Background: Mast cells express receptors for complement anaphylatoxins C3a and C5a (ie, C3a receptor [C3aR] and C5a receptor [C5aR]), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo.Objective: We sought to assess whether mouse skin mast cell expression of C3aR or C5aR influences (1) the cells' responsiveness to intradermal injections of C3a or C5a or (2) the extent of IgE-dependent mast cell degranulation and PCA in vivo.Methods: We measured the magnitude of cutaneous responses to intradermal injections of C3a or C5a and the extent of IgE-dependent mast cell degranulation and PCA responses in mice containing mast cells that did or did not express C3aR or C5aR.Results: The majority of the skin swelling induced by means of intradermal injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and IgE-dependent PCA was significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local increases in C3a levels occurred in antibody-deficient mice but not in mice deficient in Fc epsilon RI gamma.Conclusion: Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo. (J Allergy Clin Immunol 2013;131:541-8.)

Background: Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model.Methods: We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated.Results: Despite its low affinity of approximately 10(7) M-1 for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC50 of 70ng/ml [0.47nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced.Conclusion: Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.

Abstract Mast cells express receptors for complement anaphylatoxins C3a and C5a (i.e., C3aR and C5aR) and can be activated by C3a or C5a in vitro, and C3a and C5a are generated during IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent expression of C3aR or C5aR by mast cells influences either C3a- or C5a-induced cutaneous responses, or IgE-dependent passive cutaneous anaphylaxis (PCA), in vivo. In this study we assessed whether the expression of C3aR or C5aR by mouse skin mast cells influences: 1) the cells’ responsiveness to i.d. injections of exogenous C3a or C5a, or 2) the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA, in vivo. We measured the magnitude of cutaneous responses to i.d. injections of C3a or C5a, and the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA responses, in mice containing mast cells that did or did not express C3aR or C5aR. We found that the majority of the skin swelling induced by C3a or C5a required that mast cells expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and of IgE-dependent PCA were significantly reduced when mast cells lacked either C3aR or C5aR. We conclude that the expression of C3aR and C5aR by skin mast cells contributes to the ability of C3a and C5a to induce skin swelling and can enhance the extent of mast cell degranulation and inflammation during IgE-dependent PCA in vivo.

To the Editor:Anaphylaxis is a life-threatening, systemic allergic reaction that can occur suddenly after contact with an otherwise innocuous substance. In certain subjects previously sensitized to a particular antigen, re-exposure to even very small amounts of that antigen can induce the rapid and extensive activation of mast cells (MCs) and basophils. The ensuing massive release of MC- and basophil-derived mediators can then result in severe and sometimes fatal pathophysiological responses.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar It is generally accepted that anaphylaxis in human subjects is mediated by activation of MCs and basophils induced by the cross-linking of FcεRI, the high-affinity receptor for IgE, for example, with IgE and specific bivalent or multivalent antigen. Although IgE can also bind to the IgG receptors FcγRII and FcγRIII and to galectin-3, which are expressed on some MC populations, IgE is thought to influence MC and basophil functions in patients with anaphylaxis mainly through its interaction with FcεRI.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar However, it is likely that factors in addition to antigen-specific IgE can contribute to the occurrence or severity of anaphylaxis. For example, many allergic patients exhibit high levels of circulating antigen-specific IgE but never have anaphylaxis, whereas other subjects with low concentrations of specific IgE in the blood can experience anaphylactic shock.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google ScholarEndothelin-1 (ET-1), a 21-amino-acid naturally occurring peptide with potent vasoconstrictor activity, has been reported to exacerbate certain allergic reactions in rodents. For example, increased levels of ET-1 have been described in animal models of active anaphylaxis,2Sanchez-Cifuentes M.V. Rubio M.L. Ortega M. Largo R. Gomez-Garre M.D. Gonzalez Mangado N. et al.Endothelin-1 expression during early response after antigen challenge in brown-Norway rats.Pulm Pharmacol Ther. 1998; 11: 215-219Crossref PubMed Scopus (5) Google Scholar, 3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and pharmacological inhibition of the endothelin receptor ETA reportedly attenuated the magnitude of ovalbumin–induced intestinal anaphylaxis3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar or paw edema4Sampaio A.L. Rae G.A. D’Orleans-Juste P. Henriques M.G. ETA receptor antagonists inhibit allergic inflammation in the mouse.J Cardiovasc Pharmacol. 