WO1998033516A1

WO1998033516A1 - Use of il-4 and/or il-10 to treat proliferative glomerulonephritis

Info

Publication number: WO1998033516A1
Application number: PCT/US1998/001565
Authority: WO
Inventors: Peter G. Tipping; Arthur R. Kitching; Stephen R. Holdsworth
Original assignee: Schering Corporation
Priority date: 1997-02-05
Filing date: 1998-02-04
Publication date: 1998-08-06
Also published as: AU6045198A

Abstract

Disclosed are methods of treating a mammal afflicted with proliferative glomerulonephritis by administering to the mammal IL-4 and/or IL-10 in an amount effective for such treating. The method is particularly useful in treating mammals afflicted with or at risk of crescentic glomerulonephritis.

Description

USE OF IL-4 AND/OR IL-10 TO TREAT PROLIFERATTVE GLOMERULONEPHRITIS

FIELD OF THE INVENTION

The invention relates generally to the treatment of proliferative glomerulonephritis. More specifically, it relates to use of interleukin 4, interleukin 10 or combinations thereof, to treat proliferative glomerulonephritis in mammals.

BACKGROUND OF THE INVENTION

Glomerulonephritis (GN) refers to a group of inflammatory diseases of the kidneys characterized by inflammation of the small filters of the kidney known as glomeruli. GN is the most common cause of end stage kidney failure world-wide. In the U.S., it ranks second only to diabetes mellitus as the cause of end stage renal failure, which requires renal replacement therapy (i.e., renal dialysis or transplantation). These therapies are both costly and entail high risk; they are associated with considerable excess morbidity and early mortality.

There are many forms of GN. One form, termed proliferative glomerulonephritis, is characterized by glomerular hypercellularity or a proliferation of the cells in the glomerulus. Particularly severe forms of proliferative GN are characterized by collections of cells, fibrin and other proteins in the space around the glomerular tuft which is known as Bowman's space or the urinary space. Bowman's space is normally clear and allows for efficient renal function. The collection of cells and other proteins in Bowman's space often takes the shape of a crescent as viewed microscopically. Thus, severe forms of proliferative GN are often referred to as "cresentic GN". They are also known as "rapidly progressive GN". Cresentic GN is characterized by rapidly deteriorating renal function, which requires immediate treatment to prevent renal failure.

The most common forms of cresentic GN have been categorized into three groups. Type I cresentic GN is also known as anti-glomerular basement membrane (GBM) antibodv-mediated disease. This group is identified on the basis of evidence of antibodies reacting with GBM, either free in the circulation, as detected as immunoserologic assays, or bound to glomeruli. Type I crescentic GN constitutes about 10-20% of the overall group of patients suffering from cresentic GN. Patients with anti-GBM antibody-mediated disease accompanied by overt pulmonary hemorrhage are categorized separately as having Goodpasture disease. Type I GN tends to effect young or middle aged males.

Type II GN is often termed immunocomplex-mediated cresentic GN and also accounts for 10-20% of all patients with cresentic GN. The major distinguishing feature of Type II cresentic GN is the presence of extensive gloumerlar deposits of immunoglobulin and complement in a granular pattern throughout the mesangium and somewhat irregularly in the subendothelial space.

Type IE cresentic GN has been termed "pauci-immune necrotizing cresentic GN" because of the tendency for necrotizing features and the absence of immunoglobulin deposition. An overwhelming majority of patients of with Type III cresentic GN also have circulating anti-neutrophil cytoplasmic antibodies (ANCA), leading to the speculation that many of these patients may in fact be suffering from a form of vasculitis in which the kidney is the sole or predominant organ involved. This group constitutes between 50-70% of the overall group of cresentic GN cases, and tends to afflict middle-aged and older patients, with a slight predilection for males.

There is also an ongoing debate as to the existence of yet two further types of cresentic GN, known as types IV and V. Type IV GN represents a combination of Type I and Type m. It is relatively uncommon, accounting for less than 5% of all cresentic GN cases, and tends to be observed mostly in older women. Type V cresentic GN is characterized by the lesions of pauci-immunocresentic GN of Type HI, but lacks ANCA or anti-GEM antibodies. The clinical features are similar to those described for Type III cresentic GN. See, Glassock et al., "Primary Glomerulonephritis", in Brenner (ed), The Kidnpy. W.B. Saunders, Philadelphia (1996); Glassock et al., Disease-A-Month 42{£j):329-383 (1996); Glassock, Kidney International Supplement, 222:121-126 (October 1992) and Donadis et al., Am. J. Kid. Dis. 2K2):239-250 (1993). Current treatment modalities for GN may be divided into five broad categories, namely immunosuppression, plasma exchange, anticoagulant drugs, anti-hypertensives and dialysis or transplantation.

