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Local Production of Corticotropin Releasing Hormone Is Increased in Experimental Intestinal Inflammation in Rats

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Local Production of Corticotropin Releasing Hormone Is Increased in Experimental Intestinal Inflammation in Rats Gut 1996; 39: 385-392 385 Local production of corticotropin releasing hormone is increased in experimental intestinal Gut: first published as 10.1136/gut.39.3.385 on 1 September 1996. Downloaded from inflammation in rats E A F van Tol, P Petrusz, P K Lund, M Yamauchi, R B Sartor Abstract system shares bi-directional communication Background/Aimsn-Corticotropin re- pathways with both the nervous and endocrine leasing hormone (CRH) suppresses networks. 1-3 For example, peripheral and immunological functions via stimulation mucosal lymphoid organs have direct neuro- of the pituitary-adrenal axis, but is also peptidergic innervation.4 5 Not only can found in peripheral tissues. Peripheral immune cells be found in intimate association proinflammatory activity of CRH is with nerve fibres,6 they also have specific high- suggested by increased tissue concen- affinity receptors for neuropeptides and hor- trations in arthritis and in vitro inmmuno- mones.7 Furthermore, hormones and neuro- stimulatory effects. This study evaluated peptides endogenous to the gastrointestinal intestinal CRH concentrations, immuno- tract modulate the activity of immune cells localisation, and synthesis in chronic en- isolated from gut associated lymphoid tissues terocolitis and investigated in vitro and the intestinal lamina propria.8 9 A variety responsiveness of lamina propria mono- of neuropeptides can be synthesised by nuclear cells to CRH. immune cells suggesting that neuropeptides Methods-Chronic granulomatous en- have autocrine/paracrine actions on immune terocolitis was induced by intramural cells or that the immune system shares injection of peptidoglycan-polysaccharide functional homology with the neuroendocrine polymers in the ileocaecal region of Lewis system.3 l Together, these findings indicate rats. CRH protein was measured in caecal that neuropeptides and hormones can regulate specimens by immunohistochemistry and the activity of immune cells in endocrine, para- radioinmunoassay and caecal CRRH crine, and possibly autocrine fashions similar activities of mRNA expression was analysed by reverse to immunoregulatory cytokines. http://gut.bmj.com/ transcriptase polymerase chain reaction. Local production of corticotropin releasing Results-In the chronically inflamed hormone (CRH) has been associated with caecum abundant immunoreactive CRH peripheral inflammation' 1-3 suggesting that this was found in inflammatory cells, mesen- neuropeptide may have a proinflammatory role chymal cells, as well as in myenteric plexi. when produced outside the brain. This is in In contrast, only a few CRH containing sharp contrast with its well known immuno- cells were detected in normal and HSA suppressive role as the key regulator of the on September 23, 2021 by guest. Protected copyright. injected control caecums. Moreover, hypothalamus-pituitary-adrenal axis. At present caecal CRH protein levels were increased it is firmly established that the production of during chronic enterocolitis. Local CRH CRH is not confined to the hypothalamus. Center for GI Biology and Disease and synthesis as indicated by mRNA expression CRH has been demonstrated in the normal Department of was considerably increased in chronic en- spinal cord, lung, liver, spleen, stomach, Medicine terocolitis whereas it was undetectable or pancreas, ovaries, placenta, endometrium, and E A F van Tol low in uninflamed caecum. In addition, intestine with differential distribution among R B Sartor CRH stimulated in vitro proliferation of mammalian species.1421 More importantly, in Laboratory of lamina propria mononuclear cells and vitro studies almost unequivocally point to an Reproductive Biology inhibited mitogen induced proliferation. immunostimulatory effect of CRH. CRH P Petrusz Conclusion-Increased CRH protein and stimulates cytokine secretion,22-24 increases the Department of mRNA expression in chronic enterocolitis expression of interleukin 2 receptors on T Physiology and responsiveness of intestinal mono- cells,25 increases the FMLP induced production PK Lund nuclear cells to CRH indicate an immuno- of reactive oxygen metabolites by macro- Dental Research modulatory role for locally produced CRH phages,26 stimulates both spontaneous and Center in intestinal inflammation. ConA induced splenocyte proliferation,25 27 M Yamauchi (Gut 1996; 39: 385-392) (van Tol unpublished observations), and stimu- University ofNorth lates natural killer cell activity.28 Carolina, Chapel Hill, Keywords: corticotropin releasing hormone, In contrast, not much is known about the USA experimental enterocolitis, Lewis rats. role of local production of CRH in the patho- Correspondence to: Dr R B Sartor, genesis of tissue inflammation although Division of Digestive increased CRH protein or mRNA expression, Diseases and Nutrition, UNC School of Medicine, Regulation of immune reactivity to mucosal or or both, are found in human arthritis and rat 465 Burnett-Womack systemic noxious stimuli without inducing models of bacterial cell wall induced arthritis Building, CB# 7080 Chapel destructive chronic inflammation requires and carrageenin induced granulomatous Hill, NC 27599-7080, USA. 11-13 Direct evidence Accepted for publication balanced interaction between the nervous, exudative inflammation. 4 April 1996 endocrine, and immune systems. The immune for the pivotal peripheral role of CRH in 386 van Tol, Petrusz, Lund, Yamauchi, Sartor experimental inflammation came from the of 0.45 ml was divided over seven injection carrageenin model in which systemic immuno- sites including the junction of the mesentery neutralisation of CRH resulted in marked and the distal ileum (two injections X0.05 ml), suppression of inflammation." two distal ileal Peyers' patches (2X005 ml), Gut: first published as 10.1136/gut.39.3.385 on 1 September 1996. Downloaded from In this study we investigated the presence the caecal tip (lymphoid aggregate, 0.05 ml), and local production of CRH in a model of and the mid and upper caecum (two sites X0 1 chronic enterocolitis. Subserosal intramural ml). Rats in the acute inflammation group were injection of poorly degradable, purified killed 48 hours after injections whereas rats in peptidoglycan-polysaccharide from group A the chronic inflammation group were killed 29, Streptococcus pyogenes (PG-APS) in the caecum 33, or 85 days after injections. All animals were and distal ileum induces biphasic inflammation killed by overdose inhalation of CO2. Gross in genetically susceptible Lewis rats.29 Granu- inflammation was scored by a single blinded lomatous enterocolitis with associated arthritis, observer according to criteria developed and hepatic granulomas, anaemia, and leucocytosis validated for this model.29 Values of 0-4 were spontaneously develops approximately two assigned to the number of caecal nodules, weeks after a single injection of PG-APS.29 30 contraction of mesentery, number and severity Transmural granulomatous inflammation with of adhesions, and caecal wall thickening, with extensive fibrosis is characteristic for the a maximal possible summed gross gut score chronic phase of enterocolitis that persists for (GGS) of 16. at least 16 weeks in this model. In this and other experimental models of inflammation Lewis rats have been shown to be Tissue collection andprocessing a highly susceptible strain.29-3' Part of this Caecal tissue samples were snap frozen in genetic susceptibility in Lewis rats underlying liquid nitrogen and stored at -80°C for the development of exaggerated inflammation isolation of RNA and protein. The caecal tip may involve a blunted hypothalamic CRH was oriented in a plastic container with OCT response to bacterial cell wall products, cyto- (Miles Inc, Elkhart, IN), snap frozen in kines, and neurotransmitters.31" isopentane, and stored at -80°C for immuno- Here we hypothesise that Lewis rats have histochemistry studies. Total RNA was iso- increased peripheral CRH production asso- lated from samples of caecal tissue from PG- ciated with chronic inflammation, and that APS, HSA injected, and normal Lewis rats local proinflammatory effects of CRH contri- using a standard procedure.29 RNA concen- bute to the development or perpetuation of tration and purity was quantified by ab- chronic intestinal inflammation, or both, in a sorbance at 260 nm and A260/280 ratios. The susceptible host. integrity of RNA was verified by electro- phoresis of samples in 1.4% agarose gels containing ethidium bromide. http://gut.bmj.com/ Methods Bacterial cell wallpreparation Immunohistochemistry Sterile PG-APS polymers with the molecular Indirect immunohistochemical staining for rat weight of 5 X 1 o6 kDa to 5 X 1 o8 kDa were CRH was done using the avidin-biotin peroxi- prepared from group A, type 3 strain D58 dase complex (ABC) kit (Vectastain, Vector Streptococcus pyogenes as described previously34 Lab, Burlingame, CA). Sections of brain from on September 23, 2021 by guest. Protected copyright. and provided by Dr J Schwab and R Brown, a longterm adrenalectomised Lewis rat were Department of Immunology and Micro- used as controls because immunostaining of biology, University of North Carolina at CRH is increased in the brain due to lack of Chapel Hill. The preparation was sonicated to corticosteroid feedback inhibition. Ten ,um disperse aggregates immediately before use and frozen sections were cut from the brain of the the final concentration was calculated based on adrenalectomised rat and from fresh frozen rhamnose content.35 caecal tips collected from PG-APS or HSA injected or normal Lewis
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