(C) Representative purity of PB neutrophils, as shown by freshly isolated GINs from mice

(C) Representative purity of PB neutrophils, as shown by freshly isolated GINs from mice. G-CSFCinduced neutrophils (GINs). In contrast to GINs but similar to (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol PBNs, the enhanced bacterial killing by AINs accompanied both better granule maturation and greater coexpression of CD66 antigen with the integrin 2 subunit CD18. Consistently, anti-CD18 antibody neutralized Am80-induced bactericidal activities of AINs. These studies demonstrate that Am80 is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66 with CD18 to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80 as a cost-effective treatment option. Introduction Neutrophils, the most common granulocytes, constitute up to 70% of circulating leukocytes that primarily defend pathogen infections. Malignancy chemotherapy-induced neutropenia is usually a hematologic disorder marked by large decrease in the number of neutrophils in the bloodstream. It has been more than 2 decades since G-CSF was first (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol used to treat acquired and congenital neutropenia1 by promoting granulopoiesis of hematopoietic stem cells (HSCs). Despite the considerable clinic benefits of this agent when used as primary prophylaxis,2 neutropenia induced by chemotherapy in cancer patients still remains a devastating issue with substantial morbidity, mortality, (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol and cost, which places a significant burden on the individual patient and the healthcare system.1,3C6 An earlier pioneering study with G-CSF administered to normal individuals reveals that this agent adversely affects neutrophil chemotaxis and bactericidal activity against (retinoic acid (RA) through its selective binding to retinoic acid receptor (RAR),9,11 a transcription factor activated by RA12 to regulate granulocytic differentiation of both leukemic myeloblasts and HSCs.13C17 RA, a naturally occurring form of vitamin A, plays key functions in the development of the body plan and induces the differentiation of many types of normal and malignant cells.18C20 To date, RA treatment of acute promyelocytic leukemia (APL) represents the best example of successful differentiation-induction therapy in clinical oncology21; however, the side effects associated with RA therapy are generally serious and RA resistance is usually a common event.22C24 Several studies have exhibited that RAR regulates Am80-induced granulocytic differentiation.25C27 Moreover, Am80 is approximately 10-fold more efficient, with lower toxicity, than either RA or other retinoids used as differentiation therapy in APL patients.10,28 Currently, Am80 has been approved for the treatment of APL in Japan10 and tested clinically for several other cancers/diseases in the United States and Europe (http://www.cytrx.com/tamibarotene; http://clinicaltrials.gov). The advances in the use of Am80 to induce granulocytic differentiation led us to test this agent as a means to enhance neutrophil bactericidal activity arising from granulopoiesis during immune development. We report here that Am80 possesses significantly greater activity than G-CSF as an inducer of neutrophil differentiation and immune development, probably through its promotion of HSC-derived granulopoiesis by mediating the differential effects of CD66 on CD18 activation. Methods Human PBNs, cells, and cell culture Human (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol peripheral blood (PB) was taken from healthy volunteers in accord with a protocol approved by the Children’s Hospital Los Angeles/University of Southern California Keck School of Medicine (CHLA/USC) Committee on Clinical Investigations. Informed consent was obtained in accordance with the Declaration of Helsinki. Details for cells, cell culture, and purification of PBNs are available in supplemental Methods (available on the Web site; see the Supplemental Materials link at the top of the online article). For transmission electron microscopy and magnetic sorting of CD15+ neutrophils, see supplemental Methods. Phagocytotic and bacterial killing assays Each 1 105 to 2 106 of freshly purified PBNs, mouse peripheral blood neutrophils (MPBNs), cyclophosphamide (CPA)Cneutropenic mice PB neutrophils (C-MPBNs), G-CSFCinduced neutrophils (GINs), and Am80-induced neutrophils (AINs) were suspended with 500 L of DMEM with 10% FBS and incubated with log-phase DH5 (ATCC), or (ATCC) at an MOI of 5 or 10 at 37C for up to 60 minutes, whereas bacteria in the absence of neutrophils were used to determine growth. See details in supplemental Methods. Degranulation and neutrophil elastase release analyses See details in supplemental Methods. Cell proliferation, cell death, and morphologic analysis of granulocytic differentiation Experiments were performed as described17,29 and details are available in supplemental Methods. Western blotting Western blotting (WB) was performed as described.15 Antibodies for lactoferrin (Abcam), MMP-9 (Merck Chemicals), MPO (Cell Signaling Technology), and LL-37 (Biolegend) were Rabbit polyclonal to PFKFB3 used in (3β,20E)-24-Norchola-5,20(22)-diene-3,23-diol analyses. In situ bactericidal killing and anti-CD18 antibody neutralization of bactericidal activity In situ neutrophil bactericidal activities were decided using LIVE/DEAD BacLight Viability Package (Life Systems),.