Regulation of cytokine production in a hematopoietic cell

Abstract

A method useful for regulating cytokine production by a hematopoietic cell by regulating an MEKK/JNKK-contingent signal transduction pathway in such a cell is disclosed. Methods of identifying compounds capable of specifically regulating an MEKK/JNKK-contingent signal transduction pathway in hematopoietic cells, a kit for identifying cytokine regulators, methods to treat diseases involving cytokine production, and cells useful in such methods are also set forth.

What is claimed is:  
     
         1 . A method to regulate cytokine production, comprising regulating an MEKK/JNKK-contingent signal transduction pathway in a hematopoietic cell to effect regulation of cytokine production in said cell.  
     
     
         2 . The method of  claim 1 , wherein said cytokine is selected from the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β.  
     
     
         3 . The method of  claim 1 , wherein said hematopoietic cell is selected from the group consisting of a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.  
     
     
         4 . The method of  claim 1 , wherein said hematopoietic cell expresses FceRI.  
     
     
         5 . The method of  claim 1 , wherein said step of regulating said MEKK/JNKK-contingent signal transduction pathway comprises regulating a signal transduction molecule selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2.  
     
     
         6 . The method of  claim 5 , wherein said step of regulating a signal transduction molecule comprises a method selected from the group consisting of degrading said molecule, binding a regulatory compound to said molecule, inhibiting transcription of said molecule, inhibiting translation of said molecule, inhibiting activation of said molecule, and inhibiting the interaction of said molecule with another signal transduction molecule.  
     
     
         7 . The method of  claim 5 , wherein said step of regulating a signal transduction molecule results in modulation of the interaction of a transcription factor selected from the group consisting of NF-AT, AP-1, Jun, Fos, ATF-2, NFκB, and CBP, with a cytokine promoter.  
     
     
         8 . The method of  claim 5 , wherein said MEKK/JNKK-contingent signal transduction pathway is regulated by contacting said cell with an effective amount of a compound that interacts with said molecule.  
     
     
         9 . The method of  claim 1 , wherein said cytokine production is inhibited.  
     
     
         10 . The method of  claim 1 , wherein said MEKK/JNKK-contingent signal transduction pathway is activated by a PI3-K signal transduction pathway.  
     
     
         11 . The method of  claim 10 , wherein said step of regulating said MEKK/JNKK-contingent signal transduction pathway comprises regulating PI3-K.  
     
     
         12 . The method of  claim 1 , wherein said MEKK/JNKK-contingent signal transduction pathway is activated by aggregation of FceRI on said cell.  
     
     
         13 . The method of  claim 12 , wherein said method further comprises regulating a signal transduction pathway selected from the group consisting of c-kit signal transduction pathway and p38 signal transduction pathway.  
     
     
         14 . The method of  claim 12 , wherein said regulation of cytokine production does not entail regulation of the ERK signal transduction pathway.  
     
     
         15 . A method to regulate cytokine production in a hematopoietic cell expressing FceRI, comprising regulating an MEKK/JNKK-contingent signal transduction pathway in said cell to effect regulation of cytokine production in said cell, said MEKK/JNKK-contingent signal transduction pathway being activated by the PI3-K signal transduction pathway of said cell.  
     
     
         16 . The method of  claim 15 , wherein said cytokine is selected from the group consisting of TNF-α, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β.  
     
     
         17 . The method of  claim 15 , wherein said hematopoietic cell is selected from the group consisting of a mast cell, a basophil and an eosinophil.  
     
     
         18 . The method of  claim 15 , wherein said cytokine production is inhibited.  
     
     
         19 . The method of  claim 15 , wherein said step of regulating said MEKK/JNKK-contingent signal transduction pathway comprises administering to said cell an effective amount of a compound that interacts with a signal transduction molecule selected from the group consisting of PI3-K, MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2, wherein said molecule modulates the interaction of a transcription factor selected from the group consisting of NF-AT, AP-1, Jun, Fos, ATF-2, NFκB, and CBP, with a cytokine promoter.  
     
     
         20 . A method to regulate signal transduction pathways involved in cytokine production in a hematopoietic cell, comprising: 
 (a) providing a hematopoietic cell having an MEKK/JNKK-contingent signal transduction pathway;    (b) regulating signal transduction in said pathway by inhibiting the interactions between molecules in said pathway, said molecules selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2, wherein said step of regulating inhibits the production of cytokines by said cell.    
     