Immunosuppresive therapy involves the administration of immunosuppressants such as corticosteroids or cytotoxic agents. These drugs are effective in controlling some cases of proliferative GN, particularly if they are administered early in the course of disease. However, they all share a major disadvantage of being non-specific affecting virtually all aspects of the patient's immune system, rendering them immune deficient. These patients are predisposed to severe infections and a higher incidence of various forms of cancer. Immunosuppressants can further cause side effects unrelated to the immune system such as vomiting, osteoporosis, weight gain, infertility and bladder hemorrhage. Discontinuation of therapy is associated with a significant chance of relapse so immunosuppressants are most often administered on a long term basis. Thus, side effects tend to be exacerbated. Unfortunately, alternative immunosuppresive therapies, e.g., involving the administration of monoclonal antibodies specific to T-cells, have caused different, but equally severe side effects. See Chetenond, Immunol. Today 7:367-368 (1986); Abramowicz et al., Transplantation 47:606-608 (1989) and Swinnen et al., NEJM 323:1723-1728 (1990).

Plasma exchange therapy typically involves an exchange of from 2-4 liters of the patient's plasma with albumin, repeated daily for 7-14 days. This therapy is considered useful only in connection with types I and II of crescentic GN. In addition, it suffers from being highly invasive and causing potentially significant side effects, as well as requiring hospitalization and intensive monitoring. Anticoagulants have been used to prevent the formation of fibrin depositions in the glomerulus. This therapy has also been unsuccessful, due in part to a high risk of associated bleeding which is a particular concern of patients who had recent kidney biopsies and who were suffering from a pre-existing bleeding tendency due to their renal disease. Anti-hypertensives are administered to slow the rate of kidney scarring that results from cresentic GN. They also delay the onset of end stage renal failure. However, anti-hypertensives do not reverse the underlying inflammation. Finally, dialysis and kidney transplantation do not improve the function of kidneys in patients afflicted with cresentic GN. See Glassock et al., Brenner, supra.

Accordingly, there is a need in the art for a more specific form of treatment Of proliferative GN, and particularly cresentic GN, that causes fewer or less severe side effects.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method of treating a mammal afflicted with proliferative glomerulonephritis, comprising the step of administering to the mammal an amount of the cytokines interleukin-4 ("IL-4") or interleukin- 10 ("IL-10") effective for such treating.

A second aspect of the present invention is directed to the treatment of a mammal afflicted with proliferative glomerulonephritis comprising the step of co-administering to the mammal an amount of each of IL-4 and

IL-10 effective for such treating. IL-4 and IL-10 are co-administered such that they are both present in the mammalian recipient during a specified time interval. Preferably, IL-4 and IL-10 are administered simultaneously, but they may be administered sequentially provided that the administration of the second cytokine occurs within the half-life of the administration of the first cytokine. A third aspect of the present invention is directed to a method of treating a mammal at risk for crescentic glomerulonephritis, comprising the step of administering to the mammal an amount of the cytokines interleukin-4 ("IL-4") or interleukin- 10 ("IL-10"), or co-administering an amount of each of IL-4 and IL-10 effective for such treating.

In preferred embodiments of the aforementioned three aspects of the present invention, the mammal is preferably a human. The form of proliferative glomerulonephritis ("GN") to be treated is preferably crescentic GN. The preferred route of administration of IL-4, IL-10 or the combinations of IL-4 and IL-10 is parenteral, and more preferably intravenous. Human IL-4 and IL-10 are preferred; recombinant human IL- 4 and IL-10 are more preferred. The amount of IL-4 or IL-10 administered, or of each of IL-4 and IL-10 co-administered is effective to ameliorate or attenuate the development of at least one symptom, sign or histological feature of proliferative GN. Amounts generally range from about 15 μg to about 1500 μg, and more preferably from about 100 μg to about 1000 μg, per kilogram of body weight per day, for each of IL-4 and IL-10 whether administered singly or co-administered.

Administration of IL-4 or IL-10, or the co-administration of IL-4 and IL-10 to a mammal afflicted with proliferative GN, or at risk of crescentic GN, e.g., a patient suffering from proliferative GN, will cause at least one of the following effects: reduced accumulation of fibrin and proliferation of cells such as macrophages and T-cells in the glomerulus; decreased proteinuria; reduced crescent formation; and improved renal function (as measured by increased creatinine clearance, for example). The present invention also provides a more specific treatment for proliferative GN, with fewer and /or less severe side effects than those associated with prior art treatment modalities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Proliferative GN may be primary or secondary in nature, the latter being associated with infectious disease, multisystem disease or medication. See Glassock et al., supra. Mammals, preferably humans afflicted with proliferative GN may be diagnosed in accordance with standard procedures such as renal biopsy, light and immunofluorescent microscopy and serological tests. Mammals at risk of crescentic GN typically have proliferative GN which can deteriorate into crescentic GN. IL-4 (formerly B-cell stimulatory factor) is a cytokine capable of stimulating production of antibody producing B-cells and promoting growth of killer T-cells or cytotoxic T-cells. It also inhibits the activity of TH1 cells. IL-10 (formerly cytokine synthesis inhibitory factor) is a cytokine produced by T-cells and has been implicated in the control of the immune response of different classes or subsets of T helper cells. It has been reported to inhibit the synthesis of a wide spectrum of cytokines and monokines, and to exhibit growth promoting effects on murine thymocytes and T cells.

