JPH08510719A - Use of a pharmaceutical composition containing an effective amount of interleukin-10, its analogs and / or agonists - Google Patents

Abstract

  (57) [Summary] The present invention provides i) production of interleukin-5 by T lymphocytes; or ii) activation of T lymphocytes by the B7 / CD28 or ICAM-1 / LFA-1 pathway; or iii) monocyte procoagulant activity. Of interleukin-10 (IL-10) or its analogs or agonists for the manufacture of a medicament for the treatment or prevention of a disease associated with activation of iv; iv) a severe infection associated with tumor necrosis factor (TNF); Regarding use.

Description

Detailed Description of the Invention   Contains an effective amount of interleukin-10, its analogs and / or agonists Use of a pharmaceutical composition according to   The present invention provides an excess of interleukin-5, B7 / CD28 and / or ICAM-1 / LF. Excessive T cell stimulation and / or excessive monocyte coagulation stimulation by molecular interaction of A-1 A cure for a disease selected from the group consisting of diseases induced by progressive activity. Or for the prevention, manufacture of medicaments intended to treat mammals, especially humans Effective amount of interleukin-10, analogs and / or agonists thereof It relates to the use of a pharmaceutical composition containing a drug.   Human IL-10, in its mature form, contains 160 amino acids and two intermolecular dimers. It is a cytokine having a sulfide bridge. Human IL-10 is a glycosylation site Position but not glycosylated.   IL-10 is produced in the body by a number of different cell types. Immunity Cells involved in the system, namely lymphocytes T and B and monocytes / macrophages, are IL-10 Is an important origin. Besides the immune system, keratinocytes and certain tumor cells also show IL-10. Can be produced. The factors that control IL-10 synthesis are not fully understood. Absent.   First, Cytokine Synthesis Inhibitory Factor IL-10, known as (CSIF), can be used to target a number of different target cells. Exert a biological effect.   Its immunosuppressive and anti-inflammatory properties are primarily due to monocytes and The effect of this cytokine on macrophages (ie monocytes and macrophages). Inhibits the production of TH1 cytokines), but IL-10 It has recently been found that also can act directly on T lymphocytes. TH1 Cytoca Inhibition of in- (IL-2, IFN-γ) production by IL-10 is mainly due to the activity of T lymphocytes. And monocytes that act as Antigen Presenting Cells (APC) It has been observed in a system determined by macrophages. IL-10 is mainly due to APC Interfering with T lymphocytes by inhibiting the activation signal provided to T lymphocytes. It acts directly. Generation of class II major histocompatibility complex (MHC) on the APC surface Current inhibition is considered one of the relevant mechanisms.   For example, International Patent Application Publication No. WO 91/00349 describes IL-10 (CSIF) as , A mammalian cytokine that inhibits TH1 lymphocytes and regulates cellular immune responses Has been described. Using IL-10 to suppress diseases related to MHC-related immune responses Is described.   International Patent Publication No. WO 93/17698 describes graft-host correlation disease or The use of IL-10 to suppress textile rejection has been described. I in this situation The main mechanism of action of L-10 is down-regulation of MHC class II expression on monocytes. It is described as In this document, down-regulation of ICAM-1 and B7 expression on monocytes Regulation is explicitly excluded as a mechanism of action for IL-10. Accordingly Thus, IL- is associated with diseases associated with activation of T lymphocytes by the B7 / CD28 and ICAM-1 / LFA-1 pathways. The use of 10 is neither stated nor suggested. It is not.   International Patent Application Publication No. WO 93/02693, published on February 18, 1993. No. (Prior Art according to Article A.54 (3) of the European Convention) describes septic shock. For the treatment or prevention of toxic shock and toxic shock, IL-10 or analogs thereof or Described to use agonists or antagonists There is. According to this document, IL-10 was not exposed at the same time as exposure to LPS or superantigens. Or administered after that exposure. IL before exposure to endotoxin or superantigen When -10 is administered, morbidity caused by Gram-negative or Gram-positive species and There is no suggestion that mortality due to morbidity can be prevented.   International Patent Publication No. WO 93/19770 contains IL-10 in addition to IL-4. And compositions suitable for use in the treatment of acute or chronic inflammation have been described. . The use of IL-10 alone is not described.   The technical problems underlying the present invention are as follows.   i) Septic shock, preferably prior to exposure to endotoxin or superantigen Identifying effective means of treatment and prevention;   ii) It involves activation of the coagulation system by inducing expression of tissue factor on the surface of monocytes. Confirm treatment of caries disease;   iii) In a clinical setting with an alloreactive response, T Patients who show signs of rejection to standard treatments by blocking all papill activation pathways Ascertaining the means by which it can be treated; and   iv) To confirm the treatment method for diseases associated with excessive production of IL-5 by T lymphocytes. Is.   The solution to the above technical problem is to use IL-10 alone or in combination with other treatments. It was discovered by the inventors of the present invention that it was in administration. this The solution is based on the following discoveries by the inventors of the present invention. Ie   i) IL-10 is an effective inhibitor of TNF-α production by LPS-stimulated monocytes is there;   ii) IL-10 prevents the induction of monocyte procoagulant activity;   iii) IL-10 inhibits B7 and ICAM-1 expression on monocytes; and   iv) IL-10 inhibits the production of IL-5 by T lymphocytes; It is based on the discovery.   The present invention also relates to septic shock, overproduction of IL-5 by T lymphocytes, B7 / CD28 Excessive stimulation of T cells due to ICAM-1 / LFA-1 interaction or promotion of monocyte coagulation An effective amount of IL-10, an analog or agonist for the treatment of conditions associated with progressive activity A pharmaceutical composition containing the same. The present invention also administers the above pharmaceutical composition to an individual. A method for preventing, suppressing, or suppressing the above-mentioned symptoms in an individual, which comprises steps. Concerning the law.   The term "IL-10" is purified from natural sources or preferably Produced by expression of a recombinant DNA sequence encoding leukin-10 in a suitable host Means pure interleukin-10 of mammals, especially humans. The present invention IL-10 contains a viral interleukin- 10. A chimeric protein composed of human and viral sequences derived from IL-10 It is included when the characteristics of IL-10 required by the invention are maintained. Also called "IL-10" For example, PCT Patent Application Publication No. WO 91/00349 may be referred to as Cytokai. Analogs of interleukin 10 as described as inhibitors of protein synthesis and And peptides are also included (englobe).   A suitable recombinant host is a retrovirus with the proper IL-10 coding sequence. Eukaryotic cells transfected with the Ils vector or baculovirus system Can be mentioned. Retroviral vectors have been introduced into mammalian cells (eg CHO cells). Be entered. Recombinant protein obtained in supernatant is unaltered expressed in mammals Similar to sex proteins. In addition, a retrovirus carrying the IL-10 gene Particles can be packaged in packaging cells (packags) transfected with these constructs. ing cell) produced in the supernatant. Use these particles to make mammalian cells These cells can be infected to express IL-10. Baculovirus Vectors produce more recombinant protein than retroviral vectors. Used to make This expression system is glycosylated and contains disulfide bridges. Produce a recombinant protein having different post-translational modifications and different glycosylation. Induce the conversion.   The term “effective amount” of IL-10, as used herein, refers to one of the above conditions. An amount sufficient to at least ameliorate or prevent the symptoms of. Specific patient Valid for The amount depends on the condition being treated, the overall health of the patient, the mode of administration and the severity of side effects. It depends on which factor.   IL-10 analogs and / or agonists interact with IL-10 receptors as well as IL-10. It is a molecule that reacts. This analog of IL-10 and / or agonist is a class of IL-10 Captures analogs or fragments, or side epitopes of the IL-10 receptor Binds to an antibody to a ligand that interacts with the It may be an anti-idiotype antibody against a specific antibody.   Antibodies to the natural cytokines, fragments and analogs of IL-10 And recombinant forms. Besides, antibodies Can be generated against the active or inactive form of IL-10. And that The difference is that antibodies to this active cytokine are only in the active conformation. It seems to recognize the existing epitope. Anti idiota Ip antibodies can also be generated by these methods and become agonists of IL-10. Ah   Generally, IL-10 is administered as a pharmaceutical composition containing an effective amount of IL-10 and a pharmaceutical carrier. Be given. The pharmaceutical carrier is a non-compatible compatible formulation suitable for delivering the compositions of the present invention to a patient. Any toxic substance will do. Generally, compositions useful for parenteral administration of these agents Are known [eg, Remington's Pharmaceutical Science, 15th Edition (1 980), (Mack Publishing Co, Easton, PA, USA) mpany issued). Alternatively, the composition of the invention is for transplantation Alternatively, it can be introduced into the patient's body by an injectable drug delivery system. [Eg URQUHART et al., Ann. Rev. Pharmacol. Toxicol. , Volume 24, 199-2 36, 1984, edited by Lewis, Controlled Release of Pesticides and Phar maceuticals (Plenum Press, New York, USA, 1981); US patent See 3773719 and U.S. Pat. No. 3,270,960].   