adenosine agonists, cyclic AMP accumulation was inhibited with a potency order of NECA = R-PIA > CGS21680. The rat A3 receptor exhibited a unique pharmacology relative to the Al and A2 adenosine receptor subtypes and was reported not to bind the xanthine antagonists 1,3 -dipropyl- 8-phenylxanthine (DPCPX) and xanthine amine congener (XAC). Messenger RNA for the rat A3 adenosine receptor is primarily expressed in the testis.

The sheep homolog of the A3 receptor was cloned from hypophysial pars tuberalis (see Linden et al. above). The sheep receptor is 72% identical to the rat receptor, binds the radioligand 125I-ABA and is also coupled to inhibition of cyclic AMP. The agonist affinity order of the sheep receptor is I-ABA > APNEA > NECA > R-PIA CPA. The pharmacology of xanthine antagonists was extensively studied and the sheep receptor was found to exhibit high affinity for 8phenylxanthines with para-acidic substitutions. In contrast to the rat transcript, the expression of the sheep A3 adenosine receptor transcript is widespread throughout the brain and is most abundant in the lung and spleen. Moderate amounts of transcript are also observed in pineal and testis. The cloning and pharmacological profile of the human A3 adenosine receptor was disclosed by Salvatore et al., [P.N.A.S.

90:10365-10369, 1993] and is quite similar to that of the sheep A3 receptor pharmacology.

Based on the use of these cloned receptors, an assay has been described to identify adenosine receptor agonists and antagonists and determine their binding affinity (see GB 2 264 948 A, published 9/15/93, see also R.F. Bruns, et al., (1983) Proc. Natl. Acad. Sci. USA 80:2077-2080; R.F. Bruns, et al.,(1986) Mol. Pharmacol. 29:331-346; M.F. Jarvis, et al. (1989) J. Pharma. Exp. Therap. 251:888-893; K.A.

Jacobson et al., (1989) J. Med. Chem. 32:1043-1051).

Adenosine receptor agonists, antagonists and binding enhancers have been identified and implicated for usage in the treatment of physiological complications resulting from cardiovascular, pulmonary, renal and neurological disorders. Adenosine receptor agonists have been identified for use as vasodilators ((1989) FASEB. J.

3(4) Abs 4770 and 4773, (19910 J. Med. Chem. (1988) 34:2570), antihypertensive agents (D.G. Taylor et al., FASEB J. (1988) 2:1799), and anti-psychotic agents (T.G. Heffner et al., (1989) Psychopharmacology 98:31-38). Adenosine receptor agonists have been identified for use in improving renal function (R.D. Murray and P.C.

Churchill,(1985) J. Pharmacol. Exp. Therap. 232:189-193). Adenosine receptor allosteric or binding enhancers have shown utility in the treatment of ischemia, seizures or hypoxia of the brain (R.F. Bruns, et al. (1990) Mol. Pharmacol. 38:939-949; C.A. Janusz, et al., (1991) Brain Research 567:181-187). The cardioprotective agent, 5-amino-4imidazole carboxamide (AICA) ribose has utility in the treatment of ischemic heart conditions, including unstable angina and acute myocardial infarction (H.E. Gruber, et al. (1989) Circulation 80: 14001414).

Through the use of homogeonous, recombinant adenosine receptors, the identification and evaluation of compounds which have selectivity for a single receptor subtype is now possible. Because of the variable effects of adenosine documented in other species, the utilization of human adenosine receptor subtypes is advantageous for the development of human therapeutic adenosine receptor agonists, antagonists or enhancers.

The anti-inflammatory properties of adenosine have been documented. Adenosine receptor agonists inhibit TNFa production by LPS-stimulated human monocytes (Vraux, et al. 1993 Life Sci. 52:19171924) with an affinity profile which does not correspond to Al or A2a subtype pharmacology. The identification of the specific adenosine receptor subtype mediating the inhibition of TNFa has not been elucidated. With the use of affinity order profiles generated with adenosine receptor agonists, subtype selective adenosine receptor antagonists and information derived from the pharmacological characterization of the human A2b receptor cDNA stably expressed in CHO cells, I have identified the A2b adenosine receptor subtype in mediating the inhbition of TNFa in stimulated human monocytes.

The use of an A2b adenosine receptor specific agonist is advantageous over existing therapeutic agents in that a decrease or elimination of side effects experienced when non-selective agonists or the natural agonist, adenosine, are used for therapy. Allosteric effectors or enhancers of the A2b adenosine receptor would eliminate or decrease systemic side effects. Enhancers increase the binding of the native agonists and have been described for Al adenosine receptors. A2b receptor enhancers remain pharmacologically silent in the absence of adenosine and act locally at sites of inflammation where increases in adenosine concentrations are realized, thereby reducing side effects.

The use of such enhancers to inhibit TNFa production naturally forms part of the instant invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Full length amino acid sequence of human Al adenosine receptor.

Figure 2 Full length nucleotide sequence of the cloned human Al adenosine receptor complementary DNA depicted from the 5' to 3' terminus.

Figure 3 Full length amino acid sequence of human A2a adenosine receptor.

Figure 4 Full length nucleotide sequence of cloned human A2a adenosine receptor complementary DNA depicted from the 5' to 3' terminus.

Figure 5 Full length amino acid sequence of human A2b receptor.

Figure 6 Full length nucleotide sequence of cloned human A2b adenosine receptor complementary DNA depicted from the 5' to 3' terminus.

Figure 7 Saturation binding of [3H]-cyclohexyladenosine (CHA) to human Al adenosine receptor in COS7 assay.

Figure 8 Saturation binding of [3H]-CGS21680 to human A2a adenosine receptor in COS7 assay.

Figure 9 Full length amino acid sequence of human A3 adenosine receptor.

Figure 10 Full length nucleotide sequence of the cloned human A3 adenosine receptor complementary DNA depicted from the 5' to 3' terminus.

Figure 11 Adenosine agonists inhibit LPS induced TNFa production in human blood monocytes with a rank order potency of CPCA 2 NECA R-PIA > CHA > adenosine > CGS21680. Human peripheral blood mononuclear cells were cultured on plastic plates coated with fibronectin.

The. cells were treated with 100 ng/mL of LPS and the indicated concentrations of adenosine agonist. The TNFa levels were measured in cell-culture supernatant by specific ELISA after 18 hours of culture.

Figure 12 The adenosine agonist CPCA inhibits TNFa, but not ILlB or IL-6 release from LPS stimulated human monocytes.

Human peripheral blood monocytes were adhered to fibronectin coated plates and stimulated with LPS in the presence of the indicated concentrations of CPCA. Cell culture supernatant was removed after overnight incubation and tested by specific ELISA for IL-6, ILl , and TNFa.

CPCA did not inhibit IL-6 or ThlB production.

Figure 13 The Al adenosine receptor antagonist DPCPX does not affect the CPCA induced inhibition of TNFa production in LPS stimulated monocytes. Human peripheral blood monocytes were adhered to fibronectin coated plates. The cells were treated with 100 ng/mL of LPS, and the indicated concentrations of CPCA and DPCPX. TNFa production levels were measured by specific ELISA in cell culture supernatant after 18 hours of culture.

Figure 14 CG21A partially antagonizes CPCA induced inhibition of TNFa production in LPS stimulated monocytes.

Human peripheral blood monocytes were adhered to fibronectin coated plates. The cells were treated with 100 ng/mL of LPS, and the indicated concentrations of CPCA and CG21A, an adenosine A2a receptor antagonist. TNFa production levels were measured by specific ELISA in cell culture supernatant after 18 hours of culture. CGS21A inhibited TNFa production in a dose dependent manner in the absence of CPCA, consistent with the hypothesis that endogenous adenosine partially represses TNFa production in the assay.

Figure 15 The A3 adenosine receptor antagonist I-ABOPX does not affect the CPCA induced inhibition of TNFa production in LPS stimulated monocytes. Human peripheral blood monocytes were adhered to fibronectin coated plates. The cells were treated with 100 ng/mL of LPS, and the indicated concentrations of CPCA and I-ABOPX. TNFa production levels were measured by specific ELISA in cell culture supernatant after 18 hours of culture.

Figure 16 Northern blot analysis of the TNFa mRNA production in LPS stimulated monocytes treated with the adenosine agonist CPCA. Total RNA was extracted from 1 x 107 adhered human monocytes one hour following stimulation with LPS in the presence of the indicated concentrations of CPCA. Total RNA (10 Fg) was blotted using a 32p labeled cDNA probe. No significant reductions in TNFa mRNA production were observed using CPCA at levels sufficient to suppress protein production by greater than ten fold.

Figure 17 CPCA dose response of cAMP accumulation in CHO cells stably expressing the human A2b receptor.

SUMMARY OF THE INVENTION Adenosine receptor agonists have been shown to inhibit tumor necrosis factor alpha (TNFa) production in lipopolysaccharide (LPS) stimulated monocytes with an affinity order profile of CPCA 2 NECA R-PIA > CHA 2 adenosine > CGS21680. This agonist profile does not correlate with either the Al or A2a adenosine receptor subtype pharmacology. In order to define the receptor subtype mediating the inhibitory effect, adenosine receptor antagonists were evaluated for their ability to block the inhibition of TNFa production caused by CPCA in LPS-stimulated human monocytes. The involvement of the Al and A2a adenosine receptor subtypes was ruled out on the basis of the inability of DPCPX and 3-succinylaminostrylcaffine, CG21A, respectively, to appreciably antagonize the inhibition produced by CPCA. The A3 adenosine receptor specific antagonist IABOPX was also ineffective in blocking agonist induced inhibition of TNFa production. The agonist affinity order profile established for the monocyte adenosine receptor was similar to the A2b receptor in VA13 human fibroblasts and human erythroleukemic cells (HEL) defined by EC50 values for intracellular cyclic adenosine monophosphate (cAMP) accumulation. However, the potency of the agonists to inhibit TNFa production in monocytes was greater than values determined by increases in cAMP accumulation in fibroblasts or HEL cells. I have found that in stable CHO cells expresing the cloned human A2b cDNA, the potency (EC50) of CPCA to induce cAMP accumulation was similar to the value obtained for inhibition of TNFa production in LPS-stimulated human monocytes. To define which adenosine receptor subtypes are present on monocytes, Al, A2a, A2b, and A3 adenosine receptor transcripts were detected by reverse transcriptase PCR (RT-PCR) of mRNA prepared from both LPSstimulated and non-stimulated monocytes. The regulation of TNFa expression resulting from mediation at the A2b receptors is demonstrated to be consistent with a mechanism involving increased intracellular cAMP levels.

ABBREVIATIONS CHA, [3H]-cyclohexyladenosine; [3H]-NECA, [3H]-5-N-ethyl- carboxamido-adenosine; 125I-ABA, N6-(4-amino-3 l25iodobenzyl)adenosine; 125I-APNEA, N6-2-(4-amino-3 125iodophenyl)ethyladenosine; NECA, 5'-N- ethylcarboxamidoadenosine; CGS21680, 2-[4-(2 carboxyethyl)phenyl]ethylamino -5 '-N-ethylcarboxamidoadeno sine; (R,S)-PIA, (R,S)-N6-phenyl-2-propyladenosine; CPA, N6cyclopentyladenosine; CPCA, 5'-fN-cyclopropyl)- carboxamidoadenosine; CG2l A, 3-succinylaminostrylcaffine; I AB OPX, (3-(3 iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl- 1prpopylxanthine; BW-A1433, 1,3-dipropyl-8-(4acrylate)phenylxanthine; XAC, xanthine amine cogener; DPCPX, 1,3dipropyl-8-cyclopentylxanthine; GTPyS, guanosine 5'-0-3- thiotriphosphate; Gpp(NH)p, 5'-guanylimidodiphosphate; G protein, guanine nucleotide-binding proteins.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a method for achieving specific inhibition of TNFa production through agonist stimulation of the A2b adenosine receptor. TNFa is a pro-inflammatory cytokine which, among other effects, induces fever and stimulates phospholipase A2 production. Lipopolysaccharide (LPS) is a biological mediator which gives rise to a number of adverse responses. A principal mediator to these effects is TNFa. A variety of adenosine receptor agonists have been tested for their ability to block LPS-mediated TNFa production in human monocytes [Le Vraux et al., Life Sciences 52:1917-1924, 1993].

