M.Sc Thesis

M.Sc StudentReich Daphna
SubjectHypoxia Increases the Secretion of Lactate, LDH-A and
Enolase-1 from Mouse Macrophages: Possible Role in
Induction of Inflammation
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Haim Bitterman
Full Thesis textFull thesis text - English Version


Induction of inflammation depends on activation of antigen presenting cells (APCs) such as macrophages. Under homeostasis, exposure of APCs to apoptotic cells suppresses their ability to induce inflammation. Once the APCs encounter an exogenous molecule or an endogenous necrotic cell, they can induce inflammation. These are collectively called "danger signals", as they are secreted upon encountering danger, and work to initiate an immune response. Proteins and metabolites such as HMGB1 and uric acid have been recognized as danger signals. Since inflammation is often accompanied by hypoxia, anaerobic glycolysis and conversion of pyruvate to lactate are increased. We examined the possibility that certain enzymes and metabolites, such as enolase, lactate dehydrogenase (LDH) and its product lactate, may also play a role in inducing inflammation. Our work was divided into two parts: examining the effects of hypoxia and LPS stimulation on secretion of lactate, LDH and enolase in vitro in a macrophage cell line, primary murine macrophages, kidney carcinoma-derived epithelial cells, and in murine models of inflammation in vivo - double hit and LPS-induced inflammation. The second part included lactate administration to RAW 264.7 cells and its effect on several inflammatory parameters; and in two in vivo models of inflammation or angiogenesis. Our results showed that macrophages remained viable whereas RENCA cells displayed massive cell death, and the presence of inactive LDH, enolase and lactate in the supernatants was greatly increased in all cells in the hypoxic treatments, leading us to the tentative conclusion that they were secreted actively by macrophages and spilled necrotically from epithelial cells. In in vivo models of inflammation, we also observed increases in LDH, lactate and enolase.  In the second part of the work, exogenous lactate added to the cells caused cell death. When the inflammatory parameters were analyzed, we saw a biphasic effect which was not cell-death associated, in which low concentrations of lactate were pro-inflammatory and high concentrations were anti-inflammatory, a result strengthened by in vivo administration of lactate which caused recruitment of macrophages to the peritoneum at low concentrations and no recruitment at high concentrations. Lactate also proved to be angiogenic in high concentrations in an in vivo angiogenesis model. We therefore conclude that lactate may play a role both in angiogenesis and in induction of inflammation, depending on its concentration. Regarding LDH and enolase, LDH may have an additional role outside the cell, as it is secreted as an inactive enzyme in hypoxia.