When administered parenterally, IL-10 is delivered in a single-dose injectable form with a pharmaceutical carrier. (Eg, solution, suspension or emulsion). Examples of these carriers are usually Saline solution, Ringer's solution, dextrose solution and Hank's solution. Volatile Non-aqueous carriers such as foaming oils and ethyl oleate can also be used. Preferred carrier Is 5% dextrose / saline. This carrier isotonic and chemically stable Even if it contains small amounts of additives such as substances that enhance Good. IL-10 is a pure form that is substantially free of aggregates and other proteins. It is preferable to mix at a concentration within the range of about 10,000 to 100,000 U / ml. [Standard unit of activity (U) is half-maximal by MC / 9 mast cell line The amount of IL-10 required to produce a half-maximal response (THOM PSON-SNIPES et al. of Exp. Med. , 173, 507-510, 1991. )]. In most cases, IL-10 is continuously infused to patients by about 100,000- Deliver doses in the range of 100000000 U per day (ie about 1500 to 150,000 U / Kg / day) You can The daily injection volume varies based on the side effect monitoring results and the patient's blood cell count. Can be obtained. However, in some cases (eg, with treatment with anti-CD3 In order to prevent side effects), IL-10 is about 50,000 to 5,000,000 U It is preferably administered by injection.   According to the present invention, to prevent and / or treat allograft rejection, Administer IL-10, incubate cells with IL-10 or The gene can be transferred. Such gene transfer should be performed in the usual way. However, it is preferable to use one of the following methods.   1) transfer the gene encoding IL-10 into cells of a donor in vitro, then The cells are injected into the patient to induce chimerism and allograft resistance. This turn Transfer can be performed using different vectors, such as retroviral vectors. (Anderson, Science, 256, 808-813, 1992; Rosenbe rg et al., New England Journal of Medicine, 323, 570-578, 19 90 years).   2) The gene encoding IL-10 was isolated from the patient by autologous and alloreactive It metastasizes into lymphocytes in vitro. These modified cells are then delivered to the patient. Reinject to induce allograft resistance. The transfer is a retrovirus vector Can be carried out using different vectors such as   3) By perfusing the implant with a medium containing IL-10 viral particles, IL The gene encoding -10 Transfer the explants into the transplanted cells. This transfer is adenovirus-derived Can be carried out using different vectors such as T. (Rosenfeld et al., Science, 25. 2, 431-434, 1991). I-Serious infection:   Endotoxin or lipopolysaccharide (LPS) from Gram-negative bacteria causes septic shock Is a major causative agent of the mechanism of occurrence of (1). A shock-like condition is actually an animal It can be induced experimentally by a single injection of LPS. This of LPS Their toxic effects are mostly related to macrophage activation and lead to a large number of inflammatory mediators. Eater is released. Among these mediators, the major necrosis factor (TNF) ) Shows that administration of neutralizing anti-TNF antibody prevents LPS toxicity. As you can see, it seems to play an important role (2-5).   IL-10 releases TNF and associated toxicity in a mouse model of septic shock It is effective in inhibiting. Therefore, IL-10, either alone or with conventional treatments ( 26) in combination with sepsis due to Gram-negative bacterial species and its sepsis It can be used to prevent death.   The inventors of the present invention showed that pretreatment with IL-10 prevented LPS-induced toxicity. It proved that it was done. These results indicate that bacterial sepsis and related sepsis Under conditions where a congenital shock is likely to occur, such as surgery, especially field surgery ( prior to field surgery) and in other situations where bacterial infection is likely. 10 can be used prophylactically It is shown that. In these cases, in the present invention, IL-10 was exposed to bacterial microorganisms. Be given before there is a possibility   IL-10 is also used to prevent cerebral malaria (neuronal malaria) for the following reasons: Can be Ie   a) Adhesion molecules (especially ICAM-1) are expressed by infected red blood cells within the brain's microvessels. Plays an important role in cell adhesion to the skin;   b) Expression of these adhesion molecules is stimulated by malaria antigen-induced TNF; do it   c) The production of TNF induced by the malaria antigen can also be inhibited by the administration of IL-10. is there. Therefore, in the above cases, IL-10 alone or in combination, Can prevent the outbreak.   IL-10 also provides for the treatment of toxic shock syndrome according to this aspect of the invention. Or it can be used for prevention. This syndrome is caused by Gram-positive bacteria (eg Staphylococcus) It is associated with conventional toxin-induced T cell and monocyte activation. IL-10 is this professional It effectively shuts down the pathogen, either alone or in combination with conventional therapies. Can be used therapeutically for (26,27). II-Inhibition of monocyte procoagulant activity by IL-10:   The present inventors have for the first time demonstrated that IL-10 inhibits monocyte procoagulant activity. I have revealed it.   Therefore IL-10 is associated with septic shock, meningococcalemia, promonocytic leukemia, anti-C On the surface of monocytes such as the first dose response induced by D3 monoclonal antibody Tissue factor Acute diseases characterized by activation of the coagulation system with induction of expression; and antiphospholipidosis Group, hemolytic uremic syndrome, allograft rejection, disseminated intravascular coagulation and danger. Effectively used for all diseases characterized by thrombotic processes such as rugged surgery can do.   In these applications IL-10 either as the sole active ingredient or through the extrinsic pathway of the coagulation system It can be used in combination with other therapeutics that work. IL-10 is preferably a book A single cytokine present in the pharmaceutical composition of the invention.   Expression of tissue factor on the surface of human monocytes induces procoagulant activity. Expression of tissue factor is expressed by bacterial LPS (produced in septic shock), anti-CD3 monochrome Induced by a null antibody or an inflammatory mediator such as TNF or IL-1. Be done.   Induction of monocyte procoagulant by LPSIndependent of cytokinesBecause , The inhibitory effect of IL-10 on the procoagulant activity of monocytes induced by LPS Relevant to the observations made by conventional techniques that 0 inhibits monocyte cytokine synthesis None and would not be predictable from this observation.   According to a preferred embodiment of this aspect of the invention, IL-10 is anti-CD3 induced shock. It is used to manufacture a medicament for treating or preventing the syndrome. IL-10 is Has a dual function in the use of monocytes and lymphocyte cytokines (TNF- It has the effect of inhibiting both the synthesis of α and IL-1) and the procoagulant activity.   Moreover, treatment of renal transplant recipients with anti-CD3 antibody (OKT3) results in clot activity Allogeneic graft acceptance, as it can trigger hyperplasia and sometimes cause graft thrombosis. IL-10 alone or in combination with conventional therapy prior to the first injection of OKT3 in an individual May be administered for prophylaxis. III-T lymphocytes by the B7 / CD28 or ICAM-1 / LFA-1 pathway      Of IL-10 activation by IL-10:   Unlike the reports in the literature published so far (see, for example, International Patent Application Publication No. WO 93/17698 supra; Ann. Rev. Immunol., Vol. 11, 165-19 0, 1993), the inventors of the present invention showed that IL-10 expresses class II MHC. Moreover, they found that it inhibits the expression of B7 and ICAM-1 in human monocytes. In addition, this finding is based on the observation result of DING et al. (J. Immunol., 151 volumes, 1224-12). (P.34, August 1993). Ie DING et al. -10 inhibits B7 upregulation in mouse macrophages, but this They reported that they did not alter ICAM-1 expression in these cells.   B7 and ICAM-1 (intercellular adhesion molecule-1) are expressed on the membrane of antigen presenting cells (APC) Corresponding "auxiliary molecules" present on the surface of T lymphocytes, respectively. Interact with the receptors (CD28 and LFA-1) that co-stimulate T-lymphocytes (co-stim It is very important to regulate. Therefore, IL-10 is not bound by MHC II / T cell recipients. Inhibiting both the interaction of BCM / CD28 and ICAM-1 / LFA-1 Inhibits the activation of T lymphocytes. The latter action is transplantation Particularly relevant to immunity. In fact, lack of these co-stimulatory molecules Antigen-presenting cells (APCs) are ) Is triggered. In addition, ICAM-1 / LFA-1 interaction with monoclonal antibody injection In vivo inhibition for B7 / CD28 interaction with CTLA4-Ig fusion protein Internal inhibition prevents allograft rejection in vivo. Further, the invention of the present invention The authors also provided evidence that IL-10 inhibits the B7 / CD28 activation pathway in T lymphocytes. . Dual action of IL-10 in this pathway (action at the level of APC and T lymphocytes) Shows that IL-10 can be administered to increase tolerance to allografts ing.   These observations indicate that T7 lymphocytes by the B7 / CD28 or ICAM-1 / LFA-1 pathway Diseases associated with the activation of, especially allograft rejection and graft-host-related diseases Demonstrate that IL-10 can be used to treat or prevent diseases associated with alloreactive responses are doing.   According to the present invention, IL-10 is used alone or in a cell suspension derived from a donor (hemocytes, spleen). Spleen cells, bone marrow cells, bone marrow stem cells, etc.) Can be prevented or treated. IL-10 is a donor passage Grafts before transplantation to block passenger leucocytes And / or the recipient by the parenteral route. Also IL-10 To induce transplantation tolerance, add to donor cell suspension before injection into recipient May be.   In addition, the above-mentioned characteristics of IL-10 prevent acute graft-host-related disease after bone marrow transplantation. It has been shown that IL-10 can be used to prevent or treat. Prevention proto In Col, IL-10 is either added to the bone marrow cells of the donor prior to injection into the recipient, or Or, the recipient is injected after a bone marrow transplant.   