We were intrigued by the recent article by Kuijpers et al1 on the binding of annexin V to neutrophils obtained from Barth syndrome patients. The authors reported that freshly isolated neutrophils from these patients were readily labeled with annexin V (a phosphatidylserine [PS]–binding protein) in the absence of other indices of apoptosis and concluded that these cells expose an alternative ligand for annexin V that is distinct from PS. We suggest that other, recent findings could be invoked to explain these novel observations in cardiolipin (CL)–deficient Barth syndrome cells.
PS externalization is an important “eat me” signal on apoptotic cells and serves to alert neighboring macrophages. Neutropenia (reported to be a common finding in Barth syndrome, albeit not a prominent feature in the cohort examined by Kuijpers et al) could thus be linked to the aberrant exposure of PS on circulating neutrophils. Conversely, the lack of PS externalization in neutrophils from chronic granulomatous disease (CGD) patients could contribute to defective clearance and the formation of granulomas in these individuals.2 However, Kuijpers et al argue that annexin V binding to Barth syndrome neutrophils is unlikely to reflect PS externalization because macrophages failed to engulf these cells upon cocultivation in vitro. While the existence of alternative ligands for annexin V cannot be excluded (indeed, annexin V was recently found to associate with the cytoplasmic domain of the β5 integrin3 ), we wish to point out that the failure of macrophage clearance does not a priori rule out the presence of PS molecules on the cell surface. Previous studies in neutrophil-like HL-60 cells and other cell lines have shown that PS oxidation is an integral part of the apoptosis program; hence, exposition of PS alone in the absence of PS oxidation may not suffice to mediate clearance of cell corpses.4,5 Moreover, Kagan and his associates (Borisenko et al6 ) have provided strong evidence that macrophages have a sensitivity threshold for PS that facilitates reliable discrimination of apoptotic cells; it is thus conceivable that Barth syndrome neutrophils expose subthreshold amounts of PS, detectable by the annexin V assay, yet insufficient to mediate macrophage clearance. Macrophage recognition may also depend on cofactors (eg, annexin I, a caspase-dependent “eat me” signal that colocalizes with PS on apoptotic cells7 ) that are not expressed on freshly isolated Barth syndrome cells. Finally, caspase-independent PS externalization in neutrophils has been documented previously,2 and therefore the absence of other signs of apoptosis cannot be taken as evidence that cells fail to expose PS.
The findings of Kuijpers et al1 raise the interesting possibility that mitochondrial CL deficiency is directly linked to PS externalization. In support of this notion, recent studies in yeast with a disruption in the gene encoding CL synthase and therefore lacking CL in mitochondrial membranes have revealed a labilization of cytochrome c binding in these organelles.8 Moreover, recent studies in mammalian cells show that cytosolic cytochrome c may serve as a catalyst for PS oxidation with subsequent cell surface externalization of PS.9 It would therefore be of considerable interest to assess whether (constitutive) mitochondrial extrusion of cytochrome c could facilitate the egress of PS, and consequently the binding of annexin V, in CL-deficient Barth syndrome neutrophils. In addition, further analyses of yeast that lack CL synthase, or yeast that harbor a taz1 mutant and therefore display aberrant CL metabolism,10 may also shed some light on the mechanism of PS externalization in CL-deficient and -proficient cells.
Cardiolipin and annexin V: unrelated issues
The issue raised that annexin V may still recognize phosphatidylserine (PS) on the neutrophils freshly obtained from patients suffering from Barth syndrome (BTHS) is of course correct as was discussed in our paper. In particular, the point raised as to whether oxidized or nonoxidized lipids could be responsible for the basal annexin V staining has been mentioned by us as a possibility that we cannot exclude. On the other hand, oxidized PS would have been detected by the high-performance liquid chromatography–mass spectrometry (HPLC-MS) methods used. To date, we were obviously not able to detect this lipid form. We cannot answer the question of how much of the oxidized PS should be present to become detectable under conditions of an excess of nonoxidized PS or proper internal standards. However, in MS the specific position of PS would most likely easily allow for its discrimination, as well as the corresponding decrease in normal nonoxidized PS.
The issue of the lack of macrophage recognition of annexin V+ BTHS neutrophils was used by us to argue that annexin V may recognize an alternative ligand. Fadeel et al put this argument into the perspective of a certain as-yet-undefined threshold in the recognition of externalized phosphatidylserine and phagocytosis of apoptotic cells by macrophages, as put forward by Borisenko et al.1 Caspase-dependent annexin I coexpression with PS as a requirement for adequate macrophage recognition and uptake2 and the lack hereof in BTHS neutrophils makes more sense. However, the recent findings using PS receptor (PSR) knock-out mice make the interpretation again puzzling. These mice showed abnormal brain development and neonatal lethality in a fraction of PSR-/- mice.3 Subsequent studies in another murine PSR knock-out strain demonstrated similar developmental defects in brain and other organs but an intact clearance reaction of apoptotic bodies by PSR-/- macrophages.4 The latter findings question the actual role of the PSR. Its role may be far less important—if it exists—in the clearance mechanisms of apoptotic material than was suggested previously by the group of Fadok and Henson (Fadok et al5 ) because of redundancy in these biologic processes.
We do not believe that cardiolipin (CL) deficiency is directly linked to PS externalization because the circulating lymphocytes and monocytes in BTHS—being as CL-deficient as the circulating neutrophils—did not avidly bind annexin V. If this cellular difference is not taken into account, any speculation on the mechanism related to CL becomes less valid.
The suggestion of a catalyst function of cytochrome c in the cytosol to initiate PS oxidation may well exist in certain model systems6 but not in BTHS neutrophils. First, if so, this would (along the same line of reasoning of Fadeel et al) result in macrophage-mediated PS recognition, which is clearly not the case in our experiments. Second, in neutrophils there is little if any cytochrome c present,7 turning CL-defective cells into a ready-to-release state or making “labilization” of cytochrome c far less likely.
In sum, there is not yet a clear answer to the observations made in BTHS neutrophils. To date, the hypothesis of an alternative ligand fits best with our results.
Correspondence: Taco W. Kuijpers, Emma Children's Hospital, Academic Medical Center, Amsterdam, the Netherlands; e-mail: t.w.kuijpers@amc.uva.nl
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