Figure 11 summarizes the pharmacological profile of this effect [CPCA > NECA R-PIA > CHA > adenosine > CGS21680]. The conclusion reported in Le Vraux et al., based on this pharmacology, was that the inhibition of of TNFa production was probably mediated through the A3 adenosine receptor, or through an uncharacterized receptor, but not through the Al or A2 adenosine receptors. As can be seen from this data, CPCA and NECA are the most potent inhibitors of TNFa production. Both compounds have been characterized as binding both the Al and the A2 adenosine receptor subtypes with high affinity, see the table below: AFFINITY OF ADENOSINE ANALOGS FOR HUMAN ADENOSINE RECEPTOR SUBTYPES, Ki or Kd, RM *

A onists Al A2a A2h A3 NECA | 0.025 0.029 0.9 (a) 0.026 CPCA 0.006 (rat) 0.0134 (rat) 0.050(a) 1.0 CGS21680 56 0.017 1600 (b) 5.6 R-PIA 0.003 0.127 160 (b) 0.034 CHA 0.002 0.6 280 (b) n.d.

Antagonists DPCPX 0.0007 0.10 0.55 (b) 0.75 CGS21A 35 (rat) 0.143 (rat) n.d. > 50 *Values determined in rat are vindicated, otherwise all other data is from human, (a) EC50 values for cAMP accumulation in stable CHO cells expressing the human A2b cDNA; (b) EC50 values for cAMP accumulation in human erythroleukemic cells, HEL cells.

The Al adenosine receptor selective agonists R-PIA and CHA are significantly less potent than CPCA or NECA. The A2a specific agonist CGS21680 was found to be the least potent of all. The rank order of potency of the compounds to inhibit TNFa production is not like that of either Al or A2 [Le Vraux et al., Life Sciences 52:1917-1924, 1993]. The affinity order profile reported by Le Vraux et al. is similar to the agonist profile reported by Castanon and Spevak [BBRC 198:626-631, 1994] for the induction of cyclic adenosine monophosphate (cAMP) accumulation in stable CHO cell lines expressing the cloned A2b adenosine receptor. However, Castanon and Spevak did not study the role of the A2b receptor in inhibition of TNFa production. In addition, the agonist affinity order profile data reported by Le Vraux et al. for TNFa inhibition is not dissimilar from the agonist order profile reported by Salvatore et al., rP.N.A.S. 90:1036510369, 1993] for the cloned A3 adenosine receptor and suggested that the A3 receptor may be responsible for TNFa inhibition in LPSstimulated monocytes. However, the potency of CPCA for the A3 receptor was not reported by Salvatore et al. and therefore, prior to this invention, the role of A3 adenosine receptor in the inhibition of TNFa production could not be ruled out and the specific adenosine receptor subtype which is responsible for inhibition of TNFa production could not be positively identified. This patent disclosure demonstrates that CPCA has a much lower affinity for the A3 receptor than it does for the A2b receptor and by using A3 adenosine receptor specific antagonists, the involvement of A3 receptor activation in the inhibition of TNFa production is definitively ruled out. This patent disclosure demonstrates that Al and A2a adenosine receptors are not involved in the inhibition of TNFa production. This invention reveals that activation only at the A2b adenosine receptor is responsible for the inhibition of TNFa production.

The role of cAMP elevations has been correlated with the inhibition of LPS induced TNFa production defined through the use of the phosphodiesterase inhibitor pentoxifyllin [Strieter, et al., (1988) Biochem. Biophys. Res. Commun. 155: 1230-1236]. The inhibition of TNFa production through activation at A2b adenosine receptors on stimulated monocytes is therefore consistent with a mechanism resulting from increases in intracellular cAMP. Therefore, this invention comprises a method for inhibiting TNFa production specifically through A2b receptor activation.

Since Le Vraux et al., suggested that the receptor responsible for inhibition of TNFa production was possibly the A3 adenosine receptor and not the Al or A2 receptors, I initiated the following studies in order to elucidate which receptor is, in fact, responsible for inhibition of TNFa production.

I confirmed that the Al and A2a receptor subtypes are not responsible for the inhibition of TNFa production by using the Al and A2a adenosine receptor selective antagonists DPCPX and CG21A respectively. These compounds do not appreciably alter the IC50 of CPCA in antagonist competition experiments except at very high concentrations (see Figures 13 and 14). This data confirms that the Al and A2a adenosine receptor subtypes are not involved in the inhibition of TNFa production. I confirmed that the A3 receptor subtype was not responsible for the inhibition of TNFa production by using the A3 specific antagonist, I-ABOPX (Figure 15). I-ABOPX did not alter the IC50 of CPCA inhibition of TNFa production.

I further determined that the affinity of CPCA for the A3 adenosine receptor subtype is 1 ,uM and therefore, the A3 receptor cannot be responsible for the inhibition of TNFa production induced by CPCA which exhibits a much higher (20,000-fold) affinity for the A2b than the A3 adenosine receptor. I obtained the EC50 value for CPCA induced cAMP accumulation in stable CHO cell lines expressing the human A2b receptor and found that the EC50 value is the same as that obtained from the stimulated monocytes (Figure 17). I further confirmed that the effect is specific for TNFa because ILl and IL-6 production are unaffected by treatment with CPCA, (Figure 12).

Northern blot data of total RNA from LPS stimulated monocytes indicates that titration of CPCA reduces the levels of secreted TNFa protein in a dose dependent manner, Figure 16. This data indicates that adenosine agonists inhibit TNFa production primarily through post-transcriptional mechanisms. This observation is consistent with reports that TNFa mRNA contains 3'-untranslated sequences that mediate translational activation in response to specific inducing signals (e.g. LPS). Removal of these sequences has been shown to result in the inability of the mRNA to be translated. Therefore, it appears that adenosine blocks components of the LPS signal transduction pathway that are related to these 3'-untranslated elements of the TNFa gene.

To define which adenosine receptor subtypes are present on monocytes, Al, A2a, A2b, and A3 adenosine receptor transcripts were detected by reverse transcriptase PCR (RT-PCR) of mRNA prepared from both LPS-stimulated and non-stimulated monocytes. All four adenosine receptor subtypes were detected in mRNA prepared from both normal and LPS-stimulated monocytes. Even though all of the identified adenosine receptor subtypes are present on monocytes, this invention reveals that only the A2b receptor affects TNFa production.

Therefore, one embodiment of this invention is a method for identifying A2b adenosine receptor selective compounds which comprises the steps of: (a) contacting monocytes with a test compound and measuring the effect of the test compound on TNFa production; (b) contacting a test compound, identified according to step (a) as inhibiting TNFa production by the monocytes, with membranes derived from a stable cell line individually expressing each of the Al, A2a, A2b, or A3 adenosine receptor or with the whole cell individually expressing each of the Al, A2a, A2b, or A3 adenosine receptor and measuring the binding affinity of the test compound for the receptor or the effect of the test compound on cAMP production in the stable cell line; (c) selecting compounds which bind to the A2b adenosine receptor or which induces elevation in cAMP in the cell line expressing the A2b adenosine receptor and which do not bind to membranes or affect the cAMP level in the stable cell lines expressing the Al, A2a, or A3 adenosine receptor subtypes.

This invention likewise comprises the use of compounds identified according to this method which have A2b adenosine receptor enhancer or agonist activities for the inhibition of TNFa production.

This invention further comprises a method for inhibiting TNFa production by contacting monocytes with inhibitorily effective amounts of compounds that act as A2b adenosine receptor agonists. An inhibitorily effective amount of an A2b adenosine receptor agonist is, for example, 0.1 ng to 10 mg/kg per day of CPCA, NECA or a compound exhibiting similarly potent or more potent A2b adenosine receptor agonist properties.

The following examples are provided to further define but not to limit the invention defined by the foregoing description and the claims which follow: EXAMPLE 1 STEP A: In the first step of obtaining the partial cDNAs encoding the human Al and A2a adenosine receptors, total RNA was extracted by homogenizing 2.3g human ventricle in 20 ml SM guanidine isothiocyanate, 0.1M sodium citrate, pH 6.3, lmM EDTA, pH 7.0, 5% beta-mercaptoethanol, and 0.5% sodium lauryl sarcosinate. The homogenate was centrifuged for 10 min. at 10,000 rpm and the resulting supernatant was layered onto a cushion of 5.7M CsCl/0.1M EDTA, pH 7.0. After 20 hrs. of centrifugation at 24,000 rpm, the resulting pellet was precipitated one time and then passed over an oligo(dT)-cellulose (PHARMACIA, Piscataway, NJ) column to isolate poly(A)+ RNA.

An oligo(dT) primed library was synthesized from 5 Rg of the poly(A)+ human ventricle RNA using the YOU-PRIME cDNA SYNTHESIS KIT (PHARMACIA, Piscataway, NJ). See Gubler and Hoffman Gene 25:263 (1983). The resulting double-stranded cDNA was ligated into hgtl0 EcoRI arms (PROMEGA, Madison, WI) and packaged according to the GIGAPACK II GOLD PACKAGING EXTRACT protocol (STRATAGENE, La Jolla, CA). See Huynh et al.

(1985) DNA Cloning Techniques: A Practical Approach, IRL Press, Oxford, p.49 and Kretz et al. Res. 17:5409.

The E. coli strain C600Hfl (PROMEGA, Madison, WI) was infected with library phage, plated on agar plates, and incubate A2a(RDC8) sequences (F Libert, et al,(l989) Science 244:569-572).

The oligonucleotides were annealed and filled-in with a32P-dCTP (NEN, Wilmington, DE) and Klenow enzyme. The filters were hybridized with the appropriate probe in SXSSC, 30% formamide, SXDenhardt's solution, 0.1% SDS, and 0.1mg/ml sonicated salmon sperm DNA at 420C, overnight. Following hybridization the filters were washed to a final stringency of 6XSSC at 50"C and exposed to X OMAT AR film (KODAK, Rochester, NY) at -70 C. The resulting positives were plaque purified by two additional rounds of plating and hybridization. Insert DNA was excised with Notl and ligated into Notl digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA).

(Genebank &num; 52327) DNA sequences were determined by the SEQUENASE protocol (USBC, Cleveland, OH). See Tabor and Richardsaon, J. Biol. Chem. 264 pp 6447-6458. Two clones were isolated in these screens. The human ventricle Al cDNA (hval-3a) and human ventricle A2a cDNA (hva2-13) contain portions of coding sequences for proteins homologous to the reported dog Al and A2a cDNAs, respectively. The coding region of the human Al clone corresponds to nucleotides 482 through 981 (Figure 2) and is 92% identical to the dog Al sequence at the nucleotide level. The coding region of the human A2a clone corresponds to nucleotides 497 through 1239 (Figure 4), and is 90% identical to the dog A2a sequence at the nucleotide level.

STEP B: The human ventricle Al adenosine receptor partial cDNA (hvA1-3a) is a 543 bp NotI fragment containing 23 bp 3' untranslated sequence and is 460 bp short of the initiation methionine based on sequence homology to the dog Al cDNA. A modification of the 5' RACE (rapid amplification of cDNA ends) method (MA Frohman et al,(1988), Proc. Natl. Acad. Sci. USA, 85:8998-9002) was used to generate the 5' coding region of the cDNA. First strand cDNA was synthesized from 1clog of the human ventricle poly(A)+ RNA in a total volume of 40ml containing 50mM Tris, pH 8.0, 140mM KCl, 10mM MgCl2, lOmM DTT, 15mM each dNTP, 20 units RNasin (PROMEGA, Madison, WI), 20pmol human primer 79 (see Table I), and 9.2 units AMV reverse transcriptase at 37"C for 2 hrs. The reaction was then diluted to 120 Rl with 0.5 mM Tris, pH 7.6/0.05 mM EDTA and passed through a SEPHACRYL S-300 SPUN COLUMN (PHARMACIA, Piscataway, NJ). The product in the column effluent was polyadenylated in 100mM potassium cacodylate, pH 7.2, 2mM Cowl2, 0.2mM DTT, 0.15mM dATP, and 14 units terminal deoxynucleotidyl transferase in a total volume of 31cm1 for 10 min. at 37CC. The reaction was terminated by heating at 65"C for 15 min. and then diluted to 500 ml with 10 mM Tris, pH 8.0/1 mM EDTA (TE).

