Neutrophil fluorescence in clozapine users is attributable to a 14kDa secretable protein

Clozapine is the only antipsychotic agent with demonstrated efficacy in refractory schizophrenia. However, use of clozapine is hampered by its adverse effects, including potentially fatal agranulocytosis. Recently, we showed an association between neutrophil autofluorescence and clozapine use. In this study, we evaluated the subcellular localization of clozapine-associated fluorescence and tried to elucidate its source. Neutrophils of clozapine users were analyzed with fluorescence microscopy to determine the emission spectrum and localization of the fluorescence signal. Next, these neutrophils were stimulated with different degranulation agents to determine the localization of fluorescence. Lastly, isolated neutrophil lysates of clozapine users were separated by SDS-PAGE and evaluated. Clozapine-associated fluorescence ranged from 420 nm to 720 nm, peaking at 500-550 nm. Fluorescence was localized in a large number of small loci, suggesting granular localization of the signal. Neutrophil degranulation induced by Cytochalasin B/fMLF reduced fluorescence, whereas platelet-activating factor (PAF)/fMLF induced degranulation did not, indicating that the fluorescence originates from a secretable substance in azurophilic granules. SDS-PAGE of isolated neutrophil lysates revealed a fluorescent 14kDa band, suggesting that neutrophil fluorescence is likely to be originated from a 14kDa protein/peptide fragment. We conclude that clozapine-associated fluorescence in neutrophils is originating from a 14kDa soluble protein (fragment) present in azurophilic granules of neutrophils. This protein could be an autofluorescent protein already present in the cell and upregulated by clozapine, or a protein altered by clozapine to express fluorescence. Future studies should further explore the identity of this protein and its potential role in the pathophysiology of clozapine-induced agranulocytosis.