How do basophils contribute to Th2 cell differentiation and allergic responses?

How do basophils contribute to Th2 cell differentiation and allergic responses? Abstract Basophils and mast cells share some features, including basophilic granules in the cytoplasm, cell surface expression of the high-affinity IgE receptor and release of chemical mediators such as histamine. Because of this similarity and their minority status, basophils had often been erroneously considered as minor relatives or blood-circulating precursors of tissue-resident mast cells, and therefore long been neglected or underestimated in immunological studies. Taking advantage of newly developed tools, such as basophil-depleting antibodies and engineered mice deficient for only basophils, recent studies have identified previously unappreciated roles for basophils, distinct from those played by mast cells, in allergic responses, protective immunity against parasitic infections and regulation of other immune cells. In this review, we focus on two topics that we presented and discussed in the 46th Annual Meeting of the Japanese Society for Immunology held in Sendai in December 2017. The first topic is the function of basophils as antigen-presenting cells for driving Th2 cell differentiation. We demonstrated that basophils produce few or no MHC class II (MHC-II) proteins by themselves although they can acquire peptide–MHC-II complexes from dendritic cells through trogocytosis, and present them and provide IL-4 to naive CD4 T cells, promoting Th2 cell differentiation. The second topic is the basophil-specific effector molecules involved in allergic responses. Among mouse mast cell proteases (mMCPs), mMCP-8 and mMCP-11 are expressed almost exclusively by basophils. Analyses in vitro and in vivo revealed that both proteases can induce leukocyte migration through distinct mechanisms, contributing to the development of basophil-dependent allergic inflammation. allergic inflammation, antigen-presenting cell, IL-4, MHC class II, protease Introduction The 46th Annual Meeting of the Japanese Society for Immunology was held in Sendai, Japan, on December 12–14, 2017. More than 1700 participants, including 22 invited speakers from abroad, got together in the Sendai International Center to discuss hot topics in 12 symposia and in 43 workshops and poster sessions, all conducted in English. We presented our recent studies on basophils in Symposium 1 ‘Allergy: state-of-the-art’. The existence of basophils in the peripheral blood was first described by Paul Ehrlich in 1879, much earlier than the discovery of T and B lymphocytes. Although basophils are evolutionally conserved in many animal species, their functional significance in vivo remained uncertain and controversial long after their identification. Basophil research had been hindered by their rarity, accounting for <1% of peripheral blood leukocytes, and by the paucity of useful tools for analysis of their in vivo functions. To overcome these obstacles, we have developed useful analytical tools, including a basophil-depleting monoclonal antibody (mAb) (1) and a series of engineered mice for visualization and ablation of basophils or basophil-specific molecules (2–4). In this article, we provide an overview of two important issues that we presented and discussed in the meeting: firstly, the controversial issue of whether and how basophils contribute to Th2 cell differentiation; and, secondly, the effector molecules that basophils utilize to trigger basophil-dependent allergic inflammatory responses. Do basophils express MHC class II and function as antigen-presenting cells? IL-4 has been demonstrated to play a fundamental role in the differentiation of naive CD4 T cells to Th2 cells that produce Th2 cytokines such as IL-4 and contribute to protective immunity against parasitic infections and allergic responses (5). Dendritic cells (DCs) are professional antigen-presenting cells (APCs), but do not usually produce IL-4. Several cell lineages can produce IL-4, including T cells, natural killer T cells, basophils, eosinophils and mast cells. Thus, the source of ‘initial IL-4’ necessary for Th2 cell differentiation has often been a matter of debate (5). In response to various stimuli, basophils in both humans and mice readily secrete larger amounts of IL-4 than Th2 cells do on a per-cell basis (6), suggesting the possible role of basophils as IL-4 providers for Th2 cell differentiation. Indeed, Sokol et al. (7) demonstrated that subcutaneous administration of papain (which triggers allergic responses) induces migration of basophils to the T-cell zone of draining lymph nodes before Th2 differentiation starts. Depletion of basophils abolished Th2 cell differentiation in lymph nodes, indicating a crucial role of basophils in Th2 differentiation. In this case, it was interpreted that basophils provide IL-4 to T cells while DCs function as APCs. The differentiation of naive CD4 T cells toward Th1, Th2 or Th17 cells requires the interaction with APCs