Ten ,ul of the poly(A)-tailed first strand cDNA was used as template in a primary PCR amplification reaction according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT; see Saiki et al. (1988) Science 239:487-491) containing 10pmol primer 70, 25pmol primer 71, and 25pmol human primer 80 (see table I) in a total volume of 50 ml. Primer 70 is 5'-gactcgagtcgacatcga(t) 17, primer 71 is 5'-gactcgagtcgacatcga, and both are based on MA Frohman, et al (1988), Proc. Natl. Acad. Sci. USA, 85:8998-9002. One cycle of PCR was performed of 1 min at 95"C, 2 min at 50 C, 40 min at 72"C, followed by 40 cycles of 40 sec at 94"C, 2 min at 56"C, 3 min at 72"C.

The primary PCR amplification reaction product was electrophoresed through a 1.4% agarose gel and an area corresponding to approximately 600 bp was excised. The gel slice was melted and 1 Rl was used as template in a secondary PCR amplification reaction containing 100pmol primer 71 and human primer 81 (see Table I) for 30 cycles of 1 min at 94"C, 2 min at 56"C, 3 min at 72"C. The secondary PCR amplification product was digested with EcoRI and SalI and electrophoresed on a 1.4% agarose gel. An area corresponding to 500-600bp was excised and ligated into EcoRVSall digested pBLUESCRIPT H KS+ (STRATAGENE, La Jolla, CA). The sequence of the 515 bp PCR product (5'HVAl-9) was determined by the SEQUENASE protocol (USBC, Cleveland, OH). The partial human ventricle Al cDNA and the PCR product contain overlapping sequence and represent the complete coding region for the human Al receptor, including 14 and 23 bp of 5' and 3' untranslated sequences, respectively. The sequence of the human Al adenosine receptor cDNA so identified, is shown in Figure 2.

STEP C: A probe was generated by Klenow enzyme extension, including a32P-dCTP, of annealed oligonucleotides 62 and 63, and used to screen a human kidney cDNA library (CLONTECH, Palo Alto, CA).

E. coli strain C600hfl (PROMEGA, Madison, WI) was infected with library phage and grown overnight on agar plates at 37"C. Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL). The probe was incubated with the filters in 750mM NaCl, 75mM sodium citrate, 30% formamide, 0.1% sodium dodecyl sulfate, 0.5mg/mL polyvinylpyrrolidone, 0.5mg/mL bovine serum albumin, 0.5mg/mL Ficoll 400, and 0.1mg/mL salmon sperm DNA, at 42"C overnight. The filters were washed in 0.9M NaCl and 90mM sodium citrate at 50"C. A positively hybridizing phage (hkAl-14), was identified and purified by replating and screening with the probe twice more. The final phage plaque was transferred to 0.5 mL 50mM Tris, pH 7.5, 8mM MgSO4, 85 mM NaCl, lmg/mL gelatin, and 1 FL of a 1:50 dilution in water of the phage stock was used as template for PCR amplification. 50 pmol each of lamL and lamR (Table I) oligonucleotide primers were included, and subjected to 30 cycles of 40 sec at 94"C, 1 min at 55 , 3 min at 72", then a final 15 min at 72", according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT). A 2.0 kb product was identified by agarose gel electrophoresis, and this was subcloned into the EcoRI site of pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA).

Sequence analysis by the SEQUENASE protocol (USBC, Cleveland, OH) demonstrated that this cDNA was homologous to the reported dog Al clone. SmaI and EcoRI digestion released a DNA fragment containing coding sequence from base pair 76 through the translation STOP codon (Figure 2) that is identical to the human ventricle Al cDNA sequence (clones hval-3a and 5'hval-9). This fragment was used in constructiòn of the full length coding sequence (see below). The human kidney cDNA also includes about 900 bp of 3' untranslated sequence.

STEP D: The human ventricle A2a adenosine receptor partial cDNA (hvA2-13) is a 1.6 kb NotI fragment containing approximately 900 bp 3' untranslated sequence and is 496 bp short of the initiation methionine based on sequence homology to the dog A2a cDNA clone. Two consecutive rounds of 5' RACE were utilized to generate the 5' coding region of the cDNA. First strand cDNA was synthesized from 1 ,ug of the human ventricle poly(A)+ RNA in a total volume of 40 ml containing 50mM Tris, pH 8.0, 140mM KCl, 10mM MgCl2, 10mM DTT, 15mM each dNTP, 20 units RNasin, 20pmol human primer 68 or 74 (for 1st or 2nd round RACE respectively), and 9.2 units AMV reverse transcriptase at 37"C for 2 hrs. The reaction was then diluted to 120ml with 0.5 mM Tris, pH 7.6/0.05 mM EDTA and passed through a SEPHACRYL S-300 SPUN COLUMN. The products in the column effluents were polyadenylated in 100mM potassium cacodylate, pH 7.2, 2 mM CoCl2, 0.2 mM DTT, 0.15 mM dATP, and 14 units terminal deoxynucleotidyl transferase in a total volume of 31 ,ul for 10 min. at 37"C. The poly(A) tailing reaction was terminated by heating at 65"C for 15 min. and then diluted to 500 ,ul with TE.

Five or 10 ,ul (for 1st or 2nd round RACE respectively) of the poly(A) tailed first strand cDNA was used as template in the PCR amplification reaction according to the GENEAMP protocol containing 10pmol primer 70, 25 pmol primer 71 (primer 70 and 71 sequences are given above), and 25 pmol human primer 69 or 75 (1sot or 2nd round RACE respectively; see Table I) in a total volume of 50 ,ul. One cycle of PCR was performed of 1 min at 95 C, 2 min at 50"C, 40 min at 72"C, followed by 40 cycles of 40 sec at 94"C, 2 min at 56 C, 3 min at 72"C. The PCR amplification products were digested with EcoRI and SalI and electrophoresed on a 1.4% agarose gel. Areas corresponding to 200-400 bp were excised and ligated into EcoRI/SalI digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA). The sequences of the two A2a PCR products, the 332 bp 1st round RACE product (5'hvA2-14) and the 275 bp 2nd round RACE product (5'hvA2-29) were determined by the SEQUENASE (USBC, Cleveland, OH) protocol. By sequence homology comparisons with the dog A2a adenosine receptor cDNA sequence, the 1 sot round RACE product (5'hvA2-14) was 258 bp short of the initiation methionine and the second round RACE product (5'HVA2-29) was determined to extend lbp upstream of the initiation methionine. The human ventricle A2a partial cDNA clone (hvA2-13) and the human A2a PCR products (5'hvA2-14 and 5'hva2-29) contain overlapping sequence and together represent the complete coding sequence for the human adenosine A2a receptor, and include 1 bp and 0.8 kb of 5' and 3' untranslated sequence, respectively. The sequence of the human A2a adenosine receptor is shown in Figure 4.

STEPPE: A double-stranded DNA probe was generated by Klenow enzyme extension, including a32P-dCTP, of annealed oligonucleotides 66 and 67, and used to screen a human striata cDNA library (STRATAGENE, La Jolla, CA). The oligonucleotide sequence was based on a region of the human ventricle A2a cDNA sequence. E. coli strain XLl-blue (STRATAGENE, La Jolla, CA) cells were infected with library phage and grown overnight on agar plates at 37"C. Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL). The probe was incubated with the filters in 750 mM NaCl, 75 mM sodium citrate, 10% formamide, 0.5% sodium dodecyl sulfate, 0.5 mg/mL polyvinylpyrrolidone, 0.5 mg/mL bovine serum albumin, 0.5 mg/mL Ficoll 400, and 0.02 mg/mL salmon sperm DNA, at 42"C overnight.

The filters were washed in 0.9 M NaCI and 90 mM sodium citrate at 50"C, A positively hybridizing phage (hbA2-22A) was identified and purified by replating and screening with the probe twice more, and subcloned into the plasmid pBLUESCRIPT SK- by the manufacturer's protocol (STRATAGENE, La Jolla, CA). See Short et al. (1988) Nucl.

Acids Res. 16:7583-7600; Sorge (1988) Stratagies 1:3-7. The human brain A2a adenosine receptor cDNA (hbA2-22A) spans bp 43 of the A2 coding sequence (Figure 4) through the translation STOP codon, and includes about 900 bp of 3' untranslated sequence. The sequence of this human brain A2a cDNA is identical to the human ventricle A2a adenosine receptor cDNA (hvA2-13, 5'hvA2-14 and 5'hvA2-29).

STEP F: A double-stranded DNA probe was generated by Klenow enzyme extension of annealed oligonucleotides 129 and 130, including a32P-dCTP, and used to screen a human frontal cortex cDNA library (STRATAGENE, La Jolla, CA). The oligonucleotide sequence was based on a region of the human A2a and Al cDNA sequence. E. coli strain XL-1 blue (STRATAGENE, La Jolla, CA) cells were infected with library phage and grown overnight at 37"C. Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL). The probe was incubated with the filters in 750 mM Nail, 75 mM sodium citrate, 10% formamide, 0.5% sodium dodecyl sulfate, 0.5 mg/mL polyvinyl-pyrrolidone, 0.5 mg/mL bovine serum albumin, 0.5 mg/mL Ficoll 400, and 0.02 mg/mL salmon sperm DNA, at 42"C overnight.

The filters were washed in 0.9 M NaCl and 90 mM sodium citrate at 50"C. A positively hybridizing phage (hb-32c), was identified and purified by replating and screening with the probe twice more. The insert was subcloned to the plasmid pBLUESCRIPT SK- according to the manufacturer's protocol (STRATAGENE, La Jolla, CA). Sequence analysis by the SEQUENASE protocol (USBC, Cleveland, OH) demonstrated a complete open reading frame coding for amino acid sequence homologous to both of the previously isolated human Al and A2a clones. This homologous adenosine receptor subtype cDNA is the A2b subtype having the sequences in Figures 5 and 6. A 1.3 kb Smal Xmnl fragment was ligated into the SmaI site of pSVL (PHARMACIA, Piscataway, NJ), giving the full length coding sequence of the A2b adenosine receptor in a plasmid suitable for its expression in COS and CHO cells. See Sprague et al. (1983) J. Virology 45:773; Templeton and Eckhart (1984) Mol. Cell Biol. 4:817.

Table I: Sequences and directions of the primers used in the isolation of cDNA's and construction of expression plasmids, along with the positions in the clones upon which the sequences are based. Dog Al and A2a cDNA clones are from F. Libert, et al, (1989) Science 244:569-572. Primers LamL and LamR are based on the sequence of hgtl0 (T.V. Hyunh, et al. (1985) DNA Cloning: A Practical Approach, Vol 1, D. Glover, ed, IRL Press, Oxford). The A2b adenosine receptor subtype encoded by the clone hb32C was determined to be the A2b adenosine receptor subtype on the basis of the binding profile of the adenosine receptor agonist NECA and affinities for adenosine receptor antagonists measured on membranes prepared from pSVLhb32C transfected COS7, CHO or HEK 293 cells.

