WO1994007521A1

WO1994007521A1 - Methods and compositions for treatment of allergic disease

Info

Publication number: WO1994007521A1
Application number: PCT/US1993/009201
Authority: WO
Inventors: Rafeul Alam; Michael A. Lett-Brown; J. Andrew Grant
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1992-09-28
Filing date: 1993-09-28
Publication date: 1994-04-14
Also published as: AU5293293A

Abstract

Methods and compositions for treatment of allergic disorders are claimed. HRF-induced release of allergic mediators can be inhibited by exposing proallergic cells, such as mast cells and basophils, to an effective concentration of the pharmaceutical compositions claimed.

Description

METHODS AND COMPOSITIONS FOR TREATMENT

OF ALLERGIC DISEASE

Histamine releasing factors (HRF) represent a group of cytokines that release mediators from basophils and mast cells. Our prior related applications, U.S. Serial No. 143,094, International Application No. PCT/US89/00104, U.S. Serial No. 558,004, and International Application No. PCT/US91/05274 (each of which is expressly incorporated herein by reference) describe the ability of a group of cytokines (Histamine Release Inhibitory Factor or HRIF; and Interleukin-8) to inhibit histamine release induced by HRF and claim methods employing them as therapeutic agents. We have now discovered that RANTES, a recently cloned cytokine (Schall, et al., J. Immunol . 141:1018-1025, 1988) is also capable of inhibiting histamine release. Additional methods and compositions for treatment of allergic and chronic inflammatory disease and for inhibiting HRF- induced release of allergic mediators by exposing proaliergic cells, such as mast cells and basophils, to an effective concentration of RANTES or IL-8 are claimed in this application. Basophils and mast cells have been the subjects of scientific investigation since they were described by Paul Ehrlich in the 1870's. Mast cells are generally found in connective tissue; basophils, in the blood. The two cell types have many similarities. Both contain numerous metachromatically staining granules, both have cell surface receptors that bind IgE with high affinity, and both contain a myriad of diverse allergic mediators that can cause symptoms ranging from itching of the skin to the most life-threatening clinical situation, anaphylaxis. Basophils and mast cells collectively account for virtually all of the total body histamine and are collectively referred to as histamine-containing proaliergic cells. There is convincing evidence that mast cells and basophils are essential for induction of allergic hypersensitivity reactions. For example, substantial evidence suggests that mast cells are effector cells in such allergic diseases as asthma, allergic rhinitis, conjunctivitis, urticaria and anaphylaxis. Increased numbers of mast cells have been found in bronchoalveolar lavage fluid, respiratory mucosa, nasal mucosa, and biopsy specimens of urticarial lesions in these disorders. In addition, mast cell mediators, such as histamine and prostaglandin (PG) D₂, have been recovered from serum, bronchoalveolar lavage fluid, nasal washings, and skin blister fluid during natural and provoked allergic reactions. More recently, the mast cell granule-specific enzyme tryptase (not found in basophils) has been detected in serum from patients with allergic and anaphylactoid reactions. Finally, most mast cell- derived mediators, alone or in combination, can evoke such typical allergic symptoms as bronchospasm, angioedema, cough, mucus secretion, rhinorrhea, sneezing, and wheal-and-flare skin reactions and mast cell-specific degranulating agents can induce allergic reactions in vivo.

In addition, histologic evidence suggests that mast cells are involved in the pathogenesis of a number of chronic inflammatory diseases. Increased numbers of mast cells have been detected in biopsy specimens from patients with rheumatoid arthritis, ulcerative colitis, Crohn's disease, sarcoidosis, hypersensitivity pneumonitis, pulmonary fibrosis, nasal polyps, atopic dermatitis, allergic contact dermatitis, bullous pemphigoid, keloid formation, scleroderma and progressive systemic sclerosis, acute and chronic graft versus host disease, and parasitic infestation. Mast cells also have been implicated in regulation of nerve growth, and regulation of mast cell degranulation has proved useful in neurofibro atosis. Furthermore, increased levels of histamine were detected in the bronchoalveolar lavage fluid from patients with sarcoidosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis. Cromolyn sodium, a putative mast cell stabilizer, has been found beneficial to a subgroup of patients with ulcerative colitis, particularly those with proctitis.

Recent studies have also reinforced the contribution of basophils to various illnesses. For example, basophils appear to be particularly involved in some forms of allergic contact dermatitis, especially to poison ivy (Dvorak and Dvorak, Adv. Exp. Med. Biol. 29:573; 1973) and, experimentally, to dinitrochlorobenzene. Basophils represent 5% to 15% of the total infiltrating cells and are present within eight hours after application of the allergen to the skin.

The blood basophil count increases during the pollen season (Chavance M. , et al., Int. Arch. Allergy Appl. Immunol. 86:462-464; 1988), suggesting that basophilopoiesis may be influenced by environmental factors such as allergens.

The late phase allergic reaction induced by antigen challenge in highly allergic patients seems closely related to the naturally-occurring reaction in chronic allergic disorders. The number of metachromatic cells increases at the site of late allergic reactions. Since both basophils and mast cells release similar mediators, the exact contribution of each cell type originally proved difficult to determine. Naclerio, et al. [New Engl . J. Med. , 313:65-69; 1985) first discovered that the profile of mast cell/basophil-derived mediators released in the early and late phase allergic reaction is not identical. Histamine is detectable in both phases. PGD2, a major mediator produced by mast cells but not by basophils, is detectable in the early, but not the late phase allergic reaction (Id.). This observation applies to all in vivo allergic models including skin (Bascom, et al., J. Al lergy Cl in . Immunol . , 81:580; 1984), nasal (Naclerio, et al. , New Engl . J. Med . , 313:65-69; 1985) and lung (Gao, et al, J. Al lergy Cl in . Immunol . , 85:172; 1990) . Also, the morphologic characteristics of bilobed metachromatic cells identify the infiltrating cells as basophils. The metachromatic cells in the late phase reaction have an intracellular histamine content similar to blood basophils (1 pg/ml) . Finally, these intranasal cells express beta₂ integrins (CDllb/CD18) which are present on basophils, but not mast cells (Iliopoulus, et al., J. Immunol . , 148:2223-2228; 1992). All these results confirmed the active participation of basophils in the late phase allergic reaction.

