In vivo Angiotensin II AT1 receptor blockade selectively inhibits LPS-induced innate immune response and ACTH release in rat pituitary gland

Abstract

Systemic lipopolysaccharide (LPS) administration induces an innate immune response and stimulates the hypothalamic-pituitary-adrenal axis. We studied Angiotensin II AT(1) receptor participation in the LPS effects with focus on the pituitary gland. LPS (50 microg/kg, i.p.) enhanced, 3h after administration, gene expression of pituitary CD14 and that of Angiotensin II AT(1A) receptors in pituitary and hypothalamic paraventricular nucleus (PVN); stimulated ACTH and corticosterone release; decreased pituitary CRF(1) receptor mRNA and increased all plasma and pituitary pro-inflammatory factors studied. The AT(1) receptor blocker (ARB) candesartan (1mg/kg/day, s.c. daily for 3 days before LPS) blocked pituitary and PVN AT(1) receptors, inhibited LPS-induced ACTH but not corticosterone secretion and decreased LPS-induced release of TNF-alpha, IL-1beta and IL-6 to the circulation. The ARB reduced LPS-induced pituitary gene expression of IL-6, LIF, iNOS, COX-2 and IkappaB-alpha; and prevented LPS-induced increase of nNOS/eNOS activity. The ARB did not affect LPS-induced TNF-alpha and IL-1beta gene expression, IL-6 or IL-1beta protein content or LPS-induced decrease of CRF(1) receptors. When administered alone, the ARB increased basal plasma corticosterone levels and basal PGE(2) mRNA in pituitary. Our results demonstrate that the pituitary gland is a target for systemically administered LPS. AT(1) receptor activity is necessary for the complete pituitary response to LPS and is limited to specific pro-inflammatory pathways. There is a complementary and complex influence of the PVN and circulating cytokines on the initial pituitary response to LPS. Our findings support the proposal that ARBs may be considered for the treatment of inflammatory conditions.

Figure 1. Circulating cytokines

Values are mean ± SEM for groups of 7–9 animals measured individually. Cytokine levels on the saline treated groups were not detectable and were omitted from the graph. # p<0.05, ## p<0.01 as compared to vehicle-LPS treated group.

Figure 2. Hyphotalamic PVN expression of AT₁ receptors

A) Representative pictures and quantification of AT_1A in situ hybridization in PVN. B) Representative pictures and quantification of [¹²⁵I]Sar¹-Ang II binding to AT₁ receptors in PVN. Scale bar, 1 mm. All values are means ± SEM for groups of 7–9 animals measured individually. * p<0.05, ** p<0.01, *** p<0.001 as compared to vehicle-saline treated group. ### p<0.001 as compared to vehicle-LPS treated group.

Figure 3. mRNA expression of CRF in hypothalamic PVN and of CRF receptors in pituitary

A) Representative pictures and quantification of CRF in situ hybridization in PVN. Scale bar, 1 mm. B) Pituitary expression of CRF₁ and CRF₂ receptors. Data represent relative levels of mRNA normalized to 18S rRNA. All values are means ± SEM for groups of 7–9 animals measured individually. *** p<0.001 as compared to vehicle-saline treated group.

Figure 4. Pituitary expression of AT₁ receptors

A) AT_1A and AT_1B mRNA expression in pituitary. Data represent relative levels of mRNA normalized to 18S rRNA. B) Representative pictures and quantification of [¹²⁵I]Sar¹-Ang II binding to anterior pituitary AT₁ receptor. Binding was present exclusively in anterior (arrow) but not posterior (arrowhead) lobe. Scale bar, 1 mm. All values are means ± SEM for groups of 7–9 animals measured individually. * p<0.05, *** p<0.001 as compared to vehicle-saline treated group. ### p<0.001 as compared to vehicle-LPS treated group.

Figure 5. Pituitary expression of LPS receptors TLR4 and CD14

Data represent relative levels of mRNA normalized to 18S rRNA. Values are mean ± SEM for groups of 7–9 animals measured individually. *** p<0.001 as compared to vehicle-saline treated group.

Figure 6. Pituitary cytokines gene expression

Data represent relative levels of mRNA normalized to 18S rRNA. Values are mean ± SEM for groups of 7–9 animals measured individually. * p<0.05, *** p<0.001 as compared to vehicle-saline treated group; ## p<0.01, ### p<0.001 as compared to vehicle-LPS treated group.

Figure 7. Pituitary cytokines content

Data represent the cytokine protein content. Values are mean ± SEM for groups of 7–9 animals measured individually. * p<0.05 as compared to vehicle-saline treated group; ND, non detectable.

Figure 8. Pituitary nitric oxide synthase enzymes expression and activity

Data represent relative levels of mRNA normalized to 18S rRNA (A) and NOS activity measurement (B). Values are mean ± SEM for groups of 7–9 animals measured individually. * p<0.05, ** p<0.01, *** p<0.001 as compared to vehicle-saline treated group; # p<0.05, ## p<0.01 as compared to vehicle-LPS treated group. ND, non detectable.

Figure 9. Pituitary COX enzymes expression and PGE₂ content

Data represent relative levels of mRNA normalized to 18S rRNA (A) and pituitary PGE₂ content (B). Values are mean ± SEM for groups of 7–9 animals measured individually. * p<0.05, ** p<0.01 as compared to vehicle-saline treated group; # p<0.05 as compared to vehicle-LPS treated group.

Figure 10. Pituitary c-fos and IκB-α gene expression

Data represent relative levels of mRNA normalized to 18S rRNA. Values are mean ± SEM for groups of 7–9 animals measured individually. *** p<0.001 as compared to vehicle-saline treated group. ## p<0.01 as compared to vehicle-LPS treated group.

Figure 11. Circulating hormones and pituitary ACTH

A) Data represent levels of circulating ACTH and corticosterone and B) hormone content of ACTH in the anterior lobe. Values are mean ± SEM for groups of 7–9 animals measured individually. * p<0.05, ** p<0.01, *** p<0.001 as compared to vehicle-saline treated group; # p<0.05 as compared to vehicle-LPS treated group.