Intracerebroventricular injection of leukotriene B4 attenuates antigen-induced asthmatic response via BLT1 receptor stimulating HPA-axis in sensitized rats

Abstract

Background: Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immune pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB4 level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats.

Methods: Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (RL) and dynamic lung compliance (Cdyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits.

Results: Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB4 via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB4 via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10 ng), but not by BLT2 antagonist LY255283.

Conclusions: LTB4 administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB4 on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.

Figure 1

LTB₄ via i.c.v attenuates the antigen-induced changes of pulmonary function in rats, and U75302 blocks the inhibitory effect of LTB₄. On day 21 after ovalbumin sensitization, rats were challenged for 5 min with aerosolized 2% ovalbumin and the airway resistance (R_L) (A) and dynamic lung compliance (C_dyn) (B) were measured before and 5 min after the antigen challenge. Data are expressed as the mean ± S.D of vehicle-sham (n = 8), vehicle-OVA (n = 10), LTB₄-OVA (n = 9), U75302-OVA (n = 9) and U75302-LTB₄-OVA (n = 10). ^##P < 0.01 vs the vehicle-sham group; **P < 0.01 vs the vehicle-OVA group; ^††P < 0.01 vs the LTB₄-OVA group.

Figure 2

LTB₄ via i.c.v attenuates the antigen-induced increases of inflammatory cell in the BALF, and U75302 blocks the inhibitory effect of LTB₄. Total inflammatory cells in BALF were counted, and cell classification was performed on a minimum of 200 cells to classify monocytes (lymphocytes and macrophages) and polymorphonuclear cells (eosinophils and neutrophils) 24 hr after the final antigen challenge. Data are expressed as the mean ± S.D. of vehicle-sham (n = 8), vehicle-OVA (n = 10), LTB₄-OVA (n = 9), U75302-OVA (n = 9) and U75302-LTB₄-OVA (n = 10). ^##P < 0.01 vs the vehicle-sham group;**P < 0.01 vs the vehicle-OVA group; ^††P < 0.01 vs the LTB₄-OVA group.

Figure 3

Lung histopathology. Haematoxalin and eosin-stained lung tissues were evaluated and scored for eosinophil cell infiltration. Compared with vehicle-sham group (A), marked infiltration of eosinophil cells in peribronchiolar space and perivascular space were observed in the vehicle-OVA (B), there were no significant eosinophil cell infiltration presented in the group of LTB₄-OVA (C). U75302-OVA (D) and U75302-LTB₄-OVA (E) did not show any attenuation of eosinophil infiltration. Scores of eosinophil cell infiltration were graded on the basis of severity of inflammation (F). ^##P < 0.01 vs the vehicle-sham group;**P < 0.01 vs the vehicle-OVA group;^††P < 0.01 vs the LTB₄-OVA group.

Figure 4

LTB₄ via i.c.v injection increases CORT and ACTH levels in rat plasma. CORT (A) and ACTH (B) levels in plasma were measured using commercial ELISA kits at 30 min before and after LTB₄ i.c.v, and 30 min after antigen challenge. Data are expressed as the mean ± S.D (n = 6 in each group). ^##P < 0.01 vs the basal level (pretreatment); **P < 0.01 vs after antigen challenge;^$$P < 0.01 vs before antigen challenge; ^††P < 0.01 vs the LTB₄-OVA group.

Figure 5

LTB₄ via i.c.v injection increases expression of CRH in the hypothalamus in sensitized rats. Representative blots and densitometry analysis for expression of CRH mRNA by RT-PCR (A) and protein by Western blot (B). The blots are representative of three similar experiments, each run with independent samples. Data are expressed as the mean ± S.D. of each group (n = 3). ^#P < 0.05 vs the vehicle-sham group;^†P < 0.05 vs the LTB₄-OVA or LY255283-LTB₄-OVA group