Mechanisms of human neutrophil priming

• Main Author: Condliffe, Alison Mary
• Published: University of Edinburgh 1997
• Subjects: 612.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712217

Neutrophils have been implicated in the pathogenesis of many human diseases, including the adult respiratory distress syndrome (ARDS), pulmonary fibrosis and ischaemia-reperfusion injury. Priming of neutrophils by pre-exposure to bacterial products and inflammatory mediators represents a potent means of augmenting both the bactericidal capacity and injurious potential of these cells; in particular, the generation of reactive oxygen species such as the superoxide anion (O2-) may be enhanced by up to twenty-fold. Greater understanding of the signalling mechanisms underlying neutrophil priming could lead to novel therapeutic strategies aimed at preventing neutrophil-mediated tissue injury in these conditions. Exposure of neutrophils to the priming agents lipopolysaccharide (LPS), tumour necrosis factor-α (TNFα) and platelet-activating factor (PAF) was shown to modulate adhesion molecule expression and function (in particular inducing upregulation of β₂ integrins and shedding of L-selectin) in an agonist-specific fashion, with a time-course which correlated with that required to establish the primed state. Neutrophil chemoattractant receptors are coupled to intracellular signalling pathways by heterotrimeric G-proteins, principally Giα2. I have shown that TNFα, LPS and PAF increase the level of Giα2 expression detectable in neutrophil membranes, the time-course for this effect mirroring that for priming. However, the degree of G-protein upregulation is considerably less than the enhancement of the respiratory burst, suggesting that downstream signalling events play a more critical mechanistic role in priming. Two major signalling pathways implicated in the activation of the neutrophil respiratory burst were studied; the cleavage of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C to yield inositol 1,4,5-trisphosphate (Ins(l,4,5)P3), and its phosphorylation by phosphatidylinositol 3-hydroxykinase (PI3K) to phosphatidylinositol 3,4,5- trisphosphate (PtdIns(3,4,5)P3). Accumulation of neutrophil Ins(l,4,5)P3 (the signal for intracellular calcium release) in response to the secretagogue N-formyl-methionyl-leucyl-phenylalanine (fMLP) was identical in unprimed and primed neutrophils, suggesting that modulation of phospholipase C activity is not involved in signalling neutrophil priming. A small but significant enhancement of fMLP-stimulated PtdIns(3,4,5)P3 accumulation (about 25%) was seen at 10 s in TNFα-primed versus unprimed cells, but 60 s following fMLP stimulation this enhancement was 620%. This suggests that enhanced and sustained PtdIns(3,4,5)P3 generation may be important in signalling mechanisms leading to the primed respiratory burst.