Dale A. Moulding

Molecular control of neutrophil apoptosis

Human neutrophils constitutively undergo apoptosis and this process is critical for the resolution of inflammation. Whilst neutrophil apoptosis can be modulated by a wide variety of agents including GM‐CSF, LPS and TNF‐α, the molecular mechanisms underlying neutrophil death and survival remain largely undefined. Recent studies have shown the involvement of members of the Bcl‐2 protein family (especially Mcl‐1 and A1) and caspases in the regulation and execution of neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen‐activated protein kinases, also play critical roles in transducing the signals that result in neutrophil apoptosis or extended survival. This review summarises current knowledge on the molecular mechanisms and components of neutrophil apoptosis.

BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis

The human neutrophil spontaneously undergoes apoptosis, but this type of cell death can be delayed or accelerated by a wide variety of agents. There are wide discrepancies in the literature regarding the expression of the Bcl‐2 family of proteins in human neutrophils. Here, we show that A1, Mcl‐1, Bcl‐XL, and Bad are major transcripts in human neutrophils and that levels of these transcripts are cytokine regulated. However, no Bcl‐XL protein was detected in Western blots. Protein levels for the proapoptotic proteins Bad, Bax, Bak, and Bik remained constant during culture, despite changes in the levels of mRNA for these gene products. These proapoptotic proteins were extremely stable, having very long half‐lives. In contrast, A1 and Mcl‐1 transcripts were extremely unstable (with ∼3‐h half‐lives), and Mcl‐1 protein was also subject to rapid turnover. These results indicate that neutrophil survival is regulated by the inducible expression of the short‐lived Mcl‐1 and possibly the A1 gene products. In the absence of their continued expression, these prosurvival gene products are rapidly turned over, and then the activity of the stable death proteins predominates and promotes apoptosis.

The Mitochondrial Network of Human Neutrophils: Role in Chemotaxis, Phagocytosis, Respiratory Burst Activation, and Commitment to Apoptosis

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC50 = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 μg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.

In vivo localisation and stability of human Mcl‐1 using green fluorescent protein (GFP) fusion proteins

Mcl‐1 is an anti‐apoptotic member of the Bcl‐2 family of proteins. We have expressed full length and mutated GFP:Mcl‐1 fusion proteins to define structural motifs that control protein localisation and stability. When expressed in U‐937 cells, full length Mcl‐1 locates primarily within mitochondria and its half‐life was approximately 3 h, which was identical to the native, endogenously expressed protein. When the terminal 20 amino acids from the C‐terminus of the protein were detected, the protein was diffused in the cytoplasm, but its stability was unaffected. This confirms that this region is responsible for efficient targeting to mitochondria. Surprisingly, deletion of 104 amino acids (residues 79–183) that contain putative PEST sequences and other stability regulating motifs, did not affect protein stability.

Regulation of neutrophil FcgammaRIIIb (CD16) surface expression following delayed apoptosis in response to GM-CSF and sodium butyrate.

When neutrophils undergo apoptosis, they lose expression of the surface receptor CD16 (FcγRIIIb). Thus levels of surface CD16 are good indicators of apoptotic or non‐apoptotic neutrophils. Shedding of CD16 occurs via the activity of a metalloproteinase that cleaves the receptor from the plasma membrane. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and sodium butyrate both stimulate neutrophil gene expression, protect these cells from apoptosis, and maintain expression of surface CD16. In this report we have investigated whether these agents maintain surface expression of CD16 via (1) decreased shedding (2) increased mobilization of the internal pool of pre‐formed CD16, or (3) via de novo biosynthesis of new receptor molecules. Although GM‐CSF and sodium butyrate both preserved surface expression of CD16, GM‐CSF actually accelerated the rate of shedding of this receptor. Maintenance of surface levels was achieved by substantial mobilization of the internal pool of CD16. Sodium butyrate, on the other hand, maintained surface expression without extensive store depletion via a mechanism that appeared to involve a decreased rate of shedding. In these experiments we could find no evidence for de novo biosynthesis of CD16 stimulated by either GM‐CSF or sodium butyrate. These experiments indicate that multiple mechanisms exist for the maintenance of surface CD16 during rescue of neutrophils from apoptosis by different agents. J. Leukoc. Biol. 65: 875–882; 1999.

Activation of Human Neutrophils by Soluble Immune Complexes: Role of FcγRII and FcγRIIIb in Stimulation of the Respiratory Burst and Elevation of Intracellular Ca2+a

Activation of control, unprimed neutrophils with soluble immune complexes fails to generate a respiratory burst. However, if the cells are primed with either tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor prior to addition of soluble immune complexes, then a rapid and transient burst of reactive oxidant secretion is observed. In unprimed neutrophils the soluble immune complexes stimulate an intracellular Ca2+ transient that arises from the mobilization of intracellular Ca2+. However, in primed cells, an extra intracellular Ca2+ signal is observed that arises from Ca2+ influx. After removal of Fc gamma RIIIb by treatment with pronase or PI-PLC, the soluble immune complexes fail to activate a respiratory burst in unprimed neutrophils and the extra Ca2+ signal is not observed. These results indicate that during priming Fc gamma RIIIb becomes functionally activated and thence its ligation leads to stimulated Ca2+ influx and the generation of intracellular signals that lead to NADPH oxidase activation. Experiments using Fab/F(ab)2 fragments to specifically crosslink either Fc gamma RII or Fc gamma RIIIb and experiments with neutrophils from an individual with Fc gamma RIIIb gene deficiency confirm this important function for Fc gamma RIIIb in neutrophil activation.