that present complexes of MHC class II (MHC-II) plus peptide and provide co-stimulatory molecules, including CD80, CD86 and CD40, and instructive cytokines to T cells (8). It was generally thought that basophils do not express MHC-II and therefore cannot function as APCs, even though basophils express a co-stimulatory molecule and produce IL-4. This view was challenged by exciting reports from three independent groups, demonstrating that basophils, rather than DCs, function as APCs for driving Th2 cell differentiation (9–11). In all three distinct experimental settings, basophils expressed MHC-II and induced the differentiation of naive CD4 T cells to Th2 cells via basophil-derived IL-4. Ablation of basophils abolished Th2 cell differentiation in vivo. These results strongly suggested that basophils are unique APCs specialized for Th2 differentiation in contrast to DCs that are specialized for Th1 (and Th17) differentiation. This new concept of basophils, rather than DCs, being the important APCs for Th2 cell differentiation attracted much attention, but also gained severe criticism (5, 12, 13). The level of MHC-II expression on basophils is generally much lower than that on typical APCs such as DCs and B cells. This is also true for the expression of H-2M and the invariant chain that are crucial for peptide loading on MHC-II. Another concern is the possible inefficiency and off-target effects of the method used for the depletion of DCs and basophils, respectively (14, 15). Indeed, some later studies demonstrated the essential role for DCs in Th2 cell differentiation (14–17). Therefore, it remained controversial whether the MHC-II expression on basophils has a functional significance and whether basophils indeed have the capacity of antigen processing and presentation to T cells (18). To address this controversial issue, we first re-examined the MHC-II expression on basophils (19). Basophils isolated from the bone marrow and spleen expressed MHC-II at very low levels, almost negligible compared with high levels on professional APCs such as DCs and B cells. Incubation of these basophils with various cytokines including IFN-γ failed to up-regulate MHC-II expression, in spite of fact that IFN-γ is well known to induce MHC-II expression on many cell lineages. During the in vitro generation of basophils from bone marrow cells in the presence of IL-3, the addition of GM-CSF but not IFN-γ to the culture highly increased MHC-II expression on basophils. Unexpectedly, however, we could detect little or no transcriptional up-regulation of the H2Ab1 gene encoding MHC-II proteins. What causes this discrepancy? We found that the culture of bone marrow cells with IL-3 plus GM-CSF expanded DCs in addition to basophils and that depletion of DCs from the culture resulted in down-regulation of MHC-II on basophils, suggesting the transfer of MHC-II from DCs to basophils. Indeed, the co-culture of basophils and DCs carrying different MHC haplotypes clearly demonstrated that basophils acquired MHC-II molecules derived from DCs. The cell-to-cell contact between basophils and DCs is essential for MHC-II transfer, and their separation using a transwell apparatus abolished it. Confocal microscopic examination revealed that MHC-II molecules on DCs were translocated to the surface of basophils, together with patches from the DC plasma membrane. The MHC-II transfer was observed within 15 min after co-culture of basophils and DCs. All these observations are consistent with the phenomenon known as trogocytosis (20). Cell-to-cell interaction through adhesion molecules, signaling through Src and Syk kinases, and actin mobilization contribute to trogocytosis of MHC-II from DCs to basophils. Taken together, we concluded that basophils produce few or no MHC-II proteins by themselves, whereas they can acquire them from DCs through trogocytosis (19). This finding perhaps explains why the level of MHC-II expression on basophils reportedly varied under different experimental conditions, likely depending on the basophil–DC interaction and the extent of MHC-II trogocytosis. We next examined whether basophils can function as APCs, particularly for Th2 cell differentiation (19). Basophils were found to acquire not only MHC-II molecules alone but also peptide–MHC-II complexes generated by DCs. Moreover, basophils express the co-stimulatory molecule CD86, suggesting that basophils that are dressed with peptide–MHC-II can present antigens to T cells and activate them. Indeed, when peptide-specific CD4 T cells were co-cultured with basophils that were dressed with peptide–MHC-II in the absence of DCs, T cells proliferated and produced IL-4. Using mAbs that blocked MHC-II abolished the T-cell proliferation. These results clearly demonstrated that basophils that are dressed with peptide–MHC-II can function as APCs and promote Th2 cell differentiation (Fig. 1). We then sought to examine the in vivo relevance of MHC-II trogocytosis observed in vitro (19). Previous studies showed that basophils