M.Sc Thesis

M.Sc StudentAmit Cohen Bat-Chen
SubjectMacrophage Function in a Double Hit Model and Its
Regulation by Different Oxygen Concentrations
DepartmentDepartment of Medicine
Supervisors PROFESSOR EMERITUS Haim Bitterman
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Systemic inflammatory response syndrome (SIRS) often complicates the clinical course of various insults and exacerbates their prognosis. Cellular hypoxia is a common denominator that triggers inflammation in these conditions. Recently it the major part that underlying conditions play in determining the individual response to insults has been recognized. Thus models simulating single insult ("single hit" models) do not fully represent the real life clinical scenario and clinically relevant models that simulate a sequence of insults ("multiple hit" models) are needed.

Macrophages can respond to a combination of pro- and anti-inflammatory signals by a diverse range of activities and functional states. They can kill pathogens and secrete pro-inflammatory cytokines (IL-6, IL-1, TNFα) or secrete anti-inflammatory cytokines (IL-10, IL-1Ra), which down-regulate the inflammatory response, promote tissue remodeling and enhance angiogenesis.

In this study we examined the progression of the inflammatory response in a double hit model of hemorrhage (35% of total blood volume), and infection with zymosan (10mg/20gr). In order to study the specific role of macrophages in this model, peritoneal macrophages were isolated and cultured ex vivo with or without additional stimulation with LPS (1 μg/ml). In addition the double hit was simulated in vitro by incubating the RAW 264.7 macrophage cell line in hypoxia for 24 hours following addition of zymosan A (50µg/ml) for another 24 hours. Serum, peritoneal lavage and supernatants were collected and analyzed for the presence of pro- and anti-inflammatory cytokines (TNFa, IL-6, IL-10), NO production, and MMP-9 induction.

Hemorrhage followed by zymosan injection did not cause death. Mice were moderately sick in the first three days and then recovered. Despite this recovery the level of pro- and anti-inflammatory mediators (TNFα, IL-6, IL-10, MMP-9) in the peritoneal lavage was elevated in the double hit group relative to the single hit groups (p<0.01), indicating the development of a local inflammatory response after 8 days. In the serum only IL-6 was found elevated in the double hit group relative already after 3 hours. indicating a systemic inflammatory response. Both isolated peritoneal macrophages ex vivo and RAW 264.7 macrophages exhibited a similar inflammatory response in which elevated levels of TNFa, IL-6, IL-10 were elevated in the double hit group relative to the single hit groups (p<0.01). Our results demonstrate that sequential insults cause a dynamic and mixed pro- and anti- inflammatory response, both locally and systemically, and that macrophages are key regulators in this response.