Method and cell composition for screening compounds for anti-inflammatory activity

Abstract

A method and cell line for screening test compounds for antiinflammatory activity are disclosed. The cell line is a human cell line capable of producing a selected cytokine associated with an inflammatory response in humans, and transfected with (i) a vector containing DNA encoding a cytokine regulatory factor under the control of a first promoter, and (ii) a vector containing DNA encoding a detectable-marker protein, under the control of a second promoter which is responsive to cytokine induction. In the screening method, the cells are cultured under conditions of cytokine regulatory factor overexpression and cytokine induction. Addition of test compound that results in a diminution of the detectable-marker protein is evidence of anti-inflammatory activity.

1 . A method for screening test compounds for antiinflammatory activity, comprising: 
 (a) culturing a mammalian cell line capable of producing a selected cytokine associated with an inflammatory response in humans, and transfected with (i) a vector containing DNA encoding a cytokine regulatory factor under the control of a first promoter, and (ii) a vector containing DNA encoding a detectable-marker protein, under the control of a second promoter which is responsive to cytokine induction, under culture conditions in which the cytokine regulatory factor is overproduced in the transfected cells, the selected cytokine is induced, and the detectable-marker protein is produced at detectable levels,    (b) adding a test compound to the cultured cells, and    (c) observing any diminution in the level of the detectable-marker protein.    
     
     
         2 . The method of  claim 1 , wherein the cytokine regulatory factor is a mammalian PKR.  
     
     
         3 . The method of  claim 2 , wherein the cytokine regulatory factor is a human PKR.  
     
     
         4 . The method of  claim 1 , wherein the second promoter is the natural promoter of the selected cytokine.  
     
     
         5 . The method of  claim 1 , wherein the second promoter is selected from a group consisting of the promoters associated with the human gene for IL-1, IL-2, IL-3, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, TNF-beta, IL-1 receptor, and IL-8 receptor.  
     
     
         6 . The method of  claim 1 , wherein the second promoter is selected from a group consisting of the promoters associated with the human gene for IL-1, IL-6, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta.  
     
     
         7 . The method of  claim 1 , wherein the delectable marker protein is green fluorescent protein (GFP).  
     
     
         8 . The method of  claim 1 , wherein said culturing further includes adding to the cultured cells, an amount of a dsRNA effective to stimulate cytokine production in the cytokine overproducing cells in culture.  
     
     
         9 . The method of  claim 1 , wherein said culturing includes adding to the cultured cells, a priming agent selected from the group consisting of PMA, calcium ionophores, sodium butyrate, endotoxin, and cytokines.  
     
     
         10 . A cell line for use in screening test compounds for antiinflammatory activity, comprising: 
 a human cell line capable of producing a selected cytokine associated with an inflammatory response in humans, and transfected with (i) a vector containing DNA encoding a cytokine regulatory factor under the control of a first promoter, and (ii) a vector containing DNA encoding a detectable-marker protein, under the control of a second promoter which is responsive to cytokine induction,    wherein culturing the cells under culture conditions in which the cytokine regulatory factor is overproduced and cytokines are induced is effective to produce elevated levels of the detectable-marker protein, and    addition to the cell culture of a test compound that results in a diminution of the detectable-marker protein is indicative of anti-inflammatory activity.    
     
     
         11 . The cell line of  claim 10 , wherein the cytokine regulatory factor is mammalian PKR.  
     
     
         12 . The cell line of  claim 11 , wherein the cytokine regulatory factor is human PKR.  
     
     
         13 . The cell line of  claim 10 , wherein the second promoter is the natural promoter of the selected cytokine.  
     
     
         14 . The cell line of  claim 10 , wherein the second promoter is selected from a group consisting of promoters associated with the human gene for IL-1, IL-2, IL-3, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, TNF-beta, IL-1 receptor, IL-8 receptor.  
     
     
         15 . The cell line of  claim 10 , wherein the second promoter is selected from a group consisting of promoters associated with the human gene for IL-1, IL-6, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TNF-beta.  
     
     
         16 . The cell line of  claim 10 , wherein the delectable-marker protein is green fluorescent protein (GFP).