1995; 26: S416-S418PubMed Google Scholar in ovalbumin–sensitized animals. ET-1 can induce degranulation of MCs in mice,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar suggesting one mechanism by which increased levels of ET-1 might exacerbate anaphylaxis. However, the potential interactions between MC and ET-1 in anaphylaxis are complex. For example, mouse MCs can secrete ET-16Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar; MC-derived proteases, particularly MC-derived carboxypeptidase A,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 7Metz M. Piliponsky A.M. Chen C.C. Lammel V. Ǻbrink M. Pejler G. et al.Mast cells can enhance resistance to snake and honeybee venoms.Science. 2006; 313: 526-530Crossref PubMed Scopus (285) Google Scholar, 8Schneider L.A. Schlenner S.M. Feyerabend T.B. Wunderlin M. Rodewald H.R. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin.J Exp Med. 2007; 204: 2629-2639Crossref PubMed Scopus (124) Google Scholar can hydrolyze ET-1 to a biologically inactive peptide by removing the C-terminal tryptophan8Schneider L.A. Schlenner S.M. Feyerabend T.B. Wunderlin M. Rodewald H.R. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin.J Exp Med. 2007; 204: 2629-2639Crossref PubMed Scopus (124) Google Scholar; and pretreatment of in vitro–derived mouse MCs with ET-1 can significantly decrease the cells’ degranulation in response to IgE and antigen in vitro.6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar In the present study we investigated whether the severity of IgE-dependent passive systemic or cutaneous anaphylaxis is altered in mice treated with BQ-123, an antagonist of the endothelin receptor ETA.MCs are thought to represent a critical effector cell in IgE-dependent anaphylaxis.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Prior functional assays and expression analyses of various MC populations demonstrated that ETA can be expressed by connective tissue–type MCs, including peritoneal MCs.5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar, 9Metz M. Lammel V. Gibbs B.F. Maurer M. Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells.Am J Pathol. 2006; 169: 815-822Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar However, immature bone marrow–derived cultured mouse mast cells (BMCMCs), which have some similarities to mucosal-type MCs, are largely unresponsive to ET-1 and have very low levels of receptor expression.6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google ScholarWe confirmed, using flow cytometric analysis, the substantial expression of ETA and weak expression of ETB on C57BL/6 mouse peritoneal MCs (Fig 1, A), as well as the absence of detectable surface ETB and very weak expression of ETA on C57BL/6 mouse BMCMCs (Fig 1, B). When BMCMCs were induced to undergo further maturation by maintaining the cells in the presence of IL-4 and stem cell factor, in addition to IL-3, the expression of ET receptors increased to levels approaching those of freshly isolated peritoneal MCs (Fig 1, C). Taken together, these results support the notion that a robust surface expression of ETA is a characteristic of mature murine MCs.Binding of ET-1 to ETA can induce strong and rapid degranulation of MCs,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar and ET-1 levels previously have been shown to be increased in certain animal models of anaphylaxis.2Sanchez-Cifuentes M.V. Rubio M.L. Ortega M. Largo R. Gomez-Garre M.D. Gonzalez Mangado N. et al.Endothelin-1 expression during early response after antigen challenge in brown-Norway rats.Pulm Pharmacol Ther. 1998; 11: 215-219Crossref PubMed Scopus (5) Google Scholar, 3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar We therefore hypothesized that ET-1 can have effects on MC-mediated allergic reactions. To investigate whether ET-1 can contribute to features of severe IgE- and MC-dependent allergic reactions, we first analyzed IgE-dependent passive systemic anaphylaxis in mice treated with the selective ETA antagonist BQ-123. Mice were sensitized intraperitoneally with IgE anti–2,4-dinitrophenol (DNP; in 100 μL of sterile, pyrogen-free 0.9% NaCl [saline]) and challenged intraperitoneally 24 hours later with 1 mg of DNP–human serum albumin (HSA; 30-40 DNP:1 HSA; Sigma, St Louis, Mo) in 100 μL of saline to induce anaphylaxis. Mice were pretreated with BQ-123 (Calbiochem, San Diego, Calif; 20 nmol in 200 μL of vehicle [saline + 0.5% dimethyl sulfoxide]) or 200 μL of vehicle intraperitoneally 45 minutes before challenge. Mice treated with BQ-123 exhibited a statistically significant, although modest, amelioration of the allergic reaction, as assessed by monitoring the ensuing change in body temperature (Fig 2, A). Furthermore, the reduced hypothermia was associated with a modest but statistically significant reduction in the extent of degranulation of peritoneal MCs, as assessed morphologically (Fig 2, B),5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 7Metz M. Piliponsky A.M. Chen C.C. Lammel V. Ǻbrink M. Pejler G. et al.Mast cells can enhance resistance to snake and honeybee venoms.Science. 