JPH11507824A - How to inhibit phagocytosis - Google Patents

Abstract

  (57) [Summary] The present invention generally relates to a method of treating a disease resulting from an interaction between an immune complex and an Fc receptor. In particular, the present invention relates to a method for regulating the clearance of antibody-coated cells from the circulatory system by inhibiting phagocytosis, and a method for regulating the interaction between an immune complex and a tissue Fc receptor. Furthermore, the present invention relates to a method of modulating the activation of an immunological process mediated by the activation of an Fc receptor resulting from an antibody-antigen / receptor interaction.

Description

DETAILED DESCRIPTION OF THE INVENTION                           How to inhibit phagocytosis   This application is a continuation-in-part of application no. 08 / 483,530 filed on June 7, 1995. This parent application is a continuation-in-part of application no. 08 / 316,425 filed on September 30, 1994. Is a continuation-in-part application, which is further described in application Ser. No. 08 / 129,381, filed on Sep. 30, 1993. Partial continuation application. The contents of these applications are incorporated herein by reference. Department. 〔Technical field〕   The present invention generally relates to diseases resulting from the interaction between the immune complex and the Fc receptor. Regarding treatment. In particular, the present invention relates to cells coated with antibodies by phagocytosis inhibition (ant regulates clearance of ibody-coated cells), viruses, or soluble antibodies Modulating the interaction of immune complexes with cell or tissue Fc receptors How to do. The present invention also relates to the reaction between the antigen-antibody complex and the Fc receptor. It concerns the regulation of the immune response as an important starting step. [Background of the Invention]   Certain immunological disorders include monocyte or macrophage Fc (IgG) receptors. Is characterized by being disturbed. The increase in the number of Fc receptors -Elevated levels of Fc receptor mediators such as It can result from the secretion of fungal products. Decrease in the number of Fc receptors that can bind IgG Not only does the actual number of functional receptors decrease, It can result from being saturated by immune complexes. Like systemic lupus erythematosus In certain autoimmune diseases, the level of circulating immune complexes is elevated, Can cause receptor saturation.   In an autoimmune disease, the body's mechanisms for distinguishing itself from foreign invaders are normal. Does not work. Typically, the body begins to make antibodies to certain parts of itself; These antibodies elicit the immune system, which is caused by this abnormal antibody. Destroy the recognized tissue.   Autoimmune diseases have changed the focal points of an attack. Autoimmune lysis Blood anemia is a disease in which individual diseases cause one or more of their own red blood cell membrane antigens. Representative examples of a group of diseases that produce antibodies to them. Abnormal antibodies Once the red blood cells are coated, the red blood cells are then circulated by the macrophages of the spleen. From which it is subsequently destroyed in the spleen. This Typical lass diseases include immune hemolytic anemia, immune thrombocytopenic purpura and And autoimmune neutropenia. Another type of autoimmune disease is systemic There are types represented by lupus erythematosus and rheumatoid arthritis. these The disease causes chronic inflammation of joints, tendons, kidneys, lungs, heart and other organs. An example For example, in rheumatoid arthritis, the breakdown of articular cartilage into synovial fluid occurs later in the disease. Occurs in stages. However, in systemic lupus erythematosus, cartilage or bone degradation is Usually not seen. Systemic lupus erythematosus and rheumatoid arthritis are often another type Occurs in association with autoimmune diseases of the Systemic lupus erythematosus and rheumatoid arthritis In, tissue destruction is associated with the presence or absence of circulating IgG-containing complexes. Fc receptor When these complexes are identified in the tissue by cells having And possibly tissue destruction by other cells such as polymorphonuclear leukocytes in these tissues. It is believed to be started or increased. By these Fc receptors Reactions that elicit a range of immune-related reactions and have Fc receptors May damage the nearby body tissue of the vesicle.   Diseases involving interaction of IgG-containing immune complexes with macrophage Fc receptors The disease is often treated with corticosteroids or immunosuppressants. These cures Treatment can have a variety of serious side effects. The invention may be used alone or Offers alternative treatments that can be used in combination with conventional drug treatments You. [Summary of the Invention]   A general object of the present invention is to provide, for example, the phagocytic ability of cells bearing Fc receptors. Inhibits the clearance of antibody-coated cells or immune complexes To provide a way to adjust the   A specific object of the present invention is to modulate the clearance of an immune complex from a mammal. Is to provide a way to In addition, a specific object of the present invention is to provide a membrane-bound Fc receptor. Inhibits the binding of the immune complex to the receptor (and / or removes such a complex). To inhibit the sequelae of undesired tissue damage. To provide.   It is a further object of the present invention to provide suitable constructs and compounds for use in the above methods. It is to provide.   In one embodiment, the present invention provides a method for phagocytosing an immune complex (e.g., an IgG-containing immune complex). And / or intracellular biochemical activity by cells interacting with the immune complex A method for preventing secretion of a product. Examples of this method include immunocomplexes (e.g., I (gG-containing immune complex) into mammalian phagocytes in contact with Introducing endogenous kinase inhibitors into cells that activate Fc receptors The method consists of:   In another embodiment, the present invention relates to a mammal-derived immune complex (e.g., an IgG-containing (Immune complex). This method uses an immune complex Fc on the cell membrane into mammalian hematopoietic cells (eg, phagocytic cells) in contact with And introducing a molecule that specifically inhibits the expression of the receptor.   In yet another aspect, the invention relates to an immune complex (e.g., containing IgG) present in a mammal. Immunocomplexes) to bind to membrane-bound Fc receptors. You. This method involves the use of a membrane-bound Fc receptor for binding to the immune complex in the mammal. Introducing a soluble Fc receptor that competes with the sceptor. This introduction Under conditions that inhibit the binding of the immune complex to the membrane-bound Fc receptor. You.   In still another aspect, the present invention relates to a method for producing a mammalian cell having an Fc receptor. A method of inhibiting phagocytic ability. This method converts the 5 'end to the 3' end. In the shooting direction,           i) a promoter that is functional in the cell,           ii) Double-stranded DNA segment whose transcribed single strand is Fc receptor One containing a sequence complementary to the endogenous mRNA encoding           iii) Intracellular termination sequence (polyadenylation signal) Introducing into the cell a construct comprising In this construct, the complementary strand is transcribed. Reduces Fc receptor expression by binding to endogenous mRNA It is introduced under conditions that inhibit the phagocytic capacity of the plant.   Further objects or advantages of the present invention will become clear from the description hereinafter. This disclosure describes three classes of Fcγ receptors (FcγRI, FcγRII and Fcγ RIII) should be read in light of the teachings available in the art for isolation and cloning. Would be preferred. For example, Allen and Seed, Science 243: 378 (1989); Hibb s et al, Proc. Natl. Acad. Sci. USA 85: 2240 (1988); Exp. Med. 166: 1668 (1987); van de Winkle et al, FASEB J., 5: A964 (1991); Brooks et al, J., Ex. p.Med. 170: 369 (1989); Stuart et al., EMBO J. p. 8: 3657 (1989); Qui et al, Scie nce 248: 732 (1990); Simmons and Seed, Nature 333: 568 (1988); S chreiber et al, Clin. Immunol. Immunopath. 62: See also S66 (1992). [Brief description of drawings]   FIG. 1 shows a schematic of FcγRIIIAγ wild-type and mutant. Signal distribution Row (S), foreign peptide (E), transmembrane domain (TM), and cytoplasmic domain (CY) is shown above the schematic diagram of the γ chain. The enlarged area contains the conserved motif. 2 shows the region of the nucleotide sequence of the γ chain. In this figure, the murine gamma chain is shown. You. The conserved amino acid sequences of the γ and ζ chain gene families are It is underlined. Encoded by the TAC codon at nucleotides 235-237 Tyrosine close to the N-terminus (Ra et al, J. Biol. Chem. 264: 15323 (1989)) is Conservatively replaced by phenylalanine encoded by (clone M1A and Clone M1B). Similarly, close to the C-terminus encoded by TAT (168-270) Tyrosine was replaced with phenylalanine encoded by TTT (clone M 2A and clone M2B). For tyrosine double substitution mutants, N-terminal and C-terminal Were replaced with phenylalanine (clone DMA and clone D). MB). The solid line of the mutant shows the same sequence as that of the wild-type γ gene.   2A and 2B show IgG sensitization by transfected COS-1 cells. 2 shows binding and phagocytosis of RBCs (EA). Transfected COS-1 cells By EA (left figure; A, C, E and G). For transfected COS-1 cells EA phagocytosis (right figure; B, D, F, and H). (A) and (B): FcγRIIIAα and wild type Binding and phagocytosis of γ-transfected COS-1 cells. Shown using wild-type γ The three poorly eaten BCs (phagocytosed RBCs) were identified in Figures (B), (C), and (D). Indicated by arrows: transfectants containing α and γ ( MIA). (E) and (F): transfectants containing α and γ (M2A). (G) and (H): Transfectants containing α and γ (DMA). The phagocytosis of EA is not seen in D, F and H. I can't. The photo shows a 1000x magnification.   