Browsing by Subject "Mast cell"

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Immunomodulatory effects of resveratrol on human intestinal mast cell signaling in vitro and mast cell associated enteritis and colitis in mice
(2023) Bilotta, Sabrina; Lorentz, Axel
By releasing their pre-stored or de novo synthesized mediators, mast cells (MC) are important immunoregulatory cells responsible for a variety of inflammatory reactions. Although known to be major effector cells in immunoglobuline (Ig) E dependent allergic reactions, MC have been widely shown to play a role in various inflammations of the gut. Diseases of the gastrointestinal tract (GIT) are widespread and multicausal. Those affected suffer from the sometimes severe symptoms and may experience restrictions on their daily life. Even if conventional medication is applied routinely, aim of the past and current research is to establish supportive and/or alternative medication that is based on natural substances. These may be on the basis of small natural components like resveratrol, a stilbene mostly found in grapes. Numerous positive properties are attributed to resveratrol. These are anti-inflammatory, anti-cancerogenic, anti-oxidative, as well as neuroprotective effects. The use of substances of natural origin as so-called nutraceuticals can help to increase the acceptance of medication by those affected, but also to reduce and overcome the side effects associated with conventional treatment. Effects of resveratrol were examined on the reactivity of MC isolated from patients’ tissue undergoing bowel resection. The results of this work show that resveratrol exhibited potent inhibitory effects on high affinity IgE receptor mediated activation of MC, strongly inhibiting not only MC degranulation, but also gene expression of the pro-inflammatory cytokines C-X-C motif chemokine ligand (CXCL) 8, C-C motif chemokine ligand (CCL) 2, CCL3, CCL4 and tumor necrosis factor (TNF-) α. Ultimately, the intracellular signaling cascade triggered during MC activation via IgE receptor leads to mediator release. Following IgE receptor mediated activation, phosphorylation of signaling molecules like extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3, occurs. ERK1/2 was found to be responsible for phosphorylation of mitochondrial STAT3, which contributes significantly to MC degranulation. Treatment with resveratrol was able to inhibit the phosphorylation of STAT3 by more than 50 % and that of ERK1/2 by almost 100 %. Furthermore, the experiments performed succeeded in isolating the mitochondrial fraction from relatively low human intestinal MC (hiMC) numbers. Also, in this fraction we could detect phosphorylation of STAT3 and ERK1/2 after MC activation, which was reduced after treatment with resveratrol. Having shown the strong inhibitory effects in vitro, we set out to examine immunomodulatory effects of resveratrol in vivo. Presence and activity of MC are closely related to intestinal inflammations in consequence of food allergy (FA) and inflammatory bowel disease (IBD). In mice, FA can be studied using the ovalbumin (OVA)-induced allergic enteritis model and colitis can be studied using the IL-10 knockout (-/-) mice, which develop a spontaneous form of chronic colitis. We could show that the oral application of resveratrol inhibited the increase of MC numbers in the colon and duodenum of affected animals in both experimental settings. Less pronounced but still visible effects of resveratrol administration were observed in the colon with regard to epithelial damage, cell infiltration and reduction of goblet cell numbers. In all cases, based on a scoring system, the damage decreased to the level of the corresponding controls receiving no additive and in which no allergic enteritis was induced or nor colitis developed. Overall, allergic enteritis resulted in a weaker symptomatology, and IL-10-/- animals showed a delayed appearance of the typical symptoms. The results of this thesis show a strong inhibitory effect of resveratrol on hiMC. This could be detected for mediator release as well as on gene expression levels and in the phosphorylation of the signaling molecules ERK1/2 and STAT3, which we could also identify in the mitochondria of hiMC. We observed positive influences on MC-associated parameters in the OVA enteritis and IL-10-/- colitis mouse models. With regard to its use as nutraceutical, resveratrol could therefore come more of a focus in the future.
Mechanismen der Mastzellaktivierung durch gram-negative Bakterien und Bakterienprodukte aus der Darmflora.
(2006) Krämer, Sigrid; Bischoff, Stephan C.
The role of mast cells (MC) as effector cells in IgE dependent processes like the type 1 allergy has been known for a long time. During the decade, it has been shown that MC are also involved in other pathophysiological processes such as mucosal polyposis, rheumatoid arthritis, inflammatory bowl disease, tissue fibrosis, and atherosclerosis. Furthermore, MC play an important role in the regulation of host defense against microbes, tissue remodeling processes, and neuro-immunology-interaction. The first aim of the present study was to clarify the question whether human intestinal MC express toll-like receptors (TLR), which recognize conserved bacterial and viral components, and can MC be activated through TLR-ligands. The second major focus of the present study was to investigate if the stimulation of human intestinal MC with different E. coli and Shigella strains, respectively, results in an activation of MC and to identify the underlying mechanism(s). Accordingly, human intestinal MC were isolated from surgery tissue with a mechanical and enzymatical protocol. The purity of the MC cultures used in all experiments was between 98 and 100% which was achieved by positive selection (MACS). We could show, that human intestinal MC express mRNA for TLR 1, 2, 3, 4, 5, 6, 8, and 9. However, neither the stimulation with LPS (lipopolysaccharide, TLR 4 ligand), LTA (lipoteichoic acid, TLR 2 ligand), Zymosan (TLR 2 ligand), poly I:C (polyinosinic-polycytidylic acid, TLR 3 ligand), R848 (TLR 7/8 ligand), CpG (C poly G oligo-desoxy-nucleotide, TLR 9 ligand) and non CpG, respectively resulted in a release of histamine, leucotriens, TNF-alpha, or IL-8. Furthermore, mRNA expression levels of TNF-alpha and IL-8 were not induced by any of the treatments. Similar results where found when human intestinal MC were stimulated with E. coli (O101:H-) isolated from human faeces or the probiotic strain E. coli Nissle 1917. Even after stimulation with pathogenic bacteria strains such as the invasive S. flexneri M90T and the fimbriated E. coli, respectively, no induction of any of the parameters mentioned above was found. However, E. coli strains activate the intracellular signal molecule and transcription factors ERK1/2, c-Fos, and AP1, but this activation failed to induce a complete immune answer. In contrast, the hemolytic E. coli stains ATCC 25922 and ATCC 35218 provoked strong activation of intestinal MC. Using the isogenic hemolysin negative E. coli mutants and the hemolysin positive transformants of the probiotic E. coli Nissle 1917 it was shown, that human intestinal MC are sensitive target cells for E. coli alpha hemolysin. Stimulation of MC with sublytic concentration of hemolysin resulted in an induction of TNF-alpha, IL-3, IL-5, IL-6, IL-8 mRNA expression, the release of histamine as well as leucotrien. This activation was found to be regulated by calcium dependent signal cascades. Inhibition of intracellular signal molecules showed that the activation depends on L-typ calcium channels, calcineurin, NFAT and NFkappaB. Prolonged infection with hemolytic E. coli strains resulted in lysis of intestinal MC indicating a biphasic activation of hemolysin.
