WO1994027640A1

WO1994027640A1 - Method of treatment and prevention of immune complex-induced lung injury

Info

Publication number: WO1994027640A1
Application number: PCT/US1994/005806
Authority: WO
Inventors: Peter A. Ward; Michael S. Mulligan
Original assignee: The Regents Of The University Of Michigan
Priority date: 1993-05-27
Filing date: 1994-05-27
Publication date: 1994-12-08
Also published as: AU7043694A

Abstract

IL-4 and IL-10 are useful for the treatment and prevention of immune complex-induced lung injury and diseases which result in such lung inflammatory injury. Administration of IL-4 and/or IL-10 as an aerosol to the airway of the patient is particularly effective.

Description

TITLE OF THE INVENTION

METHOD OF TREATMENT AND PREVENTION OF IMMUNE COMPLEX-INDUCED LUNG INJURY This invention was made with government support under Grant Number 31963 awarded by the National Heart, Lung, and Blood institute. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention:

The present invention relates to a method of treatment and prevention of immune complex-induced lung injury and diseases which result in immune complex-induced lung injury. In particular, the present invention relates to the treatment and prevention of diseases such as Adult

Respiratory Distress Syndrome (ARDS) , chronic bronchitis, idiopathic pulmonary fibrosis, asthma, sarcoidosis, and other inflammatory disorders of the lung by the administration of either interleukin 4 (IL-4) or interleukin 10 (IL-10) , or both. The present invention also relates to pharmaceutical compositions for the treatment and prevention of inflammatory lung injury and diseases which are caused by immune reactions. The present invention also relates to dispensing devices for dispensing the present compositions.

Discussion of the Background:

There are a number of diseases which result in immunologically induced injury to the lungs. Examples of such diseases included Adult Respiratory distress Syndrome (ARDS) , chronic bronchitis, idiopathic pulmonary fibrosis, asthma, sarcoidosis, and other inflammatory diseases of the lung. These diseases manifest themselves as inflammation of the lungs. The currently available methods for reducing lung inflammation involve the systemic administration of anti-inflammatory drugs such as steroids and non-steroidal anti-inflammatory drugs. However, both classes of these drugs have significant side effects. Moreover, the non- steroidal anti-inflammatory drugs are not particularly effective.

Interleukin 4 (IL-4) and interleukin 10 (IL-10) have been recognized as having important regulatory effects on the immune and inflammatory systems. In mice, both cytokines appear to be produced by Th2 cells (Ann . Rev. Immunol . 7:145-173 (1989)). IL-4 has been shown to block the development of contact hypersensitivity reactions in mice, while administration of an antibody to IL-4 has the obverse effect (J^". Immunol . 148:1411-1415 (1992)). Similarly, anti-IL-4 antibodies are able to convert a non-protective antibody response of BALB/C mice with L. major infection to a protective, delayed-type hypersensitivity reaction (J. Exp. Med . 171:115-127(1990)). IL-4 has also been shown to reduce production of TNFα in interferon-7 (IFN7)-stimulated macrophages and, also, to reduce production of IL-1 and PGE₂ (Proc. Natl . Acad. Sci . USA 86:3803-3807 (1989)). Originally termed the cytokine synthesis inhibitory factor, IL-10 has been found to have diverse effects on immune and inflammatory cells and to perturb immune regulation. For instance, IL-10 suppresses IFN7 production by helper T cells and NK cells (J. Exp. Med . 170:2081-2095 (1989)) and suppresses the production of a series of macrophage cytokines including TNFα, IL-1, IL-lβ, IL-6, IL-8, GM-CSF, and G-CSF (J. Exp. Med . 175:1213-1220 (1993); J. Exp . Med . 174:1549-1555 (1991); J. Exp. Med . 174:1209- 1220 (1991); J. Immunol . 147:3815-3822 (1991)). IL-10 also inhibits the expression of MHC class II antigen on monocytes, in conjunction with its ability to suppress antigen-specific T cell proliferation (J. Exp . Med . 174:915-924 (1991)). Generation of nitric oxide («NO) by macrophages is also suppressed by IL-10 (Biochem . Biophys . Res . Commun . 182:1155-1159 (1992)), resulting in diminished killing of Trypanosoma, Toxoplasma and Schistosome organisms (J. Immunol . 148:1752-1756 (1992); J. Exp. Med. 175:169-174 (1992); J. Immunol . 148:3578-3582 (1992); Proc . Natl . Acad . Sci . USA 88:7011-7015 (1991)). It has been suggested that IL-10 causes increased production of IgG, IgM and IgA by B cells (J. Exp. Med . 175:671-682 (1992); Proc . Natl . Acad . Sci . USA 89:1890-1893 (1992)).

