Acute trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a 'pre-existing' nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a 'pre-existing' nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases

Triggering receptor expressed on myeloid cells-1 (TREM-1) potently amplifies acute inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Here we demonstrate that TREM-1 is also crucially involved in chronic inflammatory bowel diseases (IBD). Myeloid cells of the normal intestine generally lack TREM-1 expression. In experimental mouse models of colitis and in patients with IBD, however, TREM-1 expression in the intestine was upregulated and correlated with disease activity. TREM-1 significantly enhanced the secretion of relevant proinflammatory mediators in intestinal macrophages from IBD patients. Blocking TREM-1 by the administration of an antagonistic peptide substantially attenuated clinical course and histopathological alterations in experimental mouse models of colitis. This effect was also seen when the antagonistic peptide was administered only after the first appearance of clinical signs of colitis. Hence, TREM-1-mediated amplification of inflammation contributes not only to the exacerbation of acute inflammatory disorders but also to the perpetuation of chronic inflammatory disorders. Furthermore, interfering with TREM-1 engagement leads to the simultaneous reduction of production and secretion of a variety of pro-inflammatory mediators such as TNF, IL-6, IL-8 (CXCL8), MCP-1 (CCL2), and IL-1beta. Therefore, TREM-1 may also represent an attractive target for the treatment of chronic inflammatory disorders.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.

IL-33 is a mediator rather than a trigger of the acute allergic response in humans

BACKGROUND IL-33 enhances FcεRI-induced mediator release in human basophils without inducing degranulation itself. In contrast, studies in mice suggested that in the presence of high IgE levels, IL-33 triggers degranulation and anaphylaxis of similar severity as specific allergen. Consistent with this view, sera of atopic patients contain elevated levels of IL-33 after anaphylaxis. In this study, we determined whether IL-33 is potentially anaphylactogenic in humans with high IgE levels by regulating exocytosis independent of FcεRI cross-linking. Furthermore, we investigated whether IL-33 is released upon allergen provocation in vivo. METHODS In subjects with high serum IgE levels, we measured IL-33-induced histamine/LTC4 in vitro, CD63 translocation ex vivo, and responsiveness of mast cells in vivo by skin prick test (SPT). In asthma patients, release of IL-33 and its correlation with early (tryptase)- and late-phase markers (IL-13 levels, eosinophil numbers) of the allergic response were assessed in bronchoalveolar lavage fluids (BALFs) after allergen challenge. RESULTS IL-33 itself does not trigger basophil degranulation in vitro and ex vivo, even in subjects with high serum IgE levels, and negative SPTs demonstrate that skin mast cells do not degranulate in response to IL-33. However, in response to allergen challenge, IL-33 is rapidly released into BALFs at levels that do not correlate with other immediate- and late-phase parameters. CONCLUSION IL-33 is unlikely an independent trigger of anaphylaxis even in subjects with high IgE levels. However, the rapid release of IL-33 upon allergen provocation in vivo supports its role as a mediator of immediate allergic responses.

Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells.

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

ANAPHYLACTICALLY - MEDIATED EPITHELIAL CHLORINE ION SECRETORY RESPONSE INISOLATED JEJUNUM OF PARASITIZED GUINEA PIGS (TRICHINELLA SPIRALIS)

Electrophysiological studies were conducted to test the hypothesis that alterations in intestinal epithelial function are associated with immunological responses directed against the enteric parasite, Trichinella spirals. Trichinella antigens were used to challenge sensitized jejunum from infected guinea pigs while monitoring ion transport properties of the tissue in an Ussing-type chamber. The addition of antigen caused increases in transepithelial PD and I(,sc) that were rapidly induced, peaked at 1.5 to 2 min after antigen-challenge, and lasted 10 to 20 min thereafter. The increase in I(,sc) ((DELTA)I(,sc)) varied in a dose-dependent manner until a maximal increase of 40 (mu)A/cm('2) was obtained by the addition of 13 (mu)g of antigenic protein per ml of serosal fluid in the Ussing chamber. Trichinella antigen did not elicit alterations in either PD or I(,sc) of nonimmune tissue. Jejunal tissue from guinea pigs immunized with ovalbumin according to a protocol that stimulated homocytotropic antibody production responded electrically to challenge with ovalbumin but not trichinella antigen. Jejunal tissue which was passively sensitized with immune serum having a passive cutaneous anaphylaxis (PCA) titer of 32 for both IgE and IgG(,1) anti-trichinella anti-bodies responded electrically after exposure to trichinella antigen. Heat treatment of immune serum abolished the anti-trichinella IgE titer as determined by the PCA test but did not decrease either the electrical response of passively sensitized tissue to antigen or the anaphylactically mediated intestinal smooth muscle contractile response to antigen in the classical Schultz-Dale assay. These results strongly support the hypothesis that immunological responses directed against Trichinella Spiralis alter intestinal epithelial function and suggest that immediate hypersensitivity is the immunological basis of the response.^ Additional studies were performed to test the hypothesis that histamine and prostaglandins that are released from mucosal mast cells during IgE or IgG(,1) - antigen stimulated degranulation mediate electrophysiological changes in the intestinal epithelium that are reflective of Cl('-) secretion and mediated intracellularly by cAMP. Pharmacological and biochemical studies were performed to determine the physiological messengers and ionic basis of electrical alterations in small intestinal epithelium of the guinea pig during in vitro anaphylaxis. Results suggest that Cl('-) secretion mediated, in part, by cAMP contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting epithelial responses. ^

Increased number of non-degranulated mast cells in pancreatic ductal adenocarcinoma but not in acute pancreatitis.

