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. Author manuscript; available in PMC: 2013 Oct 12.
Published in final edited form as: Circ Res. 2012 Aug 17;111(9):1190–1197. doi: 10.1161/CIRCRESAHA.112.277475

Figure 3. Immunomodulation of IL-6 and IL-1β responses to TLR7/8 and/or TLR9 by nicotine and Ang II in vitro and in vivo.

Figure 3

Individual responses of isolated splenocytes from 4–5 week old WKY (n=5) and SHR (n=5) rats are shown in upper panels 3A to D. In vivo changes in serum levels in response to subcutaneous (s.c.) infusions of nicotine or Ang II in WKY and SHR are in the lower panels 3E to 3H. Numbers above each bar graph represent the number of animals (n) in the particular group.
  • Splenocyte responses in vitro: Splenocytes were activated by ligands to TLR7/8 (Clo97, 1.0 µM) and to TLR9 (CpG, 10 µM). IL-6 was measured in culture supernatants. Panels A - D portray responses to activation of the TLRs with exposure to nicotine (10 µM) in WKY and SHR (Panels A and B respectively) and with exposure to Ang II (1 µM) in WKY and SHR (Panels C and D, respectively). Responses of WKY are in black and white (Panels A and C) and those of SHR are in red (Panels B and D). The suppressive effect of nicotine on responses to activation of both TLRs in WKY was significant (p=<0.01), and Ang II had generally small and inconsistent effects in WKY. In contrast, a significant augmentation of the responses to activation of both TLRs by nicotine and by Ang II was consistently observed in SHR (p=<0.01).
  • In vivo serum levels: The effects of s.c. infusions of nicotine (15 mg/kg) or Ang II (0.72 mg/kg) on IL-6 and IL-1β serum levels induced by the TLR7/8 ligand (Clo97, 1 mg/kg) i.p. are shown in Panels E through H. The infusions of saline, Nic, or Ang II were maintained for 24 hours. At 20 hours, animals received i.p. injection of Clo97 or saline. Sera were collected at 24 hours and assayed for IL6 or IL-1β. Nicotine (Panels E and F) and Ang II (Panels G and H) when given with i.p. saline increased IL-6 and IL-1β in WKY and to a lesser extent in SHR. In WKY, nicotine (s.c.) suppressed significantly IL-6 and IL-1β responses to i.p. Clo97 (Panel E). In contrast, in SHR, nicotine enhanced significantly both IL-6 and IL-1 β responses (Panel F). Ang II had no effect on the responses in WKY (Panel G), yet it significantly enhanced the IL-1β response in SHR (Panel H). * indicates significant differences between responses to Nic/TLR or Ang/TLR vs. saline/TLR in both WKY and SHR (p<0.01). The anti-inflammatory effects of nicotine in WKY and pro-inflammatory effects of nicotine and Ang II in SHR seen in vitro in splenocytes were thus confirmed in vivo.