Unlike previous findings, IL-10 is known to be cyclosporin-A resistant Is now found to inhibit the active ICAM-1 / LFA-1 and B7 / CD28 pathways So as the sole active ingredient or as cyclosporin-A or other related immunity It can be used in combination with inhibitors such as FK506 or rapamycin. Therefore, to prevent or treat rejection of allografts, conventional treatment with IL Adding -10 is advantageous. Conventional treatments including cyclosporin-A IL-10 is particularly useful in patients who cannot prevent rejection by.   IL-10 offers further advantages over standard therapies. For example, IL-10 is Administration for a short period of time can prevent or treat allograft rejection. There is no need for continuous administration. Further, as described below, the inventors of the present invention have It was found that IL-10 inhibits the production of IL-5 by T lymphocytes. Good Eosinophils are induced by IL-5 and are involved in several forms of graft rejection Therefore, the beneficial effects of IL-10 during transplantation are associated with inhibition of IL-5 production . In addition, IL-10 can also be used in combination with anti-CD3 mAb. The latter combination above In addition, IL-10 enhances the immunosuppressive effect of anti-CD3 mAb To prevent its side effects associated with drug release. IV-IL-10 inhibits the production of IL-5 by T lymphocytes:   IL-10 is a cytokine produced by TH2 lymphocytes. It has been previously reported to be a potent inhibitor of IL-10 is a TH2 cytokine It has not been reported until now that it is an inhibitor.   Surprisingly, the inventors of the present invention showed that the inhibitory effect of IL-10 in T cells was IL-10 has no specific effect on the production of TH1 cytokines by these cells and Actually blocks the production of IL-5, a cytokine commonly produced by TH2 lymphocytes. He found that he could do harm. These results obtained using peripheral blood T cells Was particularly unexpected. Because similar experiments with T cell clones (manufactured Test results of a laboratory model of T lymphocytes that can provide the product: IL-10 produces IL-5 (DE WAAL MALEFYT et al., J. Immunol., 15). 0, 4754-4865, 1993).   The inventors of the present invention also found that inhibition of IL-5 production by IL-10 resulted in costimulation of T lymphocytes. I also found that it depends on. In fact, IL-10 causes T cells to have B7 / CD28 signal Inhibits the production of IL-5 by T cells when co-stimulated by T cells show IL-5 when vesicles are stimulated by PMA and A23187 calcium ignophores. Does not interfere with the production of   These observations treat diseases associated with overproduction of IL-5 by T lymphocytes Shows that IL-10 is worth it ing.   IL-5 plays an important role in diseases associated with eosinophilia, so IL-10 , To treat eosinophilia (such as those below for which there is no satisfactory cure) It is for. Examples of such diseases include allergic rhinitis and bronchial asthma. Atopic diseases including; skin diseases associated with infiltration of eosinophils such as atopic dermatitis ( Chronic eczema, erythema multiforme, herpes dermatitis, mastocytosis, bullous disorders included); Disease; bronchopulmonary aspergillosis; spanish toxic oi muscle syndrome and eosinophilia syndrome induced by L-tryptophan preparation Allergic reaction of certain medicines; tropical eosinophilia; helminth infection; Schulman (Schulman) syndrome (eosinophilic fasciitis); favorable characteristics such as Churg-Strauss syndrome Vasculitide with eosinophilia; Lefrel syndrome; chronic eosinophilic pneumonia Have eosinophilic gastroenteritis; and hypereosinophilia syndrome.   For these disorders, rIL-10 alone or with interferon alpha or It is preferably used in combination with interferon γ. Hypereosinophilia syndrome Is characterized by interstitial infiltrates in the lungs, so it is appropriate to administer IL-10. The routes are the routes using the aerosol and the routes described above. Ointment Topical formulations containing are also suitable.   In addition, the inventors of the present invention found that endogenous IL-10 produced by TH2 lymphocytes was It was also demonstrated that the same cells regulate the production of IL-5. Neutralizing anti-IL-10 monochrome Null antibody Increases IL-5 release. Therefore, according to the present invention, IL-5 from T lymphocytes Anti-IL-10 antibody can be used to treat or prevent diseases that produce insufficient production of .   Various aspects of the invention are illustrated in the drawings below.   Figure 1: Construction of mouse IL-10 retrovirus vector. In PBR322 (pCO6-HX) Cloned Harvey murine sarcoma virus (Ha-Mu The plasmid pTG1H-MIL-10 was derived from SV). v-ras^HAll coding regions of ( SacII-XhoI) and then a 560 bp fragment encoding the mouse IL-10 cDNA. It was replaced with a cement. The Ha-MuSV sequence is shown in bold and the pBR322 sequence is shown in dashed line. You Regarding the restriction endonuclease site, H indicates the Hind III site and S indicates the Sac II site. Position, X indicates an Xho I site, and B indicates a BamHI site. The black square mark is for IL-10 Indicates coding sequence, white squares are v-ras^HShows the coding sequence of, and Shaded squares indicate Ha-MuSV LTR.   Figure 2: Rectal temperature of BALB / c mice (6-21 per group) injected with: The time course of (mean ± SEM) is shown. Vehicle injection (○), LPS injection without pretreatment (100 μg intravenous injection) (●), LPS injection after pretreatment with 1000 U of IL-10 (100 μg intravenous injection) (▽) or mock-transfected (mock-tra nsfected) CHO-K1 cell-derived supernatant was pre-treated and then LPS was injected (100 μg   Intravenous injection) (▼).   Figure 3: Mortality after injection of 500 μg of LPS into 3 groups of BALB / c mice:   (●) Mice without pretreatment (n = 15);   (▽) Mice pretreated by intraperitoneal injection of 1000 U of IL-10 (n = 15 );   (▼) Inject intraperitoneally with the supernatant of mock-transfected CHO-K1 cells. Pretreated mice (n = 10).   The p-values on both sides were obtained by Fisher's direct method.   Figure 4: Human rest activated by anti-CD3 mAb and B7 / CD32 transfectants. Inhibition of IL-5 production by resting T cells by rIL-10.   Figure 5: Cytokines by T cells stimulated by PMA and anti-CD28 mAb Of rIL-10 on the secretion of LIL.   Figure 6: Systemic administration of rIL-10 induced drainage by subcutaneous injection of allogeneic cells Inhibits draining lymph node enlargement. BALB / c (similar ) Or A / J (same species) mouse-derived 2.5 x 10⁶Spleen cells from BALB / c mice Was subcutaneously injected into the toes of the right hind foot. For mice receiving allogeneic cells, days 0-5 Up to day 3, 1000 U of rIL-10 or control vehicle was injected three times per day. 5 On the day, the weight difference (delta mg) from the controlateral lymph node The swelling of the right lymph node was measured. *: Significant difference with p <0.001 compared to syngeneic injection , And ** is significantly different and p <0.05 vs. allogeneic injection It is shown that there is a significant difference at p <0.01 for the injection of species + control.                        Example Example 1: IL-10 cDNA and vector used in these examples Kector   Unless otherwise specified, the cDNAs used in these examples are as follows.   IL-10 cDNA:     For cloning of the mIL-10 (mouse IL-10) cDNA, see Example 2 and the standard. The title is “Interleukin-10 reduces the release of tumor necrosis factor and pr C. Gerard et al., "Journal of events lethality in experimental endotoxemia" Experimental Medicine, 177, 547-550, detailed in the 1993 literature. It is described in detail. The sequence of this cDNA is a single nucleotide at position 247. Found that the inventors of the present invention were "C", and International Patent Application Publication No. WO 9 Except that it is not the "T" described in 1/00349, Same as column (International Patent Application Publication No. WO 91/00349, page 29, lines 15 to 26) Is one.     For cloning of hIL-10 (human IL-10) cDNA, the title is "Interleukin-10 inhibi ts the induction of monocyte procoagulant activity by bacterial lipopoly O. Pradier et al., European Journal of Immunology, 23, 27. 00 to 2703, 1993 report. The sequence of this cDNA is public Sequence (International Patent Application Publication No. WO 91/00349).     VIL-10 (viral IL-10) DNA (Viera P. et al., P.N.A.S., Vol. 88, 1172- 1176, 1991) is a report derived from Epstein-Barr virus (EBV). Ils IL-10 Perform polymerase chain reaction (PCR) using specific oligonucleotides of DNA Cloned by. The target DNA used in this PCR was infected with EBV Were extracted from peripheral blood mononuclear cells of the patients.   The vectors used in these examples are as follows unless otherwise specified. is there.   Retrovirus vector:   The construction method of the mouse IL-10 retrovirus vector is shown in Fig. 1. d et al. Exp. Med., 177, 547-550, also described in 1993. There is.   The above vector inserts the above human IL-10 cDNA and viral IL-10 cDNA. Used for.   The second retroviral vector (pCO7-FX) is the Harvey Mouse Sarcoma Virus And derived from both the friend murine leukemia virus sequences. This vector Can achieve higher expression and higher virus titer than the first vector above . It is described by Feldman et al., Journal of Virology, 63, 5469-5474, Reported in 1989. Construction of IL-10 structure (referred to as pTG2F-IL-10) To do this, the IL-10 cDNA was inserted into the Sac II and Xho I sites of the pCO7-FX vector.   Expression of mIL-10 and hIL-10 using the baculovirus system:   The same cDNA as the above cDNA was used for the commercially available pBlueBac2 transfer vector (tramsferve vector). ctor) (Invitrogen) at the cloning site of Nhel BamHI. these In the structure, These cDNAs are under the control of the polyhedrin promoter. Transduce these cDNAs The result is a recombinant virus that can infect insect cells. Recombinant IL-10 was prepared from the cells of and purified. Example 2: IL-10 decreases TNF-α release and is lethal in experimental endotoxemia To prevent   Materials and methods   Animals: 10 obtained from KUL Proef Dieren Centrum (Leuven, Belgium) BALB / c mice aged 15 weeks were used.   Drug: LDS derived from E. coli (serotype 055: B5) is Sigma Chemical C Company (St. Louis, Missouri, USA). JESS-2A5 mAb The neutralized rat anti-mouse IL-10 IgG1 mAb was synthesized by Tim Mosmann (Edmont, Canada). (Department of Immunology, University of Alberta) (9). LO-DNP mAb or pair The rat IgG1 antibody used as a reference is H. Bazin (Brussels, Belgium) University of Catholique de Louvain) Experimental Immunology Unit) I got it.   