Jessica F. White

Regulation of Phosphatidylinositol 3-Kinase Activity and Phosphatidylinositol 3,4,5-Trisphosphate Accumulation by Neutrophil Priming Agents

Neutrophil priming by agents such as TNF-α and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-α or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10–60 s), which paralleled the augmented superoxide anion (O2−) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2− release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2− generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-α alone caused a 3-fold increase in PI3-kinase activity in p110γ PI3-kinase immunoprecipitates. p21ras activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2− generation, that PI3-kinase-γ is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21ras underlie this response.

Pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome

Rationale Acute respiratory distress syndrome (ARDS) affects over 200 000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined. Methods Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls. Main results Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis. Conclusions We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then ‘deprime’ and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.

Use of Radiolabelled Leukocytes for Drug Evaluation in Man

The endeavour to radiolabel or simply “label” autologous leukocytes has been a major clinical need. The endeavour to improve the labelling conditions and minimise interventional stresses and maintain cell functionality has been the driving objective. This chapter focuses on novel techniques used in this laboratory to radiolabel leukocytes, examples of the clinical indications that such labelled products might be informative, and how we can use these labelled cells in clinical situations to describe the life cycle behaviour (transit times and migratory capacity) of these labelled cells. Labelling techniques which preserve leukocyte functionality will assist in the development of new anti-inflammatory agents, anti-infectives, and indeed any drug or biologic where their clinical use may have an effect on these cells. Examples of labelling methods and clinical scenarios with imaging are described for conditions such as abscesses, ARDS, COPD, rheumatoid arthritis, inflammatory bowel disease, and vasculitis.

The Influence of the Spleen on Neutrophil Apoptosis in Vivo

In contrast to radiolabelled erythrocytes and platelets, radiolabelled neutrophils leave the circulating blood in an exponential manner, indicating random rather than age-dependent removal. Neutrophils transit the spleen with a range of residence times that are log normally distributed. We hypothesized that neutrophils are conditioned to undergo apoptosis to an extent that depends on their intrasplenic residence time and that this provides an explanation for the random removal of these cells from blood. Splenic venous and peripheral arterial blood was sampled simultaneously during abdominal surgery in four patients and age-dependent apoptosis assessed in whole blood using annexin V/PI staining. Apoptosis increased after 4 and 20 h ex-vivo incubation and was invariably higher in splenic venous vs arterial neutrophils. Transit through the spleen appears to promote neutrophil apoptosis, with subsequent high efficiency clearance by the liver. This may explain the mechanism underlying the random removal of neutrophils from the blood.