FIG. 3 shows wild-type and mutant by kinase assay in vitro. Fig. 7 shows the phosphorylation of tyrosine in the γ chain. This gamma chain is converted to the COS-1 transfectant Immunoprecipitation was performed using an anti-γ antiserum from the lysate. In vitro phosphorylated test The material was run on a 12.5% reducing SDS-PAGE gel. This gel is Treated with 1N KOH and dried to remove foserine and threonine After 4 days, the autoradiogram was examined. Lane 1: Fc γ RIIIA-α and γ c Pseudo-transfectants using pSVL vector without DNA inserted (Shamtra nsfectants). Lane 2: FcγRIIIAα + wild-type human γ. Lane 3: Fc γ RIIIA α + wild type mouse γ. Lane 4: FcγRIIIAα + M1A. Lane 5: FcγRIIIAα + M2A. Lane 6: FcγRIIIAα + DMA. The phosphorylated γ chain is indicated by an arrow ( (Shown on the bottom right). The arrow with an asterisk (shown in the upper right) indicates a specific tyro of about 4OKDa. Shown is the band of the Singlin protein.   FIGS.4A-4D show that Ca following FcγRIIIA stimulation²⁺Mobilization Is shown. [Ca in each cell²⁺] i is measured by cross-linking of FcγRIIIA nking). Anti-FcγRIII mAb, epinephrine (positive control () And the calcium ionophore are indicated by arrows in the respective figures. More shown. Images were obtained with either 340 nm or 380 nm excitation (emission (em isson) = 510nm). The 340/380 ratio is based on the correction using Fura-2, [Ca²⁺] i Was. The reaction of M1A, M2A and DMA transfectants It was significantly lower than that of Cactant.   FIG. 5 shows the target sequence (target sequence) for stem loop antisense ODN. Indicate option III). All Syk mRNA sequences are used to find sequences without secondary structure 3 times, RNA secondary structure predict program ion program). Each scanning is 99 nucleos Consecutive 33 bases apart in a pseudoframe (referred to as frames A, B, and C) It was done. Most open with three staggered scannings Unique sequence was selected as the target sequence. The upper rectangle with dotted line is human Sy The cDNA sequence of k mRNA is shown (Law et al, J. Biol. Chem. 269; 12310 (1994)). S Three target sites for the Semuk Syk antisense ODN are Above the column lines are shown as three short solid lines (I, II, III). Target site I, II , And III correspond to nucleotides 159 to 173 (the region surrounding the transcription initiation codon). Region), corresponding to nucleotide numbers 451 to 463 and 802 to 816, respectively I do. Target III is shown in this figure as an example. Targets I and II are the same chosen. The putative secondary structure in the Syk mRNA region containing the target III sequence is Three staggered frames of 99 nucleotides each Shown in schemes A, B, and C. In three staggered frames, The circled nucleotides are the consensus sequence of Target III with minimal secondary structure.   FIG. 6 shows the secondary structure of the stem-loop Syk antisense ODN. The 7 nuclei Otide-length stem domains at their 5 'and 3' ends from G and C only Formed by a complementary termination sequence comprising the following nucleotides: The loop domain is 3 antisense sequences; 5'-CTGTCAGCCATGCCG-3 'sequence indicated by a square Is complementary to target I in Syk mRNA (see FIG. 5) and is indicated by a triangle. The 5'-GCTTCTTGAGGAG-3 'sequence is complementary to target II and is marked with a circle. The 5'-TGTCTTGTCTTTGTC-3 'sequence shown is also indicated by a circle in FIG. Complementary to target IIIT. The three different antisense sequences correspond to targets I, III, II Are connected in tandem in the order of 5 ′ to 3 ′. ● S is phosphoro Shows modification of thiolate (phosphorothiorate), 5 'end is phosphorothiolate 1 and 2 at the 3 'end.   FIG. 7 shows inhibition of Syk antisense ODNs on phagocytosis in monocytes. Mononuclear cells (1 x 10^FiveCells / ml) is 4μg of Lipofectamine (LIPOFECTAMINE) / Ml and ODNs (linear control or linear Syk antisense ODNs are each 1.0μ M, stem loop control or stem loop Syk antisense ODN are 2. 0 μM) of the complex was incubated for 2 days, and IgG-sensitized red blood cells (EA) were Phagocytosis was examined. Phagocytosis index (PI) = RBCs taken up per 100 cells number. Each bar represents the mean ± SEM of three independent experiments.   Figure 8: Effect of Syk antisense ODNs on Syk mRNA in monocytes. Total RNA was 4 μg / ml lipofectamine (LIPOFECTAMINE) and ODNs (linear 1.0 μM each of control or linear Syk antisense ODNs Control 2 or Syloop antisense ODN 2.0 μM each) Mononuclear cells treated for 1 day (1 x 10^FiveCells / ml) and the cDNA is Synthesized from total RNA by nucleotide primers. PCR to Syk DNA The rates were performed with two Sky primers (Syk-H and Syk-M) . The PCR products are analyzed by Southern hybridization and hybridized. The shifted bands were visualized by chemifluorescence reagents.   Figure 9: Comparison of rat and human Syk antisense   Figure 10: Stem-loop rat Syk kinase expression of Syk kinase expression in RBL-2H3 cells. Inhibition by antisense oligonucleotides (ODN). A. Investigation of Syk expression by RT-PCR using rat Syk primer. Lane 1: S Cells treated with yk antisense ODN, lane 2: treated with Syk sense ODN Cells, lane 3: reagent control, lane 4: no treatment, lane 5: molecular weight mer car. B. β-actin expression by RT-PCR using rat β-actin primer Survey. Lane 1: cells treated with Syk antisense ODN, Lane 2: Syk cell. ODN treated cells, lane 3: reagent control, lane 4: no treatment, Lane 5: molecular weight marker. → = β-actin. C. Γ chain promoter in RBL-2H3 cells treated with rat Syk antisense ODN Fc by RT-PCR using primer_εInvestigation of RI gamma chain expression. Lane 1: Cells treated with Syk antisense ODN, lane 2: treated with Syk sense ODN Cells, lane 3: reagent control, lane 4: no treatment, lane 5: molecular weight Car. → = γ chain.   Figure 11: Structure of human Syk / ZAP-70 chimera [Detailed description of the invention]   The present invention provides, at least in part, cells from a mammal (e.g., For example, to methods of regulating clearance (from the mammalian circulatory system). Therefore The present invention relates to immunocomplexes with Fc receptors (e.g., those present on the surface of Autoimmunity characterized by interaction with coalescence (e.g., an IgG-containing immune complex) Those who treat immunological diseases such as diseases and immune-mediated diseases such as asthma Provide the law. The method of the invention results in the phagocytosis of cells coated with IgG antibodies. Altering the expression and / or function of the Fc receptor to reduce (Immune complex diseases such as lupus erythematosus and rheumatoid arthritis disease) The clearance of immune complexes circulating in the liver and spleen Enhanced, thereby depositing immune complexes in tissues such as the kidney and in joints It will be appreciated by those skilled in the art that procedures designed to prevent Will be. This enhancement of immune complex clearance was filed at the same time as the present application. Receptor described in co-owned application entitled "Method of stimulating phagocytosis" Liver and spleen macrophages using procedures for introducing sequences encoding Can be brought about by stimulating the page. All of the disclosure content of that separate application is Incorporated by reference in this specification.   More specifically, the present invention relates to Fc receptors required for phagocyte signaling. Inhibits the phosphorylation of protein components and associated molecules, resulting in immune complexes (e.g., IgG Soluble Fc receptor competes with membrane-bound receptor for binding of (containing immune complex) To provide a method to inhibit the function of Fc receptor by introducing into the circulation . The present invention also provides for the introduction of Fc receptor antisense constructs into receptor-producing cells. The present invention provides a method for inhibiting the expression of an Fc receptor. In addition, Akira provides a method for degrading Fc receptor RNA using, for example, ribozymes.Fc Inhibition of receptor-mediated signaling:   In one embodiment, the present invention provides a core arrangement within the cytoplasmic domain of an Fc receptor. By inhibiting the phosphorylation of the core sequence, immune complexes (e.g., IgG A method of preventing the uptake (eg, phagocytosis) of coated cells. Fc γ RII Phosphorylation of A cytoplasmic residues and the γ subunit of FcγRIIIA are involved in phagocytosis. Have been shown to be essential for signal transduction events (Indik et al, Trans. Ass. . Amer. Phys. 105: 214 (1992); Park et al, Clin. Res. 41: 324A (1993); Darby e t al, Blood 79: 352A (1992); Mitchell et al, Clin. Res. 41: 189A (1993); Huang  et al. Biol. Chem. 267: 5467 (1992); Hunter et al, Clin. Res. 41: 244A (1 993); Park et al, J. Clin. Invest. 92: 2073 (1993)). More specifically, FcγR Motif E-X8-D-X2-Y-X2-L-X12-Y-X2-L and FcRI present in the cytoplasmic domain of IIA D / E-X2, 7-D / E-Y-X2-L-X motifs present in the cytoplasmic domains of IIA γ and ζ chains Phosphorylation of tyrosine residues present within 7-Y-X2-L is required for phagocytic signaling. (The number after the letter X indicates the number of amino acids at that position; X is any amino acid X2 in Y-X2-L may be an acid, but the cytoplasmic domain of FcγRIIA or FcγRIII It is preferably an amino acid present in the Y-X2-L sequence of the γ chain of the above). These The second Y-X2-L in the sequence (motif) appears to be particularly important for phagocytosis. The present invention Intends to introduce inhibitors of kinases involved in phosphorylation into target cells. You. In specific aspects, the inhibitor is at least a functional tyrosine-containing motif. A peptide containing a similar sequence even if it is not the same as the portion (e.g., (Note the underlined part of the ream motif.) Acts as an inhibitor. In one example, the inhibitor is an Fc receptor lacking the extracellular domain. Fc receptor lacking the extracellular domain or transmembrane domain It can take the form of a scepter. Instead, the inhibitor is Or they may be structurally different from their functional parts, which are antagonistic or Phosphorylation may be inhibited non-antagonistically (for example, similar to the binding site of the active peptide). Pseudo-active peptides having various structural forms can be used). Mast (obesity) Cells, or Fc_εIn cells with receptors, mediators (e.g., Fc required for secretion of min, cytokine or leukotriene)_εSequence of the γ chain of RI Hako Can be inhibited by using the above method.   