IL-4 and IL-10 suitable for use in the present invention may be obtained from several sources. For example, they may be isolated from culture media containing activated T-cells, or via standard chemical synthesis techniques. See, e.g., Merrifield, Science 233:341-347 (1986) and Atherton et al., Solid Phase Synthesis, A Practical Approach. IRL Press, Oxford (1989). Preferably, they are obtained by recombinant techniques. General methods of molecular biology are -described, e.g., by Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual. Second ed., Cold Spring Harbor, New York and Ausubel et al., feds). Current Protocols in Molecular Biology, Green/Wiley, New York (1987 and periodic supplements). The appropriate sequences can be obtained from either genomic or cDNA libraries using standard techniques. DNA constructs encoding IL-4 and IL-10 may also be prepared synthetically by established standard methods, e.g., in an automatic DNA synthesizer, and then purified, annealed, ligated and cloned in suitable vectors. Atherton et al., supra. Polymerase chain reaction (PCR) techniques can also be used. See e.g., PCR Protocols: A Guide to Methods and Applications. 1990, Innis et al. (ed.), Academic Press, New York. Numerous complementary DNAs (cDNAs) for IL-4 have been cloned and sequenced, e.g. Yokoto et al, Proc. Natl. Acad. Sci. USA 83:5894- 5898 (1986) (human); Lee et al., Proc. Natl. Acad. Sci. USA 83:2061-2065 (1986) (mouse); Noma et al., Nature 319:640-646 (1986) (mouse); and Genzyme Corporation, Boston MA (human and mouse). Moreover, non- recombinant IL-4 has been purified from various culture supernatants, e.g. Sanderson et al, Proc. Natl. Acad. Sci. USA 23:437-440 (1986) (mouse); Grabstein et al., J. Exp. Med. 163:1405-1413 (1985) (mouse); Ohara et al., J. Immunol. 135:2518-2523 (1985) (mouse B-cell stimulatory factor (BSF)-l); Butler et al., J. Immunol. 133:251-255 (1984) (human BCGF); and Farrar et al., J. Immunol. 13.1:1838-1842 (1983) (mouse BCGF).

Preferably, the IL4 used in the present invention is human IL-4. See, e.g., U.S. Patent No. 5,552,304 to Lee et al. More preferably, it is the human IL-4 having the sequence described in Yoketo et al., Proc. Natl. Acad. Sci. USA 83:5894-5898 (1986) and PCT Patent Application No. 87/02990 published May 21, 1987, and which is expressed in E. coli (U.S. Patent No. 4,958,007). The production of human IL-4 from CHO cells is described in commonly-owned U.S. Patent Application Ser. No. 386,937, filed July 28, 1989. The production of IL-4 from E. coli is described in commonly-owned U.S. Patent 5,578,464. Human IL-10 is preferred in the present invention. In general, however, the IL-10 may be obtained from numerous other sources. For example, viral IL-10, mouse IL-10 and IL-10 from other mammalian species may be used. More preferably, the IL-10 used is recombinant human IL-10. Recombinant production of human IL-10 is described in U.S. Patent No. 5,231,012 to Mosmann et al. Preparation of human and mouse IL-10 is also described in International Application Publication No. WO 91/003249. The cloning and expression of viral IL-10 from Epstein Barr virus is disclosed in Moore et al, Science 248:1230-1234 (1990), and in EP 0506 836.

For the purposes of this invention, both glycosylated (e.g., produced in eukaryotic cells such as yeast or CHO cells) and unglycosylated (e.g., chemically synthesized or produced in E. coli) forms of IL-4 and IL-10 are equivalent, and can be used interchangeably. The terms "IL-4" and "IL-10" further embrace biologically active muteins and other fragments and analogs of IL-4 and IL-10. Muteins are typically non-naturally occurring polypeptides which differ from the native polypeptide, i.e., IL-4 and IL-10, in terms of amino acid substitutions, deletions, additions, and fusions. Preferably, amino acid substitutions will be conservative; i.e., basic amino acid residues will be replaced with other basic amino acid residues, etc. These modifications can be used in a number of combinations to produce the final modified protein chain. Amino acid sequence variants can be prepared with various objectives in mind, including increasing serum half-life, facilitating purification or preparation, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature, although others may be post- translational variants, e.g., glycosylation variants or proteins which are conjugated to polyethylene glycol (PEG), etc. Such variants can be use in this invention as long as they retain the biological activity of IL-4 or IL-10.

In general, modifications of the sequences encoding the polypeptides may be readily accomplished by standard techniques such as site-directed mutagenesis as described in Gillman et al., Gene 8:81-97 (1979) and Roberts et al., Nature 328:731-734 (1987). Most modifications are evaluated by routine screening via an assay designed to select for the desired property. Methodologies for preparing and testing IL-10 muteins are described in WO/91 /003249 and U.S. 5,231,012 to Mosmann et al., and those for IL-4 are described in U.S. 5,017,691. To prepare pharmaceutical compositions containing the peptides IL- 4 and /or IL-10, the peptides are admixed, singly or together, with a pharmaceutically acceptable carrier or excipient which is preferably inert. The preparation of such pharmaceutical compositions is known in the art. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary. Mack Publishing Company, Easton, PA (1984).