     
         21 . The method of  claim 20 , further comprising regulating other signal transduction pathways that regulate an MEKK/JNKK-contingent signal transduction pathway.  
     
     
         22 . The method of  claim 20 , further comprising regulating c-kit and p38 pathways to affect the regulation of cytokine production.  
     
     
         23 . The method of  claim 20 , wherein said step of inhibiting comprises administering to said cell an effective amount of a compound which modulates interactions between said molecules.  
     
     
         24 . The method of  claim 23 , wherein said compound modulates interactions between said molecules by a method selected from the group consisting of degrading said molecule, binding to said molecule such that the function of said molecule is inhibited, inhibiting transcription of said molecule, inhibiting translation of said molecule, and inhibiting the interaction of said molecule with another signal transduction molecule.  
     
     
         25 . The method of  claim 20 , wherein said hematopoietic cell is selected from the group consisting of a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.  
     
     
         26 . The method of  claim 20 , wherein said cytokine is selected from the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β.  
     
     
         27 . The method of  claim 20 , wherein said cytokine production is inhibited.  
     
     
         28 . The method of  claim 20 , wherein said MEKK/JNKK-contingent signal transduction pathway is activated by a PI3-K signal transduction pathway in said cell.  
     
     
         29 . The method of  claim 20 , wherein said step of regulating signal transduction comprises inhibiting the interactions between said molecules and PI3-K.  
     
     
         30 . The method of  claim 20 , wherein said MEKK/JNKK-contingent signal transduction pathway is activated by aggregation of FceRI on said cell.  
     
     
         31 . A method to identify compounds capable of regulating cytokine production in a hematopoietic cell, comprising: 
 (a) providing a hematopoietic cell having an MEKK/JNKK-contingent signal transduction pathway;    (b) contacting a putative regulatory compound with said cell; and    (c) determining whether said putative regulatory compound is capable of regulating said MEKK/JNKK-contingent signal transduction pathway to affect cytokine production by said cell. (d)    
     
     
         32 . The method of  claim 31 , wherein said hematopoietic cell is selected from the group consisting of a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.  
     
     
         33 . The method of  claim 31 , wherein said cytokine is selected from the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β.  
     
     
         34 . The method of  claim 31 , wherein said cytokine production is inhibited.  
     
     
         35 . The method of  claim 31 , wherein said regulatory compound regulates said MEKK/JNKK-contingent signal transduction pathway by modulating the interaction between signal transduction molecules selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2.  
     
     
         36 . The method of  claim 35 , wherein step of modulating the interaction between signal transduction molecules regulates the interaction of a transcription factor selected from the group consisting of NF-AT, AP-1, Jun, Fos, ATF-2, NFκB, and CBP, with a cytokine promoter.  
     
     
         37 . The method of  claim 35 , wherein said regulatory compound modulates the interaction of said molecules with PI3-K.  
     
     
         38 . A kit to identify compounds capable of regulating cytokine production in a hematopoietic cell comprising: 
 (a) a hematopoietic cell having an MEKK/JNKK-contingent signal transduction pathway, said cell producing an amount of at least one cytokine, wherein production of said cytokine by said cell is dependent upon said NEKK/JNKK-contingent signal transduction pathway; and    (b) a means for determining a change in the amount of cytokine produced by said cell after said cell is contacted with a putative regulatory compound.    
     
     
         39 . The kit of  claim 38 , wherein said cell expresses FceRI and said MEKK/JNKK-contingent signal transduction pathway is activated by aggregation of said FceRI.  
     
     
         40 . The kit of  claim 38 , wherein said hematopoietic cell is selected from the group consisting of a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.  
     
     
         41 . The kit of  claim 38 , wherein said means for determining a change in cytokine production is an assay selected from the group consisting of an immunoassay, a transcription assay and a biological assay which detects levels of cytokine production by said cell.  
     
     
         42 . The kit of  claim 41 , wherein said immunoassay is selected from the group consisting of an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, and an immunoblot assay.  
     
     
         43 . The kit of  claim 41 , wherein said immunoassay is an antibody capture enzyme-linked immunoassay.  
     
     
         44 . The kit of  claim 41 , wherein said transcription assay is a PCR-based assay for cytokine mRNA production.  
     
     
         45 . The kit of  claim 41 , wherein said biological assay comprises the use of a cell line which is responsive to a cytokine selected from the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-γ, and IFN-α/β.  
     
     
         46 . The kit of  claim 38 , wherein said cell comprises heterologous mammalian nucleic acid sequences encoding proteins involved in MEKK/JNKK-contingent signal transduction.  
     