The peptide inhibitor of the present invention or a mimetic thereof is, for example, a liposome. Can be directly introduced into target cells using a system (described in Science 26: 1877 (1993)). For administering a peptide modified to be capable of crossing a lipid membrane of a cell See also). Alternatively, the DNA sequence encoding the peptide inhibitor Can be introduced using gene therapy procedures so that it is produced intracellularly .   The inhibitor or the sequence encoding the inhibitor may be found in lung cells, including macrophages. The vesicle may be administered in the form of an aerosol. Inhibitor or inhibitor-encoding sequence Are particles that are phagocytosed by lung macrophages (e.g., liposomes, or If the sequence encodes an inhibitor, for example, Listeria It can also be present in the aerosol as a non-infectious bacterium). Phagocytosis As a result, the inhibitor or the sequence encoding the inhibitor is introduced into macrophages. Viral vectors also contain sequences encoding the peptide inhibitors of the present invention in the lung tree. It can be used to introduce into pulmonary tree cells. Vector is d Herpes or adenovirus with poor replication can also be introduced as an aerosol It can also be in the form of a vector. Also use retroviral vectors (In brief, Bajocchi et al., Nat. Genet. 3:22). 9 (1993); Lemarchand et al., Circ. Res., 72: 1132 (1993); Ram et al, Cancer Res. 53:83 (1993); Crystal, Am. J. Me d. 92: 445 (1992); Yoshimura et al., Nucl. Acids Res. 20: 3233 (1 992); Morecki et al., Cancer Immunol. Immunother. 32: 342 (199 1); Culver et al, Hum, Gene Ther. 1: 399 (1990); Culver et al. (Cu lver et al), Transplant. proc., 23: 170 (1991)).   Blood mononuclear cells can be ex vivo (ex) using sequences encoding the peptide inhibitors of the invention. (in vivo) and then redirect the patient to produce the inhibitor in vivo. May be entered.   Another approach to phosphorylation inhibition involves Fc receptor phosphorylation sites (e.g., For example, in FcγRIIA and / or in the γ subunit of FcγRIIIA). Includes the Use of Ribozymes Recognizing RNA Sequences That Identify Sequences and Enzyme Active Sites It is. The ribozyme is introduced using a carrier such as a liposome coated with IgG. , Directly into cells having the Fcγ receptor. Or I Liposomes coated with gE can be used for mast cells or basophils or associated γ IgE receptor Fc with subunit_εDirected to other cells with RI. this The method should be an appropriate approach for use in treating allergic diseases Will be appreciated by those skilled in the art. The gamma subunit of the IgE receptor is a mast cell Fc like_εSecretion of intracellular mediators by cells with RI receptor Involved in the transmission of induced signals. Destruction of γ-strand RNA causes these biological activities The secretion of the product is expected to be inhibited.   According to the above approach, ribozymes administered as described above may have some Selected sequences (e.g., specific for FcγRIIA or FcγRIIIA RNA splicing sequence or 5 'untranslated sequence) The functional sequence of the receptor required for phagocytic signal transduction The RNA to be specified is digested and removed. Mediators (e.g. histami Fc required for secretion of cytokines, cytokines or leukotrienes)_εThe sequence of the RI gamma chain The RNA sequence specified can be removed using this strategy.   The advantage of this method is that continuous production of the ribozyme in vivo can be achieved in vitro. Built packaging cells (e.g., Psi2-like cells; Miller and Rossman) Rosman), Biotechnique 7: 980, 1989 and recent techniques in molecular biology (Curr ent protocols in Molecular Biology), III: 9.1 1992 (supp. 17)). Can be performed using Like stopping ribozyme production, It will be appreciated by those skilled in the art that suicide genes can be contained in U.   For additional approaches to inhibiting receptor phosphorylation, use SyK Antisense constructs or ribosas targeting targeting sequences (see Example 5) Im use of implications. Syk other than ZAP-70 gene product of Syk kinase family The gene product is a diet of FcγRI and FcRIIIA mediated by both γ and ζ chains. It has been shown to stimulate action. (Certain Src-related tyrosine kinases ZAP-70 in the presence of ze can stimulate the phagocytosis of FcγRI and FcRIIIA. ) That is, by targeting the Syk sequence, inhibition of Syk expression and dependent phosphorus Inhibition of oxidation can be performed. Constructs and ribozymes suitable for use in this method are (Syk gene sequences, see Goat et al.) Biochem. Biophys. Res. Comm. 200: 28 (1994); Law et al. Biol. Chem. 26 9: 12310 (1994)).   Syk and ZAP-70 chimeras are related to differences in signaling between Syk and ZAP-70. Have been used to determine alternative sequences. The ZAP-70 mutant is a ZAP-70SH2 domain And the ZAP-70 intermediate region between the second SH2 domain and the catalytic domain It has been replaced by two domains and an intermediate region (FIG. 11). By the study This chimera enhances signaling of phagocytosis mediated by Fcγ receptors Indicates that it acts like Syk. In parallel, the SykSH2 domain and the Syk intermediate region Syk variants substituted with the ZAP-70SH domain and intermediate region were constructed. The chimera Works like ZAP-70 in not enhancing Fcγ receptor-mediated signaling. (Excluding COS-1 cell transfectants and phagocytic signaling). In addition, chimeras of Syk and ZAP-70 were generated. The Syk mutant has its SH_TwoDome Inn is ZAP-70 SH_TwoBuilt with domains replaced. This chimera is Syk kinase Works in a similar manner. Similarly, the ZAP-70 variant has its SH_TwoSH domain of Syk kinase_Two Built with domains replaced. This chimera acts like ZAP-70. The Syk variant is an intermediate between the second SH2 domain and the catalytic (kinase) domain. The region was constructed with the ZAP-70 and the middle region replaced. This chimera is ZAP- Acts like 70. Similarly, a ZAP-70 mutant binds the middle region of ZAP-70 to Syk kinase. It was constructed to replace the middle region. This chimera acts like Syk. Syk and ZA From these experiments with P-70 chimeras, the second SH2 domain and the catalytic (kinase) Sequences in the intermediate region between the domains correlate with Fcγ receptor signaling. It was found to be involved in the ability of Syk to interact (Park and Schreiber, Proc. N atl. Acad. Sci. USA 92: 7381 (1995) and its description of region / domain descriptions. See references cited here). Chimeras were wild-type Syk and ZAP-7. Generated using overlap PCR using 0.   The intermediate sequence of Syk kinase is involved in signaling events, including those involved in phagocytosis. Confirmation that such transduction events are selectively occurring. Script to test compounds (peptides or mimetics) by their ability to inhibit Cleaning (sieving).   For example, a test compound may be administered in the Syk intermediate region or at least 3, 5, 7, or Or larger amino acids, e.g., at least 20, 30, 50, or 100 amino acids. A portion of the Syk intermediate region consisting of succinic acid (eg, ZAP-70SH2 domain and kinase A chimera comprising a domain and a Syk intermediate sequence), and ZAP- 70 intermediate region sequences (eg, SykSH2 domain and kinase domain and ZAP-7 0 intermediate region sequence). The former, not the latter Compounds that bind to a polypeptide of the invention are signal mediated by the Syk intermediate sequence. Ring events (phagocytosis and mast cells and Fc_εFrom the cells that carry the receptor (Including mediator secretion). like this The compound may also be a cell that appears to be a Syk-deficient phagocytic cell (eg, Cells that carry the Fcγ receptor, such as COS cells, which carry the Syk intermediate sequence Construct encoding a polypeptide (e.g., encoding a chimera as described above) Construct), contacting the cells with a test compound to effect phagocytosis of the cells Phagocytosis by signaling with Syk kinase Is decrypted. Compounds that inhibit phagocytosis are mediated by the Syk intermediate region sequence. It is expected to inhibit other signaling events. So, Syk intermediate area Compounds that inhibit the phagocytic ability of cells expressing It may be tested according to the procedure. This approach also involves mast cells or other Fc_εReceptor-bearing cells, mediator secretion (e.g., histamine secretion) Also used to screen for compounds (peptides and mimetics) that inhibit DNA it can.   Identified using the above screen or identified by other methods Peptides and mimetics can be formulated as pharmaceutical compositions, for example, systemically or directly. It may be administered to the lungs (eg, by aerosol). Delivery is described here. It can be performed using the techniques described. Optimal dosages can be readily determined. (Eg, a second SH2 domain and a catalytic (kinase) domain (eg, purified form or Is in isolated form)) the Sky intermediate sequence between), or at least 5 or Part of the six amino acids or a mimetic thereof is within the scope of the present application, Can be used as described above.   As described below, the present invention relates to the Fc of mast cells and the like (see Example VI)._εReceptor -To inhibit the secretion of mediators (eg histamine) from cells carrying them The use of Syk antisense constructs is also contemplated. Histamine (mast cell Ideta) Inhibition of secretion has therapeutic significance, for example, in the treatment of asthma. Preferred targets for the Syk antisense construct are described below (Examples V and V I). The construct can be administered systemically or directly to the lung (eg, by aerosol administration). Can be given. Delivery can be performed using the techniques described herein (liposomes). Including formulation). Optimal dosage depends on patient, construct and use Will depend on the mode of administration.Soluble Fc receptor   In a further aspect, the invention relates to the use of an immune complex (e.g., an IgG-containing immune complex) with a membrane association. By inhibiting the interaction between soluble Fc receptors, their phagocytosis To cause clearance of coalescence (or to cause secretion of mediators in the cell) (Fc receptor-mediated signaling). This way Competes with the membrane-bound form of the Fc receptor for immune complex binding. Introducing a soluble form of the Fc receptor into the circulation is involved. In a soluble form Transcripts are identified in megakaryocytes and cells of the monocyte / spinal cord lineage (Rappaport et al., Exp. Hemotol. 