"Store-operated calcium entry" (SOCE) into cells is one of the major intracellular signaling responses that induce neutrophil activation. The molecular mechanism of SOCE has recently been defined through the identification of the intracellular calcium sensor proteins, STIM1 and STIM2, and the plasma membrane calcium channel proteins ORAI1, 2 and 3. In response to immune stimuli, calcium is released from intracellular "stores" in the endoplasmic reticulum, which leads to a conformational change in the STIM molecules, allowing them to physically associate with ORAI channel proteins in the plasma membrane, leading to channel opening allowing entry of extracellular calcium. In lymphocytes, the loss of SOCE results in poor cellular proliferative responses and cytokine production in response to a variety of stimuli. There have been no studies of STIM/ORAI signaling in neutrophils. Using stim1-/- bone marrow chimeric mice, we have found that loss of SOCE leads to a profound block in neutrophil activation. Our preliminary evidence suggests that PKC enzymes are the target of extracellular calcium during neutrophil activation. As a result of this defective neutrophil function, stim1-/- chimeras are protected from tissue injury in the zymosan model of acute peritonitis and show significantly reduced tissue injury in a hepatic ischemia reperfusion model. To expand on these observations, we propose a series of experiments to: 1) determine the molecular mechanisms by which SOCE leads to neutrophil activation, 2) generate neutrophil lineage specific mutants lacking individual Stim or Orai molecules, to determine which are most important in neutrophil activation, 3) develop novel single chain mAb blocking reagents, targeting Orai proteins, that will allow us to test whether cessation of SOCE in neutrophils during an ongoing inflammatory response will limit tissue injury. We will test the hypothesis that PKCs are the target of calcium in neutrophils through biochemical, genetic and chemical genetic approaches. Of the Stim and Orai proteins in mice, it is unclear which play the dominant role in SOCE in neutrophils. We will determine which of the Stim and Orai molecules are most important in neutrophils by development of neutrophil lineage specific mutants of stim1, stim2, orai1 and orai2 in mice. Finally, we will take advantage of a new UCSF / Pfizer Corp collaboration to develop novel single chain mAbs that will target Orai1, to test the hypothesis that blockade of SOCE will reverse ongoing inflammatory disease. Our goal is to determine the mechanisms and proteins involved in SOCE in neutrophils, then ask whether targeting these proteins will reverse inflammatory disease. Given the novelty of our initial findings using stim1-/- mice, achieving these goals will be a major advance in inflammation research.

Public Health Relevance

Uncontrolled activation of neutrophils is a major cause of tissue injury in inflammatory diseases such as arthritis, vasculitis and ischemia-reperfusion injury. Using gene knockout mice we have found that genetic deficiency of calcium signaling pathways in neutrophils results in a profound block in neutrophil activation and protection from tissue injury during inflammation. By studying these animals, we hope to define the target proteins inside neutrophils that are activated by calcium entry as well as develop new reagents to test whether therapeutic blockade of calcium entry can reverse ongoing inflammatory disease in mouse models.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI065495-06A1
Application #
8504418
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Nasseri, M Faraz
Project Start
2005-07-01
Project End
2018-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
6
Fiscal Year
2013
Total Cost
$368,460
Indirect Cost
$133,460
Name
University of California San Francisco
Department
Pathology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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