Mouse recombinant IL-10: cloning and expression   Synthesis of specific oligonucleotide of mouse IL-10 cDNA according to IL-10 sequence (10). The restriction site is used for subcloning the oligonucleotide It was included at the 5'end of the cord. That is, sense primer 5'-CTCCATCATGCCTGGC HincII / SacII site and antisense to TCA-3 '(nucleotides 69 to 87) SPRIMER 5'-TACACA Smal / XhoI part for CTGCAGGTGTTTTAGC-3 '(nucleotides 608 to 629) Rank. Hamster anti-CD3 mAb 145- as a non-specific stimulator of cytokine transcription Total RNA was prepared from the spleen of mice injected with 2C11 (11). As a primer Antisense oligonucleotide (1 μg), and RT buffer [50 mM KCl, 20 mM Tris HCl, pH 8.3, 2.5 mM MgCl₂, 0.1 mg / ml Acetylated BSA, 2.5 mM of each dNTP, RNasin 20U (US, Wisconsin Corp. of Promega, Madison, Nevada. Company)] in 200U Mo-MulV reverse transcription fermentation Reverse 1 μg of RNA using a final volume (RT) (Promega Corp.) of 20 μl. It was transcribed. Add 2.5 U of Taq DNA polymerase in the same buffer to the obtained solution. And 1 μg of each sense / antisense primer (final volume 100 μl) added did. DNA thermal cycler (No., Connecticut, USA) 35 cycles of PCR were carried out using Perkin-Elmer Cetus, Inc. (1) Cycles were 94 ° C. for 1 minute denaturation, 55 ° C. for 2 minutes annealing and 72 Extension for 3 minutes at ° C). 560 bp predicted size band was gotten. The specificity of the amplified cDNA was confirmed by its restriction pattern. The sequence was determined according to the dideoxynucleotide chain termination method (12). gave. Nucleotides at positions 322, 465-468, 522 and 523 Was different from the published one (10), but its amino acid was unchanged It was Sac II-Xho I restriction flag containing mouse IL-10 cDNA Instead of the coding sequence of p21ras, Harvey mouse sarcoma virus It was inserted into the derived retroviral vector pCO6-HX. Called “pTG1H-mIL-10” The IL-10 cDNA is the only functional gene in this construct. IL-10 gene A similar vector without any insert was used as a negative (mock) control. High To obtain individual colonies of cells that express levels of IL-10, pTG1H-mIL-10 was It was cotransfected with rasmid pSV2-neo. This plastic Sumido pSV2-neo contains the neomycin resistance gene as a selectable marker. Using the modified calcium phosphate method, except that the carrier DNA is not present, Transfection of CHO-K1 (Chinese Hamster Ovary K1) cell line (14). The selection of transfectants was 400 μg / ml G418 (Geneti cin; Gibco of Paisley, Scotland) did. After 10 days, individual resistant colonies were isolated and isolated from IL-10 using a specific ELISA method. Tested for current (Pharmingen, San Diego, CA, USA) Company). IL-10 concentration was expressed in the supernatant of COS cells (470 U / ml) It was measured with reference to a standard curve of IL-10 (Pharmingen). High level IL-10 (20 (0 U / ml) were cultured for 24 hours, and the collected supernatant was An outer filtration membrane (Amicon Corp., located in Dunbars, Massachusetts, USA), It was concentrated to 5000 U / IL-10 / ml. Trans with control plasmid and pSV2-neo Control (mock) supernatant from transfected CHO-K1 cells The liquor was selected, collected and then similarly concentrated. The endotoxin content of the injected preparation is 1n It was less than g / ml.   Measurement of serum levels of TNF. Samples of blood are collected by individual retro-orbital puncture for individual markers. Got from Us. Serum levels of TNF were found in WEHI-16 treated with actinomycin-D. Estimated by assaying cytotoxicity on 4 clone 13 cells (15) . Standardization of recombinant mouse TNF expressed in E. coli (NBSB, UK) The test results for cytotoxic activity are given in pg / ml (16). This formulation has a ratio Biological activity 2.25 × 10⁸It was IU / mg. This 1 IU is about 4 pg Represents mouse TNF. IL-10 was found not to interfere with this bioassay .   Experimental protocol. Mice were intraperitoneally administered CHO-IL-10 or simulated supernatant. After 30 minutes, 100 μg of LPS was intravenously injected. After attacking with LPS Serum levels of TNF were measured 1.5 hours, 3 hours, and 6 hours later, and Intestinal temperature was measured with a digital thermometer at regular time intervals. Trials of other series IL-on lethality induced by a single dose of 500 μg LPS in the study Ten effects were evaluated.   statistics. Compare TNF levels and temperatures using the Mann-Whitney U test did. Mortality data were analyzed by Fisher's direct method.   Test results and discussion   To evaluate the in vivo effect of IL-10 administration on LPS-induced TNF release. Therefore, three doses of IL-10 (10 , 100 or 1000 U) was intraperitoneally administered 30 minutes later, and 100 μg of LP S was injected intravenously. In the test so far, TNF 1.5 hours after attacking with LPS It was known that the level of (17). Pretreatment with IL-10 released it in the blood circulation after the LPS challenge. The amount of TNF released was significantly reduced as shown in Table 1. Table 1 shows similar results The results of one of the three experiments given are shown. This in vivo action of IL-10 is Already observed at doses as low as 10 U (pretreated with control vehicle Was observed at p <0.01 compared to the case where LPS was administered later). 1000 U This dose was tested further as it had more pronounced and reproducible activity Selected to. The specificity of action of the IL-10 formulation was mock-transfected in CHO-K. It was evaluated by pretreating the mice with the supernatant from 1 cell. Shown in Table 1 As such, this control formulation had no effect on TNF release. Level of TNF in serum Bell was able to attack all groups of mice 3 hours after the LPS challenge. It returned to the baseline value. This means that IL-10 simply delayed the release of TNF. Is not present (Table 1).   30 days after intraperitoneal administration of the specified dose of IL-10 to BALB / c mice (n = 5 in each group) Minutes later, 100 μg of LPS was administered. Used as a control to culture CHO-K1 cells The mice were pretreated with the vehicle or mock supernatant. Levels in serum of bioactive TNF (Average value ± SEM) is measured 1.5 and 3 hours after the LPS attack. Decided   *, ** and *** are pretreated with control medium or simulated supernatant, respectively P <0.01, p <0.05 and p <0 compared to mice challenged with LPS ． 005 indicates that there is a significant difference.   As the first method to evaluate in vivo the effect of IL-10 on LPS-induced toxicity Attack with 100 μg of LPS For 24 hours, the rectal temperature was monitored. In fact, LPS is probably associated with the release of TNF. It caused marked hypothermia (18). The data shown in Figure 2 is before IL-10. Treatment effectively prevented LPS-induced hypothermia (treated with LPS alone) Case, or when treated with LPS after pretreatment with simulated supernatant 4 hours later, there was a significant difference of p <0.0001 after 6 hours and p <0.00 after 6 hours. 5), while control from mock-transfected CHO-K1 cells The supernatant had no significant effect. 100 μg LPS, alone or in simulated There is considerable variability in the recovery of hypothermia in animals injected after clear fluid pretreatment. Was done. Specificity for the effects observed after pretreatment with CHO-IL-10 supernatant As an additional control for neutralizing anti-mouse IL-10 JESS-2A5 mAb or control rat IgG1  LO-DNP mAb was added to the 1 mg IL-10 formulation. This anti-IL-10 is an IL for hypothermia It completely inhibited the protective effect of -10, but the control rat mAb had no effect.   To evaluate the effect of IL-10 pretreatment on LPS-induced lethality, Uss were challenged with 500 μg LPS. This LPS dose was tested within 72 hours. That is the amount that will kill 50% of the animals. Mice pretreated with 1000 U of IL-10 All survived after injection of LPS, while mock-transfected CHO-K Pretreatment with 1-cell-derived supernatant did not alter LPS-induced lethality (Figure 3).   In summary, these data show that IL-10 pretreatment Shows that LPS toxicity is prevented in a mouse model of endotoxin shock There is. The beneficial effects of IL-10, at least in part, reduce TNF synthesis or release. It may be due to In fact, the same level of lethality induced by LPS Protection has been obtained by using neutralizing anti-TNF antibodies (2-5). I L-10 is another monocyte / macrophage-derived supporter involved in the development of septic shock. It can also inhibit or prevent the synthesis and / or release of itokines, especially IL-1. In vitro data suggest that this is true (1, 6, 19, 20).   Therefore, IL-10 is an anti-TNF mAb (2-5), an IL-1 receptor antagonist (21-23) , Already contains differentiation factor / leukemia inhibitory factor (24) and G-CSF (25) , Potential immune interventions for the prevention and / or treatment of septic shock (Immunointervention strategy). IL-10 is Maklov Treatment with IL-10 is of particular interest because it has the effect of inactivating Can be combined with some of the other biological agents of Example 3: Use of IL-10 in a prothrombotic state: Monocyte coagulation Inhibition of promoting activity.   LPS is primarily responsible for expressing tissue factor (TF) on monocytes and endothelial cells. It is known to induce the expression of procoagulant activity (PCA). Also TNF Inflammatory mediators such as interleukin-1 (IL-1), and LPS TF can be expressed in endothelial cells. Therefore, this prethrombogenic mechanism of IL-10 The action of cultured human Tested with left vein cells (HUVEC) and peripheral blood mononuclear cells (PBMC).   Expression of PCA and TF in monocytes induced by LPS (1 μg / ml) IL-10 (1-2.5 U / ml) was pre-incubated overnight with PBMC before adding LPS. When it was incubated, it was neutralized by 80% or more. The best way to block PCA is to It was necessary to preincubate the spheres with IL-10 for at least 6 hours. It was No effect of IL-10 on expression of LPS-induced tissue factor by EC was observed It was.   