21: 689 (1993); Warmerdam et al. Exp. Med. 172: 19 (1990)). These transcripts Lacks the sequence encoding the transmembrane receptor region, but The sequence encoding the quality domain is retained. The present invention provides an extracellular domain alone And use of soluble Fc receptors containing combinations with cytoplasmic domains Intended. Competes with membrane-bound Fc receptor for binding to cells coated with IgG A suitable receptor is suitable.   The soluble receptor of the present invention may be an FcγRI, FcγRII or FcγRIII cell Outside Can take the form of domains alone or their binding moieties (or extracellular Fc in the form of domains alone or their binding parts_εRI soluble receptor Is provided). As noted above, cytoplasmic domains or parts thereof Minutes may be present. The following are examples of possible soluble receptors. Where "I "" And "IIA" correspond to FcγRI and FcRγIIA, respectively. c Corresponds to α and γ chains of γ RIII. The first symbol is the origin of the extracellular domain And the second symbol indicates the origin of the cytoplasmic domain: I: I, I, I IA, IIA: IIA, I: IIA, α: γ, α, α: IIA, I: γ.   Soluble receptors may be chemically or recombinantly engineered, depending on their properties. (Horton et al., Biotechniques 8: 528 (19 90)). Soluble receptors are described above with respect to receptor phosphorylation inhibitors. As such, it can be administered systemically or pulmonary. Code the same thing When the synthesis of soluble receptors from existing sequences is performed in vivo, such The sequence is inserted into a suitable vector (see above) and functionally regulated in the target cell. You can link to an array.Antisense construct:   In a further aspect, the invention relates to the expression of an Fc receptor in a mammalian host cell. A method for inhibiting expression, comprising: i) a promoter functional in cells; ii) a double-stranded DN. Fragment of A (the transcribed single strand of which is the endogenous Fc receptor whose expression is to be inhibited) Iii) a termination sequence that is functional in the host cell. Comprising an antisense construct comprising in the direction of transcription from the 5 'end to the 3' end An antisense construct is introduced into said cell. This aspect of the present invention Depending on the specific Fc receptor in cells that produce many classes of receptors. It becomes possible to regulate the expression of the puter. This specificity is due to the intrinsic Fc receptor A sequence specific to the mRNA of interest for inclusion in the DNA segment ((ii) above). It is achieved by doing.   As indicated above, the present invention also targets sequences encoding Syk kinase. Antisense constructs. In such a construct, (ii) Is a segment of double-stranded DNA, and the transcribed single strand contains Syk kinase And a sequence complementary to the endogenous mRNA.   Factors affecting the effectiveness of antisense oligonucleotides include antisense oligonucleotides. Stability of oligonucleotides, delivery of cells to cytoplasm and purpose Ease of access to the mRNA. Syk antisense oligo of the present invention Nucleotides are changed in three steps to achieve these tasks. First, 5 The phosphodiester linkage at the 'or 3' end, preferably at both ends, is for example , Modified with phosphorothioates. Second, using a stem-loop structure, Protects the antisense sequence in the loop domain from creatase. The stem is , For example, a complementary termination sequence consisting only of G and C. Loop domain is an example For example, it carries three antisense sequences targeting different sites of Syk mRNA. You. mRNA forms a secondary structure by intramolecular hybridization, Secondary structure of NA prevents access of antisense oligonucleotides to target Maybe. Opening for easy access to antisense oligonucleotides To identify sequences with an “open” structure, the entire Syk mRNA sequence must have an RNA secondary structure predictor. Scanned by the measurement program. Syk mRNA is in three staggered frames (staggered  frame) and is the most open (“open ") The structure was chosen. The loop antisense oligonucleotide has three lines Shape antisense. Dramatically reduced phagocytic signal than mixtures of oligonucleotides Was. The high efficiency of stem-loop Syk antisense oligonucleotides This may be due to good stability from nuclease digestion. No. Third, Syk antisense oligonucleotides also improve cell delivery For example, it was complexed with a cationic liposome. Complexed with liposomes The stability of the stem-loop Syk antisense oligonucleotide was significantly altered Good. Fc γ RIIIA Stem-loop antiserum directed to γ subunit mRNA Sense oligonucleotides were also used. Peripheral blood mononuclear cells and stem loop Assessed by RT-PCR with the use of gamma chain antisense oligonucleotides. The mononuclear gamma chain message was reduced by> 80%. Liposomes are mononuclear cells / mac Lophage is delivered to the reticuloendothelial cell line, the main resident cell population. Lipo With thesome The complex of stem-loop Syk antisense oligonucleotides is a mononuclear cell / mac Down-regulation of Fc gamma receptor-mediated effects in lophages is required Useful as a therapeutic for immune diseases. Syk kinase also_ε It is linked to RI and B cell antigen receptors. Stemloop Syk Antisen Oligonucleotides also activate intracellular signalling through these receptors. Investigating signaling events and modulating signals mediated by these receptors It is useful for developing therapeutic agents.   According to an antisense embodiment of the present application, a sequence complementary to an endogenous mRNA target Is at least 15 nucleotides in length, preferably at least 30 nucleobases And more preferably at least 50 nucleotides. This array is Typically less than 5000 nucleotides in length, preferably less than 2000, and more desirably Is less than 1000 in length. This sequence is the transcribed region of the mRNA of interest. Or can be complementary to an untranscribed region (e.g., McKenzie et al.) , Molec. Immunol. 29: 1165 (1992), Mastuda et al, Mol. Biol. Cell 7: in pr ess, July (1996)). The length of the antisense sequence is The binding site of the mRNA depends on the nature of the antisense sequence, the mRNA site, May vary depending on the degree of inhibition. Optimization of these parameters is superfluous ( undue) It can be performed without experiment.   Suitable regulatory sequences and vectors can be selected from those known in the art. . For example, administration of an antisense construct to the lungs and spleen can be administered in vivo and It can be performed using transformation procedures both outside. Antisense transcript itself Directly into target cells using methods known in the art, including those described above. What can be done will be appreciated by those skilled in the art (Example 5-Modification of phosphorothiolate). Decorated Linear, Stem Loop Syk Antisense Oligonucleotide (ODNs) show partial resistance to serum nucleases-see also When). When complexed by liposome, phosphorothiole The antisense ODNs modified with the salt are more stable. Phosphoroty Olate-modified stem-loop Syk antisense ODNs are highly resistant in serum. Shows stability.   In addition to the above approach of inhibiting phagocytosis, the present invention provides FcγRIIA (Hunter e t al, FASEB J. June 1996, New Orleans, LA) Fc γ RIIB (e.g., Fc γ  It is also involved in the method of performing inhibition by introducing cells having RIIB2). The introduction of FcγRIIB is dependent on the sequence encoding FcγIIB or its Transfection / transformation of target cells with a partially encompassing construct (Transfecting / transforming) (Brooks et al. et al). Exp. Med. 170: 1369 (1989); Indik et al., Blood 83. : 2072 (1994)). Suitable constructs can be selected by those skilled in the art.   The following examples further illustrate certain aspects of the present invention, but are not limiting. is not. Example 1: Production of recombinant soluble FcγRIII   For the recombinant soluble FcγRIII protein, an expression vector as shown below is used. Can be produced using The soluble protein is a transmembrane protein. This corresponds to FcγRIII without the main. This construct encodes the receptor The sequence is introduced into mammalian cells under such conditions that expression of the sequence occurs. like this The recombinant protein produced in this manner was isolated from both the cell lysate and the supernatant. Separated.Transfection of adherent cells or cells in suspension:   Adherent cells (eg, CHO or COS cells) or a suitable suspension cell line Transfection can be performed. Express soluble form of Fcγ receptor Permanent transfectants that develop Achieved by filtration, calcium phosphate method or other established methods Can be done. Transfected cells were cultured for 48 hours and 2 mg / ml Geneticin (Gen eticin) (Gibco BRL, Gaithersburg, Maryland) or media containing other selective agents Can be selected in After about 12 weeks, positive colonies were isolated and cloned. Propagated for further characterization. The isolated clone is a soluble receptor To select the transfectant cell line that expresses the most Solid-phase immunoassay for enzymes using ELISA plates (Dynatech, Alexandria, Virginia) Checked by ELISA. Mass culture of adherent transfectants Is achieved by using a hollow fiber tissue culture system. Example 2: Function of soluble FcγRIII   The function of the soluble FcγRIII protein is evaluated both in vitro and in vivo. Soluble Fc receptor on binding of IgG immune complexes to cell membrane-bound receptors Influences local concentrations of ligands and soluble receptors, a table of membrane-bound receptors Areal density, valency of the ligand, and ligands for the two forms of receptor It depends on several factors, such as relative affinity. With soluble FcγRIII receptor Limiting factors in the interaction with ligands and cell membranes will make available model systems Can be used to decipher it.   