Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes

Abstract

Intracolonic administration of trinitrobenzene sulfonic acid in mice causes inflammation in the colon that is accompanied by increased expression of proinflammatory cytokines and of the substance P (SP), neurokinin 1 receptor (NK-1R) in the proximal mesenteric fat depot. We also investigated whether human mesenteric preadipocytes contain NK-1R and examined the functional consequences of exposure of these cells to SP as it relates to proinflammatory signaling. We found that human mesenteric preadipocytes express NK-1R both at the mRNA and protein levels. Exposure of human mesenteric preadipocytes to SP increased NK-1R mRNA and protein expression by 3-fold, and stimulated IL-8 mRNA expression and protein secretion. This effect was abolished when these cells were pretreated with the specific NK-1R antagonist CJ 012,255. Moreover, human mesenteric preadipocytes transfected with a luciferase promoter/reporter system containing the IL-8 promoter with a mutated NF-kappaB site lost their ability to respond to SP, indicating that SP-induced IL-8 expression is NF-kappaB-dependent. This report indicates that human mesenteric preadipocytes contain functional SP receptors that are linked to proinflammatory pathways, and that SP can directly increase NK-1R expression. We speculate that mesenteric fat depots may participate in intestinal inflammatory responses via SP-NK-1R-related pathways, as well as other systemic responses to the presence of an ongoing inflammation of the colon.

Fig. 1.

Intracolonic TNBS administration causes inflammation in mesenteric fat tissue. CD1 mice (seven to eight mice per group) were treated either with TNBS or saline for 48 h. The animals were then killed, the mesenteric fat was placed in formalin, and 10-μm-thick sections of tissue were stained with hematoxylin/eosin and observed by light microscopy. Mesenteric fat depots isolated from TNBS-treated animals exhibited venular congestion with adhesion of polymorphonuclear (PMNs) leukocytes, diapedesis (transmigration), and radial infiltration of PMNs into the perivenular adipose tissue (B). Depots isolated from saline-treated controls were normal, devoid of inflammatory cell infiltrate (A). (Scale bars: A, 50 μm; B, 100 μm.)

Fig. 2.

Intracolonic TNBS administration increases the expression of inflammatory cytokines in mesenteric fat depots. CD1 mice (seven to eight mice per group) were treated either with TNBS or saline for 48 h, and RNA was isolated from the mesenteric fat proximal to the inflamed regions. Real-time PCR analysis of mesenteric depots showed that compared with saline-exposed mice, TNBS-exposed mice had significantly increased TNF-α (A), IL-6 (B), MCP-1 (C), and KC (D) mRNA levels.

Fig. 3.

Intracolonic TNBS administration increases NK-1R mRNA and protein expression in mesenteric fat depots. TNBS was administered to CD1 mice intracolonically. After 48 h, RNA and protein lysates were collected from the mesenteric fat proximal to the inflamed regions and subjected to real-time PCR and Western blot analysis for NK-1R mRNA (n = 8 mice per group) (A) and protein (B Upper), respectively. We found increased NK-1R mRNA expression in the depots obtained from TNBS-exposed versus saline-treated mice (A). Similar results were obtained when tissue lysates were tested by Western blot analysis using an antibody directed against human NK-1R (B Upper; representative of five independent experiments). The calculated molecular mass shown on the right is estimated by the migration of molecular mass standards run simultaneously. kDa indicates kilodaltons, where k = 1,000. (B Lower) Densitometry analysis of blots shown in B Upper (n = 5 per group).

Fig. 4.

NK-1R is present in human mesenteric preadipocytes and increased by SP treatment. Human mesenteric preadipocytes express functional NK-1 receptors. (A) Primary human mesenteric preadipocytes bind ¹²⁵I BHSP and binding is diminished in the presence of excess cold SP competitor (n = 6 per group). (B) Real-time PCR analysis of RNA isolated from human mesenteric preadipocytes shows that these cells express NK-1R, and its expression is increased by exposure to SP for 4 h. SP-induced expression of NK-1R is abolished when the cells are pretreated for 20 min with the specific NK-1R inhibitor CJ 012,255 (n = 6 per group). (C) Western immunoblot analysis showing increased NK-1R expression by SP at the protein level (n = 3 independent experiments per group). The calculated molecular mass shown on the right is estimated by the migration of molecular mass standards run simultaneously. kDa indicates kilodaltons, where k = 1,000. (D) Western blot analysis of protein lysates from human mesenteric preadipocytes showing activation of ERK2 by SP after 10 min of exposure, indicating that NK-1R is functional (representative of four independent experiments).

Fig. 5.

SP increases IL-8 mRNA and protein expression in human mesenteric preadipocytes. (A) ELISA assays were performed on supernatants from human mesenteric preadipocytes treated with SP for 4 h with or without prior CJ 012,255 treatment for 20 min. SP addition caused a significant increase in the secretion of IL-8 by these cells, an effect that was abolished by the specific NK-1R inhibitor CJ 012,255 (n = 8 per group). (B) Real-time PCR analysis of RNA extracts from the same cells produced similar data. (C) When the cells were exposed to two different concentrations of SP, maximum IL-8 mRNA expression was achieved at a dose of 10⁻⁸ M (n = 6 per group). (D) In cells treated with SP for various time intervals (1, 2, 4, 6, and 24 h), maximum IL-8 mRNA expression was observed after 4 h of treatment (n = 6 per group).

Fig. 6.

SP-induced IL-8 expression in human mesenteric preadipocytes is NF-κB-dependent. Induction of SP expression in human mesenteric preadipocytes is NF-κB-dependent. (A) SP treatment of human mesenteric preadipocytes leads to decreases in IκBα protein levels within 15 min of exposure, as is evident in cell lysates subjected to Western blot analysis (representative of four independent experiments). (B) Transient transfection of human mesenteric preadipocytes with an IL-8 promoter-reporter construct (pGL2/IL-8) and subsequent treatment with SP for 4 h leads to increased IL-8 promoter activity, as measured by luciferase activity assay. Transfection with the same vector with an inactivated NF-κB site in the IL-8 promoter abolishes SP-induced activity of the promoter. Similarly, IL-8 promoter activity is lost when the cells are pretreated with CJ 012,255 for 20 min before the addition of SP (n = 5 per group).