Human recombinant IL-10 (hIL-10) was obtained as follows. Anti-CD3 monoclonal Isolation of total RNA from human PBMC stimulated in vitro by the antibody, namely OKT3, Then, reverse transcription was performed using Mo-MuLV reverse transcriptase. Specific for human IL-10 cDNA Oligonucleotide: sense primer 5'-AAGGCATGCACAGCTCAGCACTGC TC-3 '(nucleotides 26-51) and antisense primer 5'-CCACC Polymer with CTGATGTCTCAGTTTCGTATC-3 '(nucleotides 555-580) The polymerase chain reaction (PCR) was performed. Expected size and expected limit of 554 The patterned PCR product was cloned into a retroviral vector, The CHO-K1 was then transformed by the plasmid pSV2-neo containing the neomycin resistance gene. The cells were co-transfected. In selective medium (400 μg / ml G418) After 10 days of storage, individual resistant colonies were isolated and cultured for 24 hours before collection. Use the OKT3 to collect the supernatant of these colonies. It was tested for its ability to inhibit the induced production of IFN-γ by human PBMCs.   From CHO-K1 cells transfected with control plasmid and pSV2-neo A control (mock) supernatant was selected and collected in the same manner.   Bacterial LPS. E. coli 055. LPS derived from B5 was purchased from Sigma (Se, Missouri, USA). I got it from Ant Louis.   Isolation of peripheral blood mononuclear cells (PBMC):   Citrate phosphate dextrose adeni ne) (CPDA) has prevented blood clots from healthy volunteers. PBMC was prepared. Centrifuge buffy coat over Ficoll Hypaque gradient and continue. Wash with Hanks balanced salt solution (HBSS) without calcium and magnesium. Then supplemented with Hepes (20 mM), glutamine and 10% fetal calf serum (FCS) PBMCs 3 x 10 in 1 ml of RPMI1640⁶/ Ml. Last PBMC formulations are generally available in STK-S cell counter and May Gruenwald Giemsa staining 22% to 30% of monocytes and 70% to 78% of phosphorus estimated by the cell identification method using Containing less than 1% neutrophils and less than 20,000 platelets per ml It was All drugs and media were measured using the Limulus Amoebocytes Lysate assay. As a result, it contained less than 10 pg / ml of endotoxin.   Endothelial cell preparation:   Human umbilical vein endothelial cells (HUVEC) were collected with 20% of collected human serum, essential amino acids, Endothelial cell growth factor (ECGF-40 μg / m l), heparin (100 μg / ml), penicillin and streptomycin Culture was performed using the supplemented M199 medium. 96 wells of cells from the second passage Cells for 24 hours in a medium without ECGF and heparin, and then collected (at c onfluence) used.   Assay for procoagulant activity (PCA):   Single step coagulation assay for PCA on the surface of PBMC stimulated with 1 μg / mg LPS for 6 hours (Single stage clotting assay). The number of monocytes in the PBMC suspension is 5 × 10^FiveAdjusted to monocytes / ml. This cell suspension is then added to 100 μl of 25 mM CaC l₂Normal blood with citrate pooled after 1 minute incubation with Clotting was initiated by adding 100 μl of serum. Record clotting time with KC10 device A standard curve obtained by serially recording and then serially diluting commercial thromboplastin To obtain the U / ml value of PCA. 1U indicates a normal clotting time of 12.4 seconds Equivalent to the amount of rhomboplastin. Role of the tissue factor / factor VII pathway in PCA To measure normal human plasma, and in some studies factor VII deficient plasma, Pre-incubate with 12D10 (neutralizing mouse anti-factor VII MoAb) for 30 minutes With normal plasma; or neutralize these cells with mouse IgG1 anti-tissue factor Mo Incubated with Ab (4507).   PCA induced on the surface of HUVEC:   This PCA was measured after 6 hours of incubation with 10 μg / ml LPS It was In summary, calcium (1 00 μl of 30 mM CaCl₂At 37 ° C for 2 minutes), normal citrate supplemented Plasma (45 seconds at 37 ° C.) and chromogenic substrate (S2238, 0.7 mM, 10 0 μl, 37 ° C for 20 minutes), and then thrombi generated on the surface of HUVEC. Was measured. Stop the reaction with acetic acid (50 μl) and read the absorbance at 405 mm. I took it. The amount of thrombin produced in this system was determined using the standard curve obtained with purified thrombin. Calculated using 1 x 10^FiveThe values are shown per HUVEC.   Analysis of tissue factor expression by flow cytometry   Expression of TF molecules on the surface of cultured PBMCs was labeled with fluorescein. Mouse IgG1 anti-tissue factor MoAb stained and then quantified by flow cytometry It was After culturing, the cells were washed with PBS supplemented with 0.5% BSA, and the cells were washed 6 × 10 6.^FiveCells of , Anti-tissue factor MoAb, or as a control, irrelevant IgG1 MoAb at 1 ° C Incubated for hours. After washing and fixing with 1% formalin, the cells are FACS It was analyzed by a can flow cytometer. Exploits the properties of side and forward light scattering Then, the monocytes were gated. At this gate, over 70% of the cells are TF antigen , And 95% of cells carrying the TF antigen co-express the CD14 molecule. Known quantity Using flow cytometry standardized fine beads coated with fluorescein, Mean equivalents of soluble fluorescence (MESF) A calibration curve that can measure   Measurement of tissue factor mRNA expression by inverse PCR.   Test results   IL-10 inhibits LPS-induced PCA on PBMC:   Incubation of PBMC with 1 μg / ml LPS produced high levels of PCA. Reveal. Preliminary studies showed that LPS-induced PCA on PBMC peaked after 6 hours. It was confirmed to reach. Therefore, this incubation time is Selected to. PCA did not appear at all when factor VII-deficient plasma was used for coagulation assay . In addition, the PCA observed for normal plasma was measured in cells prior to performing the PCA assay. Anti-Tissue Factor MoAb was added and the plasma of the indicator together with anti-Factor VII MoAb By pre-incubating, it was possible to inhibit more than 90%. Indicates that the increase in PCA in this case is due to tissue factor. It Therefore, the inventors of the present invention tested PBMC with IL-10 overnight (18 hours). After reincubation, the cells were stimulated with LPS for 6 hours to measure PCA. As a result, PCA Was decreased depending on the dose, and various levels of IL-10 at 1.5-2.5 U / ml Maximum inhibition rates in the range of 75-90% were achieved in the test. This is a trypanbe Cell viability measured by Roux dye exclusion test exceeded 90% after culture Therefore, it was not due to the toxic effect of the IL-10 supernatant. Mock transfection Control supernatants derived from CHO cells showed no significant effect on PCA induced by LPS. Yes. Preincubation of PBMCs with IL-10 alone does not reduce basal PCA. Squid or some tests significantly reduced basic PCA.   IL-10 inhibits LPS-induced expression of tissue factor on monocytes:   PBMCs incubated with LPS for 6 hours had large amounts of tissue factor on their surface. Show. Almost all fines that are positive for TF staining The vesicles were examined by size and forward scatter properties, and double staining test with anti-CD14 MoAb. When indicated, it was a monocyte. Cells should be isotype matched and labeled with fluorescein. The above staining was not special because it was not stained at all with the sensible control MoAb. It was strange. Incubate PBMC with IL-10 overnight before adding LPS And almost completely prevented the increased expression of tissue factor on monocytes. Dosage of this phenomenon The response to was closely related to that of the inhibition of PCA by IL-10.   To inhibit LPS-induced TF expression on PCA and PBMC by IL-10, Reincubation required:   In the above test, IL-10 was incubated with PBMC for 18 hours then 6 hours After a while, I attacked with LPS. LPS after incubation with IL-10 for 6-24 hours Is optimally prevented from expressing TF on PCA and monocytes. Pre-ink When the incubation was shortened to 3 hours and 1.5 hours, the inhibitory effect of IL-10 Steadily declined. When IL-10 was added together with LPS, the expression of PCA or TF was completely reduced. Poor inhibition or minimal inhibition (<20%) was obtained.   IL-10 is unable to inhibit LPS-induced PCA on endothelial cells:   As shown in Table 1, incubation of HUVEC with LPS for 6 hours resulted in no PCA. The ability of HUVEC to generate thrombin on the membrane is greatly induced. Of dose Range is 0.2-20 U / ml and pre-incubation time is 0-18 hours Of IL-induced addition of IL-10 to HUVEC The production was unchanged.   These test results demonstrate that hIL-10 implicates the monocyte procoagulant activity induced by LPS. It has been shown to inhibit. The ability of hIL-10 to suppress the release of inflammatory mediators This direct anticoagulant effect is related to the septic system of externally administered hIL-10. It suggests that it may be a new treatment for Kock. Example 4: Interleukin-10 regulates the release of cytokines and Reduces shock syndrome induced by anti-CD3 monoclonal antibody in mice .   IL-10 is an interferon by activated TH1 cells and macrophages -Reduces secretion of γ and TNF-α. Therefore, cytokine release as well as anti-CD3 IL-10 inhibits toxicity induced by activating monoclonal antibodies Was tested for performance.   Recombinant human IL-10 (hIL-10) was added to PBMC stimulated in vitro with OKT3 did. Unstimulated PBMC and OKT3 stimulated PBMC were 150 pg / ml and 8 respectively. 000pg / It contained ml of TNF-α (measured by IRMA). hIL-10 is dose dependent The formula decreased the secretion of TNF-α. Inhibition rate of TNF secretion is 85% at the highest concentration of hIL-10 Met. HIL-10 also exceeded 90% of INF-γ release induced by OKT3 I was able to suppress it.   BALB / c by injecting 10 μg of 145-2C11 mAb of anti-mouse CD3 in vivo Recombinant mouse IL-10 (mI) for TNF-α release and hypothermia induced in mice The effect of L-10) was also tested. Serum levels of TNF-α are injected with 145-2C11 mAb And after 90 minutes undetectable (<50 IU / ml) to 795 ± 150 IU / ml (Mean ± SEM). 3 times after intraperitoneal administration of 1000 U of mIL-10 Challenge with anti-CD3 at 0 minutes could significantly reduce serum levels of TNF-α. Was achieved (p <0.05 compared to mice injected with 145-2C11 only, 90 minutes later 3 27 ± 36 IU / ml). In parallel, observed 8 hours after injection of anti-CD3 The rectal temperature decrease of 5.8 ± 1.5 ° C (mean ± SEM) was pretreated with mIL-10. It decreased to 1.2 ± 0.4 ° C. (p <0.05).   Materials and methods   Monoclonal antibody and recombinant mouse IL-10:   Hamster mAb 145-2C11 against mouse CD3 complex was used as ascites fluid in nude mice. Produced. Neutralizing anti-mouse IL-10 mAb JES5-2A5 was added to T. Dr. Mosmann (Canada, It was a gift from the University of Alberta in Edmonton. Mouse recombination IL-10 (mIL-10), as described above, Culture supernatant from CHO-K1 cells stably transfected with the corresponding cDNA I got it. Supernatant fluid collected from mock-transfected cells served as a control. I used it. The levels of endotoxin in the above mAb formulations and CHO-K1 cell supernatants were determined by Limulus amo It was less than 1 ng / ml when measured by ebocyte lysate assay.   