infiltrate and accumulate in the skin and draining lymph nodes when mice were repeatedly treated with topical application of MC903, a vitamin D3 analog, in the skin (21, 22). In this model, basophils play an important role in Th2 responses, leading to the development of atopic dermatitis-like allergic inflammation in the skin, and basophil depletion resulted in impaired Th2 cell differentiation in draining lymph nodes. We found that basophils accumulating in lymph nodes express substantial amounts of MHC-II on their surface. Confocal microscopic examination revealed that basophils are in close contact with DCs in draining lymph nodes. Importantly, when mice that were deficient for MHC-II expression only in DCs were similarly treated, the MHC-II expression on lymph node basophils was hardly detected, even though the number of basophils and DCs in draining lymph nodes was comparable between MC903-treated wild-type mice and mice with DCs deficient in MHC-II. These results suggested that the transfer of MHC-II from DCs to basophils can indeed occur in vivo as well (19) (Fig. 1). Fig. 1. View largeDownload slide Basophils can function as Th2-oriented APCs by means of trogocytosis-mediated acquisition of MHC-II from DCs and IL-4 production. Trogocytosis of peptide–MHC-II complexes from DCs to basophils in lymph nodes, together with endogenous CD86 expression in basophils, confers the APC activity on basophils. Basophils also provide IL-4 to naive CD4 T cells. Thus, basophils dressed with peptide–MHC-II can promote the differentiation of naive CD4 T cells toward Th2 cells. Fig. 1. View largeDownload slide Basophils can function as Th2-oriented APCs by means of trogocytosis-mediated acquisition of MHC-II from DCs and IL-4 production. Trogocytosis of peptide–MHC-II complexes from DCs to basophils in lymph nodes, together with endogenous CD86 expression in basophils, confers the APC activity on basophils. Basophils also provide IL-4 to naive CD4 T cells. Thus, basophils dressed with peptide–MHC-II can promote the differentiation of naive CD4 T cells toward Th2 cells. Taken together, we have defined the mechanism by which basophils display MHC-II on the cell surface at the protein level without transcription of the corresponding gene. Basophils can acquire peptide–MHC-II complexes from DCs through trogocytosis. Because basophils endogenously express CD86 and produce IL-4, basophils can function as Th2-oriented APCs only when they acquire sufficient amounts of peptide–MHC-II complexes from DCs. This appears to account for the discrepancy observed in previous studies reporting basophils as important APCs for promoting Th2 cell differentiation under some but not other experimental conditions. Further studies are needed to clarify the relative importance of basophils versus DCs as APCs in terms of Th2 cell differentiation. The close contact of T cells with basophils during antigen presentation by basophils may help basophil-derived IL-4 to efficiently stimulate T cells toward Th2 differentiation. This intimate contact may also activate T cells to produce IL-3 that in turn stimulates basophils to secrete IL-4. These conversations between basophils and T cells, if any, appear to be favorable to Th2 differentiation. Identification of basophil-specific effector molecules responsible for basophil-mediated allergic responses Both basophils and mast cells contain secretary granules, known as basophilic granules, which are stained violet with basic aniline dye. In response to various stimuli, they release materials stored in these granules, including histamine and proteases. We and others found that the repertoire of proteases in basophils and mast cells is quite distinct (23, 24). Mouse mast cell protease (mMCP)-8 is expressed specifically by basophils but not mast cells, even though it was originally identified in mouse mastocytoma cell lines (23–25). Therefore, mMCP-8 is commonly used as a specific marker to identify basophils in tissue sections (24) and to generate engineered mice expressing a gene of interest in a basophil-specific manner (2, 26, 27). mMCP-11 is the most recently discovered member of the mMCP family and shows tryptase activity (28). Although it was originally identified in mast cells (28), we demonstrated that mMCP-11 is preferentially expressed in basophils rather than mast cells (24). In contrast, the tryptase subfamily members, mMCP-6 and mMCP-7, are expressed by mast cells but not basophils (24). Unlike mast cells, basophils do not express any of the mast cell chymases. These findings suggest that distinct functions of basophils and mast cells might be attributed in part to the difference in the repertoire of proteases stored in their basophilic granules. To clarify the in vivo role of mMCP-11, we have recently established mice deficient for mMCP-11 and examined their ability to induce basophil-mediated allergic responses (3). A single intradermal administration of allergens in the ear skin of IgE-sensitized mice elicits three consecutive