The peptides may be administered in aqueous vehicles such as water, saline or buffered vehicles with or without various additives and/or diluting agents. A suspension, such as a zinc suspension can be prepared to include the peptides. Such a suspension can be useful for subcutaneous (SQ) or intramuscular (IM) injection. By adjusting the proportion of zinc and the acidity, the absorption rate of the peptide can be manipulated. The proportion of peptide and additive can be varied over a broad range so long as both are present in effective amounts. By the phrases "amount of IL-4 (IL-10 or each of IL-4 and IL-10) effective for such treating," it is meant an amount sufficient to ameliorate or attenuate at least one symptom, sign or histological feature of proliferative glomeronephritis or crescentic glomerulonephritis such as, for example, reduced renal function (as indicated by reduced creatinine clearance, for example), haematuria, hypertension, proteinuria, glomerular crescent formation, glomerular fibrin deposition and glomerular macrophage and T-cell accumulation. The amount, frequency, and period of administration will vary depending upon factors such as the disease state, the overall health and age of the patient, route of administration, severity of side effects, and the like. Determination of the appropriate dose is made by the clinician, e.g., a nephrologist, using parameters known in the art. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved. In general, the amount of each of IL-4 and IL-10 can range from about 15 μg to about 1500 μg per kilogram body weight of the patient. A preferred range is from abut 100 μg to about 1000 μg per kilogram body weight of the patient.

Compositions are usually administered orally or parenterally. Oral administration is typically in the form of a bolus. Parenteral administration is usually intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intra-articular injection or other routes could be used in appropriate circumstances. Compositions including the peptides IL-4 and/or IL-10 may also be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent 3,773,919; and U.S. Patent 3,270,960.

Preferably, the peptides are administered parenterally in a unit dosage injectable form, examples of which include solutions, suspensions and emulsions. Typically, the peptides are injected in association with a pharmaceutical carrier such as normal saline, Ringer^'s solution, dextrose solution and other aqueous carriers known in the art. Appropriate non- aqueous carriers may also be used. Examples include fixed oils and ethyl oleate. A preferred carrier is 5% dextrose in saline. It is frequently desirable to include additives in the carrier such as buffers and preservatives or other substances to enhance isotonicity and chemical stability.

Preferably, each peptide, IL-4 and/or IL-10, is formulated in purified form at a concentration of about 1 to 20 mg/ml, and substantially free of aggregates and other proteins. The concentration of each peptide in a unit dose is from about 100 micrograms to 100 milligrams varying with the application and the potency of the peptide. Most preferably, an intravenous injection delivers about 1 mg to about 100 mg of each of the peptides per day. The dose range generally ranges from about 15 μg to 1500 μg per kilogram of body weight of the recipient per day per peptide. Dosages should be varied according to side effects and blood cell counts which should be monitored frequently, preferably daily.

In preferred embodiments, IL-4 and IL-10 are co-administered. More preferably, they are administered simultaneously by mixing them prior to injection or infusion. Alternatively, the two medications may be separately infused or injected simultaneously or sequentially. In the case when they are administered separately, IL-4 and IL-10 are delivered simultaneously or as nearly so as practical. The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting as to the scope of the invention described herein, unless otherwise specified.

EXAMPLES

Two experiments were conducted using identical protocols except for the administration of the cytokines relative to the initiation of crescentic GN. In the first experiment, cytokine treatment was initiated 10 days (hours) prior to the establishment of crescentic GN, and in the second experiment, cytokine treatment was initiated 72 hours after the establishment of crescentic GN. The following protocol was followed. Induction of anti-GBM GN

Anti-GBM globulin was prepared from serum of a sheep immunized against a particulate fraction of mouse GBM by absorption with mouse red blood cells and ammonium sulfate precipitation, as previously described in Tipping et al., Kidney Intl. 46:79-88 (1994). Male C57BL/6 mice, eight to ten weeks of age (Animal Resource Centry, Canningvale, Western Australia), were sensitized by s.c. injection of a total of 2 mg of sheep globulin in 200 μl of Complete Freund's Adjuvant (CFA)(Sigma Chemical Co., St. Louis, Missouri, USA) in divided doses in each flank. Ten days later, GN was initiated by i.v. administration of 10.5 mg of sheep anti- mouse GBM globulin. Histological assessment of glomerular injury

Glomerular crescent formation: Kidney tissue was fixed in Bouin's fixative, embedded in paraffin and 3 μm tissue sections were cut and stained with periodic acid Schiff's (PAS) reagent. Glomerular crescent formation was assessed in a blinded protocol. Glomeruli were considered to exhibit crescent formation when 3 or more layers of cells were observed in Bowman's space. A minimum of 50 glomeruli were assessed to determine the crescent score for each animal.