Lung injury resulting from the deposition of IgG immune complexes has been linked to products released from neutrophils (toxic oxygen products and proteases) and lung macrophages («NO, TNFα, and IL-1) (J. Clin . Invest . 54:349- 357 (1974); J. Clin . Invest . 84:1873-1882 (1989); Proc. Natl . Acad . Sci . USA 88:6338-6342 (1991)). In IgA immune complex-induced injury, the effector cells appear to be pulmonary macrophages and injury has been related to the release of -NO, toxic products of oxygen, and generation of the monocyte chemotactic factor (MCF) (J. Clin . Invest . 74:358-369 (1984); J. Immunol . 148:3086-3092 (1992); J. Immunol . 149:2147-2154 (1992)). There remains a need for a method of blocking events that lead to inflammatory lung injury and the diseases which result in inflammatory lung injury.

SUMMARY OF THE INVENTION Accordingly, it is one object of the present invention to provide a method for the treatment and prevention of lung inflammatory injury.

It is another object of the present invention to provide a method for treating lung inflammatory disorders in a manner that has a reduced tendency to cause side effects.

It is another object of the present invention to provide a method which is highly effective for treating lung inflammatory injury.

It is another object of the present invention to provide a method, which has a reduced tendency to cause side effects and is highly effective, for treating lung inflammatory injury.

It is another object of the present invention to provide a method for treat Adult Respiratory Distress Syndrome (ARDS) .

It is another object of the present invention to provide a method for treating chronic bronchitis.

It is another object of the present invention to provide a method for treating idiopathic pulmonary fibrosis.

It is another object of the present invention to provide a method for treating asthma.

It is another object of the present invention to provide a method for treating sarcoidosis and other lung inflammatory disorders.

It is another object of the present invention to provide novel pharmaceutical compositions for the treatment and prevention of lung inflammatory injury.

It is another object of the present invention to provide novel dispensing devices for dispensing such pharmaceutical compositions.

These and other objects, which will become apparent during the following detailed description, have been achieved by the inventor's discovery that immune complex- induced lung injury may be prevented by the administration of an effective amount of either IL-4 or IL-10. BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

Figures 1A-C show the dose response relationships in the model of IgG immune complex-induced lung injury of the effects of IL-4 which was coinstilled with the anti-BSA intratracheally. Parameters of injury are albumin leakage (A) , hemorrhage (B) and MPO content in lung (C) ;

Figures 2A-C show the dose response relationship in the model of IgG immune complex-induced lung injury of the effects of IL-10. Details as described in legend to Figure 1;

Figures 3A and B illustrate the effect of 250 ng of IL-4 or 250 units of IL-10 on permeability (A) and hemorrhage (B) parameters of injury in the IgA immune complex-induced model of injury. IL-4 and IL-10 were coinstilled intratracheally with the IgA anti-DNP; Figures 4A and B show the BAL fluid content of neutrophils and alveolar macrophages in IgG (A) and IgA (B) immune complex models of lung injury as a reflection of the coinstillation intratracheally of 250 ng of IL-4 or 250 units of IL-10. Statistical comparisons are to the untreated positive control values; and Figures 5A-C illustrate the morphological changes in lungs of rats undergoing IgG immune complex-induced lung injury (at 4 hrs) . In Figure 5A, the usual features of inflammation and injury in the reference positive controls are present (intraalveolar neutrophils and fibrin and hemorrhage) . In companion animals treated with 250 ng of IL-4 (Figure 5B) , or 250 units of IL-10 (Figure 5C) , there is little evidence of intraalveolar hemorrhage or inflammation (Toluidine blue, x200) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Thus, the present invention relates to the treatment or prevention of immune complex-induced lung injury. In particular, the present method involves administering an effective amount of at least one of IL-4 and IL-10. Human IL-4 is a natural protein which was characterized by Yokota et al (Proc. Natl. Acad. Sci.. USA 83:5894-5898 (1986)), which is incorporated herein by reference. The purification of recombinant human IL-4 from a CHO-cell line is described in U.S. Patent No. 5,034,133, which is incorporated herein by reference. The extraction and purification of human IL-4 are described in U.S. Patent Nos. 4,958,007 and 5,077,388, which are incorporated herein by reference. Human IL-10 has been cloned and characterized by Vieira. P. et al. Proc. Natl . Acad . Sci . USA, 88:1172-1196 (1991). Native human IL-4 is a polypeptide which is 129 amino acid residues long. It is to be understood that the present method may employ, in addition to native human IL- 4, any derivative of native IL-4 in which up to 15 amino acid residues are deleted, added, and/or substituted, providing that such derivative substantially retains the activity of native human IL-4. By the term "substantially retains the activity of native human IL-4" it is meant that the IL-4 derivative must suppress TNFα production in stimulated macrophages by at least 50% of the activity shown for intact IL-10. Preferably, the IL-4 used in the present method is native human IL-4.

