Characterization of early cytokine responses and an interleukin (IL)-6-dependent pathway of endogenous glucocorticoid induction during murine cytomegalovirus infection

Abstract

Early infection with murine cytomegalovirus (MCMV) induces circulating levels of interleukin (IL)-12, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Studies presented here further characterize these responses by defining kinetics and extending evaluation to include IL-1, IL-6, and glucocorticoids. IL-12 p40, IFN-gamma, TNF, IL-1alpha, and IL-6 were shown to be increased, but IL-1beta was undetectable, in serum of MCMV-infected mice. The IL-12 p40, IFN-gamma, TNF, and IL-6 responses were dramatic with peak levels reaching >150-10,000 pg/ml at 32-40 h after infection and rapidly declining thereafter. Glucocorticoid induction, peaking at 36 h and reaching 30-fold increases above control values, accompanied the cytokine responses. Mice with cytokine deficiencies or neutralized cytokine function demonstrated that IL-6 was the pivotal mediator of the glucocorticoid response, with IL-1 contributing to IL-6 production. The IL-6 requirement appeared to be specific for virus-type stimuli as the synthetic analogue of viral nucleic acid, polyinosinic-polycytidylic acid, also induced IL-6-dependent glucocorticoid release, but treatments with the bacterial product lipopolysaccharide and a non-immune physical restraint stressor elicited IL-6-independent responses. Collectively, the results identify IL-6 as a primary mediator of glucocorticoid induction, and elucidate specific pathways of interactions between immune and neuroendocrine systems during viral infection.

Figure 1

Serum cytokine levels between 24 and 48 h after MCMV infection. IL-12 p40 (A), IFN-γ (B), TNF (C), IL-6 (D), and IL-1α (E) were assessed in sera collected from MCMV-infected (5 × 10⁴ PFU/mouse) and vehicle-treated C57BL/6 mice at various times between 24 and 48 h of infection by ELISA assays as described in Materials and Methods. IL-12 p40, IFN-γ, TNF, and IL-6 values are from two mice at each time point and are expressed as means ± SD. IL-1α values are plotted from individual mice at the indicated times after infection with geometric means indicated by short lines. The IL-1α samples were taken from two different experiments. Uninfected samples for all cytokines were from mice receiving control vehicle injections.

Figure 2

Serum corticosterone and ACTH levels after MCMV infection. Corticosterone and ACTH levels were measured in serum samples collected from C57BL/6 mice under low stress conditions (mice were bled within 4 min of handling for corticosterone measurement and within 2 min of handling for ACTH measurement). Serum corticosterone was measured at 12-h intervals between 12 and 72 h (A) or 2–4-h intervals between 24 and 48 h (B) and serum ACTH was measured at 4–6-h intervals between 18 and 36 h after MCMV (5 × 10⁴ PFU/mouse) infection (open circles) or vehicle injection (open squares). In A, data are mean ± SE of four mice per time point. In B, data are means ± SD of two mice per time point. For C, data are means ± SE of three mice per time point. *P <0.05, **P <0.01.

Figure 3

Corticosterone levels in mice with neutralized cytokine function. Corticosterone levels in MCMV-infected (5 × 10⁴ PFU/ mouse) (filled bar) or vehicle-injected (striped bar) IL-6–deficient (IL-6–), IFN-γ–deficient (IFN-γ⁻), IL-1ra–treated (IL-1ra) mice or respective control mice (IL-6⁺, IFN-γ⁺, PBS). Treatments were as described in Materials and Methods. Corticosterone levels were measured in serum samples collected from mice under low stress conditions (bled within 4 min of handling) 36 h after infection. Results from one of two to three experiments are shown. Data are means ± SE of three mice per group. **P <0.01.

Figure 4

Serum corticosterone levels after LPS, poly I:C, or restraint stress administration in IL-6–deficient and wild-type mice. (A) Corticosterone levels were measured in plasma collected from IL-6–deficient (IL-6⁻) (filled bar) and wild-type (IL-6⁺) (dotted bar) mice under low stress conditions (less than 4 min of handling) 2 h after injection with PBS, 50 μg LPS, or 100 μg poly I:C. (B) Corticosterone levels were measured in serum samples collected from IL-6–deficient (IL-6⁻) and wild-type (IL-6⁺) mice bled under low stress conditions (within 4 min of handling) or after 30 min of restraint. Data are means ± SE of four mice per group. **P <0.01.

Figure 5

Model for microbial stimulation of glucocorticoid induction.