Assessment of glomerular fibrin deposition: Tissue was embedded in Optimal Cutting Temperature Compound (OCT, Miles Scientific, IN), immediately frozen in liquid nitrogen and stored at -70°C. Immunofluorescence was performed on 4 μm cryostat cut tissue sections using FITC conjugated goat anti-mouse fibrin/fibrinogen serum (Nordic Immunological Laboratories, Berks, UK) at a dilution of 1 in 25.

Glomerular fibrin deposition was assessed in a blinded protocol on a minimum of 30 glomeruli per mouse and scored semiquantitatively (0-3+) as follows: 0 = no fibrin deposition; 1 = fibrin occupying up to one-third of the glomerular cross-sectional area; 2 = fibrin occupying one- third to two- thirds of the glomerulus; and 3 = greater than two-thirds of the glomerular cross-section covered by fibrin.

Glomerular T cell and macrophage accumulation: Spleen and kidney tissue was fixed in periodate/lysine/paraformaldehyde for 4 hours, washed in 7% sucrose solution, then frozen in liquid nitrogen. Tissue sections (6 μm thick) were stained to demonstrate macrophages and T cells using a three-layer immunoperoxidase technique, as described in Huang et al., Kidney Int. 46:69-78 (1994) and Tipping et al., Eur. J. Immunol. 26:454-460 (1996). The primary antibodies were GK1.5 (monoclonal anti-mouse CD4, American Type Culture Collection [ATCC], Rockville, MD) and Ml/70 (monoclonal anti-mouse Mac-1, ATCC). Sections of spleen provided a positive control for each animal and protein G purified rat immunoglobulin was substituted for primary monoclonal antibody to provide a negative control. A minimum of 20 equatorially sectioned glomeruli were assessed per animal and the results were expressed as cells per glomerular cross section (c/gcs).

Functional assessment of glomerular injury

Proteinuria: Mice were housed individually in cages to collect urine over the final 24 hours of each experiment. Urinary protein concentrations were determined by a modified Bradford method described in Bradford, Anal. Biochem. 72:248-254 (1976), adapted to a microtiter plate assay as described in Huang et al., supra. The 24 hour urinary protein excretion was calculated from the 24 hour urine volume and the urinary protein concentration. Creatinine clearance: Serum and urine creatinine concentrations were measured by the alkaline picric acid method using an autoanalyser (Cobas Bio, Roche Diagnostic, Basel, Switzerland) in mice with GN and normal male mice (N = 13). Creatinine clearance was calculated from the serum and urine creatinine values and the urine volume. Assessment of the systemic immune response DTH to sheep globulin: Mice were challenged 24 hours prior to the end of each experiment by intradermal injection of sheep globulin (50 μg in 30 μl of PBS) into the plantar surface of a hindfoot. An irrelevant antigen (horse globulin) was injected in the opposite foot pad as a control. DTH was quantified 24 hours later by measuring the difference in thickness between sheep globulin and horse globulin injected foot pads in each mouse using a micrometer (Mitutoyo Corporation, Japan). Results were expressed as the change in foot pad thickness (mm).

Measurement of IFN-γ production by splenic T cells: Spleens from sensitized mice were removed aseptically and placed in RPMI 1640 5% FCS medium. Single cell suspensions were prepared by gently teasing tissue apart. Erythrocytes were lysed by incubation in Boyle's solution (0.17 M Tris, 0.16 M ammonium chloride) for 1 min. at 37°C. Cell suspensions were washed in RPMI 1640 5% FCS, then enriched for T cells by passage through nylon wool columns. Samples of enriched T cells (4X10⁶ cells/ml in RPMI 1640 10% FCS) were incubated for 72 hours at 37°C, 5% C0₂ in 48- well tissue culture plates with sheep IgG (lOμg/ml).

In the first experiment, endogenous IL-4 production by the samples of enriched T cells as follows. IL-4 was measured by ELISA using flat- bottom polystyrene microtiter plates coated with rat anti-mouse IL-4 monoclonal antibody (mAb) (11B11, ATCC) at a concentration of 5 μg/ml and blocked with 1% BSA. Supernatant (100 μl) and recombinant mouse IL-4 standards (Genzyme, Cambridge, MA) were incubated in wells overnight at 4°C. Biotinylated rat anti-mouse IL-4 mAb (BVD6, DNAX,

Palo Alto, CA) was used as the detecting Ab at a concentration of 1 μg/ml. The plates were washed and incubated with streptavidin horseradish peroxidase complex (Silenus, Hawthorn, Victoria, Australia) at dilution of 1:1000 in 1% BSA/PBS. ABTS + H₂0₂ substrate solution was added and the reaction was stopped after 10 min with 0.1 M citric acid containing 0.1% sodium azide (Ajax Chemicals, Cheltenham, Victoria, Australia). The absorbance was read at 405 run on an ELISA plate reader. The lower limit for detection of IL-4 in this assay was 6 pg/ml.

