A rapid biochemical method for measuring antigen-induced pulmonary eosinophil margination in allergic guinea pigs.
The ability of purified guinea pig peritoneal eosinophils (EOS) to oxidise 3,3',5,5'-tetramethylbenzidine (TMB) was assessed in the presence/absence of Br- (3 mM), and compared with that of unpurified elicited peritoneal polymorphonuclear leukocytes (PMN). Br- selectively stimulated EOS peroxidase activity in a cell number-dependent manner, which was not significantly affected by the presence of diluted lung homogenate. By comparison with the peroxidase activity of added purified EOS, lung parenchyma homogenate from naive guinea pigs was estimated to contain 1.04 +/- 0.18 x 10(5) cells/mg wet tissue (n = 6), a value comparable to those calculated from published histological analyses. This was not significantly increased by ovalbumin (OA) allergen inhalation in unsensitised guinea pigs (1.4 x 10(5) EOS/mg), but was increased two-fold over the latter control to 3.0 +/- 0.18 x 10(5) cells/mg after 17 h in animals sensitised by a single injection of OA and subsequently exposed to an aerosol of bronchoactive allergen (n = 13, p < 0.05). Similar results were obtained in a parallel study using bronchoalveolar lavage (saline challenge, 20.2 +/- 2.2% EOS in lavage fluid; OA challenge, 47.1 +/- 3.6% EOS; n = 6, p < 0.05). In animals that had been doubly sensitised (two injections) to OA, the pulmonary eosinophilic response measured biochemically was more pronounced (4.9 +/- 0.2 x 10(5) cells/mg) and was significantly greater than both a non-specific protein inhalation in this sensitisation group, and OA inhalation in singly sensitised animals (n = 12, p < 0.05). Sera from the latter group was shown to contain five times less specific anti-OA IgG than the doubly sensitised animals, suggesting that EOS margination in guinea pigs is proportionate to the animals' immune status for a defined immunological challenge. These data demonstrate that in vivo EOS migration into the whole guinea pig lung can be rapidly determined by biochemical methods, and thus facilitate the in vivo assessment of novel therapeutic agents against the eosinophilic inflammation characteristic of human allergic asthma.
Tagari
P
P
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Black
C
C
Marshall
S
S
Ford-Hutchinson
A W
AW
D000818:Animals; D001249:Asthma / Q000175:diagnosis* / Q000473:pathology; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D002465:Cell Movement; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology*; D006168:Guinea Pigs; D007158:Immunologic Techniques; D008297:Male; D009504:Neutrophils / Q000276:immunology; D010544:Peroxidases / Q000032:analysis*; D011657:Pulmonary Eosinophilia / Q000175:diagnosis* / Q000473:pathology; D013379:Substrate Specificity
D047209:Eosinophil Peroxidase; D010544:Peroxidases