In addition, it has been well established that viral infection of the airways causes exacerbations of asthma, and it has been reported that incubation of basophils with viruses results in enhanced mediator release and che otaxis (Ida, et al. , J. Exp . Med . 145:192; 1977; Busse, et al., J. Al lergy Cl in . Immunol . , 71:382-388; 1983; Chonmaitree, et al. , J. Infect . Dis . , 164:592-594; 1991). Thus, these studies suggest the heightened mediator release from basophils may contribute to the exacerbation of asthma.

Several therapeutic observations emphasize the probable importance of basophils in chronic allergic inflammation, including the fact that glucocorticosteroids, a potent therapy for most allergic disorders, do not affect mediator secretion from mast cells, but have been shown to inhibit histamine release from basophils (Schleimer, et al., J. Immunol . , 143:1310; 1989). Thus, the inhibition of basophil mediator release may be one of the mechanisms by which glucocorticosteroids abolish the late phase allergic reaction and control the symptoms of chronic asthma and allergy. Basophils have also been implicated a number of other allergic disorders, such as food allergy and atopic dermatitis (May, J. Al lergy Cl in . Immunol . , 58:432-437; 1976 and Sampson, et al. , New Engl . J. Med . , 321:228; 1989) . Unfortunately, despite the convincing evidence linking mast cells and basophils to these and other disease states, the precise pathogenesis of mast cell and basophil dependent disorders, including allergic disease, is incompletely understood. For example, it is known that mast cells and basophils are stimulated to release histamine, leukotrienes, and other inflammatory mediators by the bridging of cell surface-bound IgE antibodies by appropriate allergens (or anti-IgE antibodies) , but the severity of a number of allergic diseases, for example bronchial asthma, rhinitis, and conjunctivitis, does not correlate with a patient's IgE level. Moreover, although mast cells are believed to play a role in various other diseases such as inflammatory bowel disease, rheumatoid arthritis, pulmonary fibrosis, and sarcoidosis, in the majority of these diseases, IgE antibody cannot be found. Therefore, it appears that other mechanisms of allergic mediator release play a critical role in pathogenesis of allergic diseases and other disorders mentioned above in which basophils and mast cells have been implicated. Elucidation of such mechanisms has been and remains the goal of many skilled medical scientists. One of the most exciting developments in this area was the discovery of histamine releasing activity (HRA) , cytokines designated herein as histamine releasing factor(s) (HRF), by investigators working in the laboratories of the present inventors. Thueson, et al., (J. Immunol., 123:626, 1979; J. Immunol., 123:633, 1979) and Lett-Brown, et al., [Cel l Immunol . , 87:434, 1984; Cell Immunol . , 87:445, 1984) first reported that antigen or mitogen stimulated human mononuclear cells secrete a proteinaceous factor that induces release of histamine from basophils and mast cells. Other laboratories then confirmed the synthesis of HRF by mononuclear cells. _ It has now been shown that HRF is also synthesized by B- lymphocytes and T-lymphocytes, alveolar macrophages, platelets, neutrophils, and blood monocytes cultured in vitro. The wide variety of cell types reported to secrete HRF suggests that it has considerable biologic importance. In addition to mediating histamine release, HRF has been shown to induce secretion of leukotrienes and to be chemotactic for basophils and monocytes. For a review, see Grant, et al., Fed . Proc , 45:2653, 1986, J. Al lergy Cl in . Immunol . , 77:407, 1986, and Ala , Insights in Al lergy, Vol. 2, no. 6, 1987, CV Mosby, St. Louis, and Grant, et al., J. Al lergy Cl in . Immunol . 88:683-693 (1991) all incorporated herein by reference.

It has been reported that HRF's may comprise a group of cytokines. Interleukin-3 (IL-3), granulocyte- macrophage colony stimulating factor (GM-CSF) and connective tissue activating peptide III (CTAP III) have been shown to cause degranulation of basophils from selected blood donors (Haak-Frendscho, et al. , J. Cl in . Invest . 82:17-20, 1988; Alam, R. , et al., J. Immunol . 142:3431-3435, 1989; MacDonald, et al. , J. Immunol . 142:3527-3532, 1989; Baeza, et al. , J. Cl in . Invest . 85:1516-1521, 1990), while controversy exists as to the independent histamine releasing activity of interleukin-1 (IL-1) (Subramanian and Bray, J. Immunol . 138:271-275, 1987; Massey et al. , Faseb J. 2:A1251, 1988) and interleukin-8 (IL-8) (White, et al., Immunol. Let t . 22:151-154, 1989; Dahinden, et al. , J. Exp . Med . 170:1787-1792, 1989). We have recently demonstrated that monocyte chemotactic and activating factor (MCAF) /monocyte chemoattractant protein-1 (MCP-1) is a potent histamine releasing factor for basophils. Alam et al., J. Cl in . Invest . 89:723-728, 1992. MCP-1 causes histamine release from basophils obtained from allergic as well as non-allergic healthy subjects. Histamine releasing potency of MCP-1 is comparable to anti-IgE and C5a (a fragment of the fifth component of complement. MCP-1 belongs to a broad family of 8 KD cytokines with significant structural homology (Leonard and Yoshimura, Immunol . Today. 11:97-101, 1990). MCP-1 seems to be the major component of HRF having a similar size, similar kinetics of basophil mediator release, and immunologic cross reactivity. Numerous studies provide data directly supporting the importance of HRF as a mediator of human allergic disease. For example, an HRF-like material has been obtained from skin blister fluid obtained during the late allergic reaction, now considered an important factor in the pathogenesis of chronic asthma and other allergic conditions. Increased levels of HRF have been detected in skin blister fluids obtained from urticarial lesions (Claveau, et al., J. Al l ergy Cl in . Immunol . 87:223 (abstract 1991) . HRF has also been recovered in nasal washings. In addition, HRF induces bronchoconstriction on inhalation by asthmatic subjects and a wheal-and-flare reaction in humans and non-human primates. Mononuclear cells from asthmatic patients have been shown to spontaneously produce relatively large amounts of HRF, and HRF production is enhanced on in vitro incubation with specific allergen. Moreover, the magnitude of spontaneous HRF production correlates with the severity of bronchial hyperreactivity in asthmatic patients. Alam, et al., J. Al lergy Cl in . Immunol . , 79:103, 1987. Sim, et al., [Amer . Rev. Respir . Dis . , 145:1316-1320; 1992) reported that topical beclomethasone attenuated the symptoms of patients with seasonal allergic rhinitis and simultaneously reduced the recovery of HRF from nasal washings. Furthermore, the change in symptoms was correlated with the fall in HRF. HRF has also been associated with idiopathic pulmonary fibrosis (Broide, et al., J. Immunol . 145:1838-1844; 1990).

