Interference Between AvBD6and ACTH Mediated By MCRs Modulate Lymphocytes Growth

To explore the effect and mechanism of avian β-defensin6(AvBD6) and adrenocorticotrophin (ACTH) on chicken immune system, firstly, using the model of chicken peripheral blood lymphocytes (Ch-PBL), we study the effect of AvBD6on Ch-PBL in the different immunization or infection status. In order to clarify the receptor of AvBD6or ACTH combined with Ch-PBL, we cloned the MC2R of Roman brown hens, and prepare the antibody of three linear B-cell epitope(anti-MC2R). Finally, we studied the blocking effect of anti-MC2R on the combination of AvBD6or ACTH with Ch-PBL.The results were as follows:AvBD6has different effect on the Ch-PBL at different infection or immune status.①ACTH with the concentration of0.016-0.5μg/mL, inhibited Ch-PBL proliferation significantly, and with a dose-effect dependent manner under the concentration higher than0.5μg/mL.②AvBD6with concentration below0.625μg/mL has no significant effect on Ch-PBL proliferation; but Ch-PBL proliferation was inhibited by AvBD6with concentration higher than1.25μg/mL.③AvBD6with the concentration of0.02μg/mL can synergistic stimulate Ch-PBL with LPS on the concentration of0.031～0.5μg/mL; AvBD6with the concentration of0.03～0.125μg/mL can synergistic stimulate Ch-PBL proliferation with LPS on the concentration of1μg/mL.④AvBD6with the concentration higher than0.16μg/mL can active the proliferation of Ch-PBL suppressed by ACTH with the concentration of0.01μg/mL; and AvBD6with the concentration higher than0.31μg/mL can active the proliferation of Ch-PBL suppressed by ACTH with the concentration of0.02ug/mL.We cloned and analyzed the MC2R cDNA and amino acid of Roman brown hens, then selected "15-AGQTSIPS-22,227-TSAVHQ-232,327-GEYYRSTP-334" as the epitope which have overlaps and cross with MC5R, but not overlaps with chicken TLR. The B-cell epitope antibody have positive action with adrenal cortex and adrenal gland epithelial cells with MC2R expression, and the lymphocytes of thyms, spleen, bursa, and Ch-PBL with MC5R expression.The anti-MC2R with the concentration of1/2-1/32stock solution can block the inhibition of Ch-PBL by ACTH with the concentration of0.5μg/mL; The anti-MC2R with the concentration of1/2and1/4stock solution can block the inhibition of Ch-PBL by AvBD6with the concentration of0.2μg/mL.We can initially speculate that, at early stage of the gram-negative bacteria infection with low level of LPS and AvBD6, AvBD6can activate the immune response and inflammation; rather late in infection with high level of LPS and AvBD6, AvBD6may inhibit lymphocyte proliferation and anti-inflammate; AvBD6can activate the suppressed immune system by stress. Therefore, AvBD6maybe a new molecule to maintain the homeostasis of chicken immune system; and MCRs maybe the co-receptor in the combination of AvDB6or ACTH with Ch-PBL.