Native human IL-10 is a polypeptide which is 160 amino acid residues long. It is to be understood that the present method may employ, in addition to native human IL- 10, any derivative of native IL-10 in which up to 15 amino acid residues are deleted, added, and/or substituted, providing that such derivative substantially retains the activity of native human IL-10. By the term "substantially retain the activity of native human IL-10" it is meant that the IL-10 derivative suppresses TNFα production in stimulated macrophages by at least 50% of the activity exhibited by intact human IL-10. Preferably, the IL-10 used in the present method is native human IL-10. The present method may be carried out by administering at least one of IL-4 and IL-10 in any convenient manner such as intravenous administration or administration to the airway of the patient. Preferably, the present method is carried out by administering at least one of IL-4 and IL-10 to the airway of a patient. By administering the IL-4 and/or IL-10 directly to the airway of the patient it is possible to minimize any side effects which might result from systemic administration of the IL-4 and/or IL-10. In this way, it is possible to direct the IL-4 and/or IL-10 to the lungs of the patient. The administration of IL-4 and/or IL-10 to the lungs of the patient is preferably achieved by administering the IL-4 and/or IL-10 in the form of an aerosol spray.

In the context of the present invention, the term aerosol includes compositions of matter in which particles or droplets are suspended or dispersed in a gaseous medium such as air. Thus, the term aerosol includes sprays. Apparatus and methods for forming aerosols are disclosed in Kirk-Othmer, Encyclopedia of Chemical Technology, 4th Ed., vol. 1, Wiley, New York, pp. 670-685 (1991) and Kirk- Othmer, Encyclopedia of Chemical Technology. 3rd. Ed., vol. 21, Wiley, New York, pp. 466-483 (1983), both of which are incorporated herein by reference.

The delivery of an aerosol spray which comprises at least either IL-4 or IL-10 may be conveniently carried out by means of a delivery system capable of delivering an aerosol spray. Such delivery systems include conventional nasal aerosol spray bottles, or aerosol delivery via commonly used respiratory mechanical ventilation support equipment.

Although, the exact dosage of IL-4 and 11-10 to be administered to the patient will depend, in part, on the size of the patient, the particular disease being treated, and the severity of the disease being treated, good results may be achieved by administering either IL-4 or IL-10 by having the patient inhale an aerosol containing the IL-4 and/or IL-10 in a concentration of 1 to 500 μM, preferably 10 to 50μM, 1 to 5 times per day, preferably 2 to 4 times per day. The exact dosage and frequency of treatment will depend on clinical responsiveness and other clinical parameters. Preferably, the administration of the IL-4 and/or IL- 10 is begun at the outset of the inflammatory lung injury- related disease or at the time the disorder is first diagnosed. However, good results may be obtained even if the lung inflammatory injury-related disease has progressed before the administration of IL-4 and/or IL-10 is commenced. Preferably, the administration of IL-4 and/or IL-10 is continued until the patient experiences relief from lung inflammatory injury. However, even treatment which is halted before the patient is completely free of symptoms of lung inflammatory injury is beneficial. Although good results may be obtained by administering IL-4 and/or IL-10 as the sole active ingredient, this need not preclude cotreatment with currently employed drugs (e.g., corticosteroids, aminophylline, etc). Coadministration of IL-4 and/or IL-10 via the airway may be preferred.

In another embodiment, the present invention provides pharmaceutical compositions for the treatment and prevention of lung inflammatory injury, which comprise either IL-4 or IL-10 or both. Preferably, the present compositions comprise both IL-4 and IL-10. When the present pharmaceutical composition comprises both IL-4 and IL-10, the ratio of IL-4 to IL-10 in the composition is 9:1 to 1:9, preferably 1:1. The present pharmaceutical compositions preferably are in a form which can be administered as an aerosol spray. Such compositions include aqueous solutions of IL-4 and/or IL-10. The present aqueous solutions of IL-4 and/or IL-10 may further comprise aminophylline, steroids (such as dexamethasone or methylprednisolone) and/or other bronchodilators.

The present invention may also be carried out by administering the IL-4 and/or IL-10 entrapped in a liposome. The preparation of liposomes in which IL-4 and IL-10 may be entrapped is taught in, e.g., U.S. Patent No. 5,023,087, which is incorporated herein by reference. By entrapping the IL-4 and IL-10 in a liposome it is possible to achieve the controlled release of the 11-4 and IL-10. Of course, the liposomes may also contain the additional active ingredients discussed above. In addition, the present invention may be carried out by administering mixtures of liposomes in which individual liposomes contain different active ingredients or mixtures thereof. Thus, it is possible to administer a mixture of liposomes in which some liposomes contain IL-4 and other liposomes contain IL- 10 to achieve different rates of release for the IL-4 and IL-10.

It should also be understood that the present method is useful for the treatment of any mammal. Mammals which may be treated by the present method include rats, mice, rabbits, dogs, cows, horses, sheep, monkeys, cats, pigs, and humans. Preferably, the present method is used to treat humans.