Immunotherapy has been reported to reduce the sensitivity of basophils to allergens (Evans, et al, J. Cl in . Invest . , 57:1378-1385; 1976) and the late phase allergic reaction. Kuna, et al., [J. Al l ergy Cl in . Immunol . , 83:816-824; 1989) reported that effective immunotherapy reduced the symptoms of extrinsic asthma and reduced the synthesis of HRF. It is possible that the decreased sensitivity of basophils is related to decreased synthesis of HRF and other cytokines, resulting in the attenuation of the late phase reaction following immunotherapy. These findings suggest that in asthmatic patients, increased spontaneous HRF production may cause a sustained release of allergic mediators from mast cells or basophils, resulting in chronic inflammation and ultimately leading to the development of bronchial hyperreactivity.

Given the important role played by HRF in the pathogenesis of allergic diseases and other disorders including the release of mediators from basophils and mast cells, it is likely that an agent capable of inhibiting HRF-induced mediator release could provide a valuable tool in treating mast cell/basophil dependent disorders, which include the allergic diseases. Our prior patent applications described a human histamine release inhibitory factor having a molecular weight of about 8,000 - 10,000 daltons, and inhibition of histamine release with recombinant human interleukin 8 (IL-8) (m.w. about 8,000 daltons). HRIF and IL-8 specifically antagonize cytokine mediated histamine release. IL-8 belongs to a family of cytokines that is characterized by the presence of two cysteines separated by one amino acid in their chain (Cys-X-Cys family) . Other members of this family include CTAP III, NAP-2, MIP-2, platelet factor 4 and melanocyte growth stimulatory activity (hGro) and hIP-10. RANTES is another recently cloned cytokine that is produced by T cells. Schall et al., J. Immunol . 141:1018-1025, 1988. It causes chemotaxis of monocytes and CD4+ T cells of memory phenotype. Schall et al., Na ture , 347:669-671, 1990. It belongs to the second family of low molecular weight cytokines that is characterized by the presence of a Cys-Cys sequence. The other members include MCP-1, MIP-1 alpha and beta, hLD78, hAct-2h and mTCA3. We have now shown that RANTES is capable of inhibiting allergic mediator release and may be used in therapy for allergic disorders.

The present invention provides new and useful therapeutic methods for treatment of allergic or chronic inflammatory disease. The invention employs a human cytokine designated RANTES. In a separate embodiment, the invention employs another human cytokine, interleukin-8. Each is capable of inhibiting release of mediators from mast cells or basophils. A RANTES protein is defined here to include not only the RANTES polypeptide having the sequence of the native polypeptide described by Schall et al., Na ture 347:609-671, 1990, but also to include any naturally occurring variants or alleles, as well as biologically active chemical derivatives, truncated molecules or fragments, muteins, and polypeptides to which additional amino acids have been added. Similarly, an IL-8 protein is defined here to include not only the IL-8 polypeptide having the sequence of the native polypeptide described by Yoshimura et al., Proc . Na tl . Acad . Sci . USA, 84:9233-9237, 1987, but also to include any naturally occurring variants or alleles, as well as biologically active chemical derivatives, truncated molecules or fragments, muteins, and polypeptides to which additional amino acids have been added. A RANTES or IL-8 protein is provided as the active ingredient of a pharmaceutical composition which may be used to ameliorate symptoms of allergic or chronic inflammatory disease.

The methods of treatment described may be employed in therapy for or to decrease symptoms associated with a wide variety of illnesses in which mediator release from proaliergic cells such as basophils or mast cells has been implicated. Included in this category, without limitation, are allergic and chronic inflammatory diseases such as asthma, allergic rhinitis, conjunctivitis, urticaria, anaphylaxis, contact dermatitis, food allergy, rheumatoid arthritis, ulcerative colitis, Crohn's disease, sarcoidosis, hypersensitivity pneumonitis, pulmonary fibrosis, nasal polyps, atopic dermatitis, allergic contact dermatitis, bullous pemphigoid, keloid formation, scleroderma and progressive systemic sclerosis, certain manifestations of acute and chronic graft versus host disease, neurofibromatosis, and complications resulting from parasitic infestation.

As described more fully in the Description of Preferred Embodiments, amounts of the composition effective to decrease symptomatology or pathogenesis of a given disease state may be administered to patients suffering from a given illness by known methodologies, e.g., local administration to mucous membranes of the nose, mouth, conjunctiva, vagina, rectum, etc., by intrabronchial administration (e.g., by inhalation), by topical application to allergic lesions, by local injection into such lesions, and by subcutaneous, intramuscular, intravenous, intraarticular, or intraperitoneal administration. Pharmaceutical compositions which include a pharmaceutically acceptable carrier formulated together with an amount of a RANTES protein or IL-8 protein effective for therapy to decrease symptomatology or pathology of the disease may be prepared for a given mode of administration using any of a number of procedures known to those in the pharmaceutical arts, and exemplified more fully in the Description of Preferred Embodiments. In a preferred embodiment, the composition will contain a sufficient amount of the active ingredient to facilitate convenient administration of a selected dose, e.g., in some embodiments, from 1 to 1000 mcg/m² body surface area or, more preferably, from about 10 to about 500 mcg/m² body surface area, or to achieve a serum concentration or concentration at the desired site of activity (when administered topically or locally) on the order of at least 10-⁹ M and more preferably at least 10^"8 M.

A more general embodiment of the present invention includes a method for inhibiting release of an allergic mediator from pro-allergic cells by exposing the pro- allergic cells to the active RANTES or IL-8 ingredient. In specific embodiments, the pro-allergic cells comprise mast cells, or basophils, and the allergic mediator is histamine. In a preferred embodiment the active RANTES or IL-8 ingredient is present at a concentration of at least about 10^'9 M, and more preferably, at least about 10^"8 M. In some cases, histamine release may be inhibited by at least about 10 to at least about 60 percent, at least about 15 to at least about 60 percent, and more preferably, at least about 30 to at least about 60 percent, when measured according to the protocol set forth herein. These and other aspects of the invention will become more apparent from a description of particular embodiments when read in conjunction with the drawings. Figure 1. Dose-response curves for the inhibition of MCP-1- and HRF-induced histamine release from leukocytes by IL-8 and RANTES. Leukocytes were preincubated with various concentrations of IL-8 or RANTES for 5 min and then challenged with MCP-1 or mononuclear cell-derived HRF. The percent inhibition of histamine release in the presence of IL-8 or RANTES as compared to buffer has been shown. N=20 for IL-8 against HRF, N=15 for IL-8 against MCP-1, N=9 for RANTES against both MCP-1 and HRF. Mean histamine release in the presence of buffer ranged from 30-50%.

