Eosinophil adoptive transfer system to directly evaluate pulmonary eosinophil trafficking in vivo
ABSTRACT Most in vivo studies of granulocytes draw conclusions about their trafficking based on examination of their steady-state tissue/blood levels, which result from a combination of tissue homing, survival, and egress, rather than direct examination of cellular trafficking. Herein, we developed a unique cell transfer system involving the adoptive transfer of a genetically labeled, bone-marrow-derived unique granulocyte population (eosinophils) into an elicited inflammatory site, the allergic lung. A dual polychromatic FACS-based biomarker-labeling system based on the IL4-eGFP transgene (4get) or Cd45.1 allele was used to track i.v. transferred eosinophils into the airway following allergen or TH2-associated stimuli in the lung in multiple mouse strains. The system was amenable to reverse tagging of recipients, thus allowing transfer of nonlabeled eosinophils and competitive tracking of multiple populations of eosinophils in vivo. The half-life of eosinophils in the blood was 3 h, and migration to the lung was dependent upon the dosage of transferred eosinophils, sensitive to pertussis toxin pretreatment, peaked at ∼24 h after adoptive transfer, and revealed a greater than 8-d eosinophil half-life in the lung. Eosinophil migration to the lung was dependent upon recipient IL-5 and IL-13 receptor α1 and donor eosinophil C-C chemokine receptor type 3 (CCR3) and interleukin 1 receptor-like 1 (ST2) in vivo. Taken together, this unique eosinophil transfer system provides an unprecedented opportunity to examine airway eosinophil migration without the need for extensive efforts to acquire donor source and time-consuming genetic crossing and has already been used to identify a long eosinophil half-life in the allergic lung and a definite role for ST2 in regulating eosinophil trafficking.
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ABSTRACT: Cellular microchimerism is defined as the presence of small populations of cells from one individual in another genetically distinct individual. The pivotal role of cellular microchimerism in a variety of immune settings is increasingly recognized, e.g. in context of pregnancy, transplantation and cancer. However, the detection of chimeric cells is overshadowed by technical limitations. This study aimed to overcome these limitations by testing the sensitivity and detection limit of a molecular biology approach (quantitative polymerase chain reaction, qPCR) and a cellular approach (flow cytometry) in order to identify experimentally induced cellular microchimerism in mice. Leukocytes isolated from lymph nodes or spleens of transgenic enhanced green fluorescent protein (eGFP) and CD45.1 mice respectively were used as targets to be detected as microchimeric cells among wild type (wt) or haploidentical cells. The detection limit of microchimeric cells by flow cytometry was 0.05 % or lower for the respective eGFP+ or CD45.1+ cell subsets, which equals 48 cells or fewer per 1 x 105 wt cells. The detection limit of CD45.1+ and CD45.2+ cells among haploidentical CD45.1+2+ cells by flow cytometry was 48 cells (0.05%) and 198 cells (0.2%), respectively. Using qPCR, a detection limit of 198 eGFP+ per 1 x 105 wt cells, respective 0.2 %, could be achieved. We here introduce two technical approaches to reliably detect low number of chimeric cells at a low detection limit and high sensitivity in transgenic mouse systems.Journal of immunological methods 04/2014; 406. DOI:10.1016/j.jim.2014.03.009 · 2.01 Impact Factor
- Journal of Reproductive Immunology 05/2012; 94(1):99. DOI:10.1016/j.jri.2012.03.426 · 2.37 Impact Factor
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ABSTRACT: Objectives: Interleukin (IL)-33 is a novel member of the IL-1 cytokine family and a ligand for the orphan IL-1 family receptor ST2. IL-33 induces T helper 2-type inflammatory responses and is considered to play a crucial rule in allergic inflammatory reactions, such as asthma and atopic dermatitis. However, the role of IL-33 and its receptor ST2 in chronic rhinosinusitis remains unclear. Study Design: In vitro study. Methods: The expression patterns of IL-33 and ST2 at both mRNA and protein levels in nasal polyps from eosinophilic chronic rhinosinusitis (ECRS) patients (n=10) and non-ERCS patients (n=13) as well as in seemingly normal mucosa of the uncinate processes in patients without sinusitis (control; n=5) were compared using immunohistochemical staining, enzyme-linked immunosorbent assay and real-time polymerase chain reactions. Results: ST2-positive cells in the inflammatory cells in the subepithelial layer were significantly higher in the ECRS group than other groups. The expression of ST2 mRNA in polyps of the ECRS group was significantly increased compared with controls. Many ST2 positive eosinophils were observed in the mucosa of ECRS but not of non-ECRS patients. The expression level of IL-33 mRNA was not significantly different among the three groups. Conclusions: The current study suggests that IL-33 and its receptor ST2 may play important roles in the pathogenesis of chronic rhinosinusitis, especially in ECRS, through the increased expression of ST2 in eosinophils.The Laryngoscope 04/2014; 124(4). DOI:10.1002/lary.24462 · 2.03 Impact Factor