Increasing evidence indicates that tumor microenvironment (TME) is crucial in tumor survival and metastases. Inflammatory cells accumulate around tumors and strangely appear to be permissive to their growth. One key stroma cell is the mast cell (MC), which can secrete numerous pro- and antitumor molecules. We investigated the presence and degranulation state of MC in pancreatic ductal adenocarcinoma (PDAC) as compared to acute ancreatitis (AP). Three different detection methods: (a) toluidine blue staining, as well as immunohistochemistry for (b) tryptase and (c) c-kit, were utilized to assess the number and extent of degranulation of MC in PDAC tissue (n=7), uninvolved pancreatic tissue derived from tumor-free margins (n=7) and tissue form AP (n=4). The number of MC detected with all three methods was significantly increased in PDAC, as compared to normal pancreatic tissue derived from tumor-free margins (p<0.05). The highest number of MC was identified by c-kit, 22.2∓7.5 per high power field (HPF) in PDAC vs 9.7∓5.1 per HPF in normal tissue. Contrary to MC in AP, where most of the detected MC were found degranulated, MC in PDAC appeared intact. In conclusion, MC are increased in number, but not degranulated in PDAC, suggesting that they may contribute to cancer growth by permitting selective release of pro-tumorogenic molecules.

Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo.

Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4(+/+) or il4(-/-) eosinophils. Eosinophils controlled CD103(+) dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity.

Human graft-derived mesenchymal stromal cells potently suppress alloreactive T-cell responses

After organ transplantation, recipient T cells contribute to graft rejection. Mesenchymal stromal cells from the bone marrow (BM-MSCs) are known to suppress allogeneic T-cell responses, suggesting a possible clinical application of MSCs in organ transplantation. Human liver grafts harbor resident populations of MSCs (L-MSCs). We aimed to determine the immunosuppressive effects of these graft-derived MSCs on allogeneic T-cell responses and to compare these with the effects of BM-MSCs. BM-MSCs were harvested from aspirates and L-MSCs from liver graft perfusates. We cultured them for 21 days and compared their suppressive effects with the effects of BM-MSCs on allogeneic T-cell responses. Proliferation, cytotoxic degranulation, and interferon-gamma production of alloreactive T cells were more potently suppressed by L-MSCs than BM-MSCs. Suppression was mediated by both cell-cell contact and secreted factors. In addition, L-MSCs showed ex vivo a higher expression of PD-L1 than BM-MSCs, which was associated with inhibition of T-cell proliferation and cytotoxic degranulation in vitro. Blocking PD-L1 partly abrogated the inhibition of cytotoxic degranulation by L-MSCs. In addition, blocking indoleamine 2,3-dioxygenase partly abrogated the inhibitive effects of L-MSCs, but not BM-MSCs, on T-cell proliferation. In conclusion, liver graft-derived MSC suppression of allogeneic T-cell responses is stronger than BM-MSCs, which may be related to in situ priming and mobilization from the graft. These graft-derived MSCs may therefore be relevant in transplantation by promoting allohyporesponsiveness.

Urticaria : revisión de la fisiopatología

La urticaria es una enfermedad frecuente que involucra a los mastocitos cutáneos como principal efector. Su activación puede resultar de interacciones ligando-receptor o por activación directa de señales intracelulares. Las consecuencias de la activación son la degranulación, la síntesis de leucotrienos y prostaglandinas y por último la producción de citoquinas y quimioquinas. El cuadro clínico final varía dependiendo de varios parámetros, incluyendo la naturaleza de los estímulos y la activación de los mastocitos de los pacientes.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

Neurotrophins and hyperalgesia

Nerve growth factor (NGF), a member of the neurotrophin family, is crucial for survival of nociceptive neurons during development. Recently, it has been shown to play an important role in nociceptive function in adults. NGF is up-regulated after inflammatory injury of the skin. Administration of exogenous NGF either systemically or in the skin causes thermal hyperalgesia within minutes. Mast cells are considered important components in the action of NGF, because prior degranulation abolishes the early NGF-induced component of hyperalgesia. Substances degranulated by mast cells include serotonin, histamine, and NGF. Blockade of histamine receptors does not prevent NGF-induced hyperalgesia. The effects of blocking serotonin receptors are complex and cannot be interpretable uniquely as NGF losing its ability to induce hyperalgesia. To determine whether NGF has a direct effect on dorsal root ganglion neurons, we have begun to investigate the acute effects of NGF on capsaicin responses of small-diameter dorsal root ganglion cells in culture. NGF acutely conditions the response to capsaicin, suggesting that NGF may be important in sensitizing the response of sensory neurons to heat (a process that is thought to operate via the capsaicin receptor VR1). We also have found that ligands for the trkB receptor (brain-derived neurotrophic factor and neurotrophin-4/5) acutely sensitize nociceptive afferents and elicit hyperalgesia. Because brain-derived neurotrophic factor is up-regulated in trkA positive cells after inflammatory injury and is transported anterogradely, we consider it to be a potentially important peripheral component involved in neurotrophin-induced hyperalgesia.