Figure 2. Dose-response curves for the inhibition of histamine release from purified basophils by IL-8 and RANTES. Basophils were purified by using a combination of discontinuous Percoll gradient centrifugation followed by negative selection of basophils with monoclonal antibody-coated (anti-CD2, -CD20 and -CD16) magnetic beads. The results of one (purity = 80%) of three similar experiments are shown. Mean histamine release in the presence of buffer- was 34 + 1%. Figure 3. Purification of peripheral blood mononuclear cell ("PBMC") HRF by C3 reverse phase HPLC. A sample of highly concentrated (300 x) PBMC culture fluid was applied to a semi-preparative C3 reverse phase column that was preequilibrated with 0.01% trifluoroacetic acid. The column was eluted with a step- wise gradient of 0-90% acetonitrile. Lyophilized fractions were assayed for histamine releasing activity. Three distinct peaks, HRF I (retention time, 14 min), HRF II (65 min) and HRF III (86 min) were detected. Figure 4. Inhibition of HRF I, II and Ill-induced histamine release by IL-8. Three species of HRF from several runs of C3 HPLC (see Fig. 3) were pooled and used for this experiment. Leukocytes from three donors were preincubated with IL-8 and then challenged with HRF I, II and III. The inhibition was calculated with reference to the release in the presence of buffer. The control histamine release by HRF I, II and III was 32 ± 4%, 24 ± 5% and 38 ± 3% respectively. The error bars were omitted for clarity, and SEM ranged from 4-9%.

Figure 5. Anti-MCP-1 immunoaffinity chromatography of HRF peaks from HPLC. A sample of reverse phase HPLC- purified peak I, II and III were passed through an anti- MCP-1 affinity column separately. After an extensive wash, the column was eluted with 6 M KSCN. The dialyzed samples were assayed for histamine releasing activity. Figure 6. Effect of IL-8 and RANTES on histamine release from basophils by Peripheral Blood Mononuclear Cells (PBMC) -HRF, anti-IgE, FMLP (formyl-methionyl- leucyl-phenylalanine) and C5a (N=4) . Leukocytes were preincubated with buffer, IL-8 (10^"7 M) or RANTES (lO^-8 M) for 5 min and then challenged with a predetermined dose of various secretagogues. The concentration of secretagogues was chosen to induce a histamine release in the range of 15-40%. Asterisk indicates statistical difference compared to control release at p<0.05. The present invention provides therapeutic methods and compositions for treatment of allergic and chronic inflammatory disease. The invention also encompasses methods for inhibiting release of allergic mediators from human basophils or human mast cells by exposing the cells to a composition comprising an effective amount of a substantially purified RANTES protein. In a separate embodiment, the invention encompasses methods for inhibiting release of allergic mediators from human basophils or human mast cells by exposing the cells to a composition comprising an effective amount of a substantially purified IL-8 protein. In practice of the invention, the substantially purified protein may be employed as native or naturally occurring molecules, such as alleles, biologically active proteolytic isoforms and the like, as well as biologically effective polypeptides, such as chemical derivatives, and biologically active fragments, and muteins, which have been modified in some respects but which nevertheless exhibit the histamine release inhibitory activity.

Either the RANTES or IL-8 polypeptide may be modified to produce a biologically active "chemical derivative" by a number of known procedures. For example, one or more amino acids may be modified by reaction of a functional side group including, for example, derivatization of free amino groups to form p- toluene sulfonyl groups, chloroacetyl groups, t- butyloxycarbonyl groups, carbobenzoxy groups, amine hydrochlorides, or formyl groups. In addition, carboxyl groups may be derivatized to form salts, esters, or hydrazides. Free hydroxyl groups may be derivatized to form o-acyl or o-alkyl derivatives. In addition, chemical derivatives include, without limitation, those polypeptides containing one or more naturally occurring amino acid derivatives, such as methyl histidine, hydroxyproline, hydroxylysine, or ornithine substituted for its standard counterpart amino acid, as well as polypeptides containing modifications designed to increase bioactivity or the half life of the polypeptide in vivo, for example, by the introduction of stabilizing groups capable of decreasing susceptibility to proteolysis or incorporation of one or more L-amino acids in lieu of a D-amino acid.

Also included are biologically active "fragments" of the RANTES and IL-8 polypeptides such as, for example, "truncated" polypeptides, in which one or more amino acids at the amino, and/or carboxyl terminals have been removed, (e.g., by proteolytic cleavage or by preparation of recombinant DNA vectors which have been modified, e.g. by nucleotide deletion, to direct expression of a truncated molecule or fragment or domain thereof) and biologically active polypeptides based upon a sequence of the RANTES protein which comprises an active site of the molecule. Biologically active synthetic polypeptides wholly or partially duplicative of continuous sequences of a naturally occurring or other RANTES or IL-8 polypeptide may be prepared by known methods of chemical peptide synthesis.

Of course, biologically active RANTES or IL-8 polypeptides or muteins having one or more amino acid additions or substitutions produced, e.g., by natural mutation, recombinant DNA technology, site-directed utagenesis or other mutagenic techniques, may also be used in accordance with the invention.

Methods for preparation of such chemical derivatives, fragments, and/or muteins by chemical reaction, recombinant DNA technology, or chemical synthesis (particularly for shorter peptides) are well known to those of skill in the art. Activity of a given molecule produced according to these methods may be readily ascertained with the assistance of the instant specification. Due to precautions necessarily attendant to development of every new pharmaceutical, the compositions described have not yet been tested in a clinical setting in human subjects, but the in vitro activity in inhibiting histamine release has been used to demonstrate the efficacy of these proteins as pharmacologic agents for use in treatment of allergic disease and provides a basis for formation and administration. The histamine release assay is accepted by those of skill in the art as a reliable correlate of in vivo histamine release. Based on the data set forth herein, it is reasonable to conclude that RANTES is useful for treating numerous diseases in which mast cells and basophils are involved, especially the allergic disorders. Similarly, it is reasonable to conclude that IL-8 is also useful for treating such diseases. In particular, these include, but are not limited to, allergic and chronic inflammatory diseases such as asthma, allergic rhinitis, conjunctivitis, urticaria, anaphylaxis, contact dermatitis, food allergy, rheumatoid arthritis, ulcerative colitis, Crohn's disease, sarcoidosis, hypersensitivity pneumonitis, pulmonary fibrosis, nasal polyps, atopic dermatitis, allergic contact dermatitis, bullous pemphigoid, keloid formation, scleroderma and progressive systemic sclerosis, certain manifestations of acute and chronic graft versus host disease, neurofibromatosis, and complications resulting from parasitic infestation.