2006; 313: 526-530Crossref PubMed Scopus (285) Google Scholar indicating that blockade of ETA results in decreased activation of MCs in this setting. In support of this conclusion, pretreatment with BQ-123 in mice subjected to passive systemic anaphylaxis also resulted in reduced levels of both MC-derived mouse MC protease 1 in the serum (Fig 2, C) and histamine in the peritoneal lavage fluid (Fig 2, D).Fig 2Selective blockade of ETA attenuates IgE-dependent anaphylactic reactions. A-D, Body temperature (Fig 2, A), peritoneal MC degranulation (Fig 2, B), levels of mouse mast cell protease 1 (mMCP-1) in serum (Fig 2, C), and levels of histamine in peritoneal lavage fluid (PLF; Fig 2, D) in C57BL/6 mice sensitized with IgE anti-DNP intravenously and then injected 24 hours later with DNP-HSA or vehicle (Veh) intraperitoneally 45 minutes after intraperitoneal injection of either BQ-123 or vehicle. E and F, Ear swelling (Fig 2, E) and extravasation of Evans blue dye (Fig 2, F) 1 hour after DNP-HSA challenge in mice injected intradermally with IgE anti-DNP and challenged intraperitoneally 24 hours later with DNP-HSA (in saline plus 1% Evans blue dye). Mice were injected intraperitoneally with either BQ-123 or vehicle 10 minutes before DNP-HSA challenge. Fig 2, A, ∗∗∗P < .005 versus the corresponding IgE anti-DNP plus DNP plus vehicle-challenged group (ANOVA). Fig 2, B, ∗P < .05 and ∗∗∗P < .005 for the indicated comparisons (χ2 test). Fig 2, C-F, ∗P < .05 versus the corresponding vehicle-treated group (unpaired, 2-tailed Student t test). All results are presented as means ± SEMs (Fig 2, A) or means + SEMs (Fig 3, B-F), with 8 to 10 mice per group in each panel (see the expanded Fig 2 legend in this article’s Online Repository at www.jacionline.org for more information about methods).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To assess the possible contribution of ETA to IgE- and MC-dependent allergic responses in the skin, we next induced passive cutaneous anaphylaxis by means of intradermal injection of IgE anti-DNP (20 μg in 100 μL of saline) and then challenged the mice intraperitoneally with DNP-HSA (1 mg in 100 μL of saline) or 100 μL of saline alone 24 hours later. The increase in ear thickness and the extent of plasma extravasation were then quantified as measures of allergic inflammation. Injection of BQ-123 intravenously before elicitation of the allergic reaction again resulted in a significant reduction in the assessed parameters, with about 30% to 40% reduction in the measured responses compared with those seen in the identically sensitized and challenged but vehicle-treated mice (Fig 2, E and F).Taken together, our findings show that treatment with the ETA antagonist BQ-123 can ameliorate systemic or local IgE- and anaphylactic responses in mice. We hypothesize that one mechanism that might contribute to this observation is that the ETA antagonist can reduce ETA-dependent enhancement of MC activation by endogenous ET-1 present during the allergic reaction. To the Editor: Anaphylaxis is a life-threatening, systemic allergic reaction that can occur suddenly after contact with an otherwise innocuous substance. In certain subjects previously sensitized to a particular antigen, re-exposure to even very small amounts of that antigen can induce the rapid and extensive activation of mast cells (MCs) and basophils. The ensuing massive release of MC- and basophil-derived mediators can then result in severe and sometimes fatal pathophysiological responses.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar It is generally accepted that anaphylaxis in human subjects is mediated by activation of MCs and basophils induced by the cross-linking of FcεRI, the high-affinity receptor for IgE, for example, with IgE and specific bivalent or multivalent antigen. Although IgE can also bind to the IgG receptors FcγRII and FcγRIII and to galectin-3, which are expressed on some MC populations, IgE is thought to influence MC and basophil functions in patients with anaphylaxis mainly through its interaction with FcεRI.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar However, it is likely that factors in addition to antigen-specific IgE can contribute to the occurrence or severity of anaphylaxis. For example, many allergic patients exhibit high levels of circulating antigen-specific IgE but never have anaphylaxis, whereas other subjects with low concentrations of specific IgE in the blood can experience anaphylactic shock.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Endothelin-1 (ET-1), a 21-amino-acid naturally occurring peptide with potent vasoconstrictor activity, has been reported to exacerbate certain allergic reactions in rodents. For example, increased levels of ET-1 have been described in animal models of active anaphylaxis,2Sanchez-Cifuentes M.V. Rubio M.L. Ortega M. Largo R. Gomez-Garre M.D. Gonzalez Mangado N. et al.Endothelin-1 expression during early response after antigen challenge in brown-Norway rats.Pulm Pharmacol Ther. 1998; 11: 215-219Crossref PubMed Scopus (5) Google Scholar, 3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and pharmacological inhibition of the endothelin receptor ETA reportedly attenuated the magnitude of ovalbumin–induced intestinal anaphylaxis3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar or paw edema4Sampaio A.L. Rae G.A. D’Orleans-Juste P. Henriques M.G. ETA receptor antagonists inhibit allergic inflammation in the mouse.J Cardiovasc Pharmacol. 1995; 26: S416-S418PubMed Google Scholar in ovalbumin–sensitized animals. ET-1 can induce degranulation of MCs in mice,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar suggesting one mechanism by which increased levels of ET-1 might exacerbate anaphylaxis. However, the potential interactions between MC and ET-1 in anaphylaxis are complex. For example, mouse MCs can secrete ET-16Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar; MC-derived proteases, particularly MC-derived carboxypeptidase A,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 7Metz M. Piliponsky A.M. Chen C.C. Lammel V. Ǻbrink M. Pejler G. et al.Mast cells can enhance resistance to snake and honeybee venoms.Science. 2006; 313: 526-530Crossref PubMed Scopus (285) Google Scholar, 8Schneider L.A. Schlenner S.M. Feyerabend T.B. Wunderlin M. Rodewald H.R. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin.J Exp Med. 2007; 204: 2629-2639Crossref PubMed Scopus (124) Google Scholar can hydrolyze ET-1 to a biologically inactive peptide by removing the C-terminal tryptophan8Schneider L.A. Schlenner S.M. Feyerabend T.B. Wunderlin M. Rodewald H.R. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin.J Exp Med. 2007; 204: 2629-2639Crossref PubMed Scopus (124) Google Scholar; and pretreatment of in vitro–derived mouse MCs with ET-1 can significantly decrease the cells’ degranulation in response to IgE and antigen in vitro.6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar In the present study we investigated whether the severity of IgE-dependent passive systemic or cutaneous anaphylaxis is altered in mice treated with BQ-123, an antagonist of the endothelin receptor ETA. MCs are thought to represent a critical effector cell in IgE-dependent anaphylaxis.1Summers C.W. Pumphrey R.S. Woods C.N. McDowell G. Pemberton P.W. Arkwright P.D. Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.J Allergy Clin Immunol. 2008; 121 (e2): 632-638Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Prior functional assays and expression analyses of various MC populations demonstrated that ETA can be expressed by connective tissue–type MCs, including peritoneal MCs.5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar, 9Metz M. Lammel V. Gibbs B.F. Maurer M. Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells.Am J Pathol. 2006; 169: 815-822Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar However, immature bone marrow–derived cultured mouse mast cells (BMCMCs), which have some similarities to mucosal-type MCs, are largely unresponsive to ET-1 and have very low levels of receptor expression.6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar We confirmed, using flow cytometric analysis, the substantial expression of ETA and weak expression of ETB on C57BL/6 mouse peritoneal MCs (Fig 1, A), as well as the absence of detectable surface ETB and very weak expression of ETA on C57BL/6 mouse BMCMCs (Fig 1, B). When BMCMCs were induced to undergo further maturation by maintaining the cells in the presence of IL-4 and stem cell factor, in addition to IL-3, the expression of ET receptors increased to levels approaching those of freshly isolated peritoneal MCs (Fig 1, C). Taken together, these results support the notion that a robust surface expression of ETA is a characteristic of mature murine MCs. Binding of ET-1 to ETA can induce strong and rapid degranulation of MCs,5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 6Matsushima H. Yamada N. Matsue H. Shimada S. The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.Eur J Immunol. 2004; 34: 1910-1919Crossref PubMed Scopus (68) Google Scholar and ET-1 levels previously have been shown to be increased in certain animal models of anaphylaxis.2Sanchez-Cifuentes M.V. Rubio M.L. Ortega M. Largo R. Gomez-Garre M.D. Gonzalez Mangado N. et al.Endothelin-1 expression during early response after antigen challenge in brown-Norway rats.Pulm Pharmacol Ther. 1998; 11: 215-219Crossref PubMed Scopus (5) Google Scholar, 3Shigematsu T. Miura S. Hirokawa M. Hokari R. Higuchi H. Tsuzuki Y. et al.Endothelins promote egg albumin-induced intestinal anaphylaxis in rats.Gastroenterology. 1998; 115: 348-356Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar We therefore hypothesized that ET-1 can have effects on MC-mediated allergic reactions. To investigate whether ET-1 can contribute to features of severe IgE- and MC-dependent allergic reactions, we first analyzed IgE-dependent passive systemic anaphylaxis in mice treated with the selective ETA antagonist BQ-123. Mice were sensitized intraperitoneally with IgE anti–2,4-dinitrophenol (DNP; in 100 μL of sterile, pyrogen-free 0.9% NaCl [saline]) and challenged intraperitoneally 24 hours later with 1 mg of DNP–human serum albumin (HSA; 30-40 DNP:1 HSA; Sigma, St Louis, Mo) in 100 μL of saline to induce anaphylaxis. Mice were pretreated with BQ-123 (Calbiochem, San Diego, Calif; 20 nmol in 200 μL of vehicle [saline + 0.5% dimethyl sulfoxide]) or 200 μL of vehicle intraperitoneally 45 minutes before challenge. Mice treated with BQ-123 exhibited a statistically significant, although modest, amelioration of the allergic reaction, as assessed by monitoring the ensuing change in body temperature (Fig 2, A). Furthermore, the reduced hypothermia was associated with a modest but statistically significant reduction in the extent of degranulation of peritoneal MCs, as assessed morphologically (Fig 2, B),5Maurer M. Wedemeyer J. Metz M. Piliponsky A.M. Weller K. Chatterjea D. et al.Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.Nature. 2004; 432: 512-516Crossref PubMed Scopus (256) Google Scholar, 7Metz M. Piliponsky A.M. Chen C.C. Lammel V. Ǻbrink M. Pejler G. et al.Mast cells can enhance resistance to snake and honeybee venoms.Science. 2006; 313: 526-530Crossref PubMed Scopus (285) Google Scholar indicating that blockade of ETA results in decreased activation of MCs in this setting. In support of this conclusion, pretreatment with BQ-123 in mice subjected to passive systemic anaphylaxis also resulted in reduced levels of both MC-derived mouse MC protease 1 in the serum (Fig 2, C) and histamine in the peritoneal lavage fluid (Fig 2, D). To assess the possible contribution of ETA to IgE- and MC-dependent allergic responses in the skin, we next induced passive cutaneous anaphylaxis by means of intradermal injection of IgE anti-DNP (20 μg in 100 μL of saline) and then challenged the mice intraperitoneally with DNP-HSA (1 mg in 100 μL of saline) or 100 μL of saline alone 24 hours later. The increase in ear thickness and the extent of plasma extravasation were then quantified as measures of allergic inflammation. Injection of BQ-123 intravenously before elicitation of the allergic reaction again resulted in a significant reduction in the assessed parameters, with about 30% to 40% reduction in the measured responses compared with those seen in the identically sensitized and challenged but vehicle-treated mice (Fig 2, E and F). Taken together, our findings show that treatment with the ETA antagonist BQ-123 can ameliorate systemic or local IgE- and anaphylactic responses in mice. We hypothesize that one mechanism that might contribute to this observation is that the ETA antagonist can reduce ETA-dependent enhancement of MC activation by endogenous ET-1 present during the allergic reaction. AppendixExpanded legend for Fig 1Analysis of ET receptor expression on C57BL/6 mouse MCs. IL-3 in culture medium was derived from 20% WEHI-3–conditioned medium, IL-4 (from Calbiochem) was used at 30 ng/mL, and stem cell factor (from Amgen, Thousand Oaks, Calif) was used at 50 ng/mL. We assessed surface expression of ETA and ETB using rabbit anti-mouse ETA and ETB antibodies from Santa Cruz Biotechnology (Santa Cruz, Calif; M-60 and M-74, respectively).Expanded legend for Fig 2Elicitation and assessment of IgE-dependent anaphylactic reactions. A, Passive systemic anaphylaxis was induced by sensitizing C57BL/6 mice with 20 μg of IgE anti-DNP,E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and mice were challenged 24 hours later intraperitoneally with 1 mg of DNP-HSA or vehicle. The selective ETA antagonist BQ-123 (20 nmol; Bachem, Bubendorf, Switzerland) in 200 μL of vehicle (saline [sterile, pyrogen-free 0.9% NaCl plus 0.5% dimethyl sulfoxide]) or vehicle alone was injected intraperitoneally 45 minutes before DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. B, Quantitative histomorphometry was performed on peritoneal lavage–derived MCs obtained 1 hour after DNP-HSA (30-40 DNP:1 HSA; Sigma) or saline challenge, with white bars, hatched bars, and black bars in Fig 2, B, indicating no, moderate, or extensive degranulation, respectively.E2Wershil B.K. Murakami T. Galli S.J. Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12- myristate 13-acetate.J Immunol. 1988; 140: 2356-2360PubMed Google Scholar There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. C and D, Levels of mouse MC protease 1 in the serum (Fig 2, C) were measured by means of ELISA (eBioscience, San Diego), and histamine concentrations in peritoneal lavage fluid (Fig 2, D) were measured with the Histareader 501 (RefLab, Copenhagen, Denmark), both according to the instructions of the manufacturer, 15 minutes after induction of passive systemic anaphylaxis. There were 9 to 10 (mouse MC protease 1) and 8 to 9 mice (histamine) per group pooled from 2 independent experiments, each of which produced similar results. Body temperature in the 2 treatment groups differed significantly 10 minutes after induction of passive systemic anaphylaxis (vehicle: −3.3°C ± 0.1°C vs BQ-123: −2.5°C ± 0.3°C; P < .01 unpaired Student t test, 2-tailed). E and F, Passive cutaneous anaphylaxis was induced in C57BL/6 mice by sensitizing them with 20 ng of IgE anti-DNP (in 20 μL of saline administered intradermally),E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and control C57BL/6 mice were given 20 μL of saline administered intradermally. Mice were then challenged intraperitoneally 24 hours later with 200 μg of DNP-HSA (in 200 μL of saline plus 1% Evans blue). Mice were injected intraperitoneally with either BQ-123 (20 nmol in 200 μL administered intravenously) or vehicle 10 minutes before challenge, and ear swellingE3Wershil B.K. Mekori Y.A. Murakami T. Galli S.J. 125I-fibrin deposition in IgE- dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells.J Immunol. 1987; 139: 2605-2614PubMed Google Scholar (Fig 2, E) and extravasation of Evans blue dye (absorbance of the dye; Fig 2, F),E4Inagaki N. Nagai H. Koda A. Comparative study of IgG1 and IgE antibody mediated homologous PCA in the mouse ear. Lack of cross-desensitization of IgG1 antibody mediated PCA to IgE antibody mediated PCA.Int Arch Allergy Appl Immunol. 1988; 86: 325-330Crossref PubMed Scopus (13) Google Scholar each calculated as the percentage of the mean result in the vehicle-treated group, were measured 1 hour after DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. All results are presented as means ± SEMs (Fig 2, A) or means + SEMs (Fig 2, B-F). Expanded legend for Fig 1Analysis of ET receptor expression on C57BL/6 mouse MCs. IL-3 in culture medium was derived from 20% WEHI-3–conditioned medium, IL-4 (from Calbiochem) was used at 30 ng/mL, and stem cell factor (from Amgen, Thousand Oaks, Calif) was used at 50 ng/mL. We assessed surface expression of ETA and ETB using rabbit anti-mouse ETA and ETB antibodies from Santa Cruz Biotechnology (Santa Cruz, Calif; M-60 and M-74, respectively). Analysis of ET receptor expression on C57BL/6 mouse MCs. IL-3 in culture medium was derived from 20% WEHI-3–conditioned medium, IL-4 (from Calbiochem) was used at 30 ng/mL, and stem cell factor (from Amgen, Thousand Oaks, Calif) was used at 50 ng/mL. We assessed surface expression of ETA and ETB using rabbit anti-mouse ETA and ETB antibodies from Santa Cruz Biotechnology (Santa Cruz, Calif; M-60 and M-74, respectively). Expanded legend for Fig 2Elicitation and assessment of IgE-dependent anaphylactic reactions. A, Passive systemic anaphylaxis was induced by sensitizing C57BL/6 mice with 20 μg of IgE anti-DNP,E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and mice were challenged 24 hours later intraperitoneally with 1 mg of DNP-HSA or vehicle. The selective ETA antagonist BQ-123 (20 nmol; Bachem, Bubendorf, Switzerland) in 200 μL of vehicle (saline [sterile, pyrogen-free 0.9% NaCl plus 0.5% dimethyl sulfoxide]) or vehicle alone was injected intraperitoneally 45 minutes before DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. B, Quantitative histomorphometry was performed on peritoneal lavage–derived MCs obtained 1 hour after DNP-HSA (30-40 DNP:1 HSA; Sigma) or saline challenge, with white bars, hatched bars, and black bars in Fig 2, B, indicating no, moderate, or extensive degranulation, respectively.E2Wershil B.K. Murakami T. Galli S.J. Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12- myristate 13-acetate.J Immunol. 1988; 140: 2356-2360PubMed Google Scholar There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. C and D, Levels of mouse MC protease 1 in the serum (Fig 2, C) were measured by means of ELISA (eBioscience, San Diego), and histamine concentrations in peritoneal lavage fluid (Fig 2, D) were measured with the Histareader 501 (RefLab, Copenhagen, Denmark), both according to the instructions of the manufacturer, 15 minutes after induction of passive systemic anaphylaxis. There were 9 to 10 (mouse MC protease 1) and 8 to 9 mice (histamine) per group pooled from 2 independent experiments, each of which produced similar results. Body temperature in the 2 treatment groups differed significantly 10 minutes after induction of passive systemic anaphylaxis (vehicle: −3.3°C ± 0.1°C vs BQ-123: −2.5°C ± 0.3°C; P < .01 unpaired Student t test, 2-tailed). E and F, Passive cutaneous anaphylaxis was induced in C57BL/6 mice by sensitizing them with 20 ng of IgE anti-DNP (in 20 μL of saline administered intradermally),E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and control C57BL/6 mice were given 20 μL of saline administered intradermally. Mice were then challenged intraperitoneally 24 hours later with 200 μg of DNP-HSA (in 200 μL of saline plus 1% Evans blue). Mice were injected intraperitoneally with either BQ-123 (20 nmol in 200 μL administered intravenously) or vehicle 10 minutes before challenge, and ear swellingE3Wershil B.K. Mekori Y.A. Murakami T. Galli S.J. 125I-fibrin deposition in IgE- dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells.J Immunol. 1987; 139: 2605-2614PubMed Google Scholar (Fig 2, E) and extravasation of Evans blue dye (absorbance of the dye; Fig 2, F),E4Inagaki N. Nagai H. Koda A. Comparative study of IgG1 and IgE antibody mediated homologous PCA in the mouse ear. Lack of cross-desensitization of IgG1 antibody mediated PCA to IgE antibody mediated PCA.Int Arch Allergy Appl Immunol. 1988; 86: 325-330Crossref PubMed Scopus (13) Google Scholar each calculated as the percentage of the mean result in the vehicle-treated group, were measured 1 hour after DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. All results are presented as means ± SEMs (Fig 2, A) or means + SEMs (Fig 2, B-F). Elicitation and assessment of IgE-dependent anaphylactic reactions. A, Passive systemic anaphylaxis was induced by sensitizing C57BL/6 mice with 20 μg of IgE anti-DNP,E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and mice were challenged 24 hours later intraperitoneally with 1 mg of DNP-HSA or vehicle. The selective ETA antagonist BQ-123 (20 nmol; Bachem, Bubendorf, Switzerland) in 200 μL of vehicle (saline [sterile, pyrogen-free 0.9% NaCl plus 0.5% dimethyl sulfoxide]) or vehicle alone was injected intraperitoneally 45 minutes before DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. B, Quantitative histomorphometry was performed on peritoneal lavage–derived MCs obtained 1 hour after DNP-HSA (30-40 DNP:1 HSA; Sigma) or saline challenge, with white bars, hatched bars, and black bars in Fig 2, B, indicating no, moderate, or extensive degranulation, respectively.E2Wershil B.K. Murakami T. Galli S.J. Mast cell-dependent amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12- myristate 13-acetate.J Immunol. 1988; 140: 2356-2360PubMed Google Scholar There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. C and D, Levels of mouse MC protease 1 in the serum (Fig 2, C) were measured by means of ELISA (eBioscience, San Diego), and histamine concentrations in peritoneal lavage fluid (Fig 2, D) were measured with the Histareader 501 (RefLab, Copenhagen, Denmark), both according to the instructions of the manufacturer, 15 minutes after induction of passive systemic anaphylaxis. There were 9 to 10 (mouse MC protease 1) and 8 to 9 mice (histamine) per group pooled from 2 independent experiments, each of which produced similar results. Body temperature in the 2 treatment groups differed significantly 10 minutes after induction of passive systemic anaphylaxis (vehicle: −3.3°C ± 0.1°C vs BQ-123: −2.5°C ± 0.3°C; P < .01 unpaired Student t test, 2-tailed). E and F, Passive cutaneous anaphylaxis was induced in C57BL/6 mice by sensitizing them with 20 ng of IgE anti-DNP (in 20 μL of saline administered intradermally),E1Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. et al.Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J Immunol. 1980; 124: 2728-2737PubMed Google Scholar and control C57BL/6 mice were given 20 μL of saline administered intradermally. Mice were then challenged intraperitoneally 24 hours later with 200 μg of DNP-HSA (in 200 μL of saline plus 1% Evans blue). Mice were injected intraperitoneally with either BQ-123 (20 nmol in 200 μL administered intravenously) or vehicle 10 minutes before challenge, and ear swellingE3Wershil B.K. Mekori Y.A. Murakami T. Galli S.J. 125I-fibrin deposition in IgE- dependent immediate hypersensitivity reactions in mouse skin. Demonstration of the role of mast cells using genetically mast cell-deficient mice locally reconstituted with cultured mast cells.J Immunol. 1987; 139: 2605-2614PubMed Google Scholar (Fig 2, E) and extravasation of Evans blue dye (absorbance of the dye; Fig 2, F),E4Inagaki N. Nagai H. Koda A. Comparative study of IgG1 and IgE antibody mediated homologous PCA in the mouse ear. Lack of cross-desensitization of IgG1 antibody mediated PCA to IgE antibody mediated PCA.Int Arch Allergy Appl Immunol. 1988; 86: 325-330Crossref PubMed Scopus (13) Google Scholar each calculated as the percentage of the mean result in the vehicle-treated group, were measured 1 hour after DNP-HSA or saline challenge. There were 9 mice per group pooled from 3 independent experiments, each of which produced similar results. All results are presented as means ± SEMs (Fig 2, A) or means + SEMs (Fig 2, B-F).

