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Inhibition of Neutrophil Apoptosis after Severe Trauma Is NFκβ Dependent

Nolan, Brian MD; Collette, Helen RN; Baker, Stephen MScPH; Duffy, Andrew MD; De, Mita; Miller, Carol PhD; Bankey, Paul MD, PhD

Journal of Trauma-Injury Infection & Critical Care: April 2000 - Volume 48 - Issue 4 - pp 599-605
Annual Meeting Articles

Background: Systemic inflammation may inhibit neutrophil (PMN) apoptosis and promote multiple organ dysfunction syndrome. We hypothesize that severe trauma causes dysregulation of PMN apoptosis.

Methods: Neutrophils were isolated from trauma patients (24–72 hours after injury; n = 16) and controls (healthy volunteers) and incubated for 18 hours. In separate experiments, control cells were treated ± the nuclear factor kappa beta (NFκβ) inhibitor pyrrolidinithiocarbamate then incubated with 25% patient or control plasma. Apoptosis was quantified by enzyme-linked immunosorbent assay for histone-associated DNA and annexin V fluorescence-activated cell sorter. NFκβ activation was determined by Western blot for phosphorylated Iκβ.

Results: Apoptosis was inhibited in trauma patient PMN. Neutrophil apoptosis correlated with multiple organ dysfunction syndrome score, Acute Physiology and Chronic Health Evaluation II, and platelet count. Patient plasma inhibited apoptosis and induced phosphorylation of Iκβ in control cells. Inhibition of PMN apoptosis by patient plasma was blocked by pretreatment with pyrrolidinithiocarbamate.

Conclusion: NFκβ-dependent inhibition of neutrophil apoptosis occurs after trauma. Early inhibition of PMN apoptosis is dependent on the magnitude of injury.

Neutrophil apoptosis (programmed cell death) is believed critical to the resolution of acute inflammation and prevention of secondary tissue injury. Evidence suggests that apoptosis provides a mechanism for granulocyte deactivation. 1,2 During apoptosis, PMNs lose the ability to adhere, phagocytose, degranulate, and secrete mediators in response to inflammatory stimuli. 3 As cells die by means of apoptosis, they are cleared by macrophages. Throughout this process, neutrophil membranes remain intact, preventing leakage of proinflammatory intracellular contents. 4 Furthermore, apoptotic PMNs do not stimulate cytokine secretion from ingesting macrophages. 5

The role of neutrophil apoptosis in the pathogenesis of inflammatory disease has been addressed recently. Cox et al. demonstrated an association between the clearance of apoptotic neutrophils and the resolution of acute inflammation. 6 Numerous investigations have shown an association between dysregulation of in vitro PMN apoptosis and inflammatory disease states. Delayed in vitro apoptosis has been reported in circulating PMNs from patients with burn injury, systemic inflammatory response syndrome, and sepsis. 7–10 Decreased apoptosis has also been demonstrated in both circulating and pulmonary PMNs isolated from patients with ARDS. 11,12 Serum soluble factors, including multiple cytokines, have been implicated as mediators of this effect. 7,9 Regulation of PMN apoptosis at the cellular level remains under intense investigation. Recent in vitro studies have implicated both mitogen-activated protein kinase (MAPK) and nuclear factor kappa beta (NFκβ) activation as proximal events signaling the inhibition of PMN apoptosis. 13,14

Multiple organ dysfunction syndrome (MODS) is a frequent and highly morbid complication of severe trauma and other systemic inflammatory conditions. It is generally believed that MODS results from dysregulated inflammatory responses that promote tissue damage and eventual organ dysfunction. 15 Dysfunctional neutrophil responses resulting from delayed deactivation (apoptosis) may be involved. 8 It is our hypothesis that plasma-borne factors released in response to severe trauma inhibit neutrophil apoptosis by means of activation of NFκβ.

From the Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts.

Address for reprints: Paul Bankey, MD, PhD, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655; email:

Submitted for publication September 24, 1999.

Accepted for publication January 6, 2000.

This research was supported by NIH grant 525753.

The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Presented at the 59th Annual Meeting of the American Association for the Surgery of Trauma, September 16–18, 1999, Boston, Massachusetts.

© 2000 Lippincott Williams & Wilkins, Inc.