In the second experiment, IFN-γ production by the samples of enriched T cells was measured as follows. IFN-γ in culture supernatants was measured by ELISA using flat bottom polystyrene microtiter plates (Greiner Labortechnik, Kremsmunster, Austria) coated with rat anti- mouse IFN-γ monoclonal antibody (RA-6A2, Pharmingen, San Diego, CA) at 5 μg/ml and blocked with 1% BSA. Supernatant (100 μl) or recombinant murine IFN-γ (Genzyme, Cambridge, MA) was incubated in wells overnight at 4°C. Biotinylated rat anti-mouse IFN-γ monoclonal antibody (XMG1.2, Pharmingen) was used as the detecting antibody at a concentration of 1 μg/ml. Plates were washed and incubated with streptavidin horse radish peroxidase complex (Silenus, Hawthorn, Victoria, Australia) at dilution of 1 in 2000 in 1% BSA/PBS, using 0.1M 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS, Boehringer Mannheim, Germany) in 0.02% H₂0₂ as a substrate. The absorbance at 405 nm was read on a microtiter plate reader (Dynatech Laboratories, Chantilly, VA). The lower limit for detection of IFN-γ in this assay was 6 pg/ml. Circulating mouse anti-sheep globulin antibody: Titers of mouse anti- sheep globulin antibody were measured by ELISA on serum collected at the end of each experiment. Polystyrene microtiter plates were coated with lOμg/ml normal sheep globulin in carbonate/bicarbonate buffer pH 9.6 by incubation overnight at 4°C and blocked with 1% BSA. Plates were washed, then incubated with serial dilutions of mouse serum. After further washing, bound mouse immunoglobulin was detected with horseradish peroxidase conjugated sheep anti-mouse immunoglobulin (Amersham, Little Chalfont, UK) at a dilution of 1 in 2000. ABTS substrate solution was added and the absorbance was read at 405 nm. Serum from each mouse was tested at 6 dilutions from a dilution of 1 in 100 to 1 in 102,400 and serum from 6 non-immunized mice was tested to provide normal controls. Measurement of circulating anti-sheep globulin Ig isotypes

The above ELISA technique was adapted to measure specific isotypes of mouse anti-sheep globulin antibodies. Mouse serum was incubated in wells containing bound antigen as described above using a single dilution of 1 in 200 for optimal detection of IgGl and IgG2b isotypes and 1 in 20 for optimal detection of IgG2a and IgG3 isotypes. Binding of specific mouse immunoglobulin isotypes was detected using horse radish peroxidase conjugated goat anti-mouse IgGl, IgG2a, IgG2b and IgG3 antibody (Southern Biotechnology Assoc, Birmingham, AL) at a dilution of 1 in 4000. The plates were washed and incubated with ABTS and the absorbance at 405 nm was used to compare the concentration of each isotype between treatment groups. Results were expressed as a percentage of the absorbance in control treated mice. Cytokine treatment protocols, experimental design and statistical analysis Recombinant murine IL-4 (specific activity 2.24xl0⁹ IU/mg) and IL-

10 (specific activity 6.3xl0⁷ IU/mg), (Schering Plough Research Institute, Kenilworth, NJ) were diluted in PBS and administered as a single daily i.p. dose of 2.5 μg in 100 μl. In the first experiment, treatment was initiated 1 hour prior to sensitization of mice with sheep globulin and continuing until 24 hours prior to the end of the experiment. In the second experiment, treatment was started 72 hours after initiation of disease with anti-GBM globulin and continued until 24 hours prior the end of the experiment. In both experiments, combined treatment was performed with 2.5 μg of both IL-4 and IL-10 administered in a single lOOμl i.p. injection in an identical protocol to the treatment with single cytokines.

Anti-GBM GN was induced as described above and glomerular injury was assessed 10 days after administration of anti-GBM globulin. In the first experiment, the following groups were studied: control (PBS) treatment (n=6); IL-4 treatment (n=5); IL-10 treatment (n=6); and IL-4 + IL- 10 treatment (n=6). The statistical significance of differences between groups was determined by the Mann Whitney U test.

In the second experiment, a group of mice (N=6) was killed after 3 days to determine the nature of renal injury in this model of GN prior to the commencement of treatment. Three days after initiation of GN, the remaining mice were randomly assigned to one of 4 experimental groups: control (PBS) treatment (N=7), IL-4 treatment (N=6), IL-10 treatment (N=7) and IL-4 plus IL-10 treatment (N=7). The statistical significance (at a P value of < 0.05) of differences between groups was determined by ANOVA, followed by Fisher's protected least significant differences (PLSD) test for pair-wise comparisons. RESULTS

The results of the first experiment wherein cytokine treatment was initiated prior to the establishment of crescentic GN, are set forth in Table

