Peptides 24 (2003) 1785–1794 Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry Lisa K. Ryan a,b,∗ , Gill Diamond b , Sheela Amrute a , Zhimin Feng c , Aaron Weinberg c , Patricia Fitzgerald-Bocarsly a,1 a Department of Pathology and Laboratory Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07101-1709, USA b Department of Oral Biology, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07103, USA c Department of Oral Biology, Case Western Reserve University Dental School, Cleveland, OH, USA Received 13 July 2003; accepted 11 September 2003 Abstract Production of human -defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1–0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50 ng/ml LPS for 2 h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment. © 2003 Elsevier Inc. All rights reserved. Keywords: -Defensins; Lipopolysaccharide; Monocytes; Dendritic cells; Plasmacytoid dendritic cells; Flow cytometry; Chloroquine 1. Introduction -Defensins are a class of antimicrobial peptides that have been identified in a variety of mammalian species over the last 10 years. Three human -defensin peptides (HBD1–3) have been isolated to date [6,17,20,21], with at least 30 other genes in the family identified by genomic techniques [30,41,44]. These peptides, which range from 29 to 47 aa or 3 to 5 kDa, are typically expressed by epithelial cells at mu- cosal surfaces of various tissues and were first recognized for their antimicrobial activity, but have recently been appre- ciated for their role in bridging the innate and the adaptive immune responses (for review, see [10,33]). HBD1 and 2 are predominantly expressed in epithelial cells, and expression of HBD2 (but not HBD1) mRNA is in- ducible in these cells with lipopolysaccharide (LPS) [20,52]. Recently, HBD1 and 2 gene expression was detected in peripheral blood mononuclear cells (PBMC), monocytes, alveolar macrophages (AM) and monocyte-derived den- ∗ Corresponding author. E-mail addresses: ryanlk@umdnj.edu (L.K. Ryan), bocarsly@umdnj.edu (P. Fitzgerald-Bocarsly). 1 Co-corresponding author. dritic cells (MDDC) [15,16]. Furthermore, in these studies, HBD1 mRNA was upregulated by incubating these cells with 100ng/ml of LPS. However, increases in HBD1 pep- tide levels were not studied in cells of the immune system. Typical methods for detecting -defensins include West- ern blot analysis and mass spectrometry of extracted cell lysates [35,49]. Recently, a ProteinChip ® Array followed by surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrome- try, for detecting as little as 6 fmol of HBD1 in biological fluids was described [9]. In addition, HBD2 was detected to 0.06 fmol by a radioimmunoassay (RIA) of body fluids [23] and by enzyme-linked immunosorbent assay (ELISA) to 0.5 ng/10 6 cells [25]. Immunohistochemical staining of tissues and cytospin preparations of PBMC can also detect -defensins [15,49]. However, detection of these peptides in rare cells of the immune system is difficult due to the low levels of peptide produced and the small number of some cell types in PBMC. One technique that has been used to detect cytokine levels in specific cells of a mixed cell population is flow cytome- try with cell surface and intracellular staining. For example, the plasmacytoid dendritic cell (PDC) [47] makes up only 0.1–0.5% of PBMC from normal, healthy donors, yet is 0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.09.021