name sequence position clone direction 52 ATTCGCAGCCACGTCCTGA- 1201-1260 dog A2a sense GGCGGCGGGAGCCCTTCAA- AGCAGGTGGCACCAGTGCC CGC (SEQ ID NO. 1) 53 GCGGAGGCTGATCTGCT- 1305-1246 dog A2a antisense CTCCATCACTGCCATGAG CTGCCAAGGCGCGGGCAC TGGTGCC (SEQ. ID NO. 2) 62 TCCAGAAGTTCCGGGTCA- 958-1017 dog Al sense CCTTCCTTAAGATCTGGAA TGACCACTTCCGCTGCCAGC CCA (SEQ. ID NO. 3) 63 AGTCGTGGGGCGCCTCCT- 1062-1003 dog Al antisense CTGGGGGGTCCTCGTCGAC GGGGGGCGTGGGCTGGCAG CGGA (SEQ ID NO. 4) 66 GCCTCTTTGAGGATGTGG- 500-542 5'hvA2-14 sense TCCCCATGAACTACATGGT GTACTTCA (SEQ ID NO. 5) 67 GCAGGGGCACCAGCACACA- 572-528 5'hva2-14 antisense GGCAAAGAAGTTGAAGTAC ACCATGT (SEQ ID NO.6) name sequence position clone direction 68 TCGCGCCGCCAGGAAGAT 616-599 hva2-13 antisense (SEQ ID NO 7) 69 TATATTGAATTCTAGACAC- 591-574 hva2-13 antisense CCAGCATGAGC (SEQ ID NO.8) 74 TCAATGGCGATGGCCAGG 303-286 5'hva2-14 antisense (SEQ ID NO.9) 75 TATATTGAATTCATGGA- 276-259 5'hva2-14 antisense GCTCTGCGTGAGG (SEQ ID NO. 10) 79 GTAGACCATGTACTCCAT 560-543 hval-3a antisense (SEQ ID NO. 11) 80 TATATTGAATTCTGACCT- 537-521 hval-3a antisense TCTCGAACTCGC (SEQ ID NO. 12) 81 ATTGAATTCGATCACGGG- 515-496 hval-3a antisense CTCCCCCATGC (SEQ ID NO. 13) 129 ATGGAGTACATGGTCTAC- --- --- sense TTCAACTTCTTTGTGTGGG TGCTGCCCCCGCT (SEQ ID NO. 14) name sequence position clone direction 130 GAAGATCCGCAAATAGACA- --- --- antisense CCCAGCATGAGCAGAAGCG GGGGCAGCACCC (SEQ ID NO. 15) 131 CCCTCTAGAGCCCAGCCTGT- 2-19 5'hva2-29 sense GCCCGCCATGCCCATCATGG- 1-14 5'hval-9 GCTCC (SEQ ID NO. 16) lamL CCCACCTTTTGAGCAAGTTC --- #t10 --- (SEQ ID NO. 17) lamR GGCTTATGAGTATTTCTTCC --- ;t10 (SEQ ID NO. 18) 207 CCCAAGC1TATGAAAGCCAA CAATACC (SEQ ID NO. 27) 208 TGCTCTAGACTCTGGTATCT TCACATT (SEQ ID NO. 28) EXAMPLE 2 Human Al adenosine receptor expression construct: To express the human adenosine receptor cDNA in COS, CHO and HEK 293 cells, the 118bp SalI-Smal fragment of the human ventricle Al PCR product (5'HVAl-9) was ligated together with the 1.8 SmaI-EcoRI fragment of the human kidney Al adenosine receptor cDNA (hkAl-14) and the 3.0 kb SalI-EcoRI fragment of pBLUESCRIPT II KS+, resulting in a plasmid containing the contiguous full length coding sequence for the human Al adenosine receptor cDNA and some 5' and 3' untranslated sequence. This plasmid was digested first with EcoRI, the resulting ends were filled in by Klenow enzyme extension and then the plasmid was digested with XhoI to release a fragment of 1.9 kb containing the full length human Al adenosine receptor cDNA. The fragment was subcloned into the expression vector pSVL (PHARMACIA) which had been digested with XhoI-SmaI.

Human A2a adenosine receptor expression construct: To express the human A2a adenosine receptor cDNA in COS, CHO or HEK 293 cells, a contiguous A2a cDNA sequence was constructed before subcloning into the expression vector, pSVL.

Primer 131, containing an XbaI recognition site, 14 bp of 5' untranslated sequence of human Al adenosine receptor cDNA, and the first 18 bp of human A2a adenosine receptor cDNA coding sequence was used with primer 75 in PCR with 1 ng of the plasmid containing the human ventricle A2a 2nd round RACE product (5'hvA2-29) as template. Twenty-five cycles of 40 sec at 94"C, 1 min at 55"C, and 3 min at 720C, then a final incubation of 15 min at 72"C, with 1 ng of plasmid template and 50 pmol of each primer in a volume of 50 ,uL according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT), resulted in the expected 302 bp product determined by agarose gel electrophoresis. The 172 bp XbaI-EagI digestion product of this DNA fragment was ligated together with 1125 bp EagI-BglII digestion product of the human striata A2a adenosine receptor cDNA (hbA2-22A) and XbaI-SmaI digested pSVL (PHARMACIA), generating the full length human A2a adenosine receptor cDNA coding sequence in a plasmid suitable for its expression in COS, CHO or HEK 293 cells.

Mammalian cell expression: COS7 cells (ATCC #1651-CRL) were grown in complete medium, Dulbecco's modified Eagles's medium, DMEM (GIBCO, Grand Island, NY) containing 10% fetal bovine serum, 100U/mL penicillin-streptomycin and 2 mM glutamine, in 5% CO2 at 37"C.

Transient transfection of COS7 cells was performed by the CaPO4 method (Graham,F.L. and Van Der Erb, A.J. (1973) Virology 52:456567) using the Mammalian Transfection Kit (STRATAGENE). See Chen and Okayama Mol. Cell Biol. 7:2745-2752. Plasmid DNA (15 mg) was precipitated with 125 mM CaCl2 in BBS (N,N-bis(2 hydroxyethyl)-2-aminoethanesulfonic acid buffered saline) at room temperature for 30 minutes. The DNA precipitate was added to the COS7 cells and incubated for 18h in 5% C02 at 37"C. The precipitate was removed and the cells were washed twice with serum free DMEM.

Cells were incubated in complete medium in 5% CO2 at 37"C for 48 h prior to the binding assay.

Stable expression in CHO or HEK 293 cells: To establish stable cell lines, CHO or HEK 293 cells were co-transfected with 20 ,ug of pSVL containing the adenosine receptor cDNA and lmg of pWLneo (STRATAGENE) containing the neomycin gene. See Southern and Berg (1982) J. Mol. App. Gen. 1:327-341.

Transfection was performed by the CaPO4 method. DNA was precipitated at room temperature for 30 minutes, added to the CHO cells and incubated 18h in 5% CO2 at 37"C. The precipitate was removed and the cells were washed twice with serum free DMEM.

Cells were incubated for 24h in 5% CO2 at 37 C, replated in 24-well dishes at a dilution of 1:10, and incubated an additional 24h before adding selection medium, DMEM containing 10% fetal bovine serum, 100U/mL penicllin-streptomycin, 2 mM glutamine and 0.5 mg/mL G418 (GIBCO). Transfected cells were incubated at 5% CO2, 37"C until viable colonies were visible, approximately 14-21 days. Colonies were selected and propagated. The cell clone with the highest number of human adenosine receptors was selected for subsequent application in the binding assay.

EXAMPLE 3 Binding studies: Membranes were prepared from transiently transfected COS7 cells 48 h after transfection or from G418-selected stably transfected CHO or HEK 293 cells. Cells were harvested in 1 mM EDTA in phosphate buffered saline and centrifuged at 2000 x g for 10 minutes. The cell pellet was washed once with phosphate buffered saline. The cell pellet was resuspended in 2 mL of 5 mM Tris, pH 7.6/ 5mM MgCl2. Membranes were prepared from the cells by freeze-thaw lysis in which the suspension was frozen in a dry ice/ethanol bath and thawed at 25"C twice. The suspension was homogenized after adding an additional 2 mL of 5 mM Tris, pH 7.6/5 mM MgCl2, in a glass dounce homogenizer with 20 strokes. The membranes were pelleted at 40,000 x g at 4"C for 20 minutes. The membrane pellet was resuspended at a protein concentration of 1-2 mg/mL in binding assay buffer, 50 mM Tris, pH 7.6/10 mM MgCl2. Protein concentration was determined by the method of Bradford ((1976) Anal. Biochem. 72: 248-250). Before the binding assay was performed, the membranes were incubated with adenosine deaminase (BOEHRINGER MANNHEIM), 2 U/mL for 30 minutes at 37"C. Saturation binding of [3H]-cyclohexyladenosine (CHA) was performed on membranes prepared from pSVLA1 transfected COS7 or CHO cells.

Membranes (1001lug) were incubated in the presence of 0.2 U/mL adenosine deaminase with increasing concentrations of CHA (NEN, 32 Ci/mmol) in the range of 0.62 - 30 nM for 120 minutes at 25"C in a total volume of 500 ,uL. The binding assay was terminated by rapid filtration and three washes with ice-cold 50 mM Tris,pH 7.6/10 mM MgCl2 on a SKATRON CELL HARVESTER equipped with a receptor binding filtermat (SKATRON INSTRUMENTS, INC). Nonspecific binding was determined in the presence of 100 FM N6cyclopentyladenosine (CPA). Bound radioactivity was measured by scintillation counting in READY SAFE SCINTILLATION COCKTAIL (BECKMAN). For competition binding experiments, membranes were incubated with 5 nM [3H]-CHA and various concentrations of Al adenosine receptor agonists. Saturation binding of [3H] CGS-21680 was performed on membranes prepared from pSVLA2a transfected COS7 cells. Membranes (100clog) were incubated in the presence of 0.2 U/mL adenosine deaminase with increasing concentrations of CGS21680 (NEN, 48.6 Ci/mmol) in the range of 0.62 -80 nM for 90 minutes at 25"C in a total volume of 500 ,eL. The binding assay was terminated by rapid filtration with three washes with ice-cold 50 mM Tris, pH 7.6/10 mM Mg Cl2 on a Skatron cell harvester equipped with a receptor binding filtermat (SKATRON INSTRUMENTS, INC). Non-specific binding was determined in the presence of 100,uM CPA. Bound radioactivity was measured by scintillation counting in READY SAFE LIQUID SCINTILLATION COCKTAIL (BECKMAN). For competition binding experiments, membranes were incubated with 5nM [3H]-CGS21680 and various concentrations of A2 adenosine receptor agonists.

Saturation binding of [3H]5'-N-ethylcarboxamidoadenosine (NECA) was performed on membranes (100 Fg) prepared from pSVLhb32C (A2b) transfected COS7 cells in the presence of adenosine deaminase with increasing concentrations of NECA (NEN, 15.1Ci/mmol) in the range of 1.3-106 nM for 90 minutes at 250C in a total volume of 500 ,uL. The assay was terminated by rapid filtration and three washes with ice-cold binding buffer on a cell harvester equipped with a receptor binding filtermat (SKATRON INSTRUMENTS, INC). Bound radioactivity was measured by scintillation counting. Non-specific binding was measured on membranes prepared from non-transfected COS7 cells. For competition binding experiments, membranes from transfected cells were incubated with 10 nM [3H]NECA and varying concentrations of adenosine receptor antagonists.

EXAMPLE 4 The human A3 adenosine receptor was cloned from a human striata cDNA library. Oligonucleotide probes were designed based on the rat A3 sequence of Zhou et al., Proc. Natl. Acad. Sci. 89, 7432 (1992). The complete sequence of the human A3 adenosine receptor was determined and the protein sequence deduced. The cloned human A3 adenosine receptor is expressed in a heterologous expression system in COS, CHO and HEK 293 cells. Radiolabeled adenosine receptor agonists and antagonists are used to measure the binding properties of the expressed receptor. Stable cell lines can be used to evaluate and identify adenosine receptor agonists, antagonists and enhancers.

STEP A: A synthetic probe homologous to the rat A3 adenosine receptor was generated using the polymerase chain reaction (PCR).

Three CL1 of rat brain cDNA was used as template in a PCR amplification reaction according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT) containing 50 pmol of primers 207 (5'cccaagcttatgaaagccaacaatacc) (SEQ. ID NO: 27) and 208 (5'tgctctagactctggtatcttcacatt) (SEQ. ID NO: 28) in a total volume of 50 ml. Primers 207 and 208 are based on the published rat A3 adenosine receptor sequence (Zhou, et al, (1992), Proc. Natl. Acad. Sci. USA, 89:7432-7406). Forty cycles of 40 sec at 94"C, 1 min at 55 CC, 3 min at 72"C were performed and the resulting 788 bp fragment was subcloned into HindE-XbaI digested pBLUESCRIPT II KS+ (STRATAGENE, La Jolla, CA). The sequence was verified by the SEQUENASE protocol (USBC, Cleveland, OH).

STEP B: The 788 bp PCR fragment was labeled with a32P-dCTP using the MULTIPRIME DNA LABELLING SYSTEM (AMERSHAM, Arlington Heights, IL) and used to screen a human striata cDNA library (STRATAGENE, La Jolla, CA). E. coli strain XL-1 Blue (STRATAGENE, La Jolla, CA) cells were infected with library phage and grown overnight at 37"C. Phage DNA was transferred to HYBOND-N nylon filters according to the manufacturer's protocol (AMERSHAM, Arlington Heights, IL). The probe was incubated with the filters in 5 X SSC, 30% formamide, 5 X Denhardt's solution, 0.5% sodium dodecyl sulfate, and 50 mg/ml sonicated salmon testis DNA.

The filters were washed in 2 X SSC at 55"C. A positively hybridizing phage (HS-21a) was identified and plaque purified by two additional rounds of plating and hybridization. The insert was subcloned to the plasmid pBLUESCRIPT II SK- according to the manufacturer's protocol (STRATAGENE, La Jolla, CA). Upon sequence analysis using the SEQUENASE protocol (USBC, Cleveland, OH) it was determined that clone HS-21a contained the complete open hydroxyethyl) -2-aminoethane sulfonic acid buffered saline) at room temperature for 30 minutes. The DNA precipitate was added to the COS7 cells and incubated for 18 h in 5% CO2 at 37"C. The precipitate was removed and the cells were washed twice with serum free DMEM.

Cells were incubated in complete medium in 5% CO2 at 37"C for 48 h prior to the binding assay.

Stable expression in CHO cells: To establish stable cell lines, CHO cells were cotransfected with 20 llg of pSVL containing the adenosine receptor cDNA and 1 ,ug of pWLneo (STRATAGENE) containing the neomycin gene.

Transfection was performed by the CaPO4 method. DNA was precipitated at room temperature for 30 minutes, added to the COS7 cells and incubated 18 h in 5% CO2 at 37"C. The precipitate was removed and the cells were washed twice with serum free DMEM.

Cells were incubated for 24 h in 5% CO2 at 37"C, replated in 24-well dishes at a dilution of 1:10, and incubated an additional 24 h before adding selection medium, DMEM containing 10% fetal bovine serum, 100U/mL penicillin-streptomycin, 2 mM glutamine and 1.0 mg/mL G41 8 (GIBCO). Transfected cells were incubated at 5% CO2, 37"C until viable colonies were visible, approximately 14-21 days. Colonies were selected and propagated. The cell clone with the highest number of human adenosine receptors was selected for subsequent application in the binding assay.

EXAMPLE 6 Binding assav: Membranes were prepared from transiently transfected COS7 cells 48 h after transfection or from G418-selected stably transfected CHO or HEK 293 cells. Cells were harvested in 1 mM EDTA in phosphate buffered saline and centrifuged at 2000 x g for 10 minutes. The cell pellet was washed once with phosphate buffered saline. The cell pellet was resuspended in 2 mL of 5 mM Tris, pH 7.6/ 5mM MgC12. Membranes were prepared from the cells by freeze-thaw lysis in which the suspension was frozen in a dry ice/ethanol bath and thawed at 25"C twice. The suspension was homogenized after adding an additional 2 mL of 5 mM Tris, pH 7.6/ 5mM MgCl2, in a glass dounce homogenizer with 20 strokes. The membranes were pelleted at 40,000 x g at 4"C for 20 minutes. The membrane pellet was resuspended at a protein concentration of 1-2 mg/mL in binding assay buffer, 50 mM Tris, pH 7.6/10 mM MgCl2. Protein concentration was determined by the method of Bradford ((1976) Anal. Biochem. 72: 248-250). Before the binding assay was performed, the membranes were incubated with adenosine deaminase (BOEHRINGER MANNHEIM), 2U/mL for 30 minutes at 37"C. Saturation binding of [125I]-N6-aminobenzyladenosine (125I-ABA) or [125I]-N6-2-(4-amino-3-iodophenyl)ethyl- adenosine (APNEA) was performed on membranes prepared from pSVLA3 transfected COS7 cells. Membranes (100 Rg) were incubated in the presence of 0.2U/mL adenosine deaminase with increasing concentrations of 1251-ABA in the range of 0.1-30 nM for 120 minutes at 25"C in a total volume of 500 ,eL. The binding assay was terminated by rapid filtration and three washes with ice-cold 50 mM Tris, pH 7.6/10 mM MgC12 on a Skatron cell harvester equipped with a receptor binding filtermat (SKATRON INSTRUMENTS, INC). Non-specific binding was determined on non-transfected cells. Bound radioactivity was measured by scintillation counting in Ready Safe Scintillation Cocktail (BECKMAN).

EXAMPLE 7 In vitro transcription and oocvte expression: The 1.3 kb XhoI-BamHI fragment of the pSVL expression construct (described in Example 2) containing the full length human A2a adenosine receptor coding sequence was ligated into Sall-Spel digested pGEMA (Swanson, et al, (1990) Neuron 4:929-939). The resulting plasmid, pGEMA2, was linearized with NotI, forming a template for in vitro transcription with T7 RNA polymerase. The homologous adenosine receptor subtype cDNA in pBluescript SK- was used as a template for in vitro transcription by T3 polymerase after removal of most of the 5' untranslated region, with the exception of 20 bp, as a 0.3 kb Smal fragment. The K+ channel cDNA, Kv3.2b was employed as a negative control in the cAMP accumulation assay. The generation of Kv3.2b RNA was described by Luneau, et al, ((1991) FEBS Letters 1:163-167). Linearized plasmid templates were used with the STRATAGENE mCAP kit according to the manufacturer's protocol, except that the SP6 RNA polymerase reaction was performed at 40"C. Oocytes were harvested from mature female Xenopus laevis, treated with collagenase, and maintained at 18"C in ND96 medium (GIBCO) supplemented with 1 mM sodium pyruvate and 100 mg/mL gentamycin. Fifty nanoliters (10 ng) of RNA diluted in H2O was injected and oocytes were incubated at 18"C for 48 hours.

EXAMPLE 8 cAMP accumulation assay in oocvtes: Oocytes injected with either human adenosine receptor transcript or the Kv3.2b transcript were transferred to fresh medium supplemented with 1 mM of the phosphodiesterase inhibitor, Ro 201724 (RBI, Natick, MA) and 1 mg/mL bovine serum albumin incubated for 30 minutes and transferred to an identical medium with or without the agonist adenosine (10 mM) for an additional 30 minutes at room temperature. Groups of 5-10 oocytes were lysed by transfer to ND96/100 mM HCl/1 mM Ro 20-1724 in microfuge tubes, shaken, incubated at 95"C for 3 min, and centrifuged at 12000 g for 5 min.

Supernatants were stored at -70 C before cAMP measurements. Cyclic AMP levels were determined by radioimmunoassay (RIANEN kit, DuPont/NEN) using the acetylation protocol. The adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (100 FM) was utilized to inhibit the cAMP response induced by adenosine in oocytes expressing the adenosine receptors.

EXAMPLE 9 cAMP accumulation in stable CHO cell lines: The changes in cAMP accumulation can alternatively be measured in stably transfected CHO cells expressing the human adenosine receptor subtypes. CHO cells are washed twice in phosphate buffered saline (PBS) and detached in 0.2% EDTA in PBS. The cells are pelleted at 800 rpm for 10 min and resuspended in KRH buffer (140 mM NaCl/5 mM KCl/2 mM CaCl2/1.2 mM MgSO4/1.2 mM KH2PO4/6 mM glucose/25 mM Hepes buffer, pH 7.4). The cells are washed once in KRH buffer and resuspended at 107 cells/mL. The cell suspension (100 ,uL) is mixed with 100 RL of KRH buffer containing 200 mM Ro 20-1724 and incubated at 37"C for 10 minutes. Adenosine (10 mM), NECA or CPCA was added in 200 RL KRH buffer containing 200 RM Ro 20-1724 and incubated at 37"C for 20 minutes. After the incubation, 400 mL of 0.5 mM NaOAc (pH 6.2) was added and the sample was boiled for 20 minutes. The supernatant was recovered by centrifugation for 15 minutes and stored at -70 C. cAMP levels were determined by radioimmunoassay (RIANEN kit, DuPont/NEN) using the acetylation protocol. The effect of antagonists on cAMP accumulation are measured by preincubation for 20 minutes before adding adenosine.

EXAMPLE 10 Expression Construct and Transfection The 1.7 kb HS-21a cDNA (A3) was subcloned as a SalI BamHl fragment into the expression vector pCMV5 (Mumby, S.M., Heukeroth, R.O., Gordon, J.I.and Gilman, A.G. (1990) Proc. Natl.

Acad. Sci. USA 87, 728-732.) creating the vector pCMV5-A3. CHO or HEK 293 cells stably expressing the human HS-21a cDNA were prepared by co-transfection of 15 ,ug pCMV5-A3 and 1 Fg pWLneo (Stratagene) using the calcium phosphate method. Stable cell lines were also generated using EBV based mammalian expression vectors, pREP (INVITROGEN). Neomycin resistant colonies were selected in 1 mg/mL G418 (GIBCO). Stable colonies were screened for expression of HS-21a by 125I-ABA binding.

EXAMPLE 11 Binding Studies Membranes were prepared from stable CHO cell lines in 10 mM Hepes, pH 7.4 containing 0.1 mM benzamidine and 0.1 mM PMSF as described (Mahan, L.C., et al., (1991) Mol. Pharmacol. 40, 17). Pellets were resuspended in 5 mM Hepes, pH 7.4/5 mM MgC12/0.1 mM benzamidine/0.1 mM PMSF at a protein concentration of 1-2 mg/mL and were incubated with adenosine deaminase (Boehringer Mannheim), 2U/mL at 37 oC for 20 minutes. Saturation binding of 125I- ABA was carried out on 50 mg of membranes for 120 minutes at 25 C in a total volume of 100 RL. The assay was terminated by rapid filtration and three washes with ice-cold binding buffer on a Skatron harvester equipped with a receptor binding filtermat (Skatron Instruments, INC). The specific activity of 125I-ABA, initially 2,200 Ci/mmol, was reduced to 100 Ci/mmol with nonradioactive I-ABA for saturation analysis. Nonspecific binding was measured in the presence of 1 mM I-ABA. The KD and Bmax values were calculated by the EBDA program (McPherson, G.A. (1983) Computer Programs for Biomedicine 17, 107-114). Competition binding of agonists and antagonists was determined with 125I-ABA (0.17-2.0 nM, 2000 Ci/mmol). Nonspecific binding was measured in the presence of 400 mM NECA. Binding data were analyzed and competition curves were constructed by use of the nonlinear regression curve fitting program Graph PAD InPlot, Version 3.0 (Graph Pad Software, San Diego). Ki values were calculated using the Cheng-Prusoff derivation (Cheng, Y.C.

and Prusoff, H.R. (1973) Biochem. Pharmacol. 22, 3099-3108.).

The binding properties of the receptor encoded by HS-21a were evaluated on membranes prepared from CHO cells stably expressing the HS-21a cDNA. The radioligand, 125I-APNEA, was previously used to characterize rat A3 adenosine receptors. In preliminary experiments, high non-specific 125I-APNEA binding to CHO cell membranes was observed which interfered with the measurement of specific binding to expressed receptors. Specific and saturable binding of the adenosine receptor agonist, 125I-ABA was measured on membranes prepared from the stably transfected cells (Figure 11A). The specific binding of 125I-ABA could be prevented by either 1 mM nonradioactive I-ABA or 400 RM NECA. No specific binding of 125I-ABA was measured on membranes prepared from nontransfected CHO cells. The specific binding of 125I-ABA measured in either the presence of 10 M GTPyS or 100 RM Gpp(NH)p was reduced by 56 and 44% respectively, relative to the specific binding measured in the absence of the uncoupling reagents. These results suggest that 1251 ABA exhibits some agonist activity on the receptor encoded by the HS 21 a cDNA expressed in the stable CHO cell line. 1251-ABA binds to membranes prepared from the HS-21a stable CHO cells with a dissociation constant of 10 nM (Bmax 258 fmol/mg protein) with a Hill coefficient of 0.99 indicating binding to a single class of high affinity sites (Figure 11B).

The competition of adenosine receptor agonists and antagonists for binding to HS-21a receptors was determined (Figure 12). The Ki values for agonists (top panel) were calculated to be 26 nM for NECA, 34 nM for R-PIA, 89 nM for CPA and 320 nM for S-PIA, resulting in a potency order profile of NECA > R-PIA > CPA > S-PIA.

In contrast to the insensitivity of adenosine receptor antagonists reported for the rat A3 adenosine receptor subtype, a number of xanthine antagonists exhibited competition with 125I-ABA for binding to the HS-21a receptor (Figure 12, lower panel). Studies of the sheep A3 adenosine receptor indicated that 8-phenylxanthines substituted in the para-position with acidic substituents are high affinity antagonists.

By evaluating additional xanthines in this class I-ABOPX was determined to be the highest affmity antagonist yet reported for A3 adenosine receptors. The Ki values for antagonists were calculated to be 18 nM for I-ABOPX, 55 nM for BW-A1433, 70 nM for XAC and 750 nM for DPCPX, resulting in a potency order profile of I-ABOPX > BW-A1433 > XAC > DPCPX.