Table I

Photomicrographs (not shown) of glomeruli from mice with anti- GBM GN showed a severe proliferative and crescentic pattern following control treatment with PBS, and marked attenuation of injury following treatment with IL-4, IL-10 and a combination of IL-4 and IL-10. Referring now to Table I, sensitized mice developed a severe proliferative GN after anti-GBM globulin with crescent formation in 42.5± 4.5% of glomeruli (normal 0%) and prominent deposition of fibrin in glomeruli (fibrin score 1.21+ 0.17, normal 0). This was associated with accumulation of T cells (1.39+ 0.14 c/gcs, normal O.l± 0.01 c/bcs, p < 0.001) and macrophages (4.69± 0.54 c/bcs, normal 0.1+ 0.01 c/gcs, p ( 0.001) in glomeruli. Development of GN resulted in functional evidence of severe glomerular injury with marked proteinuria (8.3± 0.9 mg/24 h, normal 0.74+ 0.08 mg/24 h, p < 0.001) and decreased creatinine clearance (93+ 12 μL/MIN, NORMAL 193±

10 μl/min, p < 0.001).

Treatment with IL-4 significantly attenuated the histological appearances of glomerular injury, abolished crescent formation (0.8± 0.5%, p < 0.006) and significantly attenuated glomerular fibrin deposition (fibrin score 0.23± 0.12, p < 0.011). T cell (0.13± 0.03 c/gcs, p > 0.011) accumulation in glomeruli was significantly reduced compared to control mice. Treatment significantly reduced proteinuria (3.6± 1.0 mg/24 h, p < 0.018) and but did not result in significant protection of renal function (creatinine clearance, 121+ 17 μl/min, p =0.2).

Similar effects on the development of GN were seen following treatment with IL-10. Histological features of disease, including crescent formation (1.2± 0.9%, p < 0.004) and glomerular fibrin deposition (fibrin score 0.18± 0.13, p < 0.006) were markedly attenuated. Glomerular accumulation of T cells (0.29+ 014 c/gcs, p < 0.004)and macrophages (0.42+ 0.12 c/gcs, p < 0.004) was also reduced. A significant decrease in proteinuria (2.2+ 0.5 mg/24 h, p < 0.004) and significant attenuation of the decline in creatinine clearance (150± 20 μl/min, p < 0.037) both indicated protection of glomerular function by treatment with IL-10.

Combined treatment with IL-4 and IL-10 markedly attenuated the histological appearance of GN such that most glomeruli appeared normal at a light microscopic level. Crescent formation (1.4± 1.0%, p < 0.004), glomerular fibrin deposition (fibrin score 0.23± 0.10, p = 0.005) and accumulation of T cells (0.24+ 0.15 c/gcs, p < 0.004) and macrophages (0.46± 0.16 c/gcs, p < 0.004) were reduced to an extent similar to that observed with treatment with IL-4 or IL-10 alone. Proteinuria (2.9+ 0.5 mg/24 h, p < 0.004) was decreased to an extent similar to treatment with a single cytokine. However, combined treatment with IL-4 and IL-10 resulted in complete protection of renal function, with creatinine clearance in treated mice (183+ 22 μl/min) the same as normal mice.

Treatment with either single cytokines or combined cytokines did not affect the overall serum titers to anti-sheep globulin AB. However, changes in the relative concentration of Ig isotypes were observed. Anti- sheep globulin of the IgG2a and IgG3 isotypes was decreased (55% and 285, respectively, compared to control treated mice) following treatment with IL-4 alone. A similar decline in the IgG2a isotype was observed with IL-10 and combined cytokine treatment (48% and 45%, respectively). IgG3 tended to show a progressively greater inhibition with IL-10 and combined cytokine treatment (51% and 75% decline, respectively). There was a small reduction in the IgGl isotype following treatment with IL-4 or IL-10 (14% and 13% reduction, respectively) and no additive effect of these cytokines was observed (16% reduction). IL-4 treatment resulted in a 31% reduction in IgG2b, but no substantial effects on this isotype were seen with IL-10 and combined cytokine treatment (4% and 4%, respectively). Treatment with both cytokines alone and in combination abolished foot swelling following cutaneous antigen challenge with sheep globulin, indicating inhibition of DTH. Production of IFN-γ by splenic T cells i n vitro was reduced in mice treated with IL-4 (73± 45 ρg/10⁶ cells per 72 h) and IL-10 (104+ 55 pg/10⁶ cells per 72 h), although these decreases compared with IFN-γ production by control treated mice (211+ 71 pg/10⁶ cells per 72 h) did not reach statistical significance. A more marked effect was observed with combined cytokine treatment (40+ 28 pg/10⁶ cell per 72 h, p = 0.087 compared with control treatment). IL-4 production was detectable in splenic T cells from control treated mice (29+ 9 pg/10⁶ cells per 72 h), but was undetectable in IL-4 and combined cytokine-treated mice. IL-4 production was detectable in two of six mice treated with IL-10 (1.2± 0.8 pg/10⁶ cells per 72 h, p < 0.009).

The results of the second experiment in which cytokine treatment was initiated 72 hours after the establishment of disease are set forth in Table π.