Cloning and expression of recombinant human IL-10:   Same as described in Example 2.   Lymphokine test:   Serum levels of TNF were measured according to the method of Example 1. IFN-γ is Db-1 And F1 rat anti-mouse IFN-γ mAb by two-site ELISA Was determined. Serum levels of IL-10 were measured by JES5-2A5 purchased from Pharmingen. And SXCl mAb were used to measure by ELISA. Serum levels of IL-6 are IL-6 dependent7 It was measured using the TD1 cell line and the hexosaminidase method. TNF, IFN-γ, IL-10 The detection limits for IL-6 and IL-6 are 2U / ml, 5U / ml, 2U / ml and 5 respectively. It was pg / ml. Exam protocol:   The mice were intraperitoneally administered with 1000 UmIL-10 or 200 μl of the simulated supernatant, Thirty minutes later, 10 μg of 145-2C11 mAb was injected intravenously. This dose of IL-10 It was chosen because it was already found to be optimal in the model of toxemia. Rectum Temperature was monitored with a digital thermometer for the next 24 hours. Blood sample At several time points after injection of the CD3 mAb, they were collected by retro-orbital puncture and It was used to measure serum levels of Cain. The blood glucose level is Gluc ostix strips and Glucometer IIM for 4 hours using standard micromethods Measured at points. To determine the effect of mIL-10 in a lethal model of anti-CD3 mAb toxicity, 1 Mice were injected ip with 0 mg D-galactosamine 90 minutes after Mice were sensitized by administering g of the mAb. In some tests, After intraperitoneal injection of crosporin A (CsA) (50 mg / kg for each administration) Attacked with anti-CD3 after 18 hours and 3 hours. Inhibits systemic release of cytokines The protocol to indicate that is described above.   statistics:   Mortality data analyzed by Fisher's one-sided direct test for group comparisons Was performed using the Student test method.   Test results     mIL-10 inhibits systemic release of IFN-γ and TNF after injection of 145-2C11 mAb Does not block systemic release of IL-6 following this injection:   Intravenous injection of 10 μg of 145-2C11 mAb into BALB / c mice resulted in a large amount in serum. Release of IFN-γ and TNF was induced and peaked at 4 hours and 90 minutes, respectively. It was Injection of 145-2C11 mAb 30 minutes after administration of 1000 U of mIL-10 It almost completely inhibits the release of IFN-γ and TN without affecting the kinetics of the response. The induction of F was significantly reduced. Pretreatment with mIL-10 is directed against IFN-γ and TNF In contrast to its inhibitory effect, it significantly affects the in vivo induction of IL-6 by the 145-2C11 mAb. I didn't. Mock-transfected CHO- administered at the same timing as mIL-10. The supernatant of K1 cells did not alter IFN-γ and TNF release, but IL-10 The specificity of action observed by the formulation is shown. In addition, puncture with 145-2C11 mAb The activity of this formulation on in vitro production of IFN-γ by stimulated spleen cells was determined by JES5-2 It was completely neutralized by the A5 anti-IL-10 mAb.     hIL-10 is specific for 145-2C11 mAb-induced IFN-γ and IL-10 release To adjust:   It was recently observed that anti-CD3 mAb induces IL-10 secretion in vivo. The effect of pretreatment with causative IL-10 on the release of this cytokine was measured. Within To avoid interference of injected IL-10 with the measurement of causative IL-10, these The test was performed with hIL-10 that was not detectable by ELISA and did not interfere with mIL-10 by ELISA. . The inventors of the present invention firstly determined that hIL-10 is a living body induced by the 145-2C11 mAb. Inhibition of IFN-γ and TNF release was significantly blocked by hIL-10 in the mouse system. Data was confirmed to be active. 145-2C11 mAb in the same test HIL-1 was evaluated at the serum peak level of IL-10 4 hours after the injection of Pretreatment with 0 did not reduce endogenous release of IL-10. IL-10 inhibitory effect Resistance of IL-10 to CsA blocked IFN-γ, TNF and IL-10 release equally It is symmetrical compared to the action.     mIL-10 reduces the acute toxicity of 145-2C11 mAb:   As previously reported, hypothermia and hypoglycemia are caused by the 145-2C11 mAb It is a sensitive parameter of the induced shock syndrome. The inventors of the present invention IL-10 significantly reduced the rectal temperature drop following injection of the 5-2C11 mAb. And found out. In a control test, mice pretreated with simulated supernatant were Showed no protection against CD3-induced hypothermia (at 6 hours Mean ± SEM: 32.5 ± 0.8 ° C for mice injected with 145-2C11 mAb only However, the temperature was 31.6 ± 1.8 ° C. P = NS). On the other hand, 145-2C11 mAb Hypoglycemia following injection of ss was unchanged by pretreatment with mIL-10.   Furthermore, the lethal potential of IL-10 was obtained in mice sensitized with D-Gal. 30% of mice sensitized with D-Gal after injection with 145-2C11 mAb Mortality was reduced to 5% in mice prematurely killed and mIL-10 pretreated .   These test results are triggered by cytokine release and anti-CD3 mAb It has been shown that in vivo toxicity is reduced by IL-10. Example 5: B7 / CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10 To be done.   This example demonstrates IL-5 production by human resting T cells isolated from peripheral blood. 2 shows the action of rIL-10. Mouse fibroblasts transfected with B7 / CD32 Activated by p.m.A with anti-CD3 mAb or anti-CD28 mAb cross-linked to IL-10 inhibits IL-5 production in activated T cells in a dose-dependent manner. Was found.   Materials and methods   reagent:   Human IL-10 (rIL-10) was cloned from previously reported IL-10 cDNA clone (Pradier , O .; C. Gerard, A .; Delvaux; M. Lybin; D. Abramowicz; P. Capel; T. Velu And M. Goldman, Eur. J. Immunol. , 23, 2700, 1993). Used to express in the baculovirus system. Ion-separation chroma Then, semi-purification was performed by gel filtration. Several tests of the invention The inventors have demonstrated that the activity of this formulation is similar to that of commercially available rIL-10. It was   Transfectant cell lines:   Human CD32 (FcγRII) alone or with B7 / BB1 antigen (designated B7) The emerging mouse fibroblast cell line 3T6 was obtained, cultured and maintained. Transfected Cells were incubated with 10 μg / ml mitomycin-C for 5 hours. And washed 3 times, then used in coculture with T cells It was   Isolation of resting T cells from peripheral blood:   Whole PB from buffy coat of healthy donors by density gradient centrifugation on Lymphoprep MC was isolated. After washing 3 times with Hanks balanced salt solution (HBSS), the cells were mM L-glutamine, gentamicin (20 μg / ml) and 10% Resuspended in tissue culture medium RPMI 1640 supplemented with FCS. 1 T lymphocyte Purified using the cyclic Lymphokwik-T procedure. The T lymphocytes are further protected against Ink with CD56, anti-CD19, anti-DR, anti-CD14 and anti-CD11b mAbs at 4 ° C for 30 minutes Incubated, washed and then incubated with magnetic beads coated with goat anti-mouse IgG. Incubated. After incubating at 4 ° C for 1 hour, the uncoated cells were magnetized. Removed using. The T cell preparations obtained were over 98% CD3 + CD28 + DR-T It contained cells but no detectable CD14 + monocytes. Several In the experiment, CD4 + T lymphocytes were subjected to magnetic immunosuppression using Dynabead coated with anti-CD8. It was further purified by the method (magnetic immunodepletion).   Stimulation of T cells:   PLA (1 ng / ml) and A23187 (0.1 μg / ml); or PMA ( 1 ng / ml) and anti-CD28 mAb (1 μg / ml); or CD32 or B7 / CD3 2 transfected fibroblasts (10^FourAnti-CD cross-linked to cells / well) 3 mAb (100 ng / ml); or phorbol 12-myristate 13-acetate Fibroblasts transfected with Tate (PMA, 1 ng / ml), and B7 / CD32. Cell (10^FourFlat-bottom 96 wells containing a total volume of 20 μl Resting T cells at 37 ° C, 5% CO₂Incubated under atmosphere. T Cells were 2.15 using transfectant cells in this experiment.^Five/ Well Vaccinated, 1.10 under other conditions^Five/ Well. Various incubation Of the culture Supernatants were collected and stored at -20 ° C until assayed for cytokine measurements.   Measurement of cytokine levels in culture supernatants:   The following anti-human IL-5 mAb or H3O rat anti-human IL-as coated mAb Using 5 mAb IgG2b and mAb7 mouse anti-human IL-5 mAb IgG1 as secondary mAb The level of IL-5 was measured by the sandwich ELISA method. The preliminary test The authors of Ming confirmed that rIL-10 does not interfere with immunoenzymatic detection of rIL-5. It was A commercial kit was used to measure IL-2 and IFN-γ. The lower limit of detection is IL-5 For 10 pg / ml, for IL-2 1 IU / ml and for IFN-γ Was 10 U / ml.   PCR analysis of IL-5 gene expression:   RNA derived from purified T cells was extracted using the guanidinium thiocyanate method and By reverse PCR, IL-5 mRNA and hypoxanthine phosphoribosyl tran Sfuerase (hprt) mRNA was analyzed. In summary, Moloney's mouse 1 μg RN using leukemia virus (MoMuLV) reverse transcriptase (200 U / assay) A was reverse transcribed. The obtained cDNA was subjected to PCR for 32 cycles. Each cycle has 9 It was carried out at 3 ° C. for 1 minute, 53 ° C. for 2 minutes and 70 ° C. for 3 minutes. IL-5 and HPRT Limer was synthesized according to the sequence of human cDNA. For IL-5, 5'sen The spurer was 5'-GCTTCTGCATTTGAGTTTGCTAGCT-3 'and 3'antiseparant was used. The primer was 5'-TGGCCGTCAATGTATTTCTTTATTAAG-3 '. PCR amplification using primers Widening gave a 291 bp fragment specific for mRNA (16).   