EXAMPLE 12 cAMP Studies Determinations were made on stably transfected CHO cells in suspension as described (Linden et al., (1993) Mol. Pharm. 44:524532). Supernatants (500 FL) were acetylated and acetylcyclic AMP was measured by automated radioimmunoassay (Hamilton, B.R. and Smith, D.O. (1991) J. Physiol. (Lond.) 432, 327-341). Antagonist dissociation constants were estimated from pA2 values as described by Schild (1957) Pharm. Rev. 9, 242-246).

EXAMPLE 13 Northern Blot Analvsis Human poly(A)+ RNA from different tissue sources (Clontech) is fractionated on a 1 % agarose-formaldehyde gel (Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Press, Cold Spring Harbor, NY), transferred to Hybond-N membranes and hybridized in SXSSPE, SXDenhardt's, 0.5% SDS, 50 mg/mL sonicated salmon testis DNA, with 30% formamide (for Al, A2a, and A2b) or 50% formamide (for HS-21a) at 42oC. DNA probes corresponding to nucleotides 512-1614, 936-2168, and 321-1540 of accession numbers X68485(A1), X68486(A2a), and X68487(A2b) respectively, and a 1.7 kb SalI-BamHI fragment of HS-21a were labeled with a32P-dCTP by the random priming method. Filters were washed under high stringency conditions in 0.1XSSC at 65oC.

EXAMPLE 14 INHIBITION OF TNFa PRODUCTION STEP A: Isolation of human peripheral blood mononuclear cells.

Human blood is obtained by venipuncture from healthy donors and collected into tubes containng 20U/mL of heparin sodium salt. The blood is diluted 1:1 with Hanks balanced salts solution containing 20 U/mL Heparin. Peripheral blood mononulear cells (PBMC) are isolated by Ficoll-Hypaque density centrifugation. The PBMC are resuspended in a small volume (2-5 mL) of RPMI + 10% autologous human serum, counted then diluted further with RPMI + 10% autologous human serum to 5 x 105 cells/mL. Subsequently the cells are plated in a six well Costar plastic plate precoated with 1 mg / mL fibronectin.

Lipopolysaccharide, as well as the appropriate adenosine agonists and antagonists, are added simultaneously. Following incubation at 37"C for 18 hours, the cell culture supernatants are harvested, clarified and tested for TNF levels by a specific trapping ELISA.

STEP B: ELISA for human TNFa.

A mouse anti-human TNFa monoclonal antibody is diluted to 0.5 mg/mL in PBS - MgCl2 - CaCl2 and added to plastic 96 - well plates.

Following a 24 hr incubation at 4"C the plates are washed with PBS Tween then treated with a solution of PBS and 1 % BSA. Following additional washing with PBS Tween, aliquots of monocytes thought to contain TNFa are added to the dish, diluted to 100 mL with PBS tween and incubated for 2 hours at 370C. The plates are further washed with PBS-Tween, then treated with a 1 to 2000 dilution of rabbit anti-human TNF polyclonal antiserum (Genzyme). The plates are incubated for 1 hour, washed then treated again with a goat anti-rabbit IgG Fabhorseradish peroxidase conjugate. The plates are incubated for one hour, washed, and the bound peroxidase is detected by the additon of a TMB peroxide mixture. TNFa levels are determined by comparison with a standard curve generated uisng pure recombinant TNFa.

EXAMPLE 15 DETECTION OF ADENOSINE RECEPTOR TRANSCRIPTS BY REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION AMPLIFICATION STEP A: Total RNA was extracted by the guanidinium isothiocyanate method (Chirgwin, J.M., et al, (1979) Biochemistry 18:5294-5299) from normal and LPS-stimulated human monocytes. First strand cDNA was reverse transcribed from 600 ng total RNA in a volume of 20 ml containing 20mM Tris-HCL (pH 8.4), 50mM KCl, 2.5mM MgCl2, 0.1mg/ml bovine serum albumin (BSA), 0.5mM dNTP's, 10 mM DTT, 10 units SUPERSCRIPT II reverse transcriptase (LIFE TECHNOLOGIES, INC., Gathersburg, MD), and 50ng random hexamers.

STEP B: Human adenosine receptor subtype transcript expression was determined using the polymerase chain reaction (PCR). Three Rl of the randomly primed first strand cDNA, prepared from monocytes (+) or (-) LPS stimulation, was used as template in a PCR amplification.

reaction according to the GENEAMP protocol (PERKIN ELMER CETUS, Norwalk, CT) containing 50pmol subtype selective primers in a total volume of 100 Ill. Primer pairs were designed to span four (Al primers) and five (A2a, A2b, A3 primers) transmembrane domains and gave no or incorrect sized PCR products when tested on human genomic DNA. Primer pairs for amplification (see Table 1) were 266+267 (awl), 253+254 (A2a), 261+262 (A2b), 230+236 (A3), and 141+142 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers 141+142 are based on the published human GAPDH sequence (Tokunaga, K., et al, (1987) Cancer Research 47:5616-5619). Cycling parameters were 1 min at 94oC, 1 min at 55oC, 3 min at 720C for 35 cycles (Al), 25 cycles (A2a), 35 cycles (A3), and 20 cycles (GAPDH).

Cycling parameters for A2b were 1 min at 94oC, 1 min at 590 C, 3 min at 720C for 30 cycles.

STEP C: Ten Rl of each PCR amplification reaction was elecrophoresed on a 1.4% agarose gel and alkaline blotted to Zeta-Probe GT membranes according to the manufacturer's protocol (BIO-RAD, Hercules, CA). Membranes were hybridized in 0.25 M sodium phosphate (pH 7.2), 0.5M NaCl,7.0% sodium dodecyl sulphate (SDS), 1 mM EDTA, 1% BSA, and 1x106 cpm/ml 32P labeled probe at 50 C.

Double-stranded DNA probes were generated by Klenow enzyme extension of annealed oligonucleotide pairs including a32P-dCTP.

Oligonucleotide pairs for probe synthesis (see Tablel) were 268+269 (Al), 66+67 (A2a), 263+264 (A2b), 259+260 (A3), and 143+144 (GAPDH). Oligonucleotides 259+260 are based on the published sheep A3 adenosine receptor (Linden, J., et al, (1993) Molecular Pharmacology 44:524-532) and 143+144 on the human GAPDH sequence (Tokunaga et al). Following hybridization membranes were washed to a final stringency of 75mM NaCl, 7.5mM sodium citrate, 0.1% SDS and exposed to autoradiography film. All four adenosine receptor subtypes were found to be present on monocytes through this analysis.

TABLE1 NAME SEQUENCE 66 5'GCCTCTTTGAGGATGTGGTCCCCATGAACTACATGGTGTACTTCA 67 5'GCAGGGGCACCAGCACACAGGCAAAGAAGTTGAAGTACACCATGT 141 5' TCACCATC?TCCAGGAGC 142 5' ACTCCrTGGAGGCCATGT 143 5'TCCTGCACCACCAACTGCTTAGCCCCCCTGGCCAAGGTCATCCAT 144 5'CATGAGCCCTTCCACGATGCCAAAGTTGTCATGGATGACCTTGGC 230 5' GTTACCTACATCACCAT6 236 5'GTTAGATAAGTTCAGACT 253 5' TCCTCGGTGTACATCACG 254 5'TCCATCTGCTTCAGCTGT 259 5'CTGGGCCTTTGCTGGCTGGTGTCATTCCTGGTGGGATTGACCCCC 260 5'TGAGGTCAGTTTCATGTTCCAGCCAAACATGGGGGTCAATCCCAC 261 5'ATGCTGCTGGAGACACAGGA 262 5' TGGTCCATCAGCTCAGTGC 263 5' GGTGGAACAGTAAAGACAGTGCCACCAACAACTGCACAGAACCCTGGGATGGAACCACGA 264 5' GGACCACATrCTCAAAGAGACACTTCACAAGGCAGCAGcTITCATrCGTGGTTCCATCCC 266 5' CTACATCGGCATCGAGGT 267 5'GAACTCGCACTTGATCAC 268 5'TGGTGGGACTGACCCCTATGTTTGGCTGGAACAATCTGAGTGCGG 269 5'TGCTGCCGTTGGCTGCCCAGGCCCGCTCCACCGCACTCAGATTGT While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, modifications, as come within the scope of the following claims and its equivalents.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Jacobson, Marlene A (ii) TITLE OF INVENTION: INHIBITION OF TNFalpha PRODUCTION BY A2b ADENOSINE RECEPTOR AGONISTS AND ENHANCERS (iii) NUMBER OF SEQUENCES: 56 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Merck & Co., Inc.

