Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic
shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma
patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate
immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit”
hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered
from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with
increased activation of the proinflammatory transcription factor NF-κB, and augmented
expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which
resides in plasma membrane lipid rafts.
The objective of this work is to define cellular mechanisms of macrophage priming by
oxidative stress following shock resuscitation. The main hypothesis investigated is that altered
cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation
strategies can diminish these effects.
AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface
levels of TLR4, which in turn results in augmented NF-κB translocation in response to small
doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in
similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the
cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation,
suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy
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transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that
oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced
movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased
responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress
can be diminished by early exposure to resuscitation regimens with direct or indirect systemic
antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline.
Hyperosmolarity was found to modulate AM TLR4 gene and protein expression.
Collectively, these studies suggest a novel mechanism whereby oxidative stress might
prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling
pathway early during shock resuscitation may represent an anti-inflammatory strategy able to
ameliorate late morbidity and mortality following S/R.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/11247 |
| Date | 01 August 2008 |
| Creators | Powers, Kinga Antonina |
| Contributors | Rotstein, Ori D. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 2436921 bytes, application/pdf |
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