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

Abstract

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.

Bacterial cyanogenesis occurs in the cystic fibrosis lung

“…In the concentrations reported, cyanide has been shown to inhibit the function of the enzyme myeloperoxidase and prevent production of hypochlorous acid during the oxidative burst [17]. Cyanide may also impair neutrophil migration and cause morphological changes related to damage to the cell cytoskeleton [18]. Interestingly, neutrophils can produce very small amounts of cyanide during chlorination of bacterial cell membranes [19], but this contribution is likely to be negligible in comparison with the high cyanide levels reported herein.…”

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

“…3,5,6 However, in neutrophils, these organelles hardly play a role in energy metabolism as they do in other cells. This conclusion was derived from the experiment shown in Figure 1, where ATP concentrations in neutrophils were measured under various conditions.…”

“…In agreement with this suggestion is the sensitivity of the neutrophil Dc m to uncouplers or K þ ionophores, such as CCCP and valinomycin, observed by us (see Figure 3) and others. 3,35 The cytochrome c deficiency raised the question about functionality of the intrinsic (mitochondrial) pathway of apoptosis in neutrophils, which has not yet been studied before. This pathway is initiated by release of cytochrome c from the mitochondria, promoting the oligomerization of Apaf-1 and recruitment of the caspase-9 zymogen.…”

“…This point of view was mainly based on the observation that mitochondrial poisons like cyanides do not influence cellular functions in neutrophils. 2 Moreover, electron microscopy studies identified hardly any mitochondria in neutrophils (see the references in Fossati et al 3 ), and mitochondrial respiration was found to be very low in these cells. 4 However, mitochondria have been recently visualized in neutrophils as a tubular network, by means of specific fluorescent dyes.…”

“…4 However, mitochondria have been recently visualized in neutrophils as a tubular network, by means of specific fluorescent dyes. 3,5 Furthermore, in neutrophils undergoing spontaneous or induced apoptosis, the mitochondria form clusters to which the proapoptotic Bax protein also is localized. [5][6][7] Such a subcellular redistribution of Bax to mitochondria upon apoptosis is known to result in the permeabilization of the outer mitochondrial membrane, with the subsequent release of additional proapoptotic proteins.…”