(B) STREET: P.O.Box 2000 (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: United States (F) ZIP: 07065 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release &num;1.0, Version &num;1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 6-MAY-1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Bencen, Gerard H (B) REGISTRATION NUMBER: 35,746 (C) REFERENCE/DOCKET NUMBER: 19222 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (908) 594-3901 (B) TELEFAX: (908)594-4720 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATTCGCAGCC ACGTCCTGAG GCGGCGGGAG CCCTTCAAAG CAGGTGGCAC CAGTGCCCGC 60 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GCGGAGGCTG ATCTGCTCTC CATCACTGCC ATGAGCTGCC AAGGCGCGGG CACTGGTGCC 60 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCCAGAAGTT CCGGGTCACC TTCCTTAAGA TCTGGAATGA CCACTTCCGC TGCCAGCCCA 60 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGTCGTGGGG CGCCTCCTCT GGGGGGTCCT CGTCGACGGG GGGCGTGGGC TGGCAGCGGA 60 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCCTCTTTGA GGATGTGGTC CCCATGAACT ACATGGTGTA CTTCA 45 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCAGGGGCAC CAGCACACAG GCAAAGAAGT TGAAGTACAC CATGT 45 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCGCGCCGCC AGGAAGAT 18 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TATATTGAAT TCTAGACACC CAGCATGAGC 30 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TCAATGGCGA TGGCCAGG 18 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TATATTGAAT TCATGGAGCT CTGCGTGAGG 30 (2) INFORMATION FOR SEQ ID NO:ll: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GTAGACCATG TACTCCAT 18 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TATATTGAAT TCTGACCTTC TCGAACTCGC 30 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATTGAATTCG ATCACGGGCT CCCCCATGC 29 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ATGGAGTACA TGGTCTACTT CAACTTCTTT GTGTGGGTGC TGCCCCCGCT 50 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GAAGATCCGC AAATAGACAC CCAGCATGAG CAGAAGCGGG GGCAGCACCC 50 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CCCTCTAGAG CCCAGCCTGT GCCCGCCATG CCCATCATGG GCTCC 45 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CCCACCTTTT GAGCAAGTTC 20 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGCTTATGAG TATTTCTTCC 20 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met Pro Pro Ser Ile Ser Ala Phe Gln Ala Ala Tyr Ile Gly Ile Glu 1 5 10 15 Val Leu Ile Ala Leu Val Ser Val Pro Gly Asn Val Leu Val Ile Trp 20 25 30 Ala Val Lys Val Asn Gln Ala Leu Arg Asp Ala Thr Phe Cys Phe Ile 35 40 45 Val Ser Leu Ala Val Ala Asp Val Ala Val Gly Ala Leu Val Ile Pro 50 55 60 Leu Ala Ile Leu Ile Asn Ile Gly Pro Gln Thr Tyr Phe His Thr Cys 65 70 75 80 Leu Met Val Ala Cys Pro Val Leu Ile Leu Thr Gln Ser Ser Ile Leu 85 90 95 Ala Leu Leu Ala Ile Ala Val Asp Arg Tyr Leu Arg Val Lys Ile Pro 100 105 110 Leu Arg Tyr Lys Met Val Val Thr Pro Arg Arg Ala Ala Val A Ala Gly Cys Trp Ile Leu Ser Phe Val Val Gly Leu Thr Pro Met Phe 130 135 140 Gly Trp Asn Asn Leu Ser Ala Val Glu Arg Ala Trp Ala Ala Asn Gly 145 150 155 160 Ser Met Gly Glu Pro Val Ile Lys Cys- Glu Phe Glu Lys Val Ile Ser 165 170 175 Met Glu Tyr Met Val Tyr Phe Asn Phe Phe Val Trp Val Leu Pro Pro 180 185 190 Leu Leu Leu Met Val Leu Ile Tyr Leu Glu Val Phe Tyr Leu Ile Arg 195 200 205 Lys Gln Leu Asn Lys Lys Val Ser Ala Ser Ser Gly Asp Pro Gln Lys 210 215 220 Tyr Tyr Gly Lys Glu Leu Lys Ile Ala Lys Ser Leu Ala Leu Ile Leu 225 230 235 240 Phe Leu Phe Ala Leu Ser Trp Leu Pro Leu His Ile Leu Asn Cys Ile 245 250 255 Thr Leu Phe Cys Pro Ser Cys His Lys Pro Ser Ile Leu Thr Tyr Ile 260 265 270 Ala Ile Phe Leu Thr His Gly Asn Ser Ala Met Asn Pro Ile Val Tyr 275 280 285 Ala Phe Arg Ile Gln Lys Phe Arg Val Thr Phe Leu Lys Ile Trp Asn 290 295 300 Asp His Phe Arg Cys Gln Pro Ala Pro Pro Ile Asp Glu Asp Leu Pro 305 310 315 320 Glu Glu Arg Pro Asp Asp 325 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 981 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATGCCGCCCT CCATCTCAGC TTTCCAGGCC GCCTACATCG GCATCGAGGT GCTCATCGCC 60 CTGGTCTCTG TGCCCGGGAA CGTGCTGGTG ATCTGGGCGG TGAAGGTGAA CCAGGCGCTG 120 CGGGATGCCA CCTTCTGCTT CATCGTGTCG CTGGCGGTGG CTGATGTGGC CGTGGGTGCC 180 CTGGTCATCC CCCTCGCCAT CCTCATCAAC ATTGGGCCAC AGACCTACTT CCACACCTGC 240 CTCATGGTTG CCTGTCCGGT CCTCATCCTC ACCCAGAGCT CCATCCTGGC CCTGCTGGCA 300 ATTGCTGTGG ACCGCTACCT CCGGGTCAAG ATCCCTCTCC GGTACAAGAT GGTGGTGACC 360 CCCCGGAGGG CGGCGGTGGC CATAGCCGGC TGCTGGATCC TCTCCTTCGT GGTGGGACTG 420 ACCCCTATGT TTGGCTGGAA CAATCTGAGT GCGGTGGAGC GGGCCTGGGC AGCCAACGGC 480 AGCATGGGGG AGCCCGTGAT CAAGTGCGAG TTCGAGAAGG TCATCAGCAT GGAGTACATG 540 GTCTACTTCA ACTTCTTTGT GTGGGTGCTG CCCCCGCTTC TCCTCATGGT CCTCATCTAC 600 CTGGAGGTCT TCTACCTAAT CCGCAAGCAG CTCAACAAGA AGGTGTCGGC CTCCTCCGGC 660 GACCCGCAGA AGTACTATGG GAAGGAGCTG AAGATCGCCA AGTCGCTGGC CCTCATCCTC 720 TTCCTCTTTG CCCTCAGCTG GCTGCCTTTG CACATCCTCA ACTGCATCAC CCTCTTCTGC 780 CCGTCCTGCC ACAAGCCCAG CATCCTTACC TACATTGCCA TCTTCCTCAC GCACGGCAAC 840 TCGGCCATGA ACCCCATTGT CTATGCCTTC CGCATCCAGA AGTTCCGCGT CACCTTCCTT 900 AAGATTTGGA ATGACCATTT CCGCTGCCAG CCTGCACCTC CCATTGACGA GGATCTCCCA 960 GAAGAGAGGC CTGATGACTA G 981 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 412 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Met Pro Ile Met Gly Ser Ser Val Tyr Ile Thr Val Glu Leu Ala Ile 1 5 10 15 Ala Val Leu Ala Ile Leu Gly Asn Val Leu Val Cys Trp Ala Val Trp 20 25 30 Leu Asn Ser Asn Leu Gln Asn Val Thr Asn Tyr Phe Val Val Ser Leu 35 40 45 Ala Ala Ala Asp Ile Ala Val Gly Val Leu Ala Ile Pro Phe Ala Ile 50 55 60 Thr Ile Ser Thr Gly Phe Cys Ala Ala Cys His Gly Cys Leu Phe Ile 65 70 75 80 Ala Cys Phe Val Leu Val Leu Thr Gln Ser Ser Ile Phe Ser Leu Leu 85 90 95 Ala Ile Ala Ile Asp Arg Tyr Ile Ala Ile Arg Ile Pro Leu Arg Tyr 100 105 110 Asn Gly Leu Val Thr Gly Thr Arg Ala Lys Gly Ile Ile Ala Ile Cys 115 120 125 Trp Val Leu Ser Phe Ala Ile Gly Leu Thr Pro Met Leu Gly Trp Asn 130 135 140 Asn Cys Gly Gln Pro Lys Glu Gly Lys Asn His Ser Gln Gly Cys Gly 145 150 155 160 Glu Gly Gln Val Ala Cys Leu Phe Glu Asp Val Val Pro Met Asn Tyr 165 170 175 Met Val Tyr Phe Asn Phe Phe Ala Cys Val Leu Val Pro Leu Leu Leu 180 185 190 Met Leu Gly Val Tyr Leu Arg Ile Phe Leu Ala Ala Arg Arg Gln Leu 195 200 205 Lys Gln Met Glu Ser Gln Pro Leu Pro Gly Glu Arg Ala Arg Ser Thr 210 215 220 Leu Gln Lys Glu Val His Ala Ala Lys Ser Leu Ala Ile Ile Val Gly 225 230 235 240 Leu Phe Ala Leu Cys Trp Leu Pro Leu His Ile Ile Asn Cys Phe Thr 245 250 255 Phe Phe Cys Pro Asp Cys Ser His Ala Pro Leu Trp Leu Met Tyr Leu 260 265 270 Ala Ile Val Leu Ser His Thr Asn Ser Val Val Asn Pro Phe Ile Tyr 275 280 285 Ala Tyr Arg Ile Arg Glu Phe Arg Gln Thr Phe Arg Lys Ile Ile Arg 290 295 300 Ser His Val Leu Arg Gln Gln Glu Pro Phe Lys Ala Ala Gly Thr Ser 305 310 315 320 Ala Arg Val Leu Ala Ala His Gly Ser Asp Gly Glu Gln Val Ser Leu 325 330 335 Arg Leu Asn Gly His Pro Pro Gly Val Trp Ala Asn Gly Ser Ala Pro 340 345 350 His Pro Glu Arg Arg Pro Asn Gly Tyr Ala Leu Gly Leu Val Ser Gly 355 360 365 Gly Ser Ala Gln Glu Ser Gln Gly Asn Thr Gly Leu Pro Asp Val Glu 370 375 380 Leu Leu Ser His Glu Leu Lys Gly Val Cys Pro Glu Pro Pro Gly Leu 385 390 395 400 Asp Asp Pro Leu Ala Gln Asp Gly Ala Gly Val Ser 405 410 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1239 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATGCCCATCA TGGGCTCCTC GGTGTACATC ACGGTGGAGC TGGCCATTGC TGTGCTGGCC 60 ATCCTGGGCA ATGTGCTGGT GTGCTGGGCC GTGTGGCTCA ACAGCAACCT GCAGAACGTC 120 ACCAACTACT TTGTGGTGTC ACTGGCGGCG GCCGACATCG CAGTGGGTGT GCTCGCCATC 180 CCCTTTGCCA TCACCATCAG CACCGGGTTC TGCGCTGCCT GCCACGGCTG CCTCTTCATT 240 GCCTGCTTCG TCCTGGTCCT CACGCAGAGC TCCATCTTCA GTCTCCTGGC CATCGCCATT 300 GACCGCTACA TTGCCATCCG CATCCCGCTC CGGTACAATG GCTTGGTGAC CGGCACGAGG 360 GCTAAGGGCA TCATTGCCAT CTGCTGGGTG CTGTCGTTTG CCATCGGCCT GACTCCCATG 420 CTAGGTTGGA ACAACTGCGG TCAGCCAAAG GAGGGCAAGA ACCACTCCCA GGGCTGCGGG 480 GAGGGCCAAG TGGCCTGTCT CTTTGAGGAT GTGGTCCCCA TGAACTACAT GGTGTACTTC 540 AACTTCTTTG CCTGTGTGCT GGTGCCCCTG CTGCTCATGC TGGGTGTCTA TTTGCGGATC 600 TTCCTGGCGG CGCGACGACA GCTGAAGCAG ATGGAGAGCC AGCCTCTGCC GGGGGAGCGG 660 GCACGGTCCA CACTGCAGAA GGAGGTCCAT GCTGCCAAGT CACTGGCCAT CATTGTGGGG 720 CTCTTTGCCC TCTGCTGGCT GCCCCTACAC ATCATCAACT GCTTCACTTT CTTCTGCCCC 780 GACTGCAGCC ACGCCCCTCT CTGGCTCATG TACCTGGCCA TCGTCCTCTC CCACACCAAT 840 TCGGTTGTGA ATCCCTTCAT CTACGCCTAC CGTATCCGCG AGTTCCGCCA GACCTTCCGC 900 AAGATCATTC GCAGCCACGT CCTGAGGCAG CAAGAACCTT TCAAGGCAGC TGGCACCAGT 960 GCCCGGGTCT TGGCAGCTCA TGGCAGTGAC GGAGAGCAGG TCAGCCTCCG TCTCAACGGC 1020 CACCCGCCAG GAGTGTGGGC CAACGGCAGT GCTCCCCACC CTGAGCGGAG GCCCAATGGC 1080 TATGCCCTGG GGCTGGTGAG TGGAGGGAGT GCCCAAGAGT CCCAGGGGAA CACGGGCCTC 1140 CCAGACGTGG AGCTCCTTAG CCATGAGCTC AAGGGAGTGT GCCCAGAGCC CCCTGGCCTA 1200 GATGACCCCC TGGCCCAGGA TGGAGCAGGA GTGTCCTGA 1239 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 332 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 216 (D) OTHER INFORMATION: /label= threonine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Leu Leu Glu Thr Gln Asp Ala Leu Tyr Val Ala Leu Glu Leu Val 1 5 10 15 Ile Ala Ala Leu Ser Val Ala Gly Asn Val Leu Val Cys Ala Ala Val 20 25 30 Gly Thr Ala Asn Thr Leu Gln Thr Pro Thr Asn Tyr Phe Leu Val Ser 35 40 45 Leu Ala Ala Ala Asp Val Ala Val Gly Leu Phe Ala Ile Pro Phe Ala 50 55 60 Ile Thr Ile Ser Leu Gly Phe Gys Thr Asp Phe Tyr Gly Cys Leu Phe 65 70 75 80 Leu Ala Cys Phe Val Leu Val Leu Thr Gln Ser Ser Ile Phe Ser Leu 85 90 95 Leu Ala Val Ala Val Asp Arg Tyr Leu Ala Ile Cys Val Pro Leu Arg 100 105 110 Tyr Lys Ser Leu Val Thr Gly Thr Arg Ala Arg Gly Val Ile Ala Val 115 120 125 Leu Trp Val Leu Ala Phe Gly Ile Gly Leu Thr Pro Phe Leu Gly Trp 130 135 140 Asn Ser Lys Asp Ser Ala Thr Asn Asn Cys Thr Glu Pro Trp Asp Gly 145 150 155 160 Thr Thr Asn Glu Ser Cys Cys Leu Val Lys Cys Leu Phe Glu Asn Val 165 170 175 Val Pro Met Ser Tyr Met Val Tyr Phe Asn Phe Phe Gly Cys Val Leu 180 185 190 Pro Pro Leu Leu Ile Met Leu Val Ile Tyr Ile Lys Ile Phe Leu Val 195 200 205 Ala Cys Arg Gln Leu Gln Arg Xaa Glu Leu Met Asp His Ser Arg Thr 210 215 220 Thr Leu Gln Arg Glu Ile His Ala Ala Lys Ser Leu Ala Met Ile Val 225 230 235 240 Gly Ile Phe Ala Leu Cys Trp Leu Pro Val His Ala Val Asn Cys Val 245 250 255 Thr Leu Phe Gln Pro Ala Gln Gly Lys Asn Lys Pro Lys Trp Ala Met 260 265 270 Asn Met Ala Ile Leu Leu Ser His Ala Asn Ser Val Val Asn Pro Ile 275 280 285 Val Tyr Ala Tyr Arg Asn Arg Asp Phe Arg Tyr Thr Phe His Lys Ile 290 295 300 Ile Ser Arg Tyr Leu Leu Cys Gln Ala Asp Val Lys Ser Gly Asn Gly 305 310 315 320 Gln Ala Gly Val Gln Pro Ala Leu Gly Val Gly Leu 325 330 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 999 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATGCTGCTGG AGACACAGGA CGCGCTGTAC GTGGCGCTGG AGCTGGTCAT CGCCGCGCTT 60 TCGGTGGCGG GCAACGTGCT GGTGTGCGCC GCGGTGGGCA CGGCGAACAC TCTGCAGACG 120 CCCACCAACT ACTTCCTGGT GTCCCTGGCT GCGGCCGACG TGGCCGTGGG GCTCTTCGCC 180 ATCCCCTTTG CCATCACCAT CAGCCTGGGC TTCTGCACTG ACTTCTACGG CTGCCTCTTC 240 CTCGCCTGCT TCGTGCTGGT GCTCACGCAG AGCTCCATCT TCAGCCTTCT GGCCGTGGCA 300 GTCGACAGAT ACCTGGCCAT CTGTGTCCCG CTCAGGTATA AAAGTTTGGT CACGGGGACC 360 CGAGCAAGAG GGGTCATTGC TGTCCTCTGG GTCCTTGCCT TTGGCATCGG ATTGACTCCA 420 TTCCTGGGGT GGAACAGTAA AGACAGTGCC ACCAACAACT GCACAGAACC CTGGGATGGA 480 ACCACGAATG AAAGCTGCTG CCTTGTGAAG TGTCTCTTTG AGAATGTGGT CCCCATGAGC 540 TACATGGTAT ATTTCAATTT CTTTGGGTGT GTTCTGCCCC CACTGCTTAT AATGCTGGTG 600 ATCTACATTA AGATCTTCCT GGTGGCCTGC AGGCAGCTTC AGCGCACTGA GCTGATGGAC 660 CACTCGAGGA CCACCCTCCA GCGGGAGATC CATGCAGCCA AGTCACTGGC CATGATTGTG 720 GGGATTTTTG CCCTGTGCTG GTTACCTGTG CATGCTGTTA ACTGTGTCAC TCTTTTCCAG 780 CCAGCTCAGG GTAAAAATAA GCCCAAGTGG GCAATGAATA TGGCCATTCT TCTGTCACAT 840 GCCAATTCAG TTGTCAATCC CATTGTCTAT GCTTACCGGA ACCGAGACTT CCGCTACACT 900 TTTCACAAAA TTATCTCCAG GTATCTTCTC TGCCAAGCAG ATGTCAAGAG TGGGAATGGT 960 CAGGCTGGGG TACAGCCTGC TCTCGGTGTG GGCCTATGA 999 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 318 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Met Pro Asn Asn Ser Thr Ala Leu Ser Leu Ala Asn Val Thr Tyr Ile 1 5 10 15 Thr Met Glu Ile Phe Ile Gly Leu Cys Ala Ile Val Gly Asn Val Leu 20 25 30 Val Ile Cys Val Val Lys Leu Asn Pro Ser Leu Gln Thr Thr Thr Phe 35 40 45 Tyr Phe Ile Val Ser Leu Ala Leu Ala Asp Ile Ala Val Gly Val Leu 50 55 60 Val Met Pro Leu Ala Ile Val Val Ser Leu Gly Ile Thr Ile His Phe 65 70 75 80 Tyr Ser Cys Leu Phe Met Thr Cys Leu Leu Leu Ile Phe Thr His Ala 85 90 95 Ser Ile Met Ser Leu Leu Ala Ile Ala Val Asp Arg Tyr Leu Arg Val 100 105 110 Lys Leu Thr Val Arg Tyr Lys Arg Val Thr Thr His Arg Arg Ile Trp 115 120 125 Leu Ala Leu Gly Leu Cys Trp Leu Val Ser Phe Leu Val Gly Leu Thr 130 135 140 Pro Met Phe Gly Trp Asn Met Lys Leu Thr Ser Glu Tyr His Arg Asn 145 150 155 160 Val Thr Phe Leu Ser Cys Gln Phe Val Ser Val Met Arg Met Asp Tyr 165 170 175 Met Val Tyr Phe Ser Phe Leu Thr Trp Ile Phe Ile Pro Leu Val Val 180 185 190 Met Cys Ala Ile Tyr Leu Asp Ile Phe Tyr Ile Ile Arg Asn Lys Leu 195 200 205 Ser Leu Asn Leu Ser Asn Ser Lys Glu Thr Gly Ala Phe Tyr Gly Arg 210 215 220 Glu Phe Lys Thr Ala Lys Ser Leu Phe Leu Val Leu Phe Leu Phe Ala 225 230 235 240 Leu Ser Trp Leu Pro Leu Ser Ile Ile Asn Cys Ile Ile Tyr Phe Asn 245 250 255 Gly Glu Val Pro Gln Leu Val Leu Tyr Met Gly Ile Leu Leu Ser His 260 265 270 Ala Asn Ser Met Met Asn Pro Ile Val Tyr Ala Tyr Lys Ile Lys Lys 275 280 285 Phe Lys Glu Thr Tyr Leu Leu Ile Leu Lys Ala Cys Val Val Cys His 290 295 300 Pro Ser Asp Ser Leu Asp Thr Ser Ile Glu Lys Asn Ser Glu 305 310 315 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 957 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATGCCCAACA ACAGCACTGC TCTGTCATTG GCCAATGTTA CCTACATCAC CATGGAAATT 60 TTCATTGGAC TCTGCGCCAT AGTGGGCAAC GTGCTGGTCA TCTGCGTGGT CAAGCTGAAC 120 CCCAGCCTGC AGACCACCAC CTTCTATTTC ATTGTCTCTC TAGCCCTGGC TGACATTGCT 180 GTTGGGGTGC TGGTCATGCC TTTGGCCATT GTTGTCAGCC TGGGCATCAC AATCCACTTC 240 TACAGCTGCC TTTTTATGAC TTGCCTACTG CTTATCTTTA CCCACGCCTC CATCATGTCC 300 TTGCTGGCCA TCGCTGTGGA CCGATACTTG CGGGTCAAGC TTACCGTCAG ATACAAGAGG 360 GTCACCACTC ACAGAAGAAT ATGGCTGGCC CTGGGCCTTT GCTGGCTGGT GTCATTCCTG 420 GTGGGATTGA CCCCCATGTT TGGCTGGAAC ATGAAACTGA CCTCAGAGTA CCACAGAAAT 480 GTCACCTTCC TTTCATGCCA ATTTGTTTCC GTCATGAGAA TGGACTACAT GGTATACTTC 540 AGCTTCCTCA CCTGGATTTT CATCCCCCTG GTTGTCATGT GCGCCATCTA TCTTGACATC 600 TTTTACATCA TTCGGAACAA ACTCAGTCTG AACTTATCTA ACTCCAAAGA GACAGGTGCA 660 TTTTATGGAC GGGAGTTCAA GACGGCTAAG TCCTTGTTTC TGGTTCTTTT CTTGTTTGCT 720 CTGTCATGGC TGCCTTTATC TATCATCAAC TGCATCATCT ACTTTAATGG TGAGGTACCA 780 CAGCTTGTGC TGTACATGGG CATCCTGCTG TCCCATGCCA ACTCCATGAT GAACCCTATC 840 GTCTATGCCT ATAAAATAAA GAAGTTCAAG GAAACCTACC TTTTGATCCT CAAAGCCTGT 900 GTGGTCTGCC ATCCCTCTGA TTCTTTGGAC ACAAGCATTG AGAAGAATTC TGAGTAG 957 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CCCAAGCTTA TGAAAGCCAA CAATACC 27 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TGCTCTAGAC TCTGGTATCT TCACATT 27 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GCCTCTTTGA GGATGTGGTC CCCATGAACT ACATGGTGTA CTTCA 45 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: GCAGGGGCAC CAGCACACAG GCAAAGAAGT TGAAGTACAC CATGT 45 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO :31: TCACCATCTT CCAGGAGC 18 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ACTCCTTGGA GGCCATGT 18 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: TCCTGCACCA CCAACTGCTT AGCCCCCCTG GCCAAGGTCA TCCAT 45 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CATGAGCCCT TCCACGATGC CAAAGTTGTC ATGGATGACC TTGGC 45 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: GTTACCTACA TCACCATG 18 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GTTAGATAAG TTCAGACT 18 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CTGACCTCAG AGTACCACAG AAATGTCACC TTCCTTTCAT GCCAA 45 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: TTGGCATGAA AGGAAGGTGA CATTTCTGTG GTACTCTGAG GTCAG 45 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CTCAGTCTGA ACTTATCTAA CTCCAAAGAG ACAGGTGCAT TTTATG 46 (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: CATAAAATGC ACCTGTCTCT TTGGAGTTAG ATAAGTTCAG ACTGAG 46 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: TCCTCGGTGT ACATCACG 18 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TCCATCTGCT TCAGCTGT 18 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CTGGGCCTTT GCTGGCTGGT GTCATTCCTG GTGGGATTGA CCCCC 45 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TGAGGTCAGT TTCATGTTCC AGCCAAACAT GGGGGTCAAT CCCAC 45 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: ATGCTGCTGG AGACACAGGA 20 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TGGTCCATCA GCTCAGTGC 19 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGTGGAACAG TAAAGACAGT GCCACCAACA ACTGCACAGA ACCCTGGGAT GGAACCACGA 60 (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GGACCACATT CTCAAAGAGA CACTTCACAA GGCAGCAGCT TTCATTCGTG GTTCCATCCC 60 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CTACATCGGC ATCGAGGT 18 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GAACTCGCAC TTGATCAC 18 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: TGGTGGGACT GACCCCTATG TTTGGCTGGA ACAATCTGAG TGCGG 45 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: TGCTGCCGTT GGCTGCCCAG GCCCGCTCCA CCGCACTCAG ATTGT 45 (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CTGAGCTCAG CAGACGAAAA CCTCACCTTC CTACCCTGCC GA 42 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: TCGGCAGGGT AGGAAGGTGA GGTTTTCGTC TGCTGAGCTC AG 42 (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: CTCAGCCAGA GCTTTTCTGG CTCCAGAGAG ACAGGCGCAT TCTATG 46 (2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (iii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CATAGAATGC GCCTGTCTCT CTGGAGCCAG AAAAGCTCTG GCTGAG 46