In another embodiment, the present invention provides dispensing devices for administering the present pharmaceutical compositions. The present dispensing devices comprise (a) a container means which contains a pharmaceutical composition comprising IL-4 and/or IL-2 and optionally another active ingredient; and (b) means for forming an aerosol of the pharmaceutical composition. Suitable container means and suitable means for forming an aerosol are describing in Kirk-Othmer, Encyclopedia of

Chemical Technology. 4th Ed., vol. 1, Wiley, New York, pp. 670-685 (1991) and Kirk-Othmer, Encyclopedia of Chemical Technology. 3rd. Ed., vol. 21, Wiley, New York, pp. 466-483 (1983) , both of which are incorporated herein by reference. Suitable container means include metal cans and glass and plastic bottles. Suitable means for forming an aerosol include combinations of propellants and valves (including an actuator and dip tube) . The propellant may be present in the container under pressure. Alternatively, the material in the container may be at atmospheric pressure and the pharmaceutical composition may be propelled by pressure created by mechanical force means such as, e.g., a bellows, bulb, or pump. Preferably, the present dispensing means is a pressurized aerosol can or an atomizer.

In the experiments described below, it has been demonstrated that, IL-4 and IL-10 are highly protective against inflammatory lung injury following deposition of IgG immune complexes and that IL-10 is highly protective in the IgA immune complex model. In the case of IgG immune complex-induced injury, the protective effects of both cytokines can be related to diminished accumulation of neutrophils in lung tissue, as defined by MPO content (Figures 1 and 2) and by BAL cell content (Figure 4) . These effects appear to correlate with the ability of both IL-4 and IL-10 to cause substantial (>75%) reductions in TNFα present in BAL fluids after intrapul onary deposition of IgG immune complexes (Table 1) . The suppressive effect of either cytokine on TNFα expression is consistent with several reports involving the use of TNFα-expressing monocytes that have been stimulated in vitro in the presence of IL-4 or IL-10 (Proc . Natl . Acad . Sci . USA 86:3803-3807 (1989); J . Exp. Med .

174:1209-1220 (1991)). Since TNFα production is required in the IgG immune complex model of injury and its blocking by antibody or with recombinant soluble TNFα receptor-1 reduces the tissue accumulation of neutrophils (J. Clin . Invest . 84:1873-1882 (1989); J. Immunol . 149:331-339

(1992)), the data in the current report are consistent with the observed effects of IL-4 and IL-10 on TNFα in the BAL fluids of animals containing intrapulmonary deposits of IgG immune complexes. Collectively, the findings underscore the role of TNFα as a pro-inflammatory factor, the function being probably linked to upregulation of E-selectin in the pulmonary vasculature. It has recently been shown that this model of lung injury is E-selectin dependent, (J^". Clin . Invest . 88:1396-1406 (1991)), and it seems reasonable to speculate that the role of TNFα relates to upregulation of vascular endothelial E-selectin, which is required for transmigration of neutrophils. E-selectin is not the sole adhesion molecule requirement in this model, since B2 integrin (CD18) also participates in this response (J. Immunol . 148:1847-1857 (1992)) . The role of TNFα in other pathophysiological events in this model is not excluded, such as priming of phagocytic cells for enhanced functional responses. Nevertheless, in the absence of available TNFα there appears to be a profound effect on neutrophil recruitment.

Since IL-10 has also been shown to reduce production of nitric oxide («NO) by stimulated macrophages (J. Exp. Med . 174:1549-1555 (1991); Biochem . Biophys . Res . Commun . 182:1155-1159 (1992)), and because «NO is an important product in events leading to lung injury in the IgG immune complex-induced model of lung injury (Proc . Natl . Acad . Sci . USA 88:6338-6342 (1991)), there are additional steps by which IL-10 may have protective functions. However, the ability of IL-4 and IL-10 to greatly reduce neutrophil accumulation in this model of injury suggests this may be an overriding mechanism of protection in the inflammatory model.

Of great interest is the ability of IL-10 but not IL-4 to protect against lung injury in the IgA immune complex model (Figure 3) . In this model of injury it is established that few neutrophils are recruited into lung, that neutrophils are not involved in the development of lung injury, that very little TNFα is found in the BAL fluids of these animals, and that treatment with antibody to TNFα or to E-selectin is not protective (J. Immunol.

148:3086-3092 (1992); Am . J . Pathol . 138:581-590 (1991); J. Immunol . 149:331-339 (1992)). Injury in this model is also known to be L-arginine dependent and likely due to the toxic effects of «NO or its derivatives such as peroxynitrite anion (ONOO^") or hydroxyl radical (HO«) (Proc . Natl . Acad . Sci . USA 88:6338-6342 (1991)). It has also been recently shown that IgA immune complex, induced lung injury is blocked by treatment of rats with antibody reactive with recombinant rat monocyte chemotactic protein-1 (MCP-1) (J. Immunol . 149:2147-2154 (1992)). Treatment with this antibody reduced vascular permeability and hemorrhage parameters by 67% and 52%, respectively.

It can be speculated that IL-10 but not IL-4 inhibits macrophage production of «NO (preventing functioning of toxic derivatives such as ONOO^") (Proc . Natl . Acad . Sci . USA 87:1620-1624 (1990)). It is also possible that IL-10 blocks transcriptional or post-transcriptional expression of lung MCP-1. If MCP-1 participates either in the recruitment of monocytes or acts as an autostimulant to pulmonary macrophages, the ability of IL-10 to intercept this function may be related to what appears to be a unique mechanism for protection against IgA immune complex-induced lung injury.

These data suggest that IL-4 and IL-10 have powerful, protective effects in inflammatory lung injury, in cases in which injury is most directly related to products of neutrophils or macrophages. The relatively low doses (ng amounts) for the effects of these cytokines indicate that these cytokines may be used as potential antiinflammatory agents. It is surprising that as little as 50 ng of IL-4 has significant protective effects against permeability and hemorrhage changes after intrapulmonary deposition of IgG immune complexes (Figure 1) . If this were to be diluted in the lung over a volume of at least 1.0 ml, the effective biological concentration would be approximately 2.5 nM or less. This indicates that these cytokines are extremely effective biological regulators of the inflammatory response. It seems likely that their production regulates further cytokine generation, in much the same way that soluble receptors and receptor antagonists are released from activated phagocytic cells in an attempt to regulate the inflammatory response. The lack of highly sensitive detectors of rat products precludes direct assessment of IL-4 and IL-10 as natural regulators of the lung inflammatory response in rats. It is also clear that the mechanisms by which IL-4 and IL-10 achieve their protective effects are diverse and not completely understood.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. EXAMPLES Materials and Methods:

Reagents. Except where noted, all reagents were purchased from the Sigma Chemical Corp. (St. Louis. MO) . Cytokines. Recombinant murine IL-4 was expressed in E. coli and purified to homogeneity and high specific activity (approximately 6.5 x 10⁶ U/mg) as described elsewhere (J. Immunol . 140:474-478 (1988)). The specific activity of murine IL-4 was evaluated in the HT-2 bioassay (J. Immunol . 140:474-478 (1988)). Recombinant murine IL-10 was expressed in E. coli and purified to homogeneity and high specific activity (approximately 1.75 x 10⁶ U/mg) exchange chromatography. The specific activity of murine IL-10 was evaluated by the cytokine synthesis inhibition assay (J^". Exp. Med. 170:2081-2095).

Animal Models of IgG and IgA Immune Complex-Induced Alveolitis. For all studies, adult male, 300-350 gram- specific pathogen-free Long-Evans rats (Charles Rivers Breeding Labs., Portage, MI) were employed. Rabbit polyclonal IgG rich in antibody to bovine serum albumin

(anti-BSA) was used in the IgG immune complex model of lung injury. Intraperitoneal ketamine and pentobarbital were administered for sedation and anesthesia. 2.5 mg rabbit IgG anti-BSA in a volume of 300 ul were individually instilled via an intratracheal catheter during inspiration after surgical exposure of the trachea. Immediately thereafter, 10 mg BSA together with trace amounts of ¹²⁵I- BSA and 51 Cr RBC (as quantitative markers of permeability and hemorrhage) were injected intravenously, as described elsewhere (Proc . Natl . Acad . Sci . USA 88:6338-6342 (1991)). Rats were sacrificed 4 hr later, and lung injury was quantitated by increases in vascular permeability and lung hemorrhage. For calculation of the permeability and hemorrhage indices, the amount of radioactivity (¹⁵I-BSA and ⁵¹Cr-RBC) remaining in the saline perfused lungs was compared to the amount of radioactivity present in 1.0 ml blood obtained at the time of sacrifice from the inferior vena cava. For calculation of percent change from the reference positive control, the negative control values were subtracted from cell positive control values (treated or untreated) in order to compute percent change

(reduction) in lung injury (Proc. Natl . Acad . Sci . USA 88:6338-6342 (1991)). For IgA immune complex-induced lung injury, murine myeloma IgA-1 MOPC-315, which contains an antigen binding site reactive with dinitrophenol (DNP) , was isolated as previously described (J. Clin . Invest .

74:358-369 (1984)). 1.0 mg IgA anti-DNP was instilled intratracheally and 3.3 mg DNP conjugated to serum albumin (DNP-BSA) were injected intravenously (along with ¹⁵I-BSA and ⁵¹Cr-RBC) . As in the IgG immune complex mode of lung injury, the tissue damage was measured 4 hr later and assessed by increases in lung vascular permeability and hemorrhage, as indicated. Calculations for percent change in injury were also made as described above. When employed, recombinant murine IL-4 or recombinant murine IL- 10 was co-instilled (in the amounts indicated) into the airways together with the IgG anti-BSA or IgA anti-DNP.

Tissue Extraction of Mveloperoxidase fMPO) Activity. Lungs obtained at the end of the reaction times (4 hr) were homogenized and sonicated in phosphate buffered (pH 7.4) saline (PBS) , and, after centrifugation, the supernatants were reacted with H₂0₂ in the presence of o-dianisidine. The change in optical density (at 460 nM) was determined per unit of tissue and extrapolated to MPO activity (J. Clin . Invest . 84:1873-1882 (1989)).

