Axel Lorentz

Human Intestinal Mast Cells Are Capable of Producing Different Cytokine Profiles: Role of IgE Receptor Cross-Linking and IL-4

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-α. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.

Mast cells are an important cellular source of tumour necrosis factor alpha in human intestinal tissue.

BACKGROUND Several inflammatory disorders of the intestine are characterised by enhanced expression of tumour necrosis factor α (TNF-α). Monocytes and macrophages have been suggested as a major cellular source of TNF-α in human gut, whereas mast cells, although known to be capable of producing TNF-α, have been poorly examined in this respect. AIMS To investigate whether human intestinal mast cells can produce TNF-α, and which factors regulate TNF-α production in these cells. METHODS Mast cells were isolated from surgery tissue specimens of patients undergoing bowel resection because of cancer. Immunohistochemical studies were performed in biopsy specimens derived from 13 patients (two healthy controls, four with Crohn’s disease, four with ulcerative colitis, three others). TNF-α mRNA and protein expression were studied in vitro by polymerase chain reaction, RNAse protection assay, western blot, and enzyme linked immunosorbent assay in isolated purified human intestinal mast cells stimulated by IgE receptor crosslinking, intestinal bacteria, and lipopolysaccharide. Cellular localisation of TNF-α was examined by immunohistochemistry. RESULTS TNF-α mRNA and protein were expressed constitutively in isolated human intestinal mast cells. Expression of TNF-α mRNA and release of TNF-α protein were substantially enhanced by IgE receptor crosslinking and by coculture of mast cells with intestinal bacteria; lipopolysaccharide had only marginal effects. Immunohistochemical studies revealed that approximately 60% of the lamina propria cells with immunoreactivity for TNF-α were mast cells. CONCLUSIONS The data show that mast cells are an important source of TNF-α in the human intestinal mucosa.

Selective expression of histamine receptors H1R, H2R, and H4R, but not H3R, in the human intestinal tract

Background and aims: Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear. Methods: We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining. Results: We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls. Conclusions: We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

IL-4-Induced Priming of Human Intestinal Mast Cells for Enhanced Survival and Th2 Cytokine Generation Is Reversible and Associated with Increased Activity of ERK1/2 and c-Fos

In synergy with stem cell factor (SCF), IL-4 strongly enhances mast cell proliferation and shifts IgE-dependent cytokine production in mature human mast cells toward an increased release of Th2 cytokines such as IL-3, IL-5, and IL-13 and a decreased IL-6 expression. In this study we analyzed the kinetics and the mechanisms of these IL-4 effects on mast cells purified from intestinal tissue. If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6. The IL-4-induced up-regulation of mast cell proliferation and FcεRI expression was also reversible if IL-4 was withdrawn for 14 days. Interestingly, in contrast to IL-4, proliferation and phenotype of human intestinal mast cells were not affected by IL-13 although both cytokines were capable of inducing STAT6 activation. Instead, IL-4 treatment (but not IL-13 treatment) was associated with an increased activity of ERK1/2 and c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1. Consistently, mast cell proliferation and cytokine expression in response to IL-4 was blocked by the MEK inhibitor PD98059. In summary, our data show that the IL-4 effects on human intestinal mast cell functions are reversible and accompanied by an increased activity of ERK1/2 and c-Fos.

Vesicle associated membrane protein (VAMP)‐7 and VAMP‐8, but not VAMP‐2 or VAMP‐3, are required for activation‐induced degranulation of mature human mast cells

Mediator release from mast cells (MC) is a crucial step in allergic and non‐allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N‐ethyl‐maleimide‐sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N‐ethylmaleide sensitive factor attachment protein (SNAP)‐23, Syntaxin (STX)‐1B, STX‐2, STX‐3, STX‐4, and STX‐6 but not SNAP‐25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)‐3, VAMP‐7 and VAMP‐8 and, in contrast to previous reports about rodent MC, only low levels of VAMP‐2. Furthermore, VAMP‐7 and VAMP‐8 were found to translocate to the plasma membrane and interact with SNAP‐23 and STX‐4 upon activation. Inhibition of SNAP‐23, STX‐4, VAMP‐7 or VAMP‐8, but not VAMP‐2 or VAMP‐3, resulted in a markedly reduced high‐affinity IgE receptor‐mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP‐7 and VAMP‐8, but not VAMP‐2, are required for rapid degranulation.

