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Down-regulated circulating PMN function after injury despite enhanced p38 MAPK activity
Ketan Sheth1, John Friel, Brian Nolan
1Department of Surgery, University of Massachusetts Medical School, Worcester, MA, USA.
Background:
Neutrophils (PMN) are initially primed by injury. However, remaining PMN found in the circulation postinjury demonstrate a down-regulated phenotype with inhibited apoptosis, CXCR2 expression, and endotoxin (LPS) responsiveness that may contribute to infectious complications and organ dysfunction. The p38 mitogen activated protein kinase (MAPK) signal transduction pathway has been implicated in regulating each of these PMN functions. We, therefore, hypothesize that p38 signaling is similarly down-regulated in postinjury circulating PMN.
Materials And Methods:
PMN were isolated from trauma patients (ISS > 20, postinjury day 3) and concurrently from healthy volunteers (control). PMN were cultured with LPS (100 ng/mL) and p38 activity assessed by Western blotting of cell lysates using a dual phosphospecific antibody for phosphorylated, active p38. In separate experiments, PMN from healthy volunteers were cultured in plasma from either healthy or injured subjects +/- LPS and p38 activity assessed by a cell-free in vitro kinase assay using the transcription factor, ATF-2, as a substrate. Apoptosis and IL-1beta secretion of the cultured PMN from each experimental condition were also quantified.
Results:
Circulating PMN from trauma patients have an upregulated LPS signaled p38 MAPK response (threefold) compared to PMN from healthy volunteers (p < 0.05). Furthermore, circulating factors present in trauma plasma can transfer this response to normal PMN. There was no significant alteration in apoptosis following LPS treatment between control and trauma patient's PMN (control: 62 +/- 3% vs. trauma: 55 +/- 5%), but there was decreased secretion of IL-1beta (control: 100 +/- 28 pg/mL vs. trauma: 12 +/- 5 pg/mL, p < 0.05).
Conclusion:
We conclude that injury down-regulates selective PMN function despite enhanced p38 activity. These results suggest a shift in the role of PMN p38 signal transduction following injury with additional critical regulation of LPS responses downstream to p38 MAPK activity.
Insights
Following injury, circulating neutrophils exhibit an enhanced p38 MAPK response to LPS, despite overall suppressed function. This suggests complex regulation of neutrophil signaling post-trauma, impacting immune responses and organ function.
Area of Science:
- Immunology
- Cell Biology
- Trauma Research
Background:
- Neutrophils (PMN) play a critical role in the immune response to injury.
- Post-injury circulating PMN display a suppressed phenotype, including reduced apoptosis and endotoxin (LPS) responsiveness.
- The p38 mitogen-activated protein kinase (MAPK) pathway is crucial for regulating PMN functions.
Purpose of the Study:
- To investigate the role of p38 MAPK signaling in post-injury circulating neutrophils.
- To determine if p38 signaling is down-regulated in PMN following severe trauma.
- To explore the impact of circulating factors in trauma plasma on PMN function.
Main Methods:
- Isolation of PMN from trauma patients and healthy volunteers.
- Assessment of p38 MAPK activity via Western blotting and in vitro kinase assays.
- Quantification of PMN apoptosis and IL-1beta secretion following LPS stimulation.
Main Results:
- Circulating PMN from trauma patients showed a threefold increased LPS-signaled p38 MAPK response compared to controls.
- Plasma from trauma patients could transfer this enhanced p38 response to normal PMN.
- While apoptosis was unchanged, IL-1beta secretion was significantly decreased in trauma patient PMN.
Conclusions:
- Injury leads to selective down-regulation of PMN function despite enhanced p38 MAPK activity.
- These findings indicate a complex shift in p38 signaling post-injury.
- Regulation of LPS responses occurs downstream of p38 MAPK activity in the context of trauma.