Claims (7)

WHAT IS CLAIMED IS:

1. A method for inhibiting ThEa production which comprises contacting the A2b subtype of the adenosine receptor with an adenosine receptor agonist.

2. A method for treating or preventing autoimmune diseases including rheumatoid arthritis, rheumatoid spondylitis, inflammatory bowl disease (ulcerative colitis and Crohns disease), intestinal pathology associated with graft vs. host disease, organ transplant reactions, septic shock, fever and myalgia due to infection and cachexia associated with chronic infections, malignancy and aquired immune deficiency syndrome, pulmonary diseases such as pulmonary sarcoidosis, silicosis, chronic pulmonary inflammatory disease, adult respiratory distress syndrome which comprises providing a sufficient quantity of an A2b adenosine receptor agonist to inhibit TNFa production.

3. A method for increasing cAMP accumulation in monocytes, and thereby inhibiting production of TNFa, which comprises contacting the monocyte A2b adenosine receptor subtype with an adenosine receptor agonist at a sufficient concentration to activate adenylate cyclase.

4. The method of any one of claims 1, 2, 3, or 4, wherein the adenosine receptor agonist is adenosine, CPCA, NECA, R PIA, or CHA.

5. A method for inhibiting TNFa production which comprises contacting the A2b subtype of the adenosine receptor with an A2b adenosine receptor enhancer.

6. A method for identifying A2b adenosine receptor agonist enhancer or A2b receptor selective compounds which comprises the steps of: (a) contacting monocytes with a test compound and measuring the effect of the test compound on TNFa production; (b) contacting a test compound, identified according to step (a) as inhibiting TNFa production by the monocytes, with membranes derived from a stable cell line individually expressing each of the Al, A2a, A2b, or A3 adenosine receptor or with the whole cell expressing each of the Al, A2a, A2b, or A3 adenosine receptor and measuring the binding affinity of the test compound for the receptor or the effect of the test compound on cAMP production in the stable cell line; (c) selecting compounds which bind to the A2b adenosine receptor or which induces elevation in cAMP in the cell line expressing the A2b adenosine receptor and which do not bind to membranes or affect the cAMP level in the stable cell lines expressing the Al, A2a, or A3 adenosine receptor subtypes.

7. A method for inhibiting production of TNFa by activated monocytes which comprises contacting monocytes with an inhibitorily effective amount of a compound identified according to Claim 6.