[The molecular mechanisms of the effects of murine interferon-gamma transgenic expression on allergen-induced allergic model via adenoviral vector].
OBJECTIVE
To investigate adenoviral vector mediated murine interferon-gamma (mIFN-gamma) transgene expression and its effect on allergen-induced airway inflammation and multiple interleukin (IL) cytokines (IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-18) expression in a murine allergic model.


METHODS
Forty-eight mice were divided into 7 groups by random digits table: a negative control group (A), allergic model groups I, II, III (B, D, F), gene therapy groups I, II, III (C, E, G). Except for group A, mice of the other groups were peritoneally sensitized with ovalbumin (OVA, 15 microg/mouse) twice on day 0 and day 5, and challenged by inhalation of 0.5% OVA (20 ml per time) twice per day from day 12 to 14. On day 15, AdCMVmIFN-gamma (5 x 10(9) PFU/mouse) solution 50 microl was administrated by nasal drip in groups C, E and G. For groups A, B, D and F, 0.9% NaCl 50 microl was administrated by nasal drip. Groups A, B and C were sacrificed on day 18. Groups D and E were sacrificed on day 21. Groups F and G were sacrificed on day 25. The concentration of mIFN-gamma in bronchioalveolar lavage (BALF) was measured by enzyme-linked immunosorbant assay. Accumulation of inflammatory cells and eosinophils (EOS) were quantified by cell count and histopathological analysis. Multi-cytokine expression was tested by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).


RESULTS
(1) mIFN-gamma was efficiently expressed after gene therapy. The concentration of mIFN-gamma in BALF was (729.0 +/- 104.7) pg/ml 3 days after gene therapy (group C); it was (984.5 +/- 119.1) pg/ml after 6 days (group E); and (310.6 +/- 59.7) pg/ml after 10 days (group G). (2) The total cell number in BALF of group B and C was (318 +/- 41) x 10(3)/ml and (137 +/- 12) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS in BALF was 0.715 +/- 0.054 and 0.452 +/- 0.016, respectively (P < 0.01). The total cell number in BALF of group D and E was (183 +/- 23) x 10(3)/ml and (92 +/- 6) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS was 0.393 +/- 0.065 and 0.083 +/- 0.038, respectively (P < 0.01). The total cell number in BALF of groups F and G was (196 +/- 7) x 10(3)/ml and (98 +/- 15) x 10(3)/ml, respectively (P < 0.01); the constituent ratio of EOS was 0.253 +/- 0.035 and 0.068 +/- 0.025, respectively (P < 0.01). (3) The histopathological results showed that in the gene therapy groups, the infiltration of inflammatory cells was markedly reduced and the damage of airway epithelium was alleviated. (4) For group D and E, the ratio of IL-10 mRNA abundance to that of home gene was 0.14 +/- 0.10 and 0.49 +/- 0.27, respectively (P < 0.01); that of IL-12 was 0.15 +/- 0.05 and 0.63 +/- 0.17, respectively (P < 0.01); that of IL-13 was 0.76 +/- 0.17 and 0.37 +/- 0.10, respectively (P < 0.01). For group F and G, the ratio of IL-10 mRNA abundance to that of home gene was 0.13 +/- 0.04 and 0.27 +/- 0.17, respectively (P = 0.019); that of IL-12 was 0.14 +/- 0.05 and 0.35 +/- 0.21, respectively (P = 0.006); that of IL-13 was 0.57 +/- 0.24 and 0.30 +/- 0.09, respectively (P = 0.003). However, there were no statistically significant changes in IL-4, IL-5, IL-6, and IL-18 in lung tissue between allergic model groups (B, D, F) and gene therapy groups (C, E, G, P > 0.05).


CONCLUSIONS
(1) mIFN-gamma gene transferred via adenoviral vector could abrogate the infiltration of EOS in OVA-induced allergic model. (2) The molecular mechanisms for effect by AdCMVmIFN-gamma transgenic therapy on murine allergic model involve the indirect action by the upregulation of IL-10 and IL-12 expression and the downregulation of IL-13 expression locally in the lungs, in addition to the direct effects of locally overexpressed mIFN-gamma.
Chen
Bin
B
Department of Respiratory Medicine, Peking University People's Hospital, Beijing 100044, China.
Gao
Zhan-cheng
ZC
D000256:Adenoviridae; D000485:Allergens / Q000235:genetics; D000818:Animals; D001249:Asthma / Q000628:therapy*; D004195:Disease Models, Animal; D004804:Eosinophils / Q000276:immunology; D005260:Female; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000235:genetics* / Q000627:therapeutic use*; D007378:Interleukins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D019076:Transgenes
D000485:Allergens; D007378:Interleukins; D007371:Interferon-gamma