[The effects of imiquimod on an animal model of asthma].
OBJECTIVE
To study the mechanism of imiquimod on asthma animals.


METHODS
(1) 40 mice and 48 rats were divided into 4 groups: control, asthma, dexamethasone and imiquimod groups. The asthma model was established. The mice and rats in the imiquimod group were exposed to an aerosol of 0.15% imiquimod. Lung inflammation and airway responsiveness were measured 24 h after the last ovalbumin (OVA) challenge. The expression of Interleukin-4 (IL-4), interferon gamma (IFN-gamma), eotaxin, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), T-bet, GATA-3, STAT6 mRNA in the lung were determined by reverse transcription polymerase chain reaction (RT-PCR). The levels of eotaxin, MDC, and TARC in sera were tested by enzyme linked immunosorbent assay (ELISA). The expression of T-bet, GATA-3 and STAT6 proteins in the lung were measured by Western blot. (2) Parabronchial lymphnodes (PBLN) were isolated and cultured. The PBLN cells were divided into blank control, positive control, dexamethasone and drug groups (1 - 3 subgroups), cultured for different hours, and the expressions of IL-4 and IFN-gamma in supernatants were determined by ELISA, The mRNA expressions of the cytokines in cells weredetected by RT-PCR. (3) Flow cytometry was used to detect intracellular IL-4 and IFN-gamma production in spleen T lymphocytes. (4) CD(4)(+) T cell of spleen pellets were subject to assessment of T-bet and GATA-3 protein and mRNA expression respectively.


RESULTS
The expiration resistance was determined before and after injection of acetylcholine chloride (20 - 160 microg/ml), and expiration resistances of the asthmatic group (6.26 +/- 0.85), (11.55 +/- 3.09), (28.74 +/- 5.94), (3710.83 +/- 197.49) cm H(2)Oxml(-1)xs(-1), were significantly elevated compared with those of the control group (1.34 +/- 0.16), (3.47 +/- 0.49), (9.29 +/- 1.27), (25.22 +/- 5.44) cm H(2)Oxml(-1)xs(-1), D = 88.98, 56.00, 45.00, 108.00, all P < 0.01). The numbers of eosinophils and lymphocytes, the thicknesses of WA/Pi and ASM/Pi in the asthmatic group [(26.0 +/- 1.6)/mm(2), (45.2 +/- 3.2)/mm(2), 12.0 +/- 1.4, 6.7 +/- 0.6] were all significantly higher than those of the imiquimod group [(12.4 +/- 2.9)/mm(2), (24.2 +/- 3.7)/mm(2), 9.2 +/- 0.6, 4.0 +/- 0.5, D or q = 193.00, 16.92, 185.50, 7.66, all P < 0.01]. In the imiquimod group, the mRNA and protein expressions of T-bet (0.48 +/- 0.08, 0.48 +/- 0.17) were significantly increased compared with those of the asthmatic group (0.08 +/- 0.12, 0.18 +/- 0.06, D = 120.96, 177.98, all P < 0.01), the mRNA and protein expressions of GATA-3 in the imiquimod group were both significantly decreased compared with those of the asthmatic group (D = 166.96, 310.97, all P < 0.01). In the control group, only low concentrations of IFN-gamma [(22 +/- 5, 31 +/- 5) pg/ml] were detected in PBLN cell cultures. After 24 or 48 h stimulation, the concentrations of IFN-gamma in drug 2 subgroup [(149 +/- 31), (154 +/- 28) pg/ml] and drug 3 subgroup [(166 +/- 30), (158 +/- 31) pg/ml] were increased significantly; Levels of IL-4 [druug 2 subgroup: (23 +/- 5), (39 +/- 11) pg/ml, drug 3 subgroup: (43 +/- 13), (56 +/- 12) pg/ml] were increased slowly compared with those in the OVA group (drug 2 subgroup 24 h IL-4, D = 9.90; drug 3 subgroup 24 h IL-4, D = 8.79, drug 2 subgroup 48 h IL-4, D = 8.80, drug 3 subgroup 48 h IL-4, D = 8.10, drug 2 subgroup 24 h IFN-gamma, q = 4.80, drug 3 subgroup 24 h IFN-gamma, q = 6.40, drug 2 subgroup 48 h IFN-gamma, q = 3.95, drug 3 subgroup 48 h IFN-gamma, q = 4.31, all P < 0.05). After imiquimod treatment, the mRNA and protein levels of T-bet in imiquimod group CD(4)(+) T cells were increased significantly compared with those in OVA group, and the mRNA and protein levels of GATA-3 were decreased significantly in CD(4)(+) T cells of imiquimod group compared with those in OVA group. The eotaxin, MDC and TARC levels of serum in asthma group [(593 +/- 41) pg/ml, (170 +/- 20) pg/ml, (221 +/- 25) pg/ml] were significant different from those in control group [(288 +/- 66) pg/ml, (100 +/- 33) pg/ml, (84 +/- 49) pg/ml], (eotaxin: q = 12.20, MDC: q = 8.00, TARC: q = 10.50, all P < 0.01). MDC and TARC levels of serum in imiquimod group [(84 +/- 13) pg/ml, (163 +/- 35) pg/ml] decreased as compared with those in asthma group (MDC: q = 9.80, TARC: q = 4.50, all P < 0.01) and MDC levels in imiquimod group were no different with normal group (q = 1.80, P > 0.05). eotaxin levels of serum in imiquimod group [(501 +/- 76) pg/ml] increased as compared with those from normal group (q = 8.50, P < 0.01), and decreased as compared with those from asthma group (q = 3.70, P < 0.05). (4) The expression of eoaxin, MDC, TARC and STAT(6) on the bronchial epithelium in imiquimod group was decreased as compared with asthma group, but increased as compared with normal group. The eotaxin, MDC and TARC mRNA expression of the lung in asthma group (0.85 +/- 0.11, 0.96 +/- 0.10, 0.94 +/- 0.28) had significant differences from those in the control group (0.45 +/- 0.08, 0.39 +/- 0.09, 0.24 +/- 0.08, eotaxin: q = 3.00, MDC: q = 15.40, TARC: q = 5.90, all P < 0.01) and those in imiquimod group (0.65 +/- 0.17, 0.66 +/- 0.12, 0.66 +/- 0.34, eotaxin: q = 1.50, MDC: q = 8.10, TARC: q = 2.40, all P < 0.05).


CONCLUSIONS
These findings suggested that imiquimod can inhibit the airway inflammation of asthma animals by reducing GATA-3 mRNA and protein expression and increasing T-bet, STAT(6) mRNA and protein expression.
Yin
Kai-sheng
KS
Department of Respiratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Jin
Shu-xian
SX
Bian
Tao
T
Wu
Qiao-zhen
QZ
Wang
Xiang
X
Yao
Xin
X
D000276:Adjuvants, Immunologic / Q000627:therapeutic use*; D000634:Aminoquinolines / Q000627:therapeutic use*; D000818:Animals; D001249:Asthma / Q000188:drug therapy*; D004195:Disease Models, Animal; D050990:GATA3 Transcription Factor / Q000235:genetics; D000077271:Imiquimod; D015847:Interleukin-4 / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017207:Rats, Sprague-Dawley; D020825:T-Box Domain Proteins / Q000235:genetics; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism; D000098815:T-bet Transcription Factor
D000276:Adjuvants, Immunologic; D000634:Aminoquinolines; D050990:GATA3 Transcription Factor; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D015847:Interleukin-4; D000077271:Imiquimod