Rolle von SNARE-Proteinen bei der Mediatorfreisetzung humaner Mastzellen
(2010) Frank, Simon P. C.; Lorentz, Axel
Mast cells are involved in a variety of physiological and pathophysiological processes in the body. They posses a wide range of stored and de novo synthesized inflammatory mediators. Therefore they play an important role not only in allergic diseases - as it was originally assumed. The aim of the present work is to acquire a more detailed insight into the processes of mediator release of human intestinal tissue mast cells. To enable the release of histamine, proteases, cytokines, chemokines and other substances, it is necessary that secretory vesicles fuse with the plasma membrane. In this process so-called SNARE proteins are essentially involved. They are generally divided into vesicle membrane (v) - and target membrane (t) -SNAREs. The present work could show that a large number of SNAREs are expressed in human intestinal mast cells. The t-SNAREs SNAP-23, syntaxin-2, -3, -4, -6, Vti1b, and the v SNAREs VAMP-2, -3, -5, -7, -8 could be clearly detected, while SNAP-25, Syntaxin-1b and VAMP-4 were expressed only slightly. A subsequent visualization of these proteins by coupling with fluorescence-marked antibodies showed their localization in the cell. There is a clear arrangement of the t-SNAREs SNAP-23, syntaxin-3, -4 and -6 at the plasma membrane, whereas syntaxin-2, Vti1b, VAMP-3, -7 and -8 are distributed in the cytoplasm in resting cells. However, after activation of the cells for 15 minutes, this arrangement changed for VAMP-7 and -8 which moved in the direction of the plasma membrane, as well as Vti1b, which shifted after a prolonged activation phase of 2 hours also towards the cell edge. In activated mast cells colocalization of SNAP-23, syntaxin-3 and -4, VAMP-7 and -8, as well as Vti1b could be demonstrated. Using co-immunoprecipitation complex formation of SNAP-23 with syntaxin-4, VAMP-7 and -8 was confirmed. For the elucidation of the functional significance of individual SNARE proteins, respective SNAREs have been turned off. After transfection of human intestinal mast cells by using different transfection reagents failed, transfection of mast cells with siRNA by electroporation succeeded. However, it turned out that the electrically treated cells lost their ability to be stimulated. In contrast, blocking of the SNARE proteins achieved by using inhibitory antibodies was successful with no deterioration of cells. Interestingly enough it was revealed that a variety of SNARE proteins participate in degranulation processes in mast cells. The release of the prestored beta-hexosaminidase was significantly reduced by antibodies against SNAP-23, syntaxin-3, -4 and -6, VAMP-7 and -8 and Vti1b after a stimulation time of one hour. It could be noted that VAMP-7 seemed to play no role anymore in the case of a prolonged activation time of six hours. The same SNAREs were also responsible for the release of LTC4. The liberation of the cytokines and chemokines IL-5, IL-6, IL-8, MCP-1, MIP-1-alpha and MIP-1-beta showed an inconsistent involvement of SNARE proteins. However, inhibition of SNAP-23, syntaxin-3 and Vti1b resulted in almost complete blockage of the release of all measured cytokines. Furthermore, antibodies against syntaxin-6 caused a significant reduction in the secretion of IL-5, IL-8 and MCP-1 and a trend towards a reduction of IL 6, MIP-1-alpha and MIP-1-beta. To different degrees both of the v-SNAREs VAMP-7 and -8 seemed to account individually for the release of different cytokines. Since release of the chemokine MIP-1-beta was not affected neither by the blockade of VAMP-7 nor by VAMP-8, another non-studied member of the VAMP family could be involved. Inhibition of syntaxin-4 led only for IL-8 to a marked and significant reduction in the release. In summary, it was shown that the release of different mediators in human intestinal mast cells is catalyzed by various different SNAREs and thus the existence of specific carriers and a targeted transport of mediators are very likely. One explanation for the involvement of a number of SNARE proteins in secretion processes may be that while some SNAREs are responsible for building up intracellular structures which are necessary for the compound exocytosis, some others are in charge of the docking of vesicles at the plasma membrane. A detailed understanding of the mechanisms and the involvement of specific SNARE proteins in the release of various mediators of human mast cells is the basis for new forms of treatments for allergies and asthma. New therapeutic approaches might aim through the temporary deactivation of certain SNARE proteins to achieve an intended reduction of mediator release.