Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70-90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors, patients with vasculitis, and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1, a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor.