Corticotropin Releasing Factor (CRF) activation of NF-kappaB-directed transcription in leukocytes

Abstract

1. The aim of this study was to test whether CRF enhanced nuclear factor kappa B (NF-kappaB)-directed gene transcription in leukocytes and the receptor specificity of the effect. Initially, we examined the ability of CRF to modulate an antigen-specific, in vitro antibody response. Since that could be mediated by NF-kappaB transcription factor activity, we tested CRF in a NF-kappaB driven luciferase gene expression reporter assay. 2. CRF enhanced the antigen-specific antibody production in a dose- and time-dependent manner. RAW 264.7 macrophage cells and splenocytes stained by immunohistochemistry were positive for CRF receptors and CRF. Expression of both was up-regulated by mitogen treatment of the splenocytes. CRF also enhanced the NF-kappaB-regulated reporter assay and this could be blocked by a CRF-R1 receptor antagonist. 3. In light of these findings, it seems likely that CRF enhanced the antigen-specific antibody response through the CRF-R1 receptor by elevation of NF-kappaB activity. This study provides further support for the concept that CRF can act as an immunomodulator mediating neuro-immune interactions.

Fig. 1.

CRF enhancement of an anti-SRBC PFC response. Mouse splenocytes were sensitized with SRBC in the presence of CRF to induce a primary IgM antibody response and measured in a plaque assay which quantified the number of plaque-forming (antibody-producing) cells as described in the Methods section. (A) Treatment with CRF added on day 0 with the SRBC antigen at the indicated doses, but without β-2-mercaptoethanol (2-ME). (B) Kinetic study where CRF (100 nM) was added on the indicated day of the assay. Open circles indicate a parallel experiment in which 2-ME (10⁻⁶ M) was present in both the control and the CRF (100 nM) treated cultures. For both panels the data are expressed as percent of the comparable control. Data are the mean ± standard deviation.

Fig. 2.

CRF-R1/2 expression on RAW 264.7 cells. (A) Cells stained with anti-CRF-R1/2, (B) isotype control stain. For greater clarity, this figure is available in color in the online version of the paper.

Fig. 3.

CRF-R1/2 expression in splenocytes. The splenocytes were cultured with a mock medium, lipopolysaccharide (10μg/mL), or concanavalin A (1μg/mL) for 48 h. Then the cultures were harvested, washed, and stained with antiserum to CRF-R1/2. (A) LPS, (B) Con A, (C) Mock, and (D) isotype control. For greater clarity, this figure is available in color in the online version of the paper.

Fig. 4.

CRF expression in splenocytes. The splenocytes were cultured with a mock medium, lipopolysaccharide (10μg/mL), or concanavalin A (1μg/mL) for 48 h. Then the cultures were harvested, washed, and stained with antiserum to CRF. (A) LPS, (B) Con A, (C) Mock, and (D) isotype control. For greater clarity, this figure is available in color in the online version of the paper.