HUMAN INTESTINAL MAST CELLS PRODUCE IL-5 IN VITRO UPON IGE RECEPTOR CROSS-LINKING AND IN VIVO IN THE COURSE OF INTESTINAL INFLAMMATORY DISEASE

IL‐5, known to be produced by T lymphocytes and eosinophils, is a key regulator of intestinal diseases such as parasitosis or eosinophilic gastroenteritis. Here we examined if mast cells contribute to the IL‐5 production in human intestinal mucosa. The number of IL‐5‐positive lamina propria cells was substantially higher in patients with intestinal inflammatory diseases (5.3 ± 4.6 %, n = 17) compared to healthy controls (0.5 ± 0.9 %, n = 8, p < 0.01). In patients, the IL‐5‐positive cells were eosinophils (70 ± 13  %) and mast cells (29 ± 14 %), whereas in controls all IL‐5‐positive cells were eosinophils. IL‐5‐positive T cells were not detected, likely because they do not store IL‐5. In vitro: studies with isolated human intestinal mast cells and eosinophils showed that mast cells do not produce IL‐5 constitutively, but release high amounts of IL‐5 (315 ± 115 pg/106 cells) following IgE receptor cross‐linking, compared to activated eosinophils (24 ± 5 pg/106 cells). Inhibitor studies suggest a regulation of IL‐5 production at the transcriptional level. In conclusion our data demonstrate that activated mast cells are a potent source of IL‐5 in the human intestinal mucosa.

Growth, phenotype, and function of human intestinal mast cells are tightly regulated by transforming growth factor β1

Background and aims: Transforming growth factor β1 (TGF-β1) is expressed in the healthy human intestine and controls mucosal immune responses and inflammation by regulating the function of lymphocytes, macrophages, dendritic cells, and eosinophils. Here, we asked whether human intestinal mast cells (MC) might also be subject to immunoregulation by TGF-β1. Methods: MC were isolated from the intestinal mucosa, purified, and cultured in vitro in the presence of stem cell factor (SCF) with or without TGF-β1. Growth characteristics, phenotype, and immunological mediator release of MC were analysed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, western blot, and different immunoassays, respectively. Results: TGF-β1 dose dependently (ED50 ≈ 0.1 ng/ml) inhibited SCF dependent growth of human intestinal MC by both enhancing apoptosis and decreasing proliferation. In parallel, TGF-β1 increased the percentage of connective tissue-type MC expressing tryptase and chymase while downregulating expression of the receptors for IgE and SCF. Furthermore, TGF-β1 dramatically changed the profile of mediators released by MC following IgE receptor crosslinking. Release of histamine, cysteinyl-leukotrienes, and tumour necrosis factor α was strongly reduced whereas prostaglandin D2 generation and cyclooxygenase 1 and 2 expression were upregulated by TGF-β1. Conclusions: Our data indicate that TGF-β1 acts as a novel potent inhibitor and modulator of human intestinal MC effector functions. The change in MC mediator release profile and protease expression induced by TGF-β1 might be of relevance for the control of MC associated disorders of the intestine such as allergic reactions, Crohn’s disease, irritable bowel syndrome, and parasitic infection.