Conformational epitopes of myelin oligodendrocyte glycoprotein (MOG) provide a major target for demyelinating autoantibodies in experimental autoimmune encephalomyelitis and recent studies indicate that a similar situation may exist in multiple sclerosis. We recently solved the crystal structure of the extracellular domain of MOG (MOGex) in complex with a Fab derived from the demyelinating mAb 8-18C5 and identified the conformational 8-18C5 epitope on MOG that is dominated by the surface exposed FG loop of MOG. To determine the importance of this epitope with regard to the polyclonal Ab response to MOGex we investigated the effects of mutating His103 and Ser104, the two central amino acids of the FG loop, on Ab binding. Mutation of these two residues reduced binding of a panel of eight demyelinating conformation-dependent mAbs to <20% compared with binding to wild-type MOGex, whereas substitution of amino acids that do not contribute to the 8-18C5 epitope had only a minor effect on Ab binding. The same restriction was observed for the polyclonal MOG-specific Ab response of MOG DNA-vaccinated BALB/c and SJL/J mice. Our data demonstrate that the pathogenic anti-MOG Ab response primarily targets one immunodominant region centered at the FG loop of MOG. Comparison of the structure of MOGex with the structures of related IgV-like domains yields a possible explanation for the focused Ab response.

BACKGROUND:TNF is thought to contribute to airway hyperreactivity (AHR) and airway inflammation in asthma. However, studies with TNF-deficient or TNF receptor-deficient mice have not produced a clear picture of the role of TNF in the AHR associated with allergic inflammation in the mouse.OBJECTIVE:We used a genetic approach to investigate the contributions of TNF to antigen-induced AHR and airway inflammation in mice on the C57BL/6 background.METHODS:We analyzed features of airway allergic inflammation, including antigen-induced AHR, in C57BL/6 wild-type and TNF(-/-) mice, using 2 different methods for sensitizing the mice to ovalbumin (OVA).RESULTS:In mice sensitized to OVA administered with the adjuvant aluminum hydroxide (alum), which develop IgE-independent and mast cell-independent allergic inflammation and AHR, we found no significant differences in OVA-induced AHR in C57BL/6-TNF(-/-) versus wild-type mice. By contrast, in mice sensitized to OVA without alum, which develop allergic inflammation that is significantly mast cell-dependent, C57BL/6-TNF(-/-) mice exhibited significant reductions versus wild-type mice in OVA-induced AHR to methacholine; numbers of lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid; levels of myeloperoxidase, eosinophil peroxidase, and the cytokines IL-4, IL-5, and IL-17 in lung tissue; and histologic evidence of pulmonary inflammation.CONCLUSION:In pulmonary allergic inflammation induced in mice immunized with OVA without alum, TNF significantly contributes to several features of the response, including antigen-induced inflammation and AHR.CLINICAL IMPLICATIONS:Our findings in mice support the hypothesis that TNF can promote the allergic inflammation and AHR associated with asthma.

Signalling through G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTK) is involved in the regulation of essential cellular processes and its deregulation is associated with tumorigenesis in vitro and in vivo. We investigated pathophysiological processes that are regulated by GPCR pathways in human kidney and bladder cancer cell lines. Our results show that GPCR ligands induce tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) as well as downstream signalling events such as recruitment of the adapter protein Shc and activation of the mitogen-activated protein kinases (MAPK) ERK1/2, JNK and p38. Moreover, we report that the EGFR transactivation signal involves the EGFR ligands amphiregulin, HB-EGF and TGFalpha as well as the metalloproteinases ADAM 10, 15 and 17, depending on the cellular system. Finally, we demonstrate that EGFR transactivation is part of a regulatory system that modulates the migratory and invasive behaviour of kidney and bladder cancer cells. In conclusion, our findings demonstrate that metalloproteinase-mediated transactivation of the EGFR is a key mechanism of the cellular signalling network that promotes MAPK activation as well as tumour cell migration and invasion in response to a variety of physiologically relevant GPCR ligands, and therefore represents a novel target for cancer intervention strategies.