The particular method and formulation advantageous for administering the pharmaceutical compositions will often vary with the particular clinical symptomatology or disease to be treated. For example, the compositions can be formulated as a component of an aerosol for intranasal or intrabronchial administration. This mode of administration may be particularly useful in treating certain allergic diseases involving the nasal passages, bronchi, and lungs, for example, allergic rhinitis and bronchial asthma. The compositions can also be administered topically to the skin or eye for treating allergic disorders at these sites. Alternatively, compositions can be formulated for intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal or intraarticular injection, and can be used to treat inflammatory reactions at these sites in which the triggering of mediator release from basophils and mast cells is involved in the pathogenesis of the illness. Various types of pharmaceutical compositions adapted for particular modes of administration are known to those of skill in the art and can be employed in accordance with the invention. For example, topical formulations, which may be suitably administered locally or to mucous membranes, include creams, lotions, gels, salves and the like contained in an aqueous or oleaginous base.

Commonly employed aqueous bases may include natural gums, cellulose derivatives, acrylic acid polymers, vinyl polymers, synthetic polysaccharides or naturally occurring polysaccharides, such as starch, dextrin, pectin, sodium alginate, etc. and mixtures thereof.

Suitable oleaginous formulations may contain various fats and oils, such as naturally occurring vegetable oils, synthetic oils, mineral oils or fats, etc., and other ingredients such as stearates, waxes and the like. Formulations for application to dermal or mucosal surfaces may contain one or more ingredients designed to facilitate transdermal delivery or transfer of active ingredient across the mucosal membrane, such as cyclodextrin, etc. Alternatively, the active ingredient may be formulated in any of a number vehicles suitable for administration by injection, most commonly including saline or other physiological buffers, which may be supplemented with additional ingredients such as dextrose, glycerol, etc. and/or other physiologically acceptable ingredients designed to stabilize the formulation or active ingredient or to preserve sterility. In other embodiments, the active ingredient may be formulated in sustained release delivery systems, including for example, suspensions and polymeric systems. See, e.g., Y. Chien, Novel Drug Del ivery Systems Marcel Dekker, Inc., N.Y. N.Y. (1982).

Information derived from studies with other cytokines (see, e.g., see the Physician ' s Desk Reference , 46th Edition. Medical Economics Data Press, 1992) provides a basis for developing a therapeutic base line dose range from which the practitioner in the pharmaceutical arts may develop a pharmaceutical unit dose for treatment of a particular disorder.

For example, intranasal alpha 2-interferon has been used to prevent viral upper respiratory infections.

Douglas, et al., [New Engl . J. Med . , 314 : 65 , 1986) and Hayden, et al. , [New Engl . J. Med . , 314:71, 1986) administered 5 X 10⁶ international units (IU) per day for an effective response. Recombinant interferon-α (specific activity of 2 x 10⁸ international units ("IU") per mg protein) is currently licensed for treatment of hairy cell leukemia at a dose of 3 X 10⁶ IU/day and for the treatment of Kaposi's Sarcoma at a dose of 36 x 10⁶ IU/day by intramuscular or subcutaneous administration. Interferon α-2B (specific activity of 2 x 10⁸ IU/mg protein) is prescribed for treatment of hairy cell leukemia at a dose of 2 x 10⁶ IU/m², for chronic hepatitis (Non A, Non B) at 3 x 10⁶ IU/m², and for AIDS-related Kaposi's Sarcoma at 30 x 10⁶ IU/m². Interferon gamma-lb is used for treatment of chronic granulomatous disease at 50 mcg/m² by subcutaneous injection. See also, Baron, et al.. The Interferon System : A Current Review to 1987 , University of Texas Press, Austin, Texas.

As reported in New Engl . J. Med . , 313:1485, 1985), interleukin-2 has been administered intravenously to patients with cancer at doses of 10⁴ to 10⁵ units per kg over eight hours and maximal dose of up to 3.3 X 10⁶ units per kg. Human Granulocyte-Colony Stimulating Factor ("G- CSF") is administered to patients with chemotherapy- induced neutropenia at a starting dosage of 5 mcg/kg/day and has been used in clinical trials at doses of at least about 70 mcg/kg/day. Human Granulocyte-Macrophage CSF ("GM-CSF") is licensed for myeloid reconstitution after bone marrow transplantation at a dose of about 250 mcg/m²/day. In line with these dosages, RANTES is expected to be administered at a dose of from about 1 mcg/m² to about 1000 mcg/m², and more preferably from about 10 mcg/m² to about 500 mcg/m². Alternatively, the RANTES protein may be formulated in a vehicle designed to deliver at least about 10^"9, and more preferably, at least ^"8 M RANTES protein to the site of action, e.g., to the surface of basophils or mast cells involved in mediating disease. The exact doses of RANTES to be used in a particular clinical application may be determined by accepted pharmaceutical methods known to those skilled in the pharmaceutical arts, for example, by measuring RANTES blood concentration and determining serum half life by enzyme linked immunoassay or other suitable assay. IL-8 may be administered at these doses as well.

Furthermore, it is also contemplated that blood concentrations of RANTES or IL-8 may be used to monitor the progress of patients treated with immunotherapy, by measuring concentration in biological fluids, for example, in blood and its components, respiratory secretions or washings, urine, or joint fluid, by radioimmunoassay, enzyme linked immunoassay, or other suitable assay procedures.

The following example describes the ability of RANTES and of IL-8 to inhibit histamine release from human proaliergic cells and may assist in understanding certain aspects of the invention. The example is not meant to limit the invention in any manner not explicitly set forth in the claims.

