Study on molecules that negatively regulate ROS production in human granulocyes

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We studied the roles of Btk and Btk-associated molecules in regulation of reactive oxygen species (ROS) production. The enzyme responsible for the respiratory burst is NADPH oxidase. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. These kinases were hyper-phosphorylated and were activated. We delved into the mechanism of PTK activation in the absence of Btk by looking into the negative regulator of PTKs (e.g. Csk/Cbp and phosphatases). We also generated wild type Mal and mutant Mal to check whether membrane translocation of Mal is critical for priming of neutrophils.Btk was also involved in regulation of over-production of ROS in eosinophils. We are now working on mast cells derived from Btk deficient patients to examine the role of Btk in production of chymase and tryptase. The role of Itk, Tec family kinase expressed mainly in T cells, in the regulation of ROS production in T cells is currently under investigation.Our data demonstrated the importance of Btk (and Mal) in negative regulation of ROS hyperproduction in granulocytes. Our data so far indicate that PH-domain defective Mal may inhibit priming of neutrophils, and thus the protein may be used for the prevention of hyper-activation of neutrophils in conditions such as sepsis.