Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice

Abstract

Corticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-alpha, IL-1beta, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.

FIG. 1.

CRH augments LPS-induced proinflammatory cytokine secretion from RAW264.7 macrophages. (A) TNF-α levels in culture medium of cells treated with CRH, LPS, and CRH plus LPS. TNF-α levels are significantly higher when cells are treated with CRH and LPS than when they are treated with LPS alone (*, P = 0.04). (B) CRH potentiates LPS-induced IL-1β secretion in a significant manner (**, P = 0.01). (C) CRH potentiates LPS-induced IL-6 secretion from RAW264.7 cells (*, P = 0.04). Unstimulated cells were unable to secrete detectable amounts of any of the above cytokines.

FIG. 2.

(A) CRH augments proinflammatory cytokines at the transcriptional level. IL-1β (upper panel), TNF-α (second panel), and IL-6 (third panel) mRNA levels were determined by a semiquantitative RT-PCR approach. CRH induces expression of all three cytokines and further potentiates the LPS-induced transcriptional activation. (B, C, and D) Densitometric analysis of the RT-PCR products of IL-1β (B), TNF-α (C), and IL-6 (D).

FIG. 3.

CRH augments LPS-induced proinflammatory cytokine expression in thioglycolate-induced peritoneal macrophages from BALB/c mice. IL-1β (A) (upper panel), TNF-α (B) (upper panel), and IL-6 (C) (upper panel) mRNA expression was estimated by RT-PCR. The RT-PCR products were quantitated by densitometry as the ratio of cytokine RNA to the actin levels (lower panels).

FIG. 4.

Corticosterone levels in LPS- and LPS-antalarmin-treated BALB/c mice. Sera were collected 1.5 h following LPS treatment.

FIG. 5.

(A) Antalarmin prolonged survival of animals treated with Salmonella serovar Enteritidis-derived LPS. Antalarmin or the antalarmin diluent was administered 1.5 h prior to LPS. Survival was observed over a period of 7 days. Control mice received the antalarmin diluent alone. Antalarmin significantly prolonged survival (P = 0.022). (B) Antalarmin prolonged survival of animals treated with E. coli-derived LPS. Antalarmin or the antalarmin diluent was administered 1.5 h prior to LPS. Survival was observed over a period of 7 days. Antalarmin significantly prolonged survival (P = 0.002).

FIG. 6.

(A) TNF-α levels in mice subjected to LPS-induced endotoxin shock. Trunk blood was collected 1 or 2 h following treatment with E. coli-derived LPS. Treatment with antalarmin significantly reduced TNF-α (n = 5 animals in each group; ***, P = 0.001). (B) IL-1β levels in mice subjected to LPS-induced endotoxin shock. Trunk blood was collected 4 or 6 h following treatment with E. coli-derived LPS. Antalarmin significantly reduced IL-1β (n = 5 animals in each group; *, P < 0.05). (C) IL-6 levels in mice subjected to LPS-induced endotoxin shock. Trunk blood was collected 4 h following treatment with E. coli-derived LPS. Antalarmin significantly reduced IL-6 (n = 5 animals in each group; ***, P < 0.0001; **, P < 0.01). (A to C) C, control; A, antalarmin.