Interleukin-6 is an essential, corticotropin-releasing hormone-independent stimulator of the adrenal axis during immune system activation

Abstract

Glucocorticoids play a critical role in control of the cytokine response after immune challenge. Conversely, cytokines modulate glucocorticoid production by the hypothalamic-pituitary-adrenal axis. To define the potency and mechanism of interleukin-6 (IL-6) for augmentation of adrenal function, we exploited mice deficient in corticotropin-releasing hormone (CRH), IL-6, or both. Mice deficient in CRH action demonstrate severely impaired glucocorticoid production in response to psychological and metabolic challenge, but near normal responses to stressors that activate the immune system. In this paper, we demonstrate that IL-6 is essential for activation of the hypothalamic-pituitary-adrenal axis during immunological challenge in the absence of hypothalamic input from CRH. IL-6 receptors are present on pituitary corticotrophs and adrenocortical cells, consistent with the ability of IL-6 to bypass CRH in augmentation of adrenal function. Plasma corticosterone levels after bacterial lipopolysaccharide injection in mice deficient in CRH or IL-6 were significantly lower than in wild-type mice but significantly greater than in mice deficient in both CRH and IL-6. A second model of immune system activation using 2C11, an antibody to the T cell receptor, demonstrated a normal corticosterone response in mice deficient in CRH or IL-6, but a markedly decreased response in mice deficient in both CRH and IL-6. Surprisingly, the relative contribution of IL-6 for modulation of the adrenal response to stress is greater in female than in male mice. This gender-specific difference in IL-6 action in mice suggests the utility of further analysis of IL-6 in determining the female predominance seen in many human inflammatory/autoimmune diseases.

Figure 1

Stimulation of plasma ACTH after IL-6 injection in WT and CRH KO mice. WT and CRH KO mice were injected with vehicle or IL-6 (n = 3–4). Noninjected mice that were bled at 08:00 for basal ACTH also are shown. *, P < 0.01 vs. WT IL-6, CRH KO vehicle, and CRH KO basal; †, P < 0.05 vs. WT vehicle and WT basal.

Figure 2

Localization of IL-6 receptor in pituitary and adrenal. (A) IL-6 receptor colocalizes with ACTH to pituitary corticotrophs. Pituitaries from WT mice were stained with immunofluorescently labeled antibodies. Staining specific for ACTH (green) is shown (Left) or IL-6 receptor (IL-6R) (red) is shown (Middle). Colocalization of both ACTH and IL-6 receptor is indicated by yellow cells (Right). (B) IL-6 receptor is located in mouse adrenal cortex. (Left) A hematoxylin/eosin stained section of WT mouse adrenal is shown. (Right) Autoradiogram of an adjacent adrenal section subjected to in situ hybridization with a probe for mouse IL-6 receptor is shown. M, medulla; ZF, zona fasciculata.

Figure 3

Plasma corticosterone response to restraint stress in female and male WT, CRH KO, IL-6 KO, and CRH KO/IL-6 KO mice (n = 3–5). Females: *, CRH KO, P < 0.001 vs. WT, P < 0.001 vs. IL-6 KO; †, IL-6 KO, P < 0.001 vs. WT; #, CRH KO/IL-6 KO, P < 0.001 vs. WT, P < 0.05 vs. CRH KO, P < 0.001 vs. IL-6 KO. Males: §, CRH KO, P < 0.01 vs. WT, P < 0.01 vs. IL-6 KO; **, CRH KO/IL-6 KO, P < 0.001 vs. WT, P < 0.05 vs. CRH KO, P < 0.001 vs. IL-6 KO. ‡, P < 0.01 WT males vs. WT females.

Figure 4

Plasma corticosterone concentration after injection of synthetic ACTH into female WT, CRH KO, IL-6 KO, and CRH KO/IL-6 KO mice. *, CRH KO, P < 0.01 vs. WT, P = 0.05 vs. IL-6 KO, P = 0.18 vs. CRH KO/IL-6 KO. †, CRH KO/IL-6 KO, P < 0.01 vs. WT, P = 0.01 vs. IL-6 KO.

Figure 5

Adrenal histology in mice with CRH and IL-6 deficiency. Hematoxylin/eosin-stained sections of adrenal from female WT (A), IL-6 KO (B), CRH KO (C) and CRH KO/IL-6 KO (D) mice are shown. The thickness of the adrenal cortex in each section is indicated by the solid line.

Figure 6

Plasma corticosterone and ACTH response to immune challenge. (A) Female and male WT, CRH KO, IL-6 KO, and CRH KO/IL-6 KO mice (n = 4–5) after injection with LPS. Females: *, CRH KO, P < 0.01 vs. WT; †, IL-6 KO, P < 0.01 vs. WT; #, CRH KO/IL-6 KO, P < 0.001 vs. WT, P < 0.01 vs. CRH KO, P < 0.001 vs. IL-6 KO. Males: **, CRH KO, P < 0.01 vs. WT, P < 0.01 vs. IL-6 KO; §, IL-6 KO, P = 0.05 vs. WT; ‡‡, CRH KO/IL-6 KO, P < 0.001 vs. WT, P < 0.001 vs. CRH KO, P < 0.001 vs. IL-6 KO; ‡, P = 0.01 WT females vs. WT males. (B) Male WT, CRH KO, IL-6 KO, and CRH KO/IL-6 KO mice (n = 3–6) after injection with PBS or 2C11. *, P < 0.01 vs. PBS of same genotype; †, P < 0.01 vs. PBS of same genotype and all other genotypes injected with 2C11. (C) Plasma ACTH after 2C11 or PBS injection in male WT, CRH KO, IL-6 KO, and CRH KO/IL-6 KO mice (n = 3–6). *, P < 0.05 vs. PBS of same genotype.

Figure 7

Plasma IL-6 levels after immune system stimulation in CRH KO and WT mice. The dotted line on each graph indicates the limit of assay sensitivity. All PBS-injected mice of each genotype (n = 3) were below the lower limit of detection. (A) Plasma IL-6 after 2C11 in male mice (n = 3–4). *, P < 0.01 vs. WT. (B) Plasma IL-6 from male and female mice after LPS injection (n = 6–9). *, P < 0.01 vs. WT.