EXAMPLE Materials and Methods

Reagents. RPMI 1640 was obtained from GIBCO Laboratories, Grand Island, NY; human serum albumin, glutamine, Histopaque-1077, Concanavalin A (Con A), penicillin, streptomycin, FMLP and recombinant C5a from Sigma Chemicals Co., St. Louis, MO; HEPES from Research Organicε, Inc., Cleveland, OH; hydroxyethyl starch (HetaStarch) from American McGaw, Irvine, CA; human recombinant IL-3, human recombinant endothelial IL-8 beta (77 amino acid peptide with alanine in its N-terminus) and recombinant monocytic IL-8 gamma (72 amino acid peptide) , human recombinant monocyte chemoattractant peptide-1 (MCP-1) and human recombinant RANTES were from Pepro Tech Inc., Rocky Hills, NJ. Stock solutions of IL-8 (6 x 10^"6 M) , MCP-1 (2 x 10"⁶ M) and RANTES (2 x 10'⁶ M) were made in HEPES-buffered saline, pH 7.4 containing 0.3% human serum albumin, 2 mM CaCl₂ and 1 m MgCl₂. Rabbit anti-human IgE serum was from Behring Diagnostics, Somerville, NY. Magnetic beads coated with anti-CD2 and CD20 monoclonal antibodies were from Dynal, Inc., Great Neck, NY and magnetic beads coated with anti-CD16 monoclonal antibodies was from Collaborative Research, Bedford, MA.

Genera tion of ERF -containing superna tant .

Leukocytes were isolated from buffy coats obtained from normal blood bank donors. PBMC were isolated by Ficoll- Hypaque gradient centrifugation as previously described (Alam, et al. , J. Cl in . Invest . 82:2056-2062, 1988) and pulsed with Con A, 25 μg/ml in RPMI 1640 medium for 4 hr, washed twice with Hanks' balanced salt solution, resuspended in RPMI 1640 medium and then cultured for 72 hr. Supernatants were harvested and concentrated 50x using an Amicon ultrafiltration chamber with YM-5 filters (MW cut-off 5,000) and ultracentrifuged. The ultracentrifuged supernatant was applied to a TSK 2000 gel filtration column. Recovery of the activity was approximately 50-60%. The fractions containing HRF activity (15-40 KD) were pooled, and aliquoted. The aliquots were frozen at -70°C and used as a source of HRF. The concentration of protein in this preparation was 8 μg/ml.

Purifica tion of HRF by reverse phase HPLC . HRF- containing PBMC supernatant was concentrated 50Ox and then applied to a C3 reverse phase semi-preparative HPLC column (Beckman Instruments) . The column was pre- equilibrated with 0.01% trifluoroacetic acid and eluted with a step-wise gradient of acetonitrile containing 0.01% trifluoroacetic acid. One milliliter fractions were collected, lyophilized and screened for HRF activity using the basophil histamine release test.

Measurement of IL- 8 in HPLC fractions . The concentration of IL-8 in the HPLC purified HRF fractions was measured using a commercially available ELISA kit (Quantikine IL-8 from R & D, Inc.). The sensitivity of the assay is 10 pg/ml. The antibody does not cross-react with IL 1 through 7, IFN, TNF and CSFs according to the manufacturer's instruction.

Isola tion of peripheral blood leukocytes . Donors for this study were selected from a large group of allergic and non-allergic subjects that were screened in our laboratory for histamine release from basophils in response to HRF, anti-IgE and C5a. Allergic status was defined by the presence of clinical symptoms, past allergic history and positive reaction to prick skin testing with a panel of local aeroallergens (32 allergens) . Venous blood from donors was anticoagulated with 10 mM EDTA and sedimented with 1.5% hydroxyethyl starch for 30 min at room temperature (Alam, et al., J. Cl in . Invest . 82:2056-2062, 1988). The leukocyte-rich buffy coat was collected and washed three times in HA buffer (HEPES buffered-saline, pH 7.4 and 0.03% human serum albumin) in a refrigerated centrifuged (4°C) at 300 x g. The washed leukocytes were suspended in HACM buffer (HEPES buffered-saline, pH 7.4, 0.03% human serum albumin, 2 mM CaCl₂) . Leukocytes from 1 ml of blood were usually used for one duplicate experiment for assaying HRF. In some experiments basophil-containing mononuclear cell suspensions were prepared by Ficoll-Hypaque gradient centrifugation, and used in the histamine release test.

Purifica tion of basophil s. For some experiments basophils were purified using a discontinuous Percoll gradient. Leonard et al., J. Leuk. Biol . 35:169-177, 1984. Partially separated basophils were further purified by negative selection of basophils using monoclonal antibody (against CD2, CD19 and CD16) -coated magnetic beads. Berman and Center, J. Immunol . 138:2100- 2103, 1987. The final purity of basophils ranged from 65-80%.

Histamine release assay. Aliquots of 50 ul of MCP-1 (10"⁷ M) , HRF (protein concentration, 8 μg/ml) , anti-IgE (1:3,000 dilution of affinity-purified antibody), recombinant IL-8 or RANTES (both 10^"11 to 10^"6 M final) were incubated with 50 ul of leukocyte suspension for 45 min in a shaking water bath at 37°C. Each experiment was done in duplicate. Four hundred microliters of HA buffer was added to each tube at the end of the incubation. The supernatants were separated from the cells by centrifugation at 600 x g for 5 min at 4°C. The histamine content of the supernatants was measured using an automated fluorometric analyzer (Alam, et al. , J.

Cl in . Invest . 82:2056-2062, 1988. Spontaneous histamine release was assessed by incubating the cells in HACM buffer alone. The total histamine content of the cells was measured by lysing the cells with 3% perchloric acid. The percentage of histamine release was calculated according to the formula: [(histamine in the supernatant) x 100] /(total histamine in the cells). Spontaneous histamine release from the cells was usually less than 5%. The values of spontaneous histamine release were subtracted from the calculated histamine 5 release.

Histamine release inhibi tion assay. For the inhibition assay, 50 ul aliquots of cells were first incubated with various dilutions of IL-8 or RANTES (10^"n

10 to 10^"7 M) for 5 min at room temperature and then challenged with 50 ul of MCP-1 (10^"7 M) , HRF, anti-IgE (1:3000 dilution), C5a (1 μg/ml) or FMLP (10^"5 M) separately (all concentrations shown are final) . The cells were then further incubated for 45 min in a water

15 bath at 37°C and the supernatants were separated by centrifugation. Histamine content of the supernatant and total cellular content were determined as described above. A typical experimental protocol included:

^{~ ~} Preincubation Challenge a. leukocytes + buffer + buffer b. leukocytes + buffer + MCP-1* c. leukocytes + IL-8 or RANTES + buffer d. leukocytes + IL-8 or RANTES + MCP-1* 5 * or other secretagogues

The percentage of inhibition of histamine release was calculated according to the formula (Alam, et al., R. , J. Cl in . Invest . 82:2056-2062, 1988: 0 t (b-a)-(d-c)] x 100/(b-a)

Most experiments were performed with so called IL-8 beta (endothelial IL-8, a 77 amino acid peptide). Results are expressed as means ± SEM. In one set of 5 experiments IL-8 gamma (monocytic IL-8, a 72 amino acid peptide) was used for comparative purposes as pointed out in the text. Results are expressed as means±SEM. Statistical analyses were done with Wilcoxon's rank sum test.