Substance P and other neuropeptides do not induce mediator release in isolated human intestinal mast cells

Abstract  Neuropeptides such as substance P (SP) and related peptides are supposed to act as mast cell agonists, and thus as mediators of neuroimmune interactions. The data supporting this hypothesis were obtained mostly from rodent experiments. Here, we studied for the first time the effect of SP and other peptides on mediator release in human intestinal mast cells, either unpurified or enriched to 85–99% purity. We found that SP at 0.1–100 μmol L−1, or other peptides including neurokinin A and B, calcitonin gene‐related peptide, vasoactive intestinal peptide and serotonin at 1 μmol L−1 do not induce release of mediators such as histamine, sulphidoleukotrienes, and tumour necrosis factor α. The peptides also failed to cause mediator release in mast cells isolated from inflamed tissue derived from Crohns disease. Using reverse transcriptase‐polymerase chain reaction, flow cytometry and immunohistochemistry, we could show that human intestinal mast cells do not express the tachykinin receptors NK‐1, NK‐2, or NK‐3 under basal conditions. However, upon stimulation by immunoglobulin E (IgE) receptor‐crosslinking, which induces an extensive mediator release reaction, a subpopulation of mast cells clearly expressed NK‐1, the SP receptor. In conclusion, our data show that SP and other neuropeptides do not act as secretagogues in human intestinal mast cells that have not been pre‐activated by IgE receptor‐crosslinking.

Cultured human intestinal mast cells express functional IL-3 receptors and respond to IL-3 by enhancing growth and IgE receptor-dependent mediator release

Mast cells are immunoregulatory effector cells capable of releasing different mediators and cytokines implicated in inflammatory tissue processes. Previous studies suggested that IL‐3 regulates growth and function of murine mast cells and human mast cell precursors, but does not affect mature human mast cells. In the present study, we found expression of IL‐3 receptors (IL‐3R) in freshlyisolated human intestinal mast cells by reverse transcriptase (RT)‐PCR and in mast cells cultured with stem cell factor (SCF) using RT‐PCR and flow cytometry. IL‐3R expression was enhanced when theculture medium was supplemented with IL‐4 in addition to SCF. In the presence of SCF, IL‐3 significantly enhanced mast cell growth in a dose‐dependent fashion (179±51% of control, p⩽0.004, n=9, ED50 ≈ 15 ng/ml) by decreasing mast cell apoptosis rather than inducing proliferation. Furthermore, IL‐3 selectively enhanced histamine (from 39.6±12.4 to 51.2±15.7% specific release, p<0.02, n=8) and leukotriene C4 (LTC4, 5.1±3.4 to 10.8±5.5 ng/106 mast cells, p<0.03, n=6) release triggered by IgE receptor cross‐linking without affecting prostaglandin D2 production. In conclusion, our data show that human intestinal mast cells express functional IL‐3R, indicating that IL‐3 not only regulates growth and function of immature, but also that of mature human mast cells.

Regulation of human intestinal mast cells by stem cell factor and IL-4.

Summary: Mature human mast cells are tissue‐residing, key effector cells of immediate allergic reactions. Moreover, mast cells have been recognized as a potent cellular source of multiple cytokines, suggesting an important role in immunoregulation and host defense. Here, we report on the regulation of mature human mast cells isolated from intestinal tissues by stem cell factor (SCF) and interleukin (IL)‐4. SCF is substantially necessary for mast cell survival and induces marginal mast cell proliferation in vitro, whereas IL‐4 by itself has no effects on mast cell survival or proliferation. Most interestingly, in synergy with SCF, IL‐4 strongly enhances mast cell proliferation. In the presence of SCF, mast cells predominantly produce pro‐inflammatory cytokines including tumor necrosis factor (TNF)‐α, IL‐1β, IL‐6, IL‐8, IL‐16, and IL‐18. Addition of IL‐4 to the culture medium induces the expression of Th2‐type cytokines (IL‐3, IL‐5 and IL‐13), and a downregulation of pro‐inflammatory cytokines, namely IL‐6. Furthermore, SCF by itself supports the predominance of the tryptase/chymase double‐positive mast cell subtype MCTC, whereas the addition of IL‐4 supports the chymase negative MCT subtype. In conclusion, SCF may primarily regulate resident mast cell survival, whereas IL‐4 may promote local proliferation of mast cells and their expression of Th2‐type cytokines.