Bronchoalveolar (BALI Lavage. At the time of sacrifice, 5 ml RPMI-1640 were repeatedly instilled and withdrawn from the lungs via an intratracheal cannula. The BAL fluid was then collected and cell counts (total and differential) were performed. After cell counts were determined, cells were removed by centrifugation and the supernatant fluids evaluated for TNFα activity using a standard LM cell cytotoxicity assay as described elsewhere (J. Clin . Invest . 84:1873-1882 (1989)).

Morphological Analysis of Lung Tissues. At the time of sacrifice, lungs were inflated with glutaraldehyde and processed in the usual manner (Proc. Natl . Acad. Sci . USA 88:6338-6342 (1991)), followed by embedding in epon, sectioning and staining with toluidine blue.

Statistical Analysis. All values are expressed as mean ± S.E.M. For all data points in the figures, n=5. Data sets are examined with one and two-way ANOVA and individual group means were then compared with student's T-tests. For calculation of percent protection, mean negative control values were first subtracted in each positive control and in each treatment group. Multiple group comparisons were made, employing the Fisher PLSD test as well as the Scheffee t test.

Results:

Protective Effects of IL-4 and IL-10 Against the Development of IgG Immune Complex-Induced Lung Injury. IgG immune complex-induced lung injury was induced in the manner described above, with sacrifice at 4 hr. The mean permeability values in the negative and positive controls were 0.16 ± 0.01 and 0.99 ± 0.02, respectively. The mean hemorrhage values for the corresponding controls were 0.04 + 0.001 and 0.22 + 0.01, respectively. Lungs were also extracted for MPO content. The values for the negative and the positive MPO controls were 0.11 ± 0.01 and 0.51 + 0.02, respectively. When employed, recombinant murine IL-4 in amounts of 10, 50, 100 or 200 ng was coinstilled into the lungs together with the anti-BSA. As is evident from the data in Figure 1, there was a dose-dependent reduction in permeability and hemorrhage values as a function of the concentration of IL-4 employed. At the doses of 10, 50, 100 and 250 ng of IL-4, the permeability values were reduced by 6% (p,N.S.), 24% (p<.001), 46% (p<.001), and 51% (p<.001), respectively (Figure 1A) . At the same doses, the hemorrhage values were reduced by 6% (p,N.S.), 28% (p=.011), 39% (p=.007), and 56% (P<.001), respectively (Figure IB) . The same range of IL-4 doses resulted in reductions of lung MPO content by 2% (p,N.S.), 34%

(p=.002), 55% (p<.001), and 69% (p<.001), respectively (Figure 1C) . Thus, the protective effects of IL-4 in this model of acute lung injury are dose-related and are reflected in reduced accumulation of neutrophils in the lung parenchyma.

Similar studies were carried out with murine recombinant IL-10 in the IgG immune complex model of lung injury. The data are shown in Figures 2A-C. At IL-10 doses of l, 10, 100 and 500 units, the permeability values were reduced by 1% (p,N.S.), 18% (p=.002), 43% (p<.001), and 63% (p<.001), respectively (Figure 2A) . When hemorrhage was employed as the endpoint, the corresponding values were reduced by 0%, 22% (p=.013), 39% (p=.01), and 61% (p<.001), respectively (Figure 2B) . In companion studies, the mean MPO values over the same range of doses of IL-10 were reduced by 0%, 31% (p=.004), 59% (P<.001), and 73% (p<.001). Thus, IL-10 is also protective in this mode of lung injury, and its protective effects correlate with diminished tissue MPO (Figure 2C) .

Selective Protective Effects of IL-4 and IL-10 on IgA Immune Complex-Induced Lung Injury. Based on the information contained in Figures 1 and 2, IgA immune complex lung injury was induced in the manner described above. For these studies, either 250 ng of IL-4 or 250 units of IL-10 were coinstilled intratracheally along with the IgA anti-DNP. DNP-BSA was then injected intravenously. Rats were sacrificed 4 hr. later and the permeability and hemorrhage measurements obtained. The mean negative and positive control permeability values were 0.15 + 0.02 and 0.50 + 0.01, respectively, while the corresponding hemorrhage values were 0.04 ± 0.002 and 0.16 ± 0.01, respectively. 250 ng of IL-4 did not measurably reduce the permeability value (-9%, p,N.S.), nor did it reduce the hemorrhage value (-8%, p,N.S.) (Figure 3A,B) . In striking contrast, 250 units of IL-10 were highly protective, reducing the permeability value by 53% (p=.001) and the hemorrhage value by 50% (p=.004) (Figures 3A and B) . The MPO values were not determined since few neutrophils accumulate in this model of lung injury, and they do not contribute to the injury (J. Clin . Invest . 74:358-369 (1984)). These data indicate selective protective effects of IL-10 but not IL-4 in IgA immune complex-induced lung injury and suggest that the mechanism of the IL-10 effect may be different from that of IL-4, at least in this model of lung injury.