RESULTS

Histamine Release . Direct histamine releasing effect of RANTES was studied on basophils from eleven allergic and four normal donors. Basophils from only four allergic donors released histamine (16 + 2%) in response to RANTES at 10^'7 M concentration. The bioactivity of RANTES was tested in the chemotaxis assay using lymphocytes. Boyden microchambers and 5 u polycarbonate membranes were used for this purpose. RANTES caused chemotaxis of lymphocytes in a dose- dependent manner and was equipotent with C5a (1 μg/ml) at the dose of 10^"8 M (48 lymphocytes/5 oil fields for RANTES, 10^"8 M vs 45 lymphocytes/5 oil fields for C5a, 1 μg/ml) . The histamine releasing activity of IL-8 beta was investigated using basophils from 20 donors including 10 allergic and 10 non-allergic healthy subjects. We used IL-8 at a concentration range of 10^'11 to 10^"6 M. IL-8 released histamine from basophils obtained from only two of ten non-allergic donors and only four of ten allergic donors at the highest concentration (10^"6 M) . The histamine release of these six donors ranged from 6-16% and the mean was 8.7±0.8% (data not shown) . The experiment was repeated on three responder donors on two separate occasions, and the results were reproduced. By increasing the concentration of IL-8 up to 3 x 10"⁶ M, we were able to observe a small increase in histamine release in the range of 15-20%. By comparison, anti-IgE and PBMC-HRF released 34 ± 7% and 51 ± 7% of histamine respectively from all donors. Inhibi tion of Histamine Release . The effect of IL-8 beta and RANTES on histamine release by MCP-1 and PBMC-HRF was studied. In preliminary studies, leukocytes were preincubated with IL-8 (10^"7 M) for different time periods and then HRF was added. The inhibition of histamine release was 60 ± 4%, 68 ± 5%, 70 ± 4% and 70 ± 4% at 5, 10, 15 and 30 min respectively (N=3) . However, leukocytes tended to release less histamine in response to HRF (as well as to MCP-1) after preincubation in the buffer alone for more than 10 min. Since the inhibition of histamine release was not significantly different at the various time points, it was decided to preincubate leukocytes with IL-8 for 5 min only. A similar preincubation time was determined for RANTES. The inhibition by IL-8 and RANTES of MCP-1 and HRF- induced histamine release is shown in Fig. 1. Both IL-8 and RANTES inhibited HRF and MCP-1 in a dose dependent manner. All inhibition curves reached a plateau at 10^"8 M concentration except for the inhibition curve of IL-8 against HRF which, was linear. Also, the inhibition of

HRF was significantly (P<0.01) higher than that of MCP-1. This is perhaps due to the inhibition of other histamine releasing cytokines, e.g., CTAP III and IL-3 that may be present in the mononuclear cell supernatant (Kuna, et al., J. Immunol . 147:1920-1924, 1991).

In three experiments basophils were purified to 65- 80% purity using a combination of discontinuous Percoll gradient centrifugation and negative selection with monoclonal antibody-coated magnetic beads. Both IL-8 and RANTES inhibited histamine release from purified basophils induced with MCP-1 and HRF (Fig. 2) . Experiments performed indicate that MCP-1 is one of the most potent histamine releasing factors for basophils. RANTES does not possess any histamine releasing activity, but antagonizes MCP-1 in this regard. There are a number of structural and functional similarities between these two cytokines. The alignment score of MCP-1 and RANTES is 12 by GENALIGN program (Wolpe and Cerami, Faseb J. 3:2565-2573, 1989). One interesting difference between these molecules is that MCP-1 has sites for N-glycosylation whereas RANTES lacks glycosylation sites (Schall, et al., Na ture , 347:669-671, 1990).

The inhibition of HRF by IL-8 was significantly (P<0.05) higher in normal subjects D(N=12) than in allergic patients (N=12) at 10^"7 and 10^"6 M. A similar trend was observed with RANTES, although a statistical analysis was not performed due to low number of subjects studied. These observations may suggest that basophils from allergic subjects not only have a heightened releasability but also a reduced sensitivity to an endogenous inhibitor.

We used IL-8 beta (endothelial) in all our experiments. In four donors the effect of IL-8 gamma was studied. A comparable dose-dependent inhibition of histamine release was observed in all donors, the maximal inhibition being 62 ± 7% and 60 ± 8% for IL-8 beta and gamma respectively.

Since we have used crude PBMC supernatant as the source of HRF for inhibition of histamine release, we next partially purified three species of HRF by a C3 reverse phase HPLC (Fig. 3) . These three peaks of HRF were then used to study the effect of IL-8. Figure 4 illustrates the results obtained from experiments done with basophils from three donors. All three peaks of HRF were inhibited by IL-8 in a dose-dependent manner, and the maximal inhibition was comparable to that obtained with crude HRF on the same donors.

In order to determine which of the three peaks include MCAF, we constructed an immunoaffinity column with purified rabbit antibody against MCP-1 as described previously (Alam, et al., J. Cl in . Invest . 89:723-728; 1992) . Each HPLC peak was applied to the column, and after an extensive wash the column was eluted with 6 M KSCN. MCP-1 was detected in peak 1 only (Fig. 5) .

The activity of IL-8 and RANTES against secretagogues such as anti-IgE, FMLP and C5a was also investigated. We used suboptimal concentrations of these agonists to induce a release in the range of 20-40%. In contrast, IL-8 and RANTES did induce the secretory response of basophils to PBMC-HRF. Neither cytokine affected histamine release induced with these secretagogues (Fig. 6) .