Table H

Development of disease in control mice Photomicrographs of glomeruli from mice with anti-GBM GN showed a severe proliferative and crescentic pattern following control treatment with PBS, and attenuation of injury following treatment with IL-10 or both IL-4 and IL-10, but did not show significant attenuation by treatment with IL-4. Three days after anti-GBM globulin, mice showed evidence of established renal injury, with proliferative changes in glomeruli with glomerular infiltration of CD4+ T cells (0.96+0.14 c/gcs) and macrophages (3.49±0.63 c/gcs), and glomerular fibrin deposition (0.45+0.11 [score 0-3+]). There was no significant glomerular crescent formation after 3 days. Proteinuria had developed in all mice after 48 hours of disease (10.7± 1.3 mg/24 hours). ^* By 10 days after anti-GBM globulin, mice treated with PBS alone from day 3 developed proliferative GN with frequent glomerular crescent formation, continuing glomerular inflammatory cell infiltrate, fibrin deposition and proteinuria. See Table π. Effect of IL-4 and/or IL-10 treatment on crescent formation and glomerular DTH

The combination of IL-4 and IL-10 administered from days 3-9 of disease had the greatest effect in preserving glomerular structure and reducing the glomerular DTH response to sheep globulin. Mice treated with both IL-4 and IL-10 had significantly reduced glomerular crescent formation (P < 0.02), fewer glomerular T cells (P < 0.001), fewer macrophages (P < 0.001) and decreased glomerular fibrin deposition (P < 0.005) at day 10 of disease. Treatment with a single cytokine had lesser effects. There was a trend towards reduced crescent formation in IL-10 treated mice (P = 0.11. These mice had fewer glomerular T cells (P < 0.02) fewer macrophages (P < 0.005) and reduced glomerular fibrin deposition (P < 0.02). IL-4 alone did not attenuate glomerular crescent formation, but did reduce glomerular T cell (P < 0.02) and macrophage (P < 0.05) infiltration to a lesser degree than treatment with either IL-10 or combined cytokine treatment. IL-4 did not significantly reduce glomerular fibrin deposition. This increasing protection from disease following treatment with IL-4, IL-10 and the combination of IL-4 and IL-10 was clearly illustrated by the photomicrographs of representative glomeruli and in the development of crescent formation, fibrin deposition, glomerular T cell infiltration and glomerular macrophage accumulation (see Table II). Effects of IL-4 and/or IL-10 treatment on renal function

Control (PBS) treated mice developed renal impairment (P < 0.05) at day 10. Treatment with IL-4 did not prevent the decline in renal function associated with crescentic GN at day 10. Treatment with either IL-10 or a combination of IL-4 and IL-10 attenuated renal impairment such that their renal function was not statistically different to normal mice (IL-10 P = 0.31, IL-4 + IL-10 P = 0.57). The trend towards progressive protection from injury paralleled the progressive attenuation of crescent formation. Cytokine treatment of established GN did not result in a significant reduction in proteinuria at day 10 (see Table II).

Effects of IL-4 and/or IL-10 on systemic immune responses

Cutaneous DTH to sheep globulin was reduced in all groups (P < 0.001). The production of IFN-γ by splenic T cells cultured with sheep IgG was not significantly altered (PBS 146± 83 pg/4xl0⁶ cells, IL-4 32+ 13, IL-10

262± 69, both IL-4 and IL-10 138± 43) although there were trends towards lower levels of IFN-γ from T cells of IL-4 treated animals and towards increased levels from T cells of mice treated with IL-10. There were no changes in total serum sheep globulin specific antibody level in treated animals, although the antibody titers of mice treated with IL-10 or both IL-4 and IL-10 tended to be greater than those of mice treated with PBS alone or IL-4. There were no changes in the relative concentrations of antigen specific IgG isotype levels between groups. The results show that in a model of proliferative and crescentic GN,

IL-4 and IL-10 were protective. That is, in administering IL-4 and/or IL-10 prior to disease induction, functional and histological indices of GN were markedly less severe than those in control treated animals. In treating established crescentic GN, the combination of IL-4 and IL-10 had the greatest effect as compared to IL-4 or IL-10 alone.

All publications and patent applications cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a mammal afflicted with proliferative glomerulonephritis, comprising the step of administering to said mammal an amount of IL-4 or IL-10 effective for such treating.

2. The method of claim 1, wherein the effective amount of IL-4 or IL-10 is from about 15 ╬╝g to about 1500 ╬╝g per kilogram body weight of said mammal.

3. The method of claim 1, wherein IL-4 is administered to said mammal.

4. The method of claim 3, wherein said IL-4 is human IL-4.

5. The method of claim 4, wherein said human IL-4 is recombinant human IL-4.

6. The method of claim 1, wherein IL-10 is administered to said mammal.

7. The method of claim 6, wherein said IL-10 is human IL-10.

8. The method of claim 7, wherein said human IL-10 is recombinant human IL-10.

9. A method of treating a mammal afflicted with proliferative glomerulonephritis, comprising the step of co-administering to said mammal an amount of IL-4 and IL-10 effective for such treating.

10. The method of claim 9, wherein said mammal is a human.