Effects of IL-4s and IL-10 on BAL Fluid Content of Cells. As indicated above, the cell content in BAL fluids of animals undergoing immune complex-induced lung injury reflects the degree of lung injury and, correspondingly, the protective effects of interventions (Proc . Natl . Acad. Sci . USA 88:6338-6342 (1991); J. Clin . Invest . 74:358-369 (1984); J. Immunol . 148:3086-3092 (1992)). In both models of lung injury, BAL neutrophils and mononuclear cells (chiefly macrophages) were quantitated. Cell content was assessed as a function of the presence or absence of 200 ng of IL-4 or 250 units of IL-10 and compared to the BAL cell content of the unprotected positive controls. All BAL samples were obtained 4 hr after initiation of lung inflammatory reactions. The results are shown in Figure 4. In the uninjured lungs of negative controls (receiving anti-BSA intratracheally and omission of intravenously administered BSA) , the mean neutrophil and mononuclear cell counts (in millions) were 0.08 ± 0.01 and 3.0 ± 0.05, respectively, while in the positive controls (with intrapulmonary deposits of IgG immune complexes) at 4 hr. the corresponding cell numbers (in millions) were 26.1 ± 1.5 and 7.0 + 0.44, respectively. The presence of 250 ng of IL-4 in the anti-BSA preparation reduced the BAL mononuclear cell content by 60% (p=.033) and the neutrophil content by 57% (p=.001). Similar reductions were found in the presence of 250 units of IL-10: mononuclear cells fell by 65% (p=.015) while neutrophil counts fell by 60% (p=.001) (Figure 4A) . Thus, reductions in the BAL cell counts, especially related to neutrophils, closely paralleled the protective effects of interventions of IL-4 and IL-10 (Figures 1 and 2) and reduction in the MPO content of lungs (Figure 1C) containing IgG immune complexes.

When BAL fluids were examined in the IgA immune complex model of lung injury, the mean neutrophil and mononuclear cell counts (in millions) in the negative controls (intratracheal administration of IgA anti-DNP but omission of intravenous DNP-BSA) were 0.09 + 0.02 and 3.2 ± 0.03, respectively, while in the positive controls 4 hr. after deposition of IgA immune complexes the corresponding mean values (in millions) for neutrophils and mononuclear cells were 1.7 ± 0.2 and 16 ± 1.3, respectively (Figure 4B) . In positive control animals treated with 250 ng of IL-4, there was a 20% decrease (p,N.S.) in neutrophil counts in the BAL fluids and no significant reduction in mononuclear cell content of BAL fluids, in keeping with the lack of protective effects of IL-4 in this model of lung injury (Figure 3) . In contrast, the presence of 250 units of IL-10 reduced the neutrophil content by 70% (p=.008) and the mononuclear cell (mostly alveolar macrophages) by 46% (p=.033), the latter falling (in millions of cells) to 10 ± 0.75. The very low neutrophil counts in the BAL fluids from lungs with IgA immune complexes should be noted as should be the fact that their presence is irrelevant to the development of lung injury (J. Clin . Invest . 74:358-369 (1984)). Thus, changes in BAL cell content in the IgA immune complex model of injury parallel the protective effects of IL-10, whereas IL-4, which was not protective in this model of lung injury (Figure 3) , failed to alter significantly the cell content of BAL fluids (Figure 4) . As stated above, these findings are consistent with changes in patterns of BAL cell content in the IgG and IgA models of lung injury in the presence of protective interventions. Morphological Features of IL-4 and IL-10 In Immune Complex-Induced Lung Injury. Lungs from rats undergoing IgG immune complex-induced lung injury were compared at 4 hr to those from companion animals to which 250 ng of IL-4 or 250 units of IL-10 had been added to the intratracheally administered anti-BSA. As shown by the data in Figures 1 and 2, substantial protection was to be expected. In Figure 5A, the usual features of alveolar injury (hemorrhage) and inflammation are present: red cells, fibrin deposits and large numbers of neutrophils. In the presence of IL-4 (Figure 5B) or IL-10 (Figure 5C) , little or no evidence of alveolar injury or inflammation is present. In both cases it was also noted that there was little evidence of neutrophil margination along venular luminal surfaces.