The requirement of preincubation of basophils with IL-8 for optimal inhibition of histamine release was studied. IL-8 did not show any inhibitory activity when added to the cells simultaneously with HRF or added 5 min after HRF (data not shown) . We also asked whether the presence of IL-8 is necessary after preincubation with the cells. For this purpose leukocytes that was preincubated with IL-8 or buffer for 5 min were washed 3x with 30 volumes of buffer. The cells were then challenged with HRF. The inhibitory effect of IL-8 was not removed by extensive washings. The histamine release was 20 ± 3% after the preincubation with buffer vs 5 ± 1% after preincubation with IL-8.

CONCLUSION

We have described a group of cytokines, HRIF, that specifically inhibits HRF-induced histamine release from basophils. Alam, et al. , J. Cl in . Invest . 82:2056-2062, 1988. Both IL-8 and RANTES resemble one form of HRIF in their molecular weights and specificity. Both have a MW of ~ 8 KD by amino acid sequencing and specifically antagonize cytokines. Like HRIF, IL-8 and RANTES do not affect histamine release by anti-IgE, C5a and FMLP. The implication of IL-8 and RANTES-induced^" inhibition of mediator release from basophils in human pathophysiology is supported by the observation of an 8 KD HRIF-like activity has been recovered from bronchoalveolar lavage fluids (Alam, et al. , Am. Rev. Respir . Dis . 141:666-671, 1990) and nasal washings (unpublished observations) . Thus, these molecules may find important therapeutic use in treatment of allergic and chronic inflammatory disease.

The foregoing description of the invention has been directed to particular preferred embodiments in accordance with the requirements of the patent statutes and for purposes of explanation and illustration. It will be apparent, however, to those skilled in the art that many modifications and changes may be made without departing from the scope and the spirit of the invention.

REFERENCES This application contains a number of references which may facilitate understanding or practice of certain aspects of the present invention. Inclusion of a reference in this application is not intended to and does not constitute an admission that such reference represents prior art with respect to the present invention.

Claims

CLAIMS :

1. A method for treatment of allergic or chronic inflammatory disease comprising administering to an individual a pharmaceutical composition comprising a biologically effective concentration of a substantially purified RANTES protein in a pharmaceutically acceptable carrier.

2. The method of claim 1 wherein the disease is asthma.

3. The method of claim 1 wherein the disease is allergic rhinitis.

4. The method of claim 1 wherein the disease is urticaria.

5. The method of claim 1 wherein the disease is contact dermatitis.

6. The method of claim 1 wherein the disease is atopic dermatitis.

7. The method of claim 1 wherein the disease is food allergy.

8. The method of claim 1 wherein the disease is idiopathic pulmonary fibrosis.

9. The method of claim 1 wherein the disease is rheumatoid arthritis.

10. The method of claim 1 wherein the disease is Crohn's disease.

11. The method of claim 1 wherein the disease is ulcerative colitis.

12. The method of claim 1 where the disease is conjunctivitis.

13. The method of claim 1 where the disease is anaphylaxis.

14. The method of claim 1 where the disease is sarcoidosis.

15. The method of claim 1 where the disease .is hypersensitivity pneumonitis.

16. The method of claim 1 where the disease is nasal polyps.

17. The method of claim 1 where the disease is bullous pemphigoid.

18. The method of claim 1 where the disease is keloid formation.

19. The method of claim 1 where the disease is selected from the group consisting of scleroderma and progressive systemic sclerosis.

20. The method of claim 1 where the disease is graft versus host disease.

21. The method of claim 1 where the disease is neurofibromatosis.

22. The method of claim 1 wherein the composition is administered by topical application.

23. The method of claim 1 wherein the composition is administered as a nasal aerosol.

24. The method of claim 1 wherein the composition is administered by direct delivery into a lesion to be treated.

25. The method of claim 1 wherein the composition is administered by subcutaneous, intramuscular or intravenous, intraarticular, or intraperitoneal injection.

26. The method of claim 1 wherein said RANTES protein is a chemical derivative of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

27. The method of claim 1 wherein said RANTES protein is a biologically active fragment of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

28. The method of claim 1 wherein said RANTES protein is a biologically active mutein of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

29. The method of claim 1 wherein said RANTES protein is administered at a dose of between 1 meg - 1000 mcg/m² of body surface area of the individual.

30. The method of claim 1 wherein said RANTES protein is administered in an amount effective to achieve a RANTES serum concentration of at least 10^"8 M in an individual to which the composition is administered

31. A pharmaceutical composition for treatment of allergic disease comprising a biologically effective concentration of a substantially purified RANTES protein in a pharmaceutically acceptable carrier.

32. The composition of claim 31 wherein said RANTES protein is a chemical derivative of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

33. The composition of claim 31 wherein said RANTES protein is a biologically active fragment of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

34. The composition of claim 31 wherein said RANTES protein is a biologically active utein of a molecule having an amino acid sequence corresponding to a naturally occurring RANTES polypeptide.

35. The composition of claim 31 wherein said RANTES protein is present in a concentration effective to achieve a RANTES serum concentration of at least 10^'8 M in an individual to which the composition is administered.

36. The composition of claim 31 containing a sufficient concentration of RANTES protein to allow administration of a single dose of between 1 meg - 1000 mcg/m² of body surface area of an individual to which the composition is administered.

37. A method for inhibiting release of an allergic mediator from human pro-allergic cells comprising exposing the pro-allergic cells to a RANTES protein.

38. The method of claim 37 wherein said pro-allergic cells comprise mast cells.

39. The method of claim 37 wherein said pro-allergic cells comprise basophils.

40. The method of claim 37 wherein said allergic mediator is histamine.

41. The method of claim 37 wherein said RANTES is present at a concentration of at least about 10^"9 M.

42. The method of claim 37 wherein said RANTES is present at a concentration of at least about 10^"8 M.

43. A method for inhibiting release of allergic mediators from human pro-allergic cells in therapy for allergic disease comprising administering to a patient having such disease a composition containing a concentration of a substantially purified RANTES protein effective to inhibit histamine release from said cells.

44. The method of claim 43 wherein the composition is administered by topical application.

45. The method of claim 43 wherein said RANTES is administered as a nasal aerosol.

46. The method of claim 43 wherein the composition is administered by direct delivery into a lesion to be treated.

47. The method of claim 43 wherein the composition is administered by subcutaneous, intramuscular or intravenous, intraarticular, or intraperitoneal injection.

48. The method of claim 43 wherein said RANTES is administered at a dose of from about 1 to about 1000 mcg/m².

49. A method for inhibiting release of allergic mediators from human pro-allergic cells comprising exposing the pro-allergic cells in vivo to a concentration of RANTES effective to inhibit release of said mediators.