Test results   Resting T cells co-stimulated by CD28 / B7 signaling express high levels of IL-5. Secrete:   The costimulation provided by the B7 molecule is the response of human resting T cells to anti-CD3 mAb. It is known to dramatically improve the answer (Annu. Rev. Immunol., Vol. 11, 191). Page, 1993, Linsley P. S. And J. A. Ledbetter's report “t he role of the CD28 receptor during T cell responses to antigen). B7 / CD Existence of fibroblasts (B7 / CD32 transfectants) transfected with 32 Expression of IL-5 by resting T cells stimulated with anti-CD3 mAb Transfected fibroblasts (CD32 transfectants) as controls It was used and evaluated. CD32 molecule (FcγR II) effectively crosslinks the TCR / CD3 complex The mAb necessary for the transfection can be bound to the transfectant. IL-5 is C Not detectable in the presence of cells transfected with D32 alone, but B7 / CD When 32 transfectants were used to co-stimulate, significant levels of IL-5 Produced with -2. IL-2 peak levels were detected at 24 hours, while Maximum levels of IL-5 were reached at 48 hours. TCR / CD3 engagement To determine whether (engagement) is required for the induction of IL-5, T cells were treated with PMA and And B7 / CD32 transfectant Or anti-CD28 mAb. IL-5 was produced in both systems, but PMA alone It was inefficient in inducing the secretion of high amounts of cytokines. Interestingly , The amount of IL-5 produced in response to PMA and calcium ionophore A23187 , Is always much lower than the amount produced in response to PMA and B7 / CD28 signaling. It was Even in these systems, the maximum level of IL-5 was 48 hours rather than 24 hours. , But IL-2 was maximal at 24 hours. Anti-CD3 mAb or PMA And IL-7 production by resting T cells stimulated by B7 / CD28 signaling Was observed in seven independent trials by the donor. As expected, CD8 + cells CD4 + cells were the major source of IL-5 because the reduction did not affect IL-5 production. It was   rIL-10 is expressed by human resting T cells co-stimulated by B7 / CD28 signaling Inhibits L-5 production:   The effects of rIL-10 on the production of IL-5 were first demonstrated by the B7 / CD32 transfectants. Were analyzed in a combined anti-CD3 mAb-based activation system. As shown in FIG. 4, rIL-1 0 inhibited IL-5 production under these conditions in a dose-dependent manner. Alongside, The inventors of the present invention show that rIL-10 inhibits the production of IL-2 by purified T cells, but IFN- Previous data was confirmed showing that it does not inhibit the production of γ. In controlled trials, rIL- 10 expresses B7 by transfected fibroblasts or by purified T cells. Neither expression of CD28 was altered. IL-2 is involved in the induction of IL-5 in other systems. The effect of rIL-10 on IL-5 was blocked by IL-2. It was confirmed that it was not due to harm. In fact, 1000 U / exogenous rIL-2 It was found that addition of up to ml did not change the inhibition of IL-5 secretion by rIL-10. It was Furthermore, the inventors of the present invention inhibit the secretion of IL-2 more effectively than rIL-10 Cyclosporine A (CsA) does not diminish IL-5 secretion but even significantly enhances it Was observed.   On secretion of cytokines by T cells stimulated with PMA and anti-CD28 mAb The effect of rIL-10 was also evaluated. As shown in FIG. 5, rIL-10 is the IL-5 under these conditions. Production was inhibited in a dose-dependent manner. IL-2 was also inhibited, but more than IL-5 The effect was low, whereas the secretion of IFN-γ was not affected by rIL-10 (Fig. 5). Together with the data above, these test results show the effect of rIL-10 on IL-5 secretion. When resting T cells are costimulated with B7 / CD28 signaling, Thing whether activated by CR / CD3 engagement or PMA It shows that they are practically the same.   rIL-10 reduced the accumulation of IL-5 mRNA in T cells activated with PMA and anti-CD28 mAb. Reduce:   To confirm whether IL-10 interfered with the expression of the IL-5 gene, resting T cells were treated with r Stimulated with PMA and anti-CD28 mAb for 24 hours in the presence or absence of IL-10 (50 U / ml). After challenge, IL-5 mRNA was analyzed by inverse PCR. rIL-10 apparently stores IL-5 mRNA Product, but has no detectable effect on HPRT housekeeping gene expression. There was no.   rIL-10 is PMA and A23187 calcium ionoph Does not inhibit IL-5 production in response to A:   In these studies, PMA and anti-CD28 mAb or PMA and A232187 calcium ion were tested. The effect of IL-10 on the production of IL-5 by T cells stimulated with onophore was compared . T cells from healthy individuals show relatively low levels of IL-5 under the latter conditions above. Produced, so secreted high levels of IL-5 under both of the above stimulatory conditions. Cells derived from patients with syndrome (severe combined immunodeficiency with hypereosinophilia) can It was included in the analysis. In fact, the profile of cytokines secreted by CD4 + cells in this patient The file conforms to the TH2-like profile. Of healthy individuals and Omen syndrome In both patients, rIL-10 produced PM-5 + A23187-induced IL-5 production. It did not deteriorate the life, but rather increased it. In contrast, rIL-10 responded to PMA + anti-CD28 mAb. The level of IL-5 produced in response was dramatically reduced. The degree of inhibition is It was similar (about 70%) in patients with Menmen syndrome.   Endogenous IL-10 down-regulates IL-5 production by resting T cells:   Since peripheral blood T cells are known to secrete IL-10 when activated, The effect of endogenously produced IL-10 on IL-5 secretion was investigated. Neutralizes IL-10 mAb The mAb used to drive the B7 / CD32 transfectants captures CD32. This test was performed with PMA and anti-CD28 mAb because it interferes with cross-linking of the anti-CD3 mAb against it. Performed on sexualized T cells. Anti-IL-10 mAb was added in three experiments performed. When added, IL-5 production was 114%, 48% and 64 % Increase but no isotype-matched control mAb It was. This means that the production of IL-5 is regulated by endogenous IL-10 in this system. Is shown. Example 6: IL-10 inhibits B7 and ICAM-1 expression on human monocytes.   In this example, IL-10 regulated expression of accessory molecules B7 and ICAM-1 on monocytes. Is shown. This property is due to the immunosuppressive property of IL-10.   Materials and methods   Recombinant cytokines:   Recombinant IL-10 transfected with expression vector containing human IL-10 cDNA Obtained from the supernatant of CHO (Chinese Hamster Ovary-K1) cells. versus Illuminated (simulated) supernatants were cloned with a similar vector without the IL-10 gene inserted. Harvested from transfected CHO-K1 cells.   Cell preparation and culture:   By centrifuging heparinized blood from healthy donors with Lymphoprep. Thus, peripheral blood mononuclear cells (PBMC) were isolated. RP supplemented with 5% bovine fetal serum PMBC in MI 1640 in the presence or absence of 250 U / ml IFN-γ 24 hours, 1 × 10⁶/ Ml. Serial dilution of IL-10 or simulated supernatant The cells were stimulated with IFN-γ 1 hour after the addition of the solution to the cells.   Analysis by flow cytometry:   Cells were treated with 0.5% BSA and 10 mM NaN₃Washed with PBS supplemented with and then 10 pg / ml primary mouse monochrome Incubate with internal antibody (mAb) for 30 minutes at 4 ° C. Primary mAb is B7-2 4 Anti-B7 mAb (18), anti-ICAM-1 mAb, anti-HLA-DR mAb or irrelevant mouse IgG mAb Of F (ab) ′₂It was a fragment. The binding of these antibodies is free from rabbit anti-mouse. FITC-coupled F (ab) 'of globulin antiserum₂Fragment and Revealed by incubation for 30 minutes at 4 ° C. After washing, cells Were reincubated with irrelevant mouse IgG for 10 minutes at 4 ° C and then filtered. Add incoerythrin (PE) conjugated anti-CD14 mAb and incubate at 4 ° C for 30 min. I started. Expression of B7, ICAM-1 and HLA-DR on the surface of CD14 positive cells was analyzed by FACScan. It measured using the float cytometer. IFN-γ and IL-10 are both It did not interfere with the gating. Coated with a certain amount of fluorescein Using standardized fine beads for flow cytometry, the mean fluorescence channel (mean  Number of fluorescein molecules captured per cell (fluorescence channel) It was confirmed that there was a linear relationship with. From this, the measured average fluorescence Channels can be converted to Mean Equivalents of Soluble Fluorescence (MESF). Antigen-specific mean fluorescence intensity Degree (MESF) was calculated by subtracting non-specific fluorescence.   Test results   IL-10 inhibits ICAM-1 expression on monocytes:   MHC class II on monocytes both in basal state and after stimulation with IFN-γ First, the data showing that IL-10 reduced the expression of the molecule (HLA-DR) was confirmed. I accepted. next The effect of IL-10 on the expression of ICAM-1 on these cells was tested. FAC obtained S-histogram showed that IL-10 reduced ICAM-1 expression in resting monocytes . Three experiments performed on three different donors showed that basic ICAM-1 expression Percentage inhibition was in the range of 50-65%. Simulated supernatant has no effect Therefore, the IL-10 formulation proved to have specificity. By IFN-γ stimulation, ICAM-1 expression on monocytes is clearly upregulated compared to vehicle alone However, the isotype control tinnings did not change. IL-10 (3U / ml) IFN-γ-induced upregulation of ICAM-1 on the surface of monocytes when added Was hindered. In the case of the simulated supernatant, no inhibitory effect was found Thus, the inhibitory effect of the IL-10 formulation was dose-dependent and specific.   IL-10 blocks the upregulation of B7 on monocytes stimulated by IFN-γ. Harm:   Incubation of PBMC with IFN-γ (250 U / ml) for 24 hours resulted in simple A clear upregulation of B7 expression on the surface of the sphere was induced. IFN-γ to IL Addition of -10 (3 U / ml) markedly upregulated B7 on monocytes. It dropped. IFN-γ or IFN-γ + IL-10 are both F (ab) ′₂Absence of anti-B7 Below, the control sting (co) observed with FITC-conjugated anti-mouse immunoglobulin antiserum ntrol staining) was not changed. Three generations of three different donors B7-staining MESF values in a tabular study showed that the inhibitory effect of IL-10 was dose-dependent Sex specific It shows that. In these three studies, the percentage inhibition of B7 expression by IL-10 The rate was in the range of 60-97%.   These test results indicate that IL-10 blocks ICAM-1 and B7 expression on the surface of monocytes. It is shown to do harm. The in vivo relevance of these observations is allograft Rejection by anti-ICAM-1 mAb and anti-LFA-1 mAb or T cell activation Model prevented by CTLA-4-Ig blocking the B7 / CD28-CTLA-4 pathway in Escherichia coli (Isobe et al., Science, 2545, 1125, 1992; Lenschow et al., Science, 357, p. 759, 1992). Example 7: Systemic administration of rIL-10 results in alloreactive response in vivo.  response), it inhibits the formation of edema and the synthesis of IFN-γ.   IL-10, in addition to inhibiting the expression of MHC, also on macrophages or monocytes Down-regulates B7 molecule and ICAM-1 expression. These properties of IL-10 are 10 is capable of inducing acute cell transplant rejection and inhibiting the effector phase. It suggests that seed graft resistance can be increased. First to this problem As a method of the present invention, the inventors of the present invention induce by injecting allogeneic cells locally. Analysis of the effect of systemic administration of rIL-10 in an in vivo model of alloreactivity generated did.   The results obtained show that the main effect of rIL-10 is that water induced by alloantigen attack. Prevents tumor formation and IFN-γ synthesis It means that it is.   Materials and methods   mouse:   BALB / c (H-2 at 10 to 12 weeks of age^d), A / J (H-2^k) And C57 / BL6 (H- Two^b) Harlan CPB (Zeist, The Netherlands), Center National d e la Recherche Scientifique (Orleans, France) and Iffa Credo Reims, L'Arbresle).   Mouse rIL-10 adjustments:   Mouse rIL-10 was cloned as previously described (Example 1) and then Invi. Bars obtained from trogen Corp. (San Diego, Calif., USA) The culovirus expression vector pBlueBac2 was used to produce in Sf9 insect cells. Ph Using the JES 5-2A5 anti-IL-10 monoclonal antibody obtained from armingen, rIL-10 Affinity purification was performed. For administration in vivo, rIL-10 was added to 0.1% Dilute with saline buffer containing serum (vehicle for rIL-10) to a final concentration of 10 000 U / ml (measured by ELISA).   Lymphadenopathy assay and test protocol:   2.5 × 10 in RPMI1640 (0.1 ml)⁶Syngeneic (BALB / c) or half A suspension containing allogeneic (A / J x BALB / c) F1 spleen cells was applied on day 1 with BALB / c It was injected into the toes of the right hind foot of Us. From day 0 to day 5, 1000 U for mice RIL-10 or control vehicle was injected ip every 8 hours. This dose of IL-10 In two different organisms In the model, that is, shock due to endogenous toxin and anti-CD3 monoclonal antibody -Induced activation of polyclonal T cells blocks cytokine release. It was selected because it was found to be effective in harming. On the fifth day, left and right The lymph node was surgically removed from the popliteal fossa and weighed. The test results are on the right (Note The data representing the weight difference between the lymph nodes on the left side (the side not injected) and the left side (the side not injected). Data (in mg).   Lymph node cell count and histological analysis:   A cell suspension count for each lymph node was performed, then lymph node cells from each group Phycoerythrin-conjugated anti-CD4 mono obtained from Becton Dickinson Clonal antibody (L3T4) or FITC-conjugated anti-CD8 monoclonal antibody (Lyt 2 ) And then the percentage of CD4 + and CD8 + cells was determined by flow cytometry. I have   Analysis of IFN-γ mRNA expression by inverse PCR:   MRNA derived from popliteal lymph node cells was obtained from Invitrogen Corp. Micro-Fast  Extraction was performed using the Track (registered trademark) mRNA isolation kit. 1 μg of each RNA sample, It was reverse transcribed into cDNA using an oligo dT primer. Then IFN-γ or hypoxa N-phosphoribosyl transferase (HPRT) specific primer PCR amplification was performed. Amplified products are separated on a 2% agarose gel and then odor It was visualized by staining with ethidium iodide.   Test results   rIL-10 is a draining lymph node ( Inhibits swelling of the draining lymph node).   Draining lymph node swelling is alloreactive response induced by subcutaneous injection of allogeneic cells The inventors of the present invention, firstly, for this parameter, The effect of rIL-10 administration was evaluated. Injection of allogeneic cells into the toes of the right foot as shown in FIG. Then, the drainage popliteal lymph node was measured by the weight difference from the left popliteal lymph node. Caused significant swelling (average delta value ± SD: 5.0 ± 1.8 mg) . Administration of rIL-10 significantly reduced lymph node swelling (mean delta ± SD : 2.8 ± 1.7 mg, p <0.00 compared to mice injected with allogeneic cells only 1 is significant). Increased cells in draining lymph nodes of mice injected with allogeneic cells The number did not decrease significantly when rIL-10 was administered (Table 2). The effect of a may not be due to inhibiting the recruitment of cells. In addition, drainage The average percentage of CD4 + and CD8 + cells in the lymph node was that mice showed rIL-10 as allogeneic cells. They were similar whether they were injected together or with allogeneic cells (CD4 35%: 43% and 11%: 14% for + cells and CD8 + cells, respectively. ). These data show that the inhibition of lymphadenopathy by administration of rIL-10 is mainly Suggest that it was due to reduced edema formation. This is Confirmed by histological analysis of draining lymph nodes (Table 3).   Administration of rIL-10 prevents IFN-γ mRNA accumulation: Since the production of IFN-γ is a crucial element of the alloreactive response, the inventors of the present invention , R for induction of IFN-γ mRNA in draining lymph nodes of mice injected with allogeneic cells The effect of IL-10 was measured. Inverse PCR experimental data show that administration of rIL-10 resulted in β-activities. In these mice without altering the expression of the mouse mRNA or housekeeping gene. IFN-γ mRNA accumulation was dramatically reduced. Cited references

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI A61K 38/00 ACJ A61K 37/02 ACD ADV ACJ ADZ ABN AEB AEB 38/21 ABY ADZ 39/395 ADV (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AU, BB, BG, BR, CA, CN, CZ, FI, GE, HU, JP, KP, KR, KZ, LK, LV, MG, MN, MW, NO, NZ, PL, RO, RU, SD, SK, UA, US, UZ, VN (72) Inventor Ve , Thierry Belgium, BE-1070 Brussels, Root Drenic 808 Campus Erasm, Eleven (72) Inventor Abramowitz, Daniel Belgium, BE-1070 Brussels, Root Drennik 808 Servis de Munologi, Optimal Erasm (72) Inventor Bruins, Catherine Belgium, Be-1070 Brussels, Root Drenick 808 Campus Erasm, Eleven (72) Inventor Capel, Paul Belgium, Beer-1070 Brussels, Root Drennik 808 Servis de Munology, Optics Erasm (72) Invention Delvo, Anne Belgian, Belgium 1070 Brussels, Root Drenic 808 Campus Erasm, Eleven (72) Inventor Donquier, Vincent Belgium, Bay 1070 Brussels, Root Drenic 808 Servi Demulogology, optical Erasm (72) Inventor Gerard, Catherine Belgium, Bé 1070 Brussels, Root Drennick 808 Campus Erasm, Eleven (72) Inventor Marcian, Arnault Belgium, Bé 1070 Brussels, Root Drennick 808 Servis de Munologis, Opital Erasm (72) Inventor Pradiere, Olivier Belgium, Be-1070 Brussels, Root Drenic 808 Servis de Munology, Optics Erasm (72) Inventor Chandené, Lillian Belgium, Be-1070 Brussels, Root Drenick 808 Servis de Munology, Optics Erasm (72) Inventor Willem, Fabien Belgium, Be 1070 Brussels, Root Drennick 808 Servis de Munology, Optics Erasm

Claims (1)

[Claims]   1. i) interleukin-5 production by T lymphocytes; or   ii) activation of T lymphocytes by the B7 / CD28 or ICAM-1 / LFA-1 pathway; or   iii) diseases associated with activation of monocyte procoagulant activity;   iv) Severe infectious disease with tumor necrosis factor (TNF) production; Interleukin-10 (IL -10) or an analogue or agonist thereof.   2. Overproduction of IL-5 by T lymphocytes such as eosinophilia-related diseases The method according to claim 1, which is used for producing a medicament for treating or preventing a disease associated with Use of IL-10.   3. Atopic diseases including allergic rhinitis and bronchial asthma; atopic skin Skin diseases associated with eosinophil infiltration such as inflammation (chronic eczema, erythema multiforme, herpes dermatitis, fertilizer) (Including eucytosis, bullous disorder); Crohn's disease; bronchopulmonary aspergillosis; Intoxicated oil syndrome; muscle pain induced by L-tryptophan preparation, eosinophilia symptom Group; allergic reaction of certain medications; tropical eosinophilia; helminthic infections; Syndrome (eosinophilic fasciitis); Eosinophilia such as Churg-Strauss syndrome With vasculitis; Lefrel syndrome; chronic eosinophilic pneumonia; eosinophilic gastroenteritis; Drug used for treating or preventing a disease selected from the group consisting of eosinophilia syndrome Use according to claim 2 for the manufacture of.   4. Claim 2 wherein IL-10 is the only active ingredient of the drug ~ Use according to any one of 3 to.   5. IL-10 was found to be interferon-α (IFN-α) and interferon in medicine. Any one of claims 2 to 3 in combination with feron-γ (IFN-γ) Use as described in.   6. Diseases associated with T lymphocyte activation by the B7 / CD28 or ICAM-1 / LFA-1 pathway Medicine used for treating or preventing a patient, for example, a disease associated with alloreactive response Use of IL-10 according to claim 1 for the manufacture of.   7. Host-graft-related diseases, such as allograft rejection, and graft-host-related diseases For producing a medicament for treating or preventing a disease selected from the group consisting of Use according to claim 6.   8. The use according to claim 6 or 7, wherein IL-10 is the only active ingredient of the medicament. for.   9. IL-10 is a drug in the drug cyclosporin A or other related immunosuppressants For example, a contract in combination with FK506, rapamycin or anti-CD3 monoclonal antibody. Use according to claim 6 or 7.   Ten. Accompanied by activation of monocyte procoagulant activity by inducing surface tissue factor expression A method according to claim 1 for producing a medicament for use in preventing or treating a disease. Use of IL-10.   11. Induced by septic shock, meningococcalemia, promonocytic leukemia, anti-CD3 antibody Reactions, cerebral malaria, antiphospholipid syndrome, hemolytic uremic syndrome, disseminated Prevents diseases selected from the group consisting of intravascular coagulation and high-risk surgery Alternatively, the use according to claim 10 for producing a medicament for use in treating.   12. To produce a medicament used to prevent severe infections associated with TNF production Use of IL-10 according to claim 1.   13. Septic shock induced by Gram-negative bacteria and cerebral malarial A contract for producing a medicine used for preventing a disease selected from the group consisting of Use according to claim 12 of the application.   14. Use of IL-10 according to claim 1, wherein IL-10 is recombinant human IL-10.   15． IL-10 is viral IL-10, IL-10 analog, IL-10 agonist, IL-10 peptide , Or a chimeric protein derived from both cellular IL-10 and viral IL-10 Use according to claim 14 or 1.