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.
