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Regulation of interleukin-8 from macrophages by acute hypoxia and hyperoxia : a role in the pathogenesis of the acute respiratory distress syndrome (ARDS)

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HiraniNA_2002redux.pdf (40.44Mb)
Date
2002
Author
Hirani, Nikhil A.
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Abstract
 
 
The acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung inflammation. Patients with ARDS require support on the intensive care unit (ICU) and the associated mortality approaches 50%. ARDS represents the severe end of a spectrum of lung injury that evolves over a period of hours or days in a subgroup of patients following a major insult such as multiple trauma, sepsis or aspiration. Professor Haslett's group in Edinburgh have undertaken clinical studies in patients in the very early at-risk period of ARDS, soon after the initiating insult. We have shown that in patients with multiple trauma, raised levels of intrapulmonary interleukin-8 (IL-8), but not other inflammatory cytokines, are associated with subsequent progression to ARDS (n=56, P<0.001). IL-8 is a potent chemoattractant and activator of neutrophils, considered to be the primary injurious cell in ARDS. The high IL-8 levels were detected within a few hours (range 0.75 - 4 hr) of the trauma incident. Immunohistochemical analysis implicated the alveolar macrophage as a potent source of intrapulmonary IL-8. The mechanisms by which IL-8 may be rapidly generated in this clinical setting are unknown.
 
Our clinical observations suggest that events occurring in the immediate aftermath of a trauma incident contribute to the generation of IL-8 in macrophages. I hypothesised that clinically relevant physiological events may include:
 
1) A neuro-endocrine 'stress' response to major trauma. This would result in the rapid intrapulmonary and systemic release of clinically relevant stress mediators including catecholamines and neuropeptides that may stimulate the macrophage to generate IL8.
 
1) A neuro-endocrine 'stress' response to major trauma. This would result in the rapid intrapulmonary and systemic release of clinically relevant stress mediators including catecholamines and neuropeptides that may stimulate the macrophage to generate IL8.
 
2) Acute tissue hypoxia and hyperoxia. By the time of sampling, the trauma victims were likely to have undergone a period of sustained tissue hypoxia secondary to headinjury, atelectasis and lung contusion and subsequent resuscitation with delivery of high flow oxygen. I hypothesised that hypoxia / hyperoxia was as direct multiplestimuli or 'hits' to generate IL-8 in macrophages.
 
I aimed to test these hypotheses in studies of cultured human monocyte-derived macrophages and in a novel animal model of acute lung injury.
 
In human-monocyte derived macrophages, I have shown that the stress mediators adrenalin, substance P and macrophage migration inhibitory factor (MIF) do not increase IL-8 production at an early time-point (2 hr). Compared to normoxic controls, acute hypoxia (PO2 ~ 3.5 KPa) increased IL-8 protein release by 1.8-fold by 2 hours and steady-state IL-8 111RNA expression by 30 mins. The multiple hit of hypoxia / hyperoxia was found to be a more potent stimulus for IL-8 generation than hypoxia or hyperoxia alone.
 
The effects of hypoxia / hyperoxia on IL-8 generation were studied in a rabbit model of acute lung injury. Localised bronchoscopic instillation of HC1 into the left lower lobe of an anaesthetised ventilated rabbit resulted in significantly increased IL-8 mRNA and protein expression, neutrophil infiltration into alveolar airspaces and lung in the directly injured lung but not the contralateral 'indirectly' injured lung. Systemic hypoxaemia was induced by reduction in the inspiratory oxygen fraction. Compared to normoxic controls III (arterial PaC>2 ~ 11 KPa), acute hypoxia (Pa02 ~ 5 KPa) for up to 2 hours increased intrapulmonary IL-8 mRNA but not protein expression in the acid-injured lung. Delivery of 100% oxygen for 2 hours (PaC>2 ~ 60 KPa) following acute hypoxia (a multiple-hit), increased both intrapulmonary IL-8 mRNA and IL-8 protein levels. The increase in IL-8 protein was attenuated if the reoxygenation phase was controlled to return arterial PO2 to normoxic levels (-11 KPa).
 
The mechanisms by which hypoxia may rapidly increase IL-8 mRNA expression in monocyte-derived macrophages was further studied in vitro. The rapidity of the response (30 mins) suggested an increase in gene transcription. Electromobility gel-shift assay revealed that hypoxia increased nuclear levels of the IL-8 promoter-binding transcription factors AP-1 and CEBP-P, but not NF-kB, by 15 min exposure. Hypoxia induced macrophage expression of HIF-la, a critical regulator of hypoxic adaptive responses. However cobalt chloride and desferrioxamine, HIF-la-inducing hypoxia mimics, did not upregulate IL-8, suggesting that IL-8 transcription may be HIF-1 independent. Finally it was demonstrated that in contrast to IL-8, hypoxia inhibited expression of a panel of chemokines and cytokines including MCP-1, MlP-la, MIP-ip and TNF-a. Both the pattern of chemokine expression and transcription factor activation with hypoxia differed from that induced by bacterial lipopolysaccharide (LPS), which potently activated NF-kB and upregulated several inflammatory genes.
 
These data support the hypothesis that acute hypoxia / hyperoxia act as multiple-hits in the generation of macrophage-derived IL-8 in vitro and intrapulmonary IL-8 in vivo, representing a potential mechanism for our observation of elevated alveolar IL-8 levels patients with multiple trauma that progress to ARDS. The observation that hypoxia alone rapidly and selectively increased IL-8 mRNA expression suggests that hypoxia may represent a 'priming' stimulus in macrophages, 'arming' the cell for a subsequent second hit such as hyperoxia. Furthermore, the specific chemokine response to hypoxia differs markedly with that observed with LPS implying potentially distinct adaptive responses to hypoxia and infection in the macrophage.
 
URI
http://hdl.handle.net/1842/28236
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