     
         47 . The kit of  claim 38 , wherein said cell comprises heterologous mammalian nucleic acid sequences encoding proteins involved in signal transduction through FceRI.  
     
     
         48 . A method to treat a disease involving cytokine production in an animal, comprising regulating an MEKK/JNKK-contingent signal transduction pathway to affect cytokine production by a hematopoietic cell.  
     
     
         49 . The method of  claim 48 , wherein said disease is selected from the group consisting of allergic diseases, anaphylaxis, diseases involving defects in hematopoietic cells, inflammation, mast cell disorders, sepsis and cancer.  
     
     
         50 . The method of  claim 48 , wherein said disease is allergic inflammation.  
     
     
         51 . The method of  claim 48 , wherein said step of regulating comprises administering to said animal an effective amount of a compound which interacts with a signal transduction molecule in said NEKK/JNKK-contingent signal transduction pathway selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2, said interaction resulting in the regulation of cytokine production by a hematopoietic cell in said animal.  
     
     
         52 . The method of  claim 51 , wherein said compound is selected from the group consisting of an MEKK inhibitor, a JNKK inhibitor, and a JNK inhibitor.  
     
     
         53 . The method of  claim 48 , wherein said step of regulating comprises administering to said animal an effective amount of a compound which inhibits PI3-K.  
     
     
         54 . A method to treat allergic inflammation in an animal, comprising regulating cytokine production in a hematopoietic cell in said animal, said cell expressing FceRI, wherein said step of regulating comprises regulating an MEKK/JNKK-contingent signal transduction pathway.  
     
     
         55 . The method of  claim 54 , wherein said cell is selected from the group consisting of a mast cell, a basophil and an eosinophil.  
     
     
         56 . The method of  claim 54 , wherein said cytokine production is inhibited.  
     
     
         57 . The method of  claim 54 , wherein said step of regulating MEKK/JNKK-contingent signal transduction pathway comprises administering to said animal an effective amount of a compound which interacts with a signal transduction molecule in said MEKK/JNKK-contingent signal transduction pathway, said interaction resulting in the regulation of cytokine production by said hematopoietic cell in said animal.  
     
     
         58 . The method of  claim 54 , wherein said animal is a mammal.  
     
     
         59 . The method of  claim 54 , wherein said animal is a human.  
     
     
         60 . A compound for regulating cytokine production in a hematopoietic cell, wherein said compound interacts with a signal transduction molecule in an MEKK/JNKK-contingent signal transduction pathway, said molecule selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2.  
     
     
         61 . The compound of  claim 60 , wherein said compound does not regulate an ERK-dependent signal transduction pathway.  
     
     
         62 . A compound for regulating cytokine production in a hematopoietic cell identified by the method comprising: 
 (a) providing a hematopoietic cell having an MEKK/JNKK-contingent signal transduction pathway, wherein production of cytokines by said cell is dependent solely on said pathway;    (b) contacting said compound with said cell; and    (c) identifying said compound by its ability to regulate said MEKK/JNKK-contingent signal transduction pathway.    
     
     
         63 . The compound of  claim 62 , wherein said method further comprises determining the ability of said compound to inhibit cytokine production in said cell.  
     
     
         64 . A cell used in a method to identify compounds capable of regulating cytokine production, comprising a cell having at least one heterologous mammalian nucleic acid sequence encoding at least one protein involved in an MEKK/JNKK-contingent signal transduction pathway.  
     
     
         65 . The cell of  claim 64 , wherein said cell is capable of producing cytokines and production of said cytokines is regulated by said MEKK/JNKK-contingent signal transduction pathway.  
     
     
         66 . A method of using a hematopoietic cell to screen putative regulatory compounds for their ability to regulate cytokine production in said cell comprising: 
 (a) providing a hematopoietic cell having an MEKK/JNKK-contingent signal transduction pathway;    (b) contacting said cell with a putative regulatory compound; and    (c) determining whether said putative regulatory compound is capable of modulating the amount of cytokine produced by said cell.    
     
     
         67 . The method of  claim 66 , wherein said cell is selected from the group consisting of a mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and a natural killer cell.  
     
     
         68 . The method of  claim 66 , said cell having at least one heterologous mammalian nucleic acid sequence encoding at least one protein involved in an MEKK/JNKK-contingent signal transduction pathway.  
     
     
         69 . The method of  claim 66 , wherein said cell lacks an ERK-dependent signal transduction pathway.