The in vitro assay system consists of a labeled IgG ligand and an IgG-coated red Based on competition between soluble and cell membrane receptors for blood cells (EA) . Cells without Fc γ receptors (Fc γ receptor-negative cells) Retains functional ability to bind and take up cells containing immune complexes and antibodies Transmembrane FcγRIII molecules have been transfected Ruiz and Schreiber, J. et al. Clin. Invest 88: 149 (1991)). these Using the assay, the function of the soluble receptor and the The ability to interfere with the detection of oligomers of EA and IgG by a protein is examined. soluble The function of sex FcγRIII can also be examined in vivo. In these studies, Using an established experimental animal model, soluble FcγRIII administered in vivo It is examined whether it changes the clearance of cells covered by the body (see Ruiz and Schreiber, J. Clin. Invest 88: 149 (1991)) . The immunomodulatory capacity of soluble FcγRIII is examined in a similar manner. Example 3: Cytoplasmic tyrosine residues required for phagocytic signal mediationExperimental procedure:   Plasmid construction and introduction of point mutations:   pSVL eukaryotic expression vector (Pharmacia LKB, Piscataway, NJ) is used in COS-1 cells FcγRIIIA was used for expression. huFc γ RIIIA α cDNA is derived from pSVL XbaI and It was cloned into the BamHI cloning site. Similarly, muFc γ RIIIA γ cDNA Was cloned into the XhoI and BamHI cloning sites. TCR / Fc γ RIIIA ζ was cloned into the XbaI and BamHI cloning sites of pSVL. gamma chain Conservative substitution of cytoplasmic tyrosine with phenylalanine is a two-step overlapping extension polymer. Rase chain reaction (PCR) (Horton et al, Biotechniques 8: 528 (1990)). The double tyrosine sub mutant Institution mutants) were replaced with N-terminal tyrosine residues followed by C-terminal tyrosine residues. Constructed sequentially by substituting groups. 6 from each mutant Clones were isolated and subjected to DNA sequencing. The correct DNA sequence Of several clones, each with tyrosine substitutions for further study Two clones from (body) were randomly selected.   Transient transfection:   FcγRIIIA isoform, FcγRIIIA-γγ, FcγRIIIAζζ Cotransfection of 1 cell with γ or c cDNA and α cDNA (cotran sfection). Transfection of cDNA is improved DEAE -Performed using the dextran (DEAE-Dextran) method. That is, 300,000 COS- One cell was seeded in a 35 mm well plate 24 hours before transfection. 7 0-80% confluence plates are washed twice, prior to transfection. Because Dulbecco's Modified of Eagles's Medi um) (DMEM, Gibco BRL, Grand Island, NY) for 30 minutes. 4 μg of plasmid DNA (0.5 μg / μl) in Nu medium (10% Nu serum [Collaborative Biomedi cal, Two oak Park, Bedford, MA], 1 mg / ml DEAE dextran and 100 μM 1 ml of transfection buffer containing DMEM with Roquine (Chloroquine) Was added slowly. Transfection buffer containing DNA in COS-1 cells And incubated for 4 hours at 37 ° C. Cells are then shocked with 10% DMSO in phosphate buffered saline (PBS) for 2 minutes. They were then washed twice with DMEM and grown in DMEM supplemented with Nu serum. Cells They were examined 48 hours after transfection.   Immunofluorescence staining and flow cytometry:   Transfected cells were stained with a transfer pipette using staining buffer (0.02% nitric acid). (PBS containing sodium chloride and 0.1% BSA). Cells are centrifuged and 60 Resuspended in μl staining buffer, anti-FcγRIIIA mAb, 3G8 (Ankeres et al. eless et al), Annu. Rev. Immunol. 6: 251 (1988)) or an isotype control (isoty pe control) for 30 minutes at 4 ° C. Wash cells Goat anti-mouse IgG purified and conjugated with fluorescein (Tago Inc. Burlingame, (CA). Stained cells are from FACstar flow cytometer. Measured using a tomometer (Becton Dickinson Co., Mountain View, CA) .   Binding and phagocytosis of IgG-sensitized RBCs (EA):   Sterilized sheep red blood cells in PBS without calcium and magnesium (109 / ml) Is a rabbit anti-sheep RBS antibody (Cappel L) equivalent to the subagglutinating titer aboratories, Cochranville, PA) Sexualized. IgG-sensitized RBCs (EA) are washed twice with PBS, and Final concentration to overlay the affected COS-1 cells.⁹/ ml Was resuspended. Cells were expressed as previously shown (Indik et al., J. Clin. Invest. 88: A66 (1991)), setting (> 10EA per COS-1 cell) Phagocytosis was examined. For analysis of phagocytosis, COS-1 cells bound to EA (3 Short wash) with hypotonic PBS to remove EA bound to the surface. A hypotonic shock (35 seconds) was given. The cells are then stained with a light-Giemsa staining solution. And phagocytosis (EA ingested) was examined under a light microscope. Result obtained Was analyzed by Student's T-test.   In vitro kinase analysis:   Transfected cells (2x10⁷Cells) are washed once with PBS, 5 μg / ml 10 min on ice with anti-FcγRIIIA mAb and goat anti-mouse IgG And incubated. Cells are washed once with PBS and phosphatase and plate Lysis buffer containing a protease inhibitor (150 mM sodium chloride, 25 mM Hepes [pH 7.4] and 1% polyoxyethylene 10 oleyl ether [BRIJ-96; Sigma, St. Louis MO ]) Was incubated at room temperature for 3 minutes before adding 1.5 ml. Phosphata Protease and protease inhibitors (1 mM EGTA, 1 mM sodium orthovanadate (Naorth ovanadate), 1 mM PMSF, 10 μg / ml aprotinin, 50 μg / ml leupeptin and 10 0 μg / ml soybean trypsin inhibitor) was added fresh to the lysis buffer. 15 minutes on ice After lysis (reaction), the cell lysate is centrifuged at 4 ° C for 30 minutes to clean Was done. FcγRIIIA-γ chain is used as anti-human γ antiserum (Gene-Pierre) in lysis buffer. Kinet (Jean-Pierre Kinet), NIAID-NIH, Rockville, MD) and protein Immunoprecipitated using A-Sepharose CL4B (Sigma, St. Louis, MO). Pele The wells are washed three times in lysis buffer and mixed with low salt buffer (100 mM sodium chloride, 25 mM (Pes, pH 7.4 and 5 mM manganese chloride). Pellet is 25 mM Hepes, p H7.4, 5 mM manganese chloride, 5 mM p-nitrophenyl phosphate, 1 μM cold ATP (Boe hringer Mannheim, Indianapolis, IN) and 5μCi γ- [32p] ATP (6000 Ci or Ink with 30 μl of the mixture containing 222 TBq / mmol; Dupont NEN, Boston, MA) (20 ° C, 10 minutes). Add reducing SDS-PAGE sample buffer This stops the reaction and the labeled protein is reduced to 12.5% cing) Separated on SDS-PAGE gel. The gel is fixed in a methanol / acetic acid solution. Treated with 1N potassium hydroxide to remove phosphoserine and threonine. (2 hours at 55 ° C.), dried and autoradiographed for 4 days.   [Ca²⁺] iMobilization:   COS-1 cells placed on a cover glass were 2 μM Fura-2 / AM (Calbiochem. San Di ego, CA) for 30 minutes and washed twice. After that, Bargrass was used for multiple single-cell measurements of [Ca2 +] i. measurements) for the Leidem cell chamber (Medical S ystems, Greenville, NY). FcγRIIIA receptor is biotinylated Streptavidin was added with anti-FcγRIII Alternatively, it was cross-linked with anti-FcγRIII mAB 3G8 whole IgG. 10 as positive control μM epinephrine crosslinks epinephrine receptor expressed on COS cells Added to remove Calcium imaging was performed using the Image 1AT quantitative fluorescence system. Nikon Diaphoto using stem (Universal Imaging, West Chester, PA) Use a 4Ox fluorescent objective on a microscope (Nikon Diaphot microscope). Was done. Images were obtained with either 340 nm or 380 nm excitation (emission ) = 510 nm). The 340/380 ratio image is calculated based on (the size of) 1 pixel x 1 pixel, The average 340/380 ratio in each cell was determined each hour. 340/380 ratio is play [Ca based on solution correction using isolated Fura-2 acid²⁺] i was converted.Fc Phagocytosis mediated by γ RIIIAα and associated γ and ζ chains   The wild-type γ and ζ cDNAs of Fc γ RIIIA are transferred to COS-1 cells together with the Fc γ RIIIA α chain. Measures the ability of EA (sensitized RBC) to induce phagocytosis when co-transfected Was done. The surface expression of FcγRIIIA is determined by flow cytometry, The effect was the same when co-transfection was performed in any of (1) and (2) (Table 1). Mean fluorescence intensity for co-transfected cells stained with anti-FcγRIIIAmAB Degree (EMI) is somewhere in comparison to cells stained with the IgG isotype control Compared to mock transfected cells stained with anti-γ RIIIA mAB. By a factor of two (Table 1). Transfectants have linked IgG sensitized RBCs (EA) And were examined for their ability to exert phagocytosis. About 50% COS-1 transfector Phagocytosed EA (Table 1). Transfected by wild-type gamma In microscopic observation of the isolated COS-1 cells, the uptake of EA was consistently It showed 0 ± 5% (p <0.02). Thus, phagocytosis of EA is dependent on COS-1 cells bound to EA. It was detected in about 40% of the vesicle transfectants. In contrast, those containing 含 む chains In transfectants, 3.8% of the cells had EA incorporated (p <0. 02) (Table 1). Furthermore, in ζ-containing cells that showed phagocytosis, Average number of incorporated EAs decreased in less than half of the levels observed for γ Was. In COS-1 cells in which all three cDNAs, α, γ and ζ, were transfected, Show that cells that take up EA show 16%, consistently attenuating phagocytosis (Table 1). In contrast, a pseudotransfectant carrying EA was also identified as E (non-sensitized R). Transfectants carrying BC) also showed no binding or phagocytosis.   Transfection efficiency was determined by flow cytometry. Average fluorescence intensity ( MFI) showed for one of three experiments that showed similar results. Internalized The PBCs were observed under a microscope (1000x). The results indicate the phagocytosis and binding of EA (b) (Zet formation) is shown as the average SEM. At least for each clone Three separate experiments were performed. 5 randomly selected 1500 cells for each experiment It was measured at the site where it was performed. * Average fluorescence intensity.^§-PI (phagocytosis index): COS-1 fine Number of internalized RBCs per 100 cells.Two gamma-chain cytoplasmic tyrosines are required for phagocytosis   Study the effect of two conserved γ-chain tyrosine on FcγRIIIA-mediated phagocytosis Therefore, the N-terminus (clone M1A and M1B) or near the C-terminus (clone M2A and M1A) And M2B) nearby tyrosine were each replaced with phenylalanine. 2-tyrosine substitution For variants with, both tyrosines were replaced with phenylalanine (DMA and Yo And DMB) (Fig. 1).   