Effects of IL-4 and IL-10 On BAL Fluid Content Of TNFα. It is now established that in IgG immune complex- induced lung injury TNFα appears in substantial amounts in BAL fluids (J. Clin . Invest . 84:1873-1882 (1989)). Furthermore, blocking of TNFα by systemic treatment of rats with anti TNFα is significantly protective against lung injury in a manner related to reduction in numbers of neutrophils accumulating in the lung tissue (J. Clin . Invest . 84:1873-1882 (1989)). On the basis of this information, we examined TNFα in the BAL fluid 4 hr after deposition of IgG and IgA immune complexes in animals treated either with IL-4 (250 ng) or with IL-10 (250 units) , as described above. As shown by the data in Table 1, in the IgG immune complex model, IL-4 reduced the BAL content of TNFα by 78% (p<.001) while IL-10 caused a similar reduction (77%, p<.001) in TNFα content. In the IgA immune complex model of lung injury, the rise in TNFα content in BAL fluids was very low (from 0.03 ± 0.01 to 3.4 ± 0.51 units). Although IL-4 and IL-10 both markedly interfered with this small rise (Table 1) , this information is probably not relevant to events leading to injury, since in the IgA immune complex model of injury that blocking of what little TNFα is present does not protect against injury (Am . J. Pathol . 138:581-590 (1991); J. Immunol . 149:331-339 (1992)). These data imply that the reduction of TNFα by IL-4 and IL-10 in the IgG immune complex model is probably directly linked to the protective effects of these cytokines, the results of which are associated with reduced lung content of neutrophils (and MPO) . In contrast, in the IgA immune complex model, the protective function only afforded by IL-10 is almost surely related to a mechanism other than its effect on the low levels of TNFα in the BAL fluids.

Table 1 Effects of IL-4 and IL-10 on BAL Fluid Content of TNFα

BAL TNFα

Condition srvention¹ units reduction p value

Negative Control none 0.03 ± 0.01 IgG Immune Complexes none 287 ± 11.0

II II II IL-4 63.3 ± 7.1 78% <.001

IL-10 66.7 ± 18.0 77% <.001

IgA Immune Complexes none 3.4 ± 0.51

II II II IL-4 0.93 ± 0.04 73% .017

10 IL-10 0.22 ± 0.06 94% .007 t 250 ng of IL-4 or 250 units of IL-10 were mixed with IgG anti-BSA or IgA anti-DNP immediately prior to airway instillation

* Units are represented as the mean ± S.E.M.

** Anti-BSA was instilled into the airway in the absence of intravenous injection of the

15 BSA

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

WHAT IS CLAIMED AS NEW AND DESIRED TO BE SECURED BY LETTERS PATENT OF THE UNITED STATES IS:

1. A method for treating or preventing lung inflammatory injury in a mammal, comprising administering an effective amount of a compound selected from the group consisting of IL-4 and IL-10 to a mammal in need thereof.

2. The method of Claim 1, wherein IL-4 and IL-10 are coadministered to said mammal.

3. The method of Claim 1, wherein said compound is administered to the air way of said mammal.

4. The method of Claim 1, wherein said compound is administered to the airway of said patient in an aerosol comprising said compound in a concentration of 1 to 500 μM, 1 to 5 times per day.

5. The method of Claim 1, wherein an additional active ingredient selected from the group consisting of aminophylline, dexamethasone, and methylprednisolone is coadministered with said compound.

6. The method of Claim 1, wherein said lung inflammatory injury is the result of a disease process selected from the group consisting of Adult Respiratory Distress Syndrome, chronic bronchitis, idiopathic pulmonary fibrosis, asthma, sarcoidosis, and other lung inflammatory disorders.

7. The method of Claim 6, wherein said disease is Adult Respiratory Distress Syndrome.

8. The method of Claim 6, wherein said disease is chronic bronchitis.

9. The method of Claim 6, wherein said disease is sarcoidosis.

10. The method of Claim 6, wherein said disease is idiopathic pulmonary fibrosis.

11. A pharmaceutical composition, comprising: (a) a compound selected form the group consisting of IL-4 and IL- 10; (b) an active ingredient selected from the group consisting of aminophylline, dexamethasone, and methylprednisolone; and (c) a physiologically acceptable carrier.

12. The pharmaceutical composition of Claim 11, which comprises both IL-4 and IL-10.

13. The pharmaceutical composition of Claim 11, which is in a form suitable for administration to an air way of a mammal as an aerosol.

14. A pharmaceutical composition, comprising: (a) IL-

4; (b) IL-10; and (c) a physiologically acceptable carrier.

15. The pharmaceutical composition of Claim 14, further comprising an active ingredient selected form the group consisting of aminophylline, dexamethasone, and methylprednisolone.

16. The pharmaceutical composition of Claim 14, which is in a form suitable for administration to an air way of a mammal as an aerosol.

17. The pharmaceutical composition of Claim 14, wherein said IL-4 and IL-10 are present in a ratio of 9:1 to 1:9.

18. A device comprising: (a) a container means containing a pharmaceutical composition comprising at least one compound selected from the group consisting of IL-4 and IL-10; and

(b) means for converting said pharmaceutical composition into an aerosol comprising said compound.

19. The device of Claim 18, wherein said pharmaceutical composition comprises IL-4 and IL-40.

20. The device of Claim 18, wherein said pharmaceutical composition comprises an additional active ingredient selected from the group consisting of aminophylline, dexamethasone, and methylprednisolone.