The overall objective of this grant proposal is to investigate how perivascular mast cells modulate microvascular endothelial interactions with circulating eosinophils. The ability of anti-IgE activated mast cells in human lung, nasa mucosa, or skin to express a 'TH2' cytokine profile (IL-4+, IL-5+, GM-CSF+, gammaIFN, IL-2) able to influence endothelial cell adhesion molecule expression (IL-4) and eosinophil function (IL-5, GM-CSF) will be assessed by combining in situ hybridization to identify cytokine mRNA with immunostaining (tryptase or chymase) to identify T or TC mast cells. In selected experiments, purified populations of mast cells will be in situ hybridized with both a non-radioactively labelled digoxigenin-11-UTP gammaIFN RNA probe and an S labelled IL-4 RNA probe to determine whether individual mast cells co-express IL-4 and/or gamma interferon. The ability of stimulated mast cell supernatant applied to mesenteric blood vessels to effect the rolling, adhesion and transmigration across endothelium of fluorescently labelled eosinophils in vivo in a rabbit mesentery model will be studied utilizing intravital video microscopy. The relative importance to eosinophil adhesion of individual mediators in mast cell supernatant will be studied using defined mast cell mediators (IL-4, histamine, LTC), as well as mast cell lysates, and supernatant studies in the presence of specific mediator and cytokine antagonists. Unidentified factors in stimulated mast cell supernatant (which induce eosinophil adhesion) will be purified to homogeneity to determine if they are novel cytokines. Eosinophils which have rolled, adhered and transmigrated in vivo will be recovered and studied by in situ hybridization for IL-5 and GM-CSF mRNA expression. In in vitro studies, peripheral blood eosinophils will be purified utilizing a MACS (magnetic cell separator) and separated according to density on Pecoll gradients. hypodense and normodense eosinophils will be stimulated with either the calcium ionophore A23187 or mast cell supernatant for varying time periods. Cytokine (IL-5, GM-CSF) mRNA expression by hypodense and normodenses eosinophils will be assessed by combining staining with carbol chromotrope 2R (to identify eosinophils) and in situ hybridization to identify cytokine mRNA. Supernatant will be assessed for LTC as well as GM-CSF and IL-5 protein using bioassays and an immunoassay. Overall, these studies will define whether mediators derived from perivascular mast cells communicate directly or indirectly (via activation of endothelium) with circulating eosinophils to influence their adhesion to influence their adhesion and state of activation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI033977-02
Application #
2069031
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1994-05-01
Project End
1997-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Lim, Dae Hyun; Cho, Jae Youn; Miller, Marina et al. (2006) Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice. Am J Physiol Lung Cell Mol Physiol 291:L265-71
Broide, David H (2005) DNA vaccines: an evolving approach to the treatment of allergic disorders. Allergy Asthma Proc 26:195-8
Cho, Jae Youn; Miller, Marina; Baek, Kwang Je et al. (2004) Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest 113:551-60
Youn, Cho Jae; Miller, Marina; Baek, Kwang Je et al. (2004) Immunostimulatory DNA reverses established allergen-induced airway remodeling. J Immunol 173:7556-64
Cho, Jae Youn; Miller, Marina; Baek, Kwang Je et al. (2004) Immunostimulatory DNA inhibits transforming growth factor-beta expression and airway remodeling. Am J Respir Cell Mol Biol 30:651-61
Ikeda, Reid K; Miller, Marina; Nayar, Jyothi et al. (2003) Accumulation of peribronchial mast cells in a mouse model of ovalbumin allergen induced chronic airway inflammation: modulation by immunostimulatory DNA sequences. J Immunol 171:4860-7
Ikeda, Reid K; Nayar, Jyothi; Cho, Jae Youn et al. (2003) Resolution of airway inflammation following ovalbumin inhalation: comparison of ISS DNA and corticosteroids. Am J Respir Cell Mol Biol 28:655-63
Miller, Marina; Cho, Jae Youn; Baek, Kwang Je et al. (2002) Plasmid DNA encoding the respiratory syncytial virus G protein protects against RSV-induced airway hyperresponsiveness. Vaccine 20:3023-33
Broide, David H; Miller, Marina; Castaneda, Diego et al. (2002) Core 2 oligosaccharides mediate eosinophil and neutrophil peritoneal but not lung recruitment. Am J Physiol Lung Cell Mol Physiol 282:L259-66

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