MFI and rosette formation positive cells measured by flow cytometry. All transfectants carrying γ mutant and wild-type γ Surface expression of the receptor complex was demonstrated to be similar in Tanto (Table 2). Comparing these levels of expression, tyrosine residues in the cytoplasmic tail of the gamma chain Is not required for the formation of the FcγRIIIA receptor complex required for surface expression. Do you get it. The results summarized in Table 2 are as follows. M1γ mutants have phagocytic fingers Showed a decrease in phagocytic activity of more than 99% as represented by the Transfectants carrying EA and minimal EA per cell <1% of Tanto) (p <0.02). Mutants of M2 and DMγ are substantially phagocytic (1 out of 5000 cells examined) (Table 2, FIG. 2) Inhibition of phagocytosis by tyrphostin 23   To investigate whether phagocytosis requires phosphorylation of tyrosine residues, Fcγ COS-1 cells cotransfected with RIIIA and wild-type gamma were ostin) 23 (tyr23), that is, increasing the concentration of tyrosine kinase inhibitor Incubation was performed (Yaish et al, Science 242: 933 (1988)). Tyr23, dosage Phagocytosis, with 50% inhibition at 25 μM and complete inhibition at 200-400 μM Inhibition was shown (p <0.01) (Table 3). In contrast, tyr23 does not affect EA binding Was. Transfectant diet washed after pretreatment with tyr23 (400 μM) Inhibition of phagocytosis reduces survival, as the activity of the drug has been partially or completely restored. It was not related to small numbers. (Washed overnight, no data displayed). Tyrosine residue of γ subunit is phosphorylated in vitro   Potential phosphorylation of gamma-chain tyrosine residues with COS-1 transfectants It was determined by the in vitro kinase assay used. The results shown in Table 4 indicate that the wild-type γ chain Has been demonstrated to be phosphorylated in vitro. In contrast, the mutant γ-chain transfectants and pseudo-transfectants Showed no oxidation. For single tyrosine substitution mutants (M1A and M2A), Since no phosphorylation occurred at the rosin residue, either of the two tyrosine residues was Apparently one phosphorylation appears to require the completion of another tyrosine residue (Table 3). These phosphorylation data show the substitution of one of the two tyrosine residues. Induces a phagocytosis signal because phagocytosis is greatly cleared by the exchange It correlates well with the ability of the γ chain (Table 2, FIG. 2).   For in vitro kinase analysis, all lanes except for the mock transfectants Showed a clear band of about 40 kDa. This band is bound phosphate that co-precipitates with γ May indicate protein.γ cytoplasmic tyrosine is required for Ca ²⁺ mobilization   To determine if γ-chain tyrosine is required for calcium mobilization, Transfected cells (WT, M1A, M2A, or DMA) using a digital video microscope. Calcium response was measured after FcγRIIIA crosslinking (Table 4). In COS cells Ca²⁺Epinephrine, which elicits a signal, serves as a positive control in all experiments. Used. Transfectants with the WT receptor complex are biotin After cross-linking with stabilized anti-FcγmRIII and adding streptavidin, or Anti-FcγRIII showed typical transient calcium rise after cross-linking with whole IgG . In 5 consecutive experiments (169 cells), 58% of cells were induced with 10 μM epinephrine The anti-FcγmRIII by at least 50% calcium signal Responded (Table 4, FIG. 4). In contrast, traffic with either M1A, M2A, or DMA Transfected COS cells markedly increase the calcium response to anti-FcγRIII Although reduced, in one of four experiments, M1A transfected COS-1 cells Marked calcium mobilization occurred. Example IV:                Macrophage FC γ RIII signaling                Induces protein tyrosine kinase activation   Certain tyrosine residues within the intracellular FC γ RIII γ subunit What is required for subsequent effector action is that chimeric or mutant receptors Confirmed using transfected NK cells and lymphocytes or fibroblasts (Darby et al. Blood 79: 352A Nov (1992)). Macrophage signaling Contains protein-related tyrosine kinases (PTK) and phosphotyrosine Lung macrophages or cultured mononuclear cells (M) to study The above natural FcγRIII was examined. After Fc γ RIII cross-links with Fab antibody Within a few seconds, Western blots reveal a characteristic pattern of phosphotyrosine substrates. Appeared. This response is transient, peaking at 5 minutes for many substrates, and 10-20 minutes later. Decrease. Phosphotyrosine patterns are new macrophages and cultured Mononuclear cells could not be distinguished. This makes the latter a useful in vitro model Proven. It is not GAP itself but p120^rasA protein that binds to GAP P62 is identified as one of these phosphotyrosine substrates by specific immunoprecipitation. It has been certified. The second substrate is the hematopoietic carcinogenic product p95^vavAnd p95^vavIs , TCR, slg and Fc_εWe know that it is also tyrosine phosphorylated after RI activation Was. The kinase PTK72 / Syk has been used to date for B cell Slg and mast cell Fc_εRI Signal N However, the primary role of macrophage FcγRIII after activation was It is also a phosphotyrosine substrate. In vitro kinase analysis of anti-Syk immune complexes Syk autophosphorylation showed a 3-4 fold increase 5-10 minutes after scepter ligation. Sky is It has also been discovered that γ-chain FcγRIIIA is present in immunoprecipitates. this This suggests that Sky is linked to the phosphorylated γ chain. Example V: Antisense oligonucleotides   Two antisense oligonucleotides (ODNs) for human Syk mRNA Was done. A linear antisense ODN targeting the region surrounding the transcription initiation codon used. Other antisense ODNs hybridize with three different sites in human Sky mRNA. Designed to have a stem loop structure that can be rediscovered. These Sky Apps Antisense ODNs inhibit Fcγ receptor-mediated phagocytosis in cultured mononuclear cells. It was used to investigate the effect of Syk tyrosine kinase in the application signal.Construction of antisense oligodeoxynucleotides:   Antisense or cross-over (scramble) ODNs protect this from nucleases. Qualified to protect. One phosphodiester backbone at the 5 'end The backbone of two phosphodiesters at the 3 'end is And modified using Secondary structure of Syk mRNA and ODNs (Law et al. J. Biol. Che m. 269: 12310 (1994)) is based on MacD on a Macintosh computer. Implemented using NASIS program (Hitachi, Software, San Bruno, CA) . 5'-CGCTGTCAGCCATGCCG-3 'Linear 17mer Syk antisense SODN targets the region surrounding the transcription start codon of Syk mRNA. Stemle Antisense ODN contains three different target sites for Syk mRNA: target I (transcription initiation region, nucleotide numbers 159 to 173), target II (4 51-463), and complementary to target III (802-816) (Law et al. J. Biol. Chem. 269: 12310 (1994)). Table 5). Stem-loop Syk antisense ODNs are themselves stalks and stems. Loop) to form a structure and include the smallest intramolecular secondary structure in the loop domain (Figure 6). Stem loop Syk antisense ODN is 5'-GGG GGGGCTGTCAGCCATGCCGTGTCTTGTCTTTGTCGCTTCTTGAGGAGCCCCCCC-3 ′. linear 17mer Syk antisense ODN has an arbitrary sequence 5'-GCCCAAGATGATTCCAG-3 ' One. The stem loop 61mer control ODN contains any sequence 5 in the loop domain. '-ATGGAATCATCTTGGGCATTCATTCGTTCCTCAAAGAAGAATATGAA-3'. The linear And the stem-loop control ODN also have phosphorylation at the 5 'and 3' ends. Oet qualified.Preparation of liposomes   Control scrambled phosphodiester ODNs, linear and stem loop 1 μg of each Syk antisense ODNs was added to 4 μg (2 μl) in 50 μl of PBS. Lipofectamine (LIPOFECTAMINE)^TM(GIBCO BRL, Life Technologies Inc. Gai thersburg, MD). The ODN liposome complex was added at room temperature for 4 hours. Formed in 5 minutes.Mononuclear cell isolation and culture:   Peripheral blood mononuclear cells from healthy individuals are collected using standard adherence pro cedure) (Darby et al. J. Immunol. 152; 5429 (1994)). You In other words, heparinized blood was subjected to the Ficoll-Hypaque method (Lymphocyte Separation Med). ium; Organon Teknika, Durham, NC), and wash the interface cells twice with PBS. Was cleaned. Mononuclear cells were RP with 10% heat inactivated FCS and 2 mM L-glutamine. In complete medium containing MI1640 (GIBCO, Life Technologies Inc. Gaithersburg, MD) Was resuspended. Cells are plated at 37 ° C. on tissue culture flasks previously coated with FCS. Attached. After 45-90 minutes, non-adherent cells are removed by a crude wash in HBSS did. Cells were harvested by vigorous shaking. The yield of monocytes is 2-6x 10⁷Cells / 500 ml blood. Monocytes are always trypan blue clear orchid More than 98% of the animals were alive as determined by the method. Isolated mononuclear cells Shows RPMI supplemented with L-glutamine (2 mM) and 10% heat inactivated FCS. 37 degrees 5% CO in 1640_TwoHeld in.Oligodeoxynucleotide treatment of cells   1x10^FiveMononuclear cells were treated with 2 μg / ml lipofectamine, 0.5 μM linear control. 0.5 μM linear Syk antisense ODN or 0.1 μM stem loop controller Roll, ODN-liposome complex containing 0.1 μM stem-loop Syk antisense ODN With 24 well plates (Falcon; Becton Dickinson Labware, Lincoln Pa.) (rk, NJ) in a FCS-free 0.3 ml RPMT1640 medium at 37 ° C. for 4 hours. same A quantity of the ODN liposome mixture was also added to each well on the second day. Medium 10% The cells were added to a final volume of 1 ml with RPMI1640 containing FCS and the cells were incubated at 37 ° C. Incubated for 2 days. This gives 1x10^FiveFor mononuclear cells, 4 μg / ml Pofectamine and 1.0 μM linear control, 1.0 μM linear Syk en Chicken ODN or 0.2 μM stem loop control or 0.2 μM Sky Incubate for 2 days with ODN liposome complex containing Muroop Antisense ODN You have been beatted.Preparation of IgG sensitized red blood cells (EA):   1x10⁹  Sheep red blood cells (RBCs) / ml (Rockland Inc., Gilbertville, P. A) with the same amount of rabbit anti-sheep RBC antibody (Cappel Laboratories, West Chest er PA) at 37 ° C for 30 minutes at the highest subagglutinating concentration I was sensitized. The IgG-sensitized RBCs are washed twice and in PBS as described above. Final concentration 1x10⁹RBCs / ml (Schreiber et al, J. Clin. Invest. 56: 1 18981975)).IgG Phagocytosis of sensitized RBCs (EA):   Mononuclear cells treated with antisense ODNs were incubated at 37 ° C for 30 minutes with EA (100 ratio of EA to mononuclear cells : 1). Briefly contact cells with hypotonic solution PBS and attach The sex EA was removed. Next, the cells were stained with Light Giemsa, and phagocytosed RB Cs was measured with a microscope (x1000). 