waves of ear swelling (29). The first two waves (early-phase and late-phase ear swelling) are mast cell-dependent, immediate-type allergic reactions, whereas the third one is delayed-onset swelling and becomes apparent on day 2, peaking on day 4, accompanied by massive accumulation of pro-inflammatory cells, including eosinophils and macrophages, in the ear skin lesion. We designated this delayed-onset allergic response as ‘IgE-mediated chronic allergic inflammation (IgE-CAI)’ (29). Deficiency of basophils but not mast cells completely abolished the development of IgE-CAI, demonstrating a crucial role for basophils, not mast cells, in this response (1, 29). mMCP-11-deficient mice displayed no apparent anomaly in basophils in terms of the number, morphology, cell surface markers and expression of mMCP-8. mMCP-11-deficient basophils normally migrate in response to the chemokine MIP-2, degranulate and produce cytokines such as IL-4 when activated. Nevertheless, mMCP-11-deficient mice showed an ameliorated IgE-CAI response with a reduction in ear swelling, microvascular hyperpermeability and accumulation of pro-inflammatory cells in the skin lesion (3). Moreover, repeated intradermal injection of recombinant mMCP-11 in wild-type mice resulted in infiltration and accumulation of pro-inflammatory cells, including neutrophils, eosinophils, basophils, monocytes and macrophages. Importantly, a protease-dead mutant of mMCP-11 did not show such a leukocyte-recruiting activity, demonstrating the essential role of protease activity of mMCP-11 in this chemotactic function. We then examined the mechanism underlying mMCP-11-elicited leukocyte recruitment by using the transwell migration assay in vitro (3). Consistent with the in vivo observation, enzymatically active, but not inactive, mMCP-11 induced migration of eosinophils, basophils and macrophages. Pre-treatment of these leukocytes with pertussis toxin (which prevents G-proteins from interacting with G-protein-coupled receptors) inhibited the mMCP-11-elicited leukocyte migration. Thus, mMCP-11 functioned as a protease and induced leukocyte migration through G-protein-coupled receptors. As expected, the addition of nafamostat, a protease inhibitor, efficiently blocked the leukocyte migration. Curiously, when culture medium was pre-incubated with mMCP-11 at 37°C for 2 h, followed by addition of nafamostat to inactivate the mMCP-11 activity, the treated culture medium was able to induce leukocyte migration. Moreover, serum depletion from culture medium abolished the mMCP-11-elicited leukocyte migration. These results suggested that mMCP-11 acts on a serum protein(s) present in culture medium, and its proteolytic product rather than mMCP-11 itself induces leukocyte migration. In the IgE-CAI setting in vivo, mMCP-11 released from activated basophils may cleave some as-yet-unidentified proteins present in the affected skin tissue, and their proteolytic proteins show chemotactic activity to recruit leukocytes including basophils (Fig. 2). Such recruited basophils can be activated to release more mMCP-11. This appears to drive a kind of amplification cycle that exacerbates and prolongs the allergic inflammation. Fig. 2. View largeDownload slide Basophil-derived mMCP-11 is an effector molecule contributing to the development of basophil-mediated allergic inflammation. (1) Basophils that are armed with IgE via the high-affinity IgE receptor (FcεRI) migrate into the skin where corresponding antigens have been administered. (2) Basophils are activated with antigens and release mMCP-11 molecules that are stored in the secretory granules. (3) Released mMCP-11 acts on a target protein(s) present in the skin tissue. (4) Proteolytic products of the target protein(s) show chemotactic activity and promote infiltration of leukocytes, leading to inflammation in the skin. Fig. 2. View largeDownload slide Basophil-derived mMCP-11 is an effector molecule contributing to the development of basophil-mediated allergic inflammation. (1) Basophils that are armed with IgE via the high-affinity IgE receptor (FcεRI) migrate into the skin where corresponding antigens have been administered. (2) Basophils are activated with antigens and release mMCP-11 molecules that are stored in the secretory granules. (3) Released mMCP-11 acts on a target protein(s) present in the skin tissue. (4) Proteolytic products of the target protein(s) show chemotactic activity and promote infiltration of leukocytes, leading to inflammation in the skin. As mentioned above, mMCP-8 is expressed specifically by basophils but not mast cells. It shows sequence similarity with mouse granzyme B in the region critical for substrate specificity, but its substrates remain to be identified (25, 30). To explore possible functions of mMCP-8, we prepared recombinant forms of mMCP-8 and found that mMCP-8 can cleave α-tubulin in vitro (31). This is the first demonstration that mMCP-8 indeed has a proteolytic activity, even though α-tubulin may not be a physiologically relevant