Kazushige Obata

Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks

Ticks are ectoparasitic arthropods that can transmit a variety of microorganisms to humans and animals during blood feeding, causing serious infectious disorders, including Lyme disease. Acaricides are pharmacologic agents that kill ticks. The emergence of acaricide-resistant ticks calls for alternative control strategies for ticks and tick-borne diseases. Many animals develop resistance to ticks after repeated infestations, but the nature of this acquired anti-tick immunity remains poorly understood. Here we investigated the cellular and molecular mechanisms underlying acquired resistance to Haemaphysalis longicornis ticks in mice and found that antibodies were required, as was IgFc receptor expression on basophils but not on mast cells. The infiltration of basophils at tick-feeding sites occurred during the second, but not the first, tick infestation. To assess the requirement for basophil infiltration to acquired tick resistance, mice expressing the human diphtheria toxin receptor under the control of the mast cell protease 8 (Mcpt8) promoter were generated. Diphtheria toxin administration to these mice selectively ablated basophils. Diphtheria toxin-mediated basophil depletion before the second tick infestation resulted in loss of acquired tick resistance. These data provide the first clear evidence, to our knowledge, that basophils play an essential and nonredundant role in antibody-mediated acquired immunity against ticks, which may suggest new strategies for controlling tick-borne diseases.

Nonredundant Roles of Basophils in Immunity

Basophils are the rarest granulocytes and represent less than 1% of peripheral blood leukocytes. They are evolutionarily conserved in many animal species, but their functional significance remained an enigma long after their discovery by Paul Ehrlich in 1879. Studies of basophils were hindered by their rarity, by difficulties in identifying them, and by the paucity of useful analytical tools. Because basophils display several characteristics shared by tissue-resident mast cells, they were often considered minor and possibly redundant relatives of mast cells or even blood-circulating precursors of mast cells. However, newly developed tools for their functional analysis, including basophil-depleting antibodies and genetically engineered mice deficient only in basophils, have fueled basophil research and defined previously unrecognized functions of basophils. We now appreciate that basophils play nonredundant roles in acquired immunity regulation, protective immunity to pathogens, and immunological disorders such as allergy and autoimmunity.

Newly discovered roles for basophils: a neglected minority gains new respect

Basophils are the least common type of granulocyte and they account for less than 1% of peripheral blood leukocytes. Because of this minority status and a phenotype that is similar to mast cells, basophils have often been neglected in immunological studies or considered to have minor, redundant roles in immune responses in vivo. However, recent studies have now defined previously unrecognized roles for basophils in both immune regulation and allergic responses, and have shown that basophils and mast cells have distinct roles in immune responses.

Mast Cells and Basophils Are Selectively Activated In Vitro and In Vivo through CD200R3 in an IgE-Independent Manner

Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via FcεRI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.

Basophils preferentially express mouse mast cell protease 11 among the mast cell tryptase family in contrast to mast cells

Tryptases and chymases are the major proteins stored and secreted by mast cells, and they have various biological functions. However, the nature of proteases produced by basophils has been poorly characterized, particularly in mice. mMCP‐11 is the most recently discovered mast cell tryptase in mice and was originally identified as Prss34, which is transcribed in some mast cell‐like cell lines and at the early stage in the culture of BMMC with IL‐3. Curiously, Prss34 is preferentially expressed in the BM and spleen among normal tissues in contrast to other mast cell tryptases. Therefore, it remains elusive what types of cells express mMCP‐11 in vivo. Here, we show that mMCP‐11 is highly expressed by primary basophils and to a much lesser extent, by some mast cells. Prss34 transcripts were detected abundantly in primary and cultured basophils and very weakly in peritoneal mast cells or cultured BMMC. Conversely, transcripts for mMCP‐6 and mMCP‐7 tryptases were preferentially expressed by cultured and peritoneal mast cells but not basophils. We established a mMCP‐11‐specific mAb and showed that mMCP‐11 proteins are indeed expressed by primary basophils and those infiltrating the affected tissues during allergic inflammation and parasitic infections. Some primary mast cells also expressed mMCP‐11 proteins, albeit at a much lower level. Thus, basophils rather than mast cells are the major source of mMCP‐11. This is the first study to demonstrate that mouse basophils produce a trypsin‐like protease.

Newly appreciated roles for basophils in allergy and protective immunity

To cite this article: Karasuyama H, Obata K, Wada T, Tsujimura Y, Mukai K. Newly appreciated roles for basophils in allergy and protective immunity. Allergy 2011; 66: 1133–1141.

Emerging roles of basophils in protective immunity against parasites

Basophils, the least common type of granulocyte, have long been considered as minor effector cells in allergic responses because of their ability to release allergy-inducing chemical mediators such as histamine and leukotriene C4. However, it is unlikely that many animal species evolutionarily conserve basophils to only elicit allergic responses without any host-beneficial function. The study of basophils has been hampered by their rarity and difficult identification, as well as the lack of suitable animal models. Recent studies using novel analytical tools, including basophil-depleting antibodies and genetically engineered mice deficient only in basophils, have illuminated the crucial and nonredundant roles for basophils in protective immunity against both ecto- and endoparasites.

IgG-mediated systemic anaphylaxis to protein antigen can be induced even under conditions of limited amounts of antibody and antigen.

Systemic anaphylaxis is an acute, severe, and potentially fatal allergic reaction. Two classes of antibodies, IgE and IgG, contribute to the development of anaphylaxis in mice, through different mechanisms with distinct usage of effector cells and chemical mediators. Larger quantities of antibody and antigen are reportedly required to induce IgG-mediated anaphylaxis than IgE-mediated one, suggesting that the former may not happen as frequently as the latter in real life. To readdress this issue, we established in the present study a novel mouse model of passive IgG-mediated systemic anaphylaxis to a native protein antigen, ovalbumin (OVA), rather than artificially haptenated protein antigens used in previous studies. Passive sensitization of mice with a cocktail of but not individual IgG1 mAbs specific to distinct OVA epitopes elicited systemic anaphylaxis in response to OVA challenge. Importantly, much smaller doses of antibody and antigen than previously reported were sufficient for the induction of IgG-mediated systemic anaphylaxis. Moreover, a relatively small dose of antigen could induce severe anaphylaxis through both IgE- and IgG-mediated mechanisms when mice had been passively sensitized with antigen-specific IgE and IgG. These results strongly suggest that IgG-mediated systemic anaphylaxis is not rare among antibody-mediated systemic anaphylaxis, in contrast to previous thought, and significantly contributes to active systemic anaphylaxis in real life, at least in mice.

Basophil-derived mouse mast cell protease 11 induces microvascular leakage and tissue edema in a mast cell-independent manner.

Mouse mast cell protease 11 (mMCP-11) is the most recently identified member of the mouse mast cell tryptase family. This tryptase is preferentially produced by basophils in contrast to other members that are expressed by mast cells but not basophils. Although blood-circulating basophils have long been considered as minor and redundant relatives of tissue-resident mast cells, recent studies illustrated that basophils and mast cells play distinct roles in vivo. To explore the in vivo role of basophil-derived mMCP-11, here we prepared recombinant mMCP-11 and its protease-dead mutant. Subcutaneous injection of the wild-type mMCP-11 but not the mutant induced edematous skin swelling with increased microvascular permeability in a dose-dependent manner. No apparent infiltration of proinflammatory cells including neutrophils and eosinophils was detected in the skin lesions. The cutaneous swelling was abolished by the pretreatment of mice with indomethacin, a cyclooxygenase inhibitor, suggesting the major contribution of prostaglandins to the microvascular leakage. Of note, the cutaneous swelling was elicited even in mast cell-deficient mice, indicating that mast cells are dispensable for the mMCP-11-induced cutaneous swelling. Thus, basophil-derived mMCP-11 can induce microvascular leakage via prostaglandins in a mast cell-independent manner, and may contribute to the development of basophil-mediated inflammatory responses.

Basophils and mast cells play critical roles for leukocyte recruitment in IgE-mediated cutaneous reverse passive Arthus reaction

BACKGROUND The pathogenic role of IgE has been implicated in a variety of allergic and inflammatory diseases. We have previously established an IgE-mediated cutaneous reverse passive Arthus model in which eosinophil infiltration is a prominent feature. This uniquely provides a model of type III hypersensitivity in which Fc classes of Ig that forms immune complex differentially determine the disease manifestation. OBJECTIVE To investigate the mechanisms of how mast cells and basophils regulate this IgE-mediated Arthus reaction. METHODS IgE-mediated cutaneous reverse passive Arthus reaction was induced in wild-type C57BL/6 or WBB6F1-+/+ mice and mast-cell-deficient WBB6F1-W/W(v) mice by intradermal injection of IgE anti-trinitrophenyl antibodies followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. Basophils were depleted in vivo using anti-CD200R3 monoclonal antibody prior to the IC challenge. RESULTS Hemorrhage and infiltration of eosinophils, neutrophils, and basophils were significantly reduced but were not completely abrogated in WBB6F1-W/W(v) mice compared with those in wild-type WBB6F1-+/+ mice. Wild-type C57BL/6 mice treated by basophil-depleting mAb also showed significantly decreased hemorrhage and inflammatory cell infiltration, especially that of eosinophils, compared with control mice. Furthermore, basophil depletion in WBB6F1-W/W(v) mice led to nearly complete inhibition of eosinophil recruitment. By contrast, basophil depletion did not further decrease neutrophil infiltration in WBB6F1-W/W(v) mice. CONCLUSION While mast cells play a central role, basophils also have an important function, especially for eosinophil recruitment, in IgE-mediated cutaneous reverse passive Arthus reaction.