100 monocytes randomly selected and internalized EA was expressed as an indicator of phagocytosis.Flow cytometry analysis:   Mononuclear cells were treated with anti-FcγRI (32.2) Indik et al, Blood 83: 2072 (1994)) or anti-Fc  γ RII (IV.3) (Indik et al, Blood 83: 2072 (1994)) or anti-Fc γ RIII (3G8) moAb (Park et al, J. Clin. Invest. 92: 1967 (1993)), Park et al. Clin. Invest. 92: 2073 (1993)) at 30 ° C. for 30 minutes. Calcium-free magnesium containing albumin (BSA) and 0.02% sodium nitride Washed twice with PBS and FITC-conjugated goat anti-mouse F (ab ') 2 IgG (Tago (Bullingamne, CA) at 4 ° C. for 30 minutes. The cells are then washed and 1% paraffin Fixed with formaldehyde. Fluorescence was measured using FACSar (Becton Dickinson, Mountain View , CA) and average fluorescence intensity data and summary plots (Consort) 30 Obtained using software. 10,000 events are logarithmic for all samples Recorded on light scale.Reverse transcribed polymerase chain reaction (RT-PCR):   Total RNA treated with scrambled control and Syk antisense ODNs From isolated monocytes. The cDNA is ligated to any 6 nucleotide primer (Boehri nger Mannheim, Indianapolis, IN). PCR Using the synthesized cDNA as a template, the following two primers:       Nucleotide of Syk mRNA (Law et al, J. Biol. Chem. 269: 12310 (1994)) Syk-H primers corresponding to numbers 122 to 139:             5′-GGTGTGTGCCCTCCGGCC-3 ′, and       Syk-M primer:             5'-CTGCAGGTTCCATGT-3 '(nucleotide number 550-564) Was performed using The PCR product is obtained by Southern hybridization. Was analyzed.Southern Hybridization   RT-PCR products were electrophoresed on a 1.5% agarose gel. DNA is Niro Implanted on membranes (NEN Research Products, Boston, MA). The implanted membrane , 6X SSPE and 50% formamide in a biotinylated internal probe (Sy k-pS: high at 5'-GGGAGTGGTAGTGGCAGAGG-3 ', nucleotide numbers 408-427) It was bridged. After washing the membrane in 0.1 × SSC at 50 ° C., it was hybridized. Band is a chemifluorescence detection reagent (PROTOGENE^TM, Nucleic Acid Detection System , GIBCO BRL Life Technologies Inc. Gaithersburg, MD).result   Mononuclear cells incubated with linear Syk antisense ODN (1 μM) There was a reduction in the level of phagocytosis. Phagocytosis is calculated from the phagocytosis index (PI, 220 ± 8.8) To 113 ± 12.3), a decrease of 49%. Mononuclear stem loop Syk Incubation with antisense ODN (0.2 μM) increased phagocytosis to 89% Was also reduced (PI, from 220 ± 8.8 to 24 ± 4.2) (FIG. 7). Scrambled Kong Both trawl ODNs and linear or stem-loop (0.2 μM) ODNs are available for FcγRIIA It did not seriously affect mediated phagocytosis. Similarly, even with liposomes alone, It did not reduce phagocytosis mediated by FcγRIIA. Expression of FcγRIIA As measured by flow cytometry analysis on cultured mononuclear cells, It did not change with any treatment. This result shows that Syk tyrosine quina Is a major signal for FcγRIIA-mediated phagocytosis in monocytes. Transducer has been shown to be And the interaction between FcγRIIA and Syk kinase (as well as tyrosine phosphorylation) Was shown to be important.   Next, is reduced phagocytosis in monocytes correlated with Syk mRNA levels? Was decided. Quantitative RT-PCR (reverse transcribed polymerase chain reaction) ) Was used to determine cytoplasmic levels of Syk mRNA in monocytes. Syk total RNA Mononuclear cells treated with antisense ODNs (1x10^FiveCells / ml) and the first strand Used to synthesize cDNA. As shown in FIG. 8, the linear Syk antisense ODN (1.0 μM) substantially reduced Syk mRMA. Stem loop Syk antisense Syk mRMA (FIG. 8) was completely removed by ODN (0.2 μM). In contrast, Two scrambled control ODNs (1.0 μM linear control, Or 0.2 μM stem-loop control) and Syk mRMA alone Did not decrease. These results show that Syk antisense ODNs have a linear Both mulops can degrade Syk mRMA in mononuclear cells with sequence-specific manners. I knew it would work. Furthermore, stem-loop Syk antisense ODN targets mRNA. It was more effective than the linear antisense molecule for getting. Example VI: Stem-loop Syk antisense oligonucleotides (ODNs)               Inhibits histamine secretion   Experimental designCell culture : 17% of RBL-2H3 cells (rat mast cells containing histamine) Fetal calf serum, 100 U penicillin and 100 μg strept per ml 5% at 37 ° C. in minimal essential medium supplemented with mycin and 4 mM glutamine CO_TwoGrew up in. Cells are placed on a 1.6 cm plate or 24 hours prior to analysis. 1x10 on well plate^FiveSeeded at a cell / well concentration.Construction of antisense ODNs : Antisense to protect against nuclease digestion And ODNs are 5 'of the phosphodiester backbone. Repair by adding one phosphorothioate at the end and two at the 2 3 'end. Decorated. Three linear Syk antisense ODNS: target I linear Syk antisense ODNS DN (5'ATTGCCCGCCATCTCT3 ', nucleotide containing the translation initiation codon of Syk mRNA 319 to 333), target II (5'GATTTGATTCTTGAG3 ', nucleotide 1175-11) 89), target III (5'ATTTGGTAGTATCCCT3 ', nucleotides 1465 to 1479) Was designed. Stem-loop Syk antisense ODN has three targets. (See FIG. 9) (see also Example V above).ODN treatment of cells : 5 x 10 before lipofection details^FourAlso Is 1x10^FiveRBL-2H3 cells were seeded in each well of a 24-well plate . ODN liposome complexes were added twice, once on the second day and once on the third day. every time 4 μl of DOTAP (Lipofectamine) (1 μg / μl stock) and 2 Complexes of μl ODN (1 μg / μl) were formed in EMEM (75 μl total volume). ODN liposome complex is added to each well containing 175 μl of serum-free culture medium Was done. The cells were incubated at 37 ° C. for 24 hours. The second ODN liposo Medium complex (75 μl) was added and the culture medium was added to 5% FCS (final volume 1.0 ml). Conditioned and transfected RBL-2H3 cells were incubated at 37 ° C. with histamine Incubated for another day before ligation analysis.Reverse transcribed polymerase chain reaction (RT-PCR):   Total RNA was treated with Syk sense control or Syk antisense ODN Isolated from RBL-2H3. The cDNA is combined with any 6 nucleotide primers and Synthesized from total RNA by oligo (d) T. PCR synthesized cDNA Two primers as rate: rat-5Syk 5'-TTTGGCAACA TCACCCGC-3 '(nucleotide numbers 368-386) and rat-3Sy k primer: 5'-ACTTATGATGGCTTGCTC-3 '(nucleo (748-762). Connect gamma and beta actin primers Used as a troll.Histamine secretion analysis : Histamine secretion analysis was performed using rat RBL- as described below. 2H3 cell IgE receptor Fc_εCrosslink RI and use an enzyme immunoassay kit (immunote ch, France) using histamine secretion. Well 1x10^Five24 well plates containing RBL-2H3 cells in 1.0 ml EMEM Incubated overnight at 37 ° C. The cells are washed once with PBS and 1.0 m on ice. 1 PAGCM (standard histamine secretion buffer) and 100 μl Fc_ε30 minutes with RI antibody For a while. After gently washing once with PBS, RBL-2H3 cells are washed as follows. That is, 1.0 ml of PAGCM buffer alone (negative control), 10 μl of cal 1.0 ml of PAGCM buffer (podium containing 50 μg / ml stock) Active control) or 10 μl of goat anti-mouse antibody (1 mg / ml) Incubate with 1.0 ml of PAGCM for 30 minutes at 37 ° C. PA containing histamine GCM buffer was removed from the cells and analyzed by enzyme analysis. Create a standard curve 100 μl of standard was included for this purpose.   result   The results presented in FIG. 10A show that Syk expression in RBL-2H3 cells Significantly inhibited by the presence of antisense ODN but not by sense ODN The thing is proved. RBL-2H3 cells treated with Syk antisense ODN It does not affect tin expression (FIG. 10B). Similarly, replace RBL-2H3 cells with Syk Treatment with antisense ODN has no effect on gamma chain expression. Fc_εAfter cross-linking the RI and goat anti-mouse antibodies, the stem-loop Syk antisense RBL-2H3 cells treated with SODNs were treated with control DNA-treated control DNA. Secreted histamine as much as 74% less than histamine.                        Anti Fc_εRI and goat anti-mouse antibodies     Histamine secretion of 1.9ng sense DNA     Antisense 0.5ng histamine secretion (74% inhibition)   All documents cited above are incorporated herein by reference. The present invention relates to what is presently considered to be the most practical and preferred embodiment. Although described, it is not intended to limit the invention to the disclosed embodiments. But On the contrary, various modifications and variations are included within the spirit and scope of the appended claims. And equivalent adaptations are intended to be covered.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 48/00 ABG C12P 21/02 C C12N 5/10 C12Q 1/68 A C12P 21/02 C12N 5/00 B C12Q 1/68 A61K 37/02 ACD (72) Inventor Park, Jong-gu, Drexel Hill, Drexel Brook Drive 19013, Pennsylvania, USA 6813

Claims (1)

[Claims] 1. A method for preventing the phagocytosis of an immune complex in a mammal, comprising the step of: providing a kinase endogenous to the cell associated with an Fc receptor present on a membrane of the cell in a phagocyte of the mammal in contact with the immune complex. Wherein the introduction is carried out under conditions such that phagocytic ability is inhibited. 2. 2. The method of claim 1, wherein said immune complex is an IgG-containing immune complex. 3. 2. The method of claim 1, wherein the inhibitor is a peptide or mimetic Deal method. 4. 4. The method according to claim 3, wherein the peptide is introduced directly into the cell. 5. 4. The method of claim 3, wherein said peptide is incorporated into liposomes prior to introduction into said cells. 6. 4. The method of claim 3, wherein the DNA sequence encoding the peptide is introduced into the cell under conditions such that expression of the DNA sequence produces the peptide. 7. The method of claim 3, the peptide, the methods include a sequence corresponding to the tyrosine-containing motif gamma chain of the cytoplasmic domain Fc gamma RIIA or Fc gamma RIIIA or FC _epsilon RI. 8. 4. The method according to claim 3, wherein said peptide comprises the sequence Y-X2-L, wherein X2 is any two amino acids. 9. The method according to claim 8, X2 is a method Fc gamma RIIA or Fc gamma RIIIA properly is showing the amino acids Y-X2-L of the cytoplasmic domain of gamma chain of FC _epsilon RI. 10. A method for preventing the clearance of an immune complex from a mammal, comprising specifically locating a transcript encoding an Fc receptor present on the membrane of the mammal in contact with the immune complex in phagocytes of the mammal. A method comprising introducing a molecule that degrades. 11. 11. The method of claim 10, wherein the immune complex is an IgG-containing immune complex. 12. 11. The method according to claim 10, wherein said molecule is a ribozyme. 13. A method of inhibiting the binding of an immune complex present in a mammal to a membrane-bound Fc receptor, comprising introducing into said mammal a soluble Fc receptor that competes with the membrane-bound Fc receptor for binding to said immune complex. And wherein the introduction is performed under conditions where binding of an immune complex to the membrane-bound Fc receptor is prevented. 14． 14. The method of claim 13, wherein said immune complex is an IgG-containing immune complex. 15. 14. The method of claim 13, wherein the soluble receptor consists essentially of the extracellular domain of an Fcγ receptor or a binding portion thereof. 16. The method of claim 13, said soluble receptor is characterized by consisting of the extracellular domain and the second Fc gamma receptor type from the cytoplasmic domain from a first Fcγ-receptor or Fc _epsilon receptor type, A method wherein at least one of said first and second receptor types is an α or γ chain of FCγRI or FcγRIII. 17． A method for inhibiting the phagocytic ability of a mammalian cell that expresses an Fc receptor, comprising: in the cell, in the direction of transcription from the 5 'end to the 3' end, i) a promoter functional in the cell; A) segments of double-stranded DNA whose transcribed single-strand comprises a sequence complementary to the endogenous mRNA encoding the Fc receptor; iii) a termination sequence functional in the cell. And wherein the construct comprises the complementary strand being transcribed and binding to the endogenous mRNA, thereby reducing expression of the Fc receptor, and enhancing the phagocytic capacity of the cell. A method introduced under conditions that inhibit the activity. 18. 18. The method of claim 17, wherein the sequence that is complementary to the endogenous mRNA is complementary to an untranslated region of the mRNA. 19. A method of inhibiting the phagocytic potential of a mammalian cell that expresses an Fc receptor, wherein the promoter is functional in the cell in the direction of transcription from the 5 'end to the 3' end, ii) A segment of double-stranded DNA whose transcribed single strand contains a sequence complementary to the endogenous mRNA encoding Syk kinase; iii) a termination sequence functional in the cell. Introducing a construct having the formula: wherein the complementary strand is transcribed and binds to the endogenous mRNA, thereby reducing the expression of the Sky kinase and increasing the signaling potential of the cell. A method that is introduced under conditions that are inhibited. 20. 20. The method of claim 19, wherein said Fc receptor is FcγRI, FcγRIIA or FcγRIIIA. 21. A method of inhibiting the phagocytic potential of a mammalian cell that expresses an Fc receptor, comprising introducing into the cell a nucleic acid that is complementary to an endogenous mRNA encoding Syk kinase, wherein the nucleic acid comprises: A method wherein the method is introduced under conditions such that it binds to the mRNA and thereby inhibits transcription of the mRNA to Syk kinase. 22. 22. The method of claim 21, wherein said Fc receptor is Fc [gamma] RI, Fc [gamma] RIIA, or Fc [gamma] RIIIA. 23. 20. The method of claim 19, wherein the sequence is complementary to a region of the endogenous mRNA that has no secondary structure. 24. 22. The method of claim 21, wherein said nucleic acid is complementary to said mRNA region without secondary structure. 25. 22. The method according to claim 21, wherein the nucleic acid has a stem-loop structure. 26. 22. The method according to claim 21, wherein the nucleic acid has the sequence shown in Figs. 27. A method of inhibiting the phagocytic ability of a mammalian cell that expresses an Fc receptor, comprising introducing a nucleic acid complementary to an endogenous mRNA encoding an Fc receptor into the cell, wherein the nucleic acid comprises the nucleic acid. Is introduced under conditions such that it binds to the mRNA, thereby inhibiting transcription of the mRNA to the FC receptor. 28. 28. The method according to claim 27, wherein said nucleic acid is an RNA molecule. 29. 28. The method of claim 27, wherein said nucleic acid is complementary to an untranslated region of said mRNA. 30. A method of inhibiting the signal transduction of γ-subunit of IgE receptor Fc _epsilon RI, the cells that担時the receptor, of the cells that activate signal transduction of the Fc _epsilon RI receptors or a γ subunit A method comprising introducing an inhibitor of an endogenous kinase, wherein said introduction is performed under conditions such that said signaling is inhibited. 31. 31. The method according to claim 30, wherein said inhibitor is a peptide or a mimetic thereof. 32. 32. The method of claim 31, wherein said peptide comprises the sequence Y-2XL (where X2 represents any amino acid). 33. 33. The method according to claim 32, wherein X2 represents an amino acid of the Y-X2-L sequence of the cytoplasmic domain of γ-chain _FCεRI . 34. i) a promoter, ii) a segment of double-stranded DNA whose transcribed single strand contains a sequence complementary to Fc receptor mRNA, and iii) a termination sequence, A construct provided in the direction of transcription to the 'end, wherein the promoter, double-stranded DNA and termination sequence are operably linked. 35. 35. A cell comprising the construct of claim 34, wherein said promoter and said termination sequence are functional in said cell. 36. Essentially, Fc.gamma. Or Fc _epsilon receptor or soluble Fc receptor consisting of the extracellular domain of the binding site. 37. A soluble receptor comprising an extracellular domain from a first Fcγ receptor type and a cytoplasmic domain from a second Fcγ receptor type, wherein at least one of the first and second receptor types is an α or γ chain of FcγRI or FcγRIII. Is a soluble receptor. 38. A DNA molecule encoding the soluble receptor according to claim 36. 39. A DNA molecule encoding the soluble receptor of claim 37. 40. Essentially, Fc _gamma RIIA or peptide consisting of a tyrosine comprising motif of the cytoplasmic domain or a functional part of the gamma chain of Fc gamma RIIIA or Fc _epsilon RI. 41. A DNA molecule encoding the peptide of claim 40. 42. A cell comprising the peptide of claim 40. 43. A peptide that inhibits Medeieta secreted from phagocytosis or mast cells, sequence Y-X2-L (X2 is the _gamma chain of Fc _gamma RIIA or Fc _gamma RIIIA or F c _epsilon RI cytoplasmic domain -X2- two shows the amino acid) peptide comprising a portion of the Fc gamma RIIA or Fc gamma RIIIA or the gamma chain of Fc _epsilon RI cytoplasmic domain containing the L sequence. 44. 2. The method of claim 1, wherein said inhibiting reduces or prevents local tissue damage resulting from monocyte or neutrophil activity. 45. A method for inhibiting phagocytosis of a Syk-producing cell, wherein the cell comprises an antisense construct or ribozyme targeting a syk-encoding sequence present in the cell, under conditions that inhibit Syk production. A method comprising introducing. 46. A method of inhibiting phagocytic ability of the cells, the method, in the cells, the method comprising introducing the Fc _gamma RIIB under conditions such that the inhibition is executed. 47. 31. The method according to claim 30, wherein said kinase is Syk kinase. 48. A method of inhibiting the signal transduction of γ-subunit of IgE receptor Fc _epsilon RI, the cells that担時the receptor, an antisense construct which targets a sequence encoding a Syk present in the cell, the signal A method comprising introducing under conditions such that transmission is inhibited. 49. 49. The method of claim 48, wherein said cells are present in the lungs of an asthmatic patient. 50. 50. The method of claim 49, wherein the introducing is performed by administering the construct to the lungs of the patient by aerosol or systemic administration. 51. 50. The method of claim 47, wherein the inhibitor targets an intermediate region between the second SH domain and the catalytic (kinase) domain of Syk kinase. 52. A method for screening (sieving) a test compound for the ability to selectively inhibit signaling mediated by the Syk kinase intermediate region, comprising the steps of: isolating a compound comprising a Syk intermediate sequence and a ZAP70 intermediate sequence; And determining whether the compound binds to the Syk intermediate sequence or Zap70 intermediate region, wherein the compound binds to the Syk intermediate region but not to the Zap70 intermediate region sequence. The method wherein the compound has selective inhibitory ability. 53. A method of inhibiting secretion of a mediator from a Syk producing cell, the method comprising the steps of: A method comprising introducing an agent that selectively inhibits transmission under conditions such that the inhibition is effected. 54. 54. The method of claim 53, wherein said cells are present in the lungs of an asthmatic patient. 55. A nucleic acid carrying the sequence described in FIG. 5 or FIG. 56. i) a promoter and ii) a segment of double-stranded DNA whose transcribed single strand is complementary to Syk kinase mRNA operably linked to the promoter, from the 5 'end. An antisense construct comprising in the direction of transcription to the 3 'end. 57. 57. The construct of claim 56, wherein the sequence that is complementary to Syk kinase mRNA is complementary to a region of Syk kinase mRNA that surrounds or encloses the transcription initiation codon. 58. 57. The construct of claim 56, wherein the sequence that is complementary to Syk kinase mRNA is complementary to a region of Syk kinase mRNA that has minimal secondary structure. 59. A pharmaceutical composition comprising a Syk kinase intermediate region or a part or mimetic consisting of at least 6 amino acids, and a pharmaceutically acceptable carrier.