substrate. A single intradermal injection of recombinant mMCP-8 induced cutaneous swelling with increased microvascular permeability through cyclo-oxygenase (COX)-mediated prostaglandin production. Multiple intradermal administrations of mMCP-8 resulted in infiltration and accumulation of leukocytes, predominantly neutrophils, and, to a lesser extent, monocytes/macrophages and eosinophils (31). Heat-treated mMCP-8 failed to do so, suggesting the importance of the protease activity in mMCP-8-mediated leukocyte recruitment. We then examined possible mechanisms underlying the action of mMCP-8 (31). In contrast to mMCP-11, mMCP-8 did not induce any significant migration of leukocytes when assessed with the transwell migration assay, implying that mMCP-8 evokes leukocyte migration through a mechanism distinct from that used by mMCP-11. We found that multiple intradermal administrations of mMCP-8 up-regulated the expression of genes encoding chemokines such as CXCL1, CCL2 and CCL24, which are known to attract neutrophils, monocytes/macrophages and eosinophils, respectively. On the basis of these observations, we assume that mMCP-8 may act on skin-resident cells such as fibroblasts to induce their production of chemokines, which in turn attract leukocytes (Fig. 3). Fig. 3. View largeDownload slide Basophil-derived mMCP-8 also induces inflammation with leukocyte infiltration, but through a mechanism distinct from that used by mMCP-11. (1, 2) Basophils migrate into the skin and are activated as described in Fig. 2. (3) Activated basophils release mMCP-8 that in turn acts on skin-resident cells such as fibroblasts. (4) Skin-resident cells activated with mMCP-8 produce chemokines that induce leukocyte infiltration to the skin. Fig. 3. View largeDownload slide Basophil-derived mMCP-8 also induces inflammation with leukocyte infiltration, but through a mechanism distinct from that used by mMCP-11. (1, 2) Basophils migrate into the skin and are activated as described in Fig. 2. (3) Activated basophils release mMCP-8 that in turn acts on skin-resident cells such as fibroblasts. (4) Skin-resident cells activated with mMCP-8 produce chemokines that induce leukocyte infiltration to the skin. Considering that basophil-selective expression of mMCP-8 and mMCP-11 and their pro-inflammatory activity observed in our studies, both proteases likely contribute to the non-redundant role of basophils, distinct from that played by mast cells, in various immune responses, including allergic inflammation and protective immunity against pathogens, particularly parasites. Our studies have cast new light on the functional significance of proteases produced by activated basophils. Conclusion We addressed the controversial issue of whether basophils indeed function as APCs for driving Th2 cell differentiation and defined the mechanism by which basophils display MHC-II on the cell surface at the protein but not transcriptional level. Trogocytosis-mediated acquisition of peptide–MHC-II complexes from DCs can confer the APC activity on basophils and, together with IL-4 production by basophils, make it possible for basophils to promote Th2 cell differentiation. This finding appears to reconcile some discrepancies observed in previous studies demonstrating that basophils expressed MHC-II on the cell surface and function as APCs under some but not other experimental conditions. We also demonstrated the important role of proteases, mMCP-8 and mMCP-11, that are expressed almost exclusively by basophils in contrast to other members of the mMCP family, in triggering allergic inflammation. Taken together, our studies have demonstrated that basophils are key players in Th2 immune responses even though they account for <1% of peripheral blood leukocytes. Basophils and their products could be therapeutic targets for the treatment of Th2 immune disorders including allergy, even though further studies are necessary to verify that the findings in mouse models are indeed relevant to human disease pathology. Funding This work is supported by a research grant, 15H05786, from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Conflicts of Interest statement The authors declared no conflicts of interest. References 1 Obata, K., Mukai, K., Tsujimura, Y., et al.   2007. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood  110: 913. Google Scholar CrossRef Search ADS PubMed  2 Wada, T., Ishiwata, K., Koseki, H., et al.   2010. 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Google Scholar CrossRef Search ADS PubMed  31 Tsutsui, H., Yamanishi, Y., Ohtsuka, H., Sato, S., Yoshikawa, S. and Karasuyama, H. 2017. The Basophil-specific protease mMCP-8 provokes an inflammatory response in the skin with microvascular hyperpermeability and leukocyte infiltration. J. Biol. Chem . 292: 1061. Google Scholar CrossRef Search ADS PubMed  © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Immunology Oxford University Press

How do basophils contribute to Th2 cell differentiation and allergic responses?

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Abstract

Abstract Basophils and mast cells share some features, including basophilic granules in the cytoplasm, cell surface expression of the high-affinity IgE receptor and release of chemical mediators such as histamine. Because of this similarity and their minority status, basophils had often been erroneously considered as minor relatives or blood-circulating precursors of tissue-resident mast cells, and therefore long been neglected or underestimated in immunological studies. Taking advantage of newly developed tools, such as basophil-depleting antibodies and engineered mice deficient for only basophils, recent studies have identified previously unappreciated roles for basophils, distinct from those played by mast cells, in allergic responses, protective immunity against parasitic infections and regulation of other immune cells. In this review, we focus on two topics that we presented and discussed in the 46th Annual Meeting of the Japanese Society for Immunology held in Sendai in December 2017. The first topic is the function of basophils as antigen-presenting cells for driving Th2 cell differentiation. We demonstrated that basophils produce few or no MHC class II (MHC-II) proteins by themselves although they can acquire peptide–MHC-II complexes from dendritic cells through trogocytosis, and present them and provide IL-4 to naive CD4 T cells, promoting Th2 cell differentiation. The second topic is the basophil-specific effector molecules involved in allergic responses. Among mouse mast cell proteases (mMCPs), mMCP-8 and mMCP-11 are expressed almost exclusively by basophils. Analyses in vitro and in vivo revealed that both proteases can induce leukocyte migration through distinct mechanisms, contributing to the development of basophil-dependent allergic inflammation. allergic inflammation, antigen-presenting cell, IL-4, MHC class II, protease Introduction The 46th Annual Meeting of the Japanese Society for Immunology was held in Sendai, Japan, on December 12–14, 2017. More than 1700 participants, including 22 invited speakers from abroad, got together in the Sendai International Center to discuss hot topics in 12 symposia and in 43 workshops and poster sessions, all conducted in English. We presented our recent studies on basophils in Symposium 1 ‘Allergy: state-of-the-art’. The existence of basophils in the peripheral blood was first described by Paul Ehrlich in 1879, much earlier than the discovery of T and B lymphocytes. Although basophils are evolutionally conserved in many animal species, their functional significance in vivo remained uncertain and controversial long after their identification. Basophil research had been hindered by their rarity, accounting for <1% of peripheral blood leukocytes, and by the paucity of useful tools for analysis of their in vivo functions. To overcome these obstacles, we have developed useful analytical tools, including a basophil-depleting monoclonal antibody (mAb) (1) and a series of engineered mice for visualization and ablation of basophils or basophil-specific molecules (2–4). In this article, we provide an overview of two important issues that we presented and discussed in the meeting: firstly, the controversial issue of whether and how basophils contribute to Th2 cell differentiation; and, secondly, the effector molecules that basophils utilize to trigger basophil-dependent allergic inflammatory responses. Do basophils express MHC class II and function as antigen-presenting cells? IL-4 has been demonstrated to play a fundamental role in the differentiation of naive CD4 T cells to Th2 cells that produce Th2 cytokines such as IL-4 and contribute to protective immunity against parasitic infections and allergic responses (5). Dendritic cells (DCs) are professional antigen-presenting cells (APCs), but do not usually produce IL-4. Several cell lineages can produce IL-4, including T cells, natural killer T cells, basophils, eosinophils and mast cells. Thus, the source of ‘initial IL-4’ necessary for Th2 cell differentiation has often been a matter of debate (5). In response to various stimuli, basophils in both humans and mice readily secrete larger amounts of IL-4 than Th2 cells do on a per-cell basis (6), suggesting the possible role of basophils as IL-4 providers for Th2 cell differentiation. Indeed, Sokol et al. (7) demonstrated that subcutaneous administration of papain (which triggers allergic responses) induces migration of basophils to the T-cell zone of draining lymph nodes before Th2 differentiation starts. Depletion of basophils abolished Th2 cell differentiation in lymph nodes, indicating a crucial role of basophils in Th2 differentiation. In this case, it was interpreted that basophils provide IL-4 to T cells while DCs function as APCs. The differentiation of naive CD4 T cells toward Th1, Th2 or Th17 cells requires the interaction with APCs that present complexes of MHC class II (MHC-II) plus peptide and provide co-stimulatory molecules, including CD80, CD86 and CD40, and instructive cytokines to T cells (8). It was generally thought that basophils do not express MHC-II and therefore cannot function as APCs, even though basophils express a co-stimulatory molecule and produce IL-4. This view was challenged by exciting reports from three independent groups, demonstrating that basophils, rather than DCs, function as APCs for driving Th2 cell differentiation (9–11). In all three distinct experimental settings, basophils expressed MHC-II and induced the differentiation of naive CD4 T cells to Th2 cells via basophil-derived IL-4. Ablation of basophils abolished Th2 cell differentiation in vivo. These results strongly suggested that basophils are unique APCs specialized for Th2 differentiation in contrast to DCs that are specialized for Th1 (and Th17) differentiation. This new concept of basophils, rather than DCs, being the important APCs for Th2 cell differentiation attracted much attention, but also gained severe criticism (5, 12, 13). The level of MHC-II expression on basophils is generally much lower than that on typical APCs such as DCs and B cells. This is also true for the expression of H-2M and the invariant chain that are crucial for peptide loading on MHC-II. Another concern is the possible inefficiency and off-target effects of the method used for the depletion of DCs and basophils, respectively (14, 15). Indeed, some later studies demonstrated the essential role for DCs in Th2 cell differentiation (14–17). Therefore, it remained controversial whether the MHC-II expression on basophils has a functional significance and whether basophils indeed have the capacity of antigen processing and presentation to T cells (18). To address this controversial issue, we first re-examined the MHC-II expression on basophils (19). Basophils isolated from the bone marrow and spleen expressed MHC-II at very low levels, almost negligible compared with high levels on professional APCs such as DCs and B cells. Incubation of these basophils with various cytokines including IFN-γ failed to up-regulate MHC-II expression, in spite of fact that IFN-γ is well known to induce MHC-II expression on many cell lineages. During the in vitro generation of basophils from bone marrow cells in the presence of IL-3, the addition of GM-CSF but not IFN-γ to the culture highly increased MHC-II expression on basophils. Unexpectedly, however, we could detect little or no transcriptional up-regulation of the H2Ab1 gene encoding MHC-II proteins. What causes this discrepancy? We found that the culture of bone marrow cells with IL-3 plus GM-CSF expanded DCs in addition to basophils and that depletion of DCs from the culture resulted in down-regulation of MHC-II on basophils, suggesting the transfer of MHC-II from DCs to basophils. Indeed, the co-culture of basophils and DCs carrying different MHC haplotypes clearly demonstrated that basophils acquired MHC-II molecules derived from DCs. The cell-to-cell contact between basophils and DCs is essential for MHC-II transfer, and their separation using a transwell apparatus abolished it. Confocal microscopic examination revealed that MHC-II molecules on DCs were translocated to the surface of basophils, together with patches from the DC plasma membrane. The MHC-II transfer was observed within 15 min after co-culture of basophils and DCs. All these observations are consistent with the phenomenon known as trogocytosis (20). Cell-to-cell interaction through adhesion molecules, signaling through Src and Syk kinases, and actin mobilization contribute to trogocytosis of MHC-II from DCs to basophils. Taken together, we concluded that basophils produce few or no MHC-II proteins by themselves, whereas they can acquire them from DCs through trogocytosis (19). This finding perhaps explains why the level of MHC-II expression on basophils reportedly varied under different experimental conditions, likely depending on the basophil–DC interaction and the extent of MHC-II trogocytosis. We next examined whether basophils can function as APCs, particularly for Th2 cell differentiation (19). Basophils were found to acquire not only MHC-II molecules alone but also peptide–MHC-II complexes generated by DCs. Moreover, basophils express the co-stimulatory molecule CD86, suggesting that basophils that are dressed with peptide–MHC-II can present antigens to T cells and activate them. Indeed, when peptide-specific CD4 T cells were co-cultured with basophils that were dressed with peptide–MHC-II in the absence of DCs, T cells proliferated and produced IL-4. Using mAbs that blocked MHC-II abolished the T-cell proliferation. These results clearly demonstrated that basophils that are dressed with peptide–MHC-II can function as APCs and promote Th2 cell differentiation (Fig. 1). We then sought to examine the in vivo relevance of MHC-II trogocytosis observed in vitro (19). Previous studies showed that basophils infiltrate and accumulate in the skin and draining lymph nodes when mice were repeatedly treated with topical application of MC903, a vitamin D3 analog, in the skin (21, 22). In this model, basophils play an important role in Th2 responses, leading to the development of atopic dermatitis-like allergic inflammation in the skin, and basophil depletion resulted in impaired Th2 cell differentiation in draining lymph nodes. We found that basophils accumulating in lymph nodes express substantial amounts of MHC-II on their surface. Confocal microscopic examination revealed that basophils are in close contact with DCs in draining lymph nodes. Importantly, when mice that were deficient for MHC-II expression only in DCs were similarly treated, the MHC-II expression on lymph node basophils was hardly detected, even though the number of basophils and DCs in draining lymph nodes was comparable between MC903-treated wild-type mice and mice with DCs deficient in MHC-II. These results suggested that the transfer of MHC-II from DCs to basophils can indeed occur in vivo as well (19) (Fig. 1). Fig. 1. View largeDownload slide Basophils can function as Th2-oriented APCs by means of trogocytosis-mediated acquisition of MHC-II from DCs and IL-4 production. Trogocytosis of peptide–MHC-II complexes from DCs to basophils in lymph nodes, together with endogenous CD86 expression in basophils, confers the APC activity on basophils. Basophils also provide IL-4 to naive CD4 T cells. Thus, basophils dressed with peptide–MHC-II can promote the differentiation of naive CD4 T cells toward Th2 cells. Fig. 1. View largeDownload slide Basophils can function as Th2-oriented APCs by means of trogocytosis-mediated acquisition of MHC-II from DCs and IL-4 production. Trogocytosis of peptide–MHC-II complexes from DCs to basophils in lymph nodes, together with endogenous CD86 expression in basophils, confers the APC activity on basophils. Basophils also provide IL-4 to naive CD4 T cells. Thus, basophils dressed with peptide–MHC-II can promote the differentiation of naive CD4 T cells toward Th2 cells. Taken together, we have defined the mechanism by which basophils display MHC-II on the cell surface at the protein level without transcription of the corresponding gene. Basophils can acquire peptide–MHC-II complexes from DCs through trogocytosis. Because basophils endogenously express CD86 and produce IL-4, basophils can function as Th2-oriented APCs only when they acquire sufficient amounts of peptide–MHC-II complexes from DCs. This appears to account for the discrepancy observed in previous studies reporting basophils as important APCs for promoting Th2 cell differentiation under some but not other experimental conditions. Further studies are needed to clarify the relative importance of basophils versus DCs as APCs in terms of Th2 cell differentiation. The close contact of T cells with basophils during antigen presentation by basophils may help basophil-derived IL-4 to efficiently stimulate T cells toward Th2 differentiation. This intimate contact may also activate T cells to produce IL-3 that in turn stimulates basophils to secrete IL-4. These conversations between basophils and T cells, if any, appear to be favorable to Th2 differentiation. Identification of basophil-specific effector molecules responsible for basophil-mediated allergic responses Both basophils and mast cells contain secretary granules, known as basophilic granules, which are stained violet with basic aniline dye. In response to various stimuli, they release materials stored in these granules, including histamine and proteases. We and others found that the repertoire of proteases in basophils and mast cells is quite distinct (23, 24). Mouse mast cell protease (mMCP)-8 is expressed specifically by basophils but not mast cells, even though it was originally identified in mouse mastocytoma cell lines (23–25). Therefore, mMCP-8 is commonly used as a specific marker to identify basophils in tissue sections (24) and to generate engineered mice expressing a gene of interest in a basophil-specific manner (2, 26, 27). mMCP-11 is the most recently discovered member of the mMCP family and shows tryptase activity (28). Although it was originally identified in mast cells (28), we demonstrated that mMCP-11 is preferentially expressed in basophils rather than mast cells (24). In contrast, the tryptase subfamily members, mMCP-6 and mMCP-7, are expressed by mast cells but not basophils (24). Unlike mast cells, basophils do not express any of the mast cell chymases. These findings suggest that distinct functions of basophils and mast cells might be attributed in part to the difference in the repertoire of proteases stored in their basophilic granules. To clarify the in vivo role of mMCP-11, we have recently established mice deficient for mMCP-11 and examined their ability to induce basophil-mediated allergic responses (3). A single intradermal administration of allergens in the ear skin of IgE-sensitized mice elicits three consecutive waves of ear swelling (29). The first two waves (early-phase and late-phase ear swelling) are mast cell-dependent, immediate-type allergic reactions, whereas the third one is delayed-onset swelling and becomes apparent on day 2, peaking on day 4, accompanied by massive accumulation of pro-inflammatory cells, including eosinophils and macrophages, in the ear skin lesion. We designated this delayed-onset allergic response as ‘IgE-mediated chronic allergic inflammation (IgE-CAI)’ (29). Deficiency of basophils but not mast cells completely abolished the development of IgE-CAI, demonstrating a crucial role for basophils, not mast cells, in this response (1, 29). mMCP-11-deficient mice displayed no apparent anomaly in basophils in terms of the number, morphology, cell surface markers and expression of mMCP-8. mMCP-11-deficient basophils normally migrate in response to the chemokine MIP-2, degranulate and produce cytokines such as IL-4 when activated. Nevertheless, mMCP-11-deficient mice showed an ameliorated IgE-CAI response with a reduction in ear swelling, microvascular hyperpermeability and accumulation of pro-inflammatory cells in the skin lesion (3). Moreover, repeated intradermal injection of recombinant mMCP-11 in wild-type mice resulted in infiltration and accumulation of pro-inflammatory cells, including neutrophils, eosinophils, basophils, monocytes and macrophages. Importantly, a protease-dead mutant of mMCP-11 did not show such a leukocyte-recruiting activity, demonstrating the essential role of protease activity of mMCP-11 in this chemotactic function. We then examined the mechanism underlying mMCP-11-elicited leukocyte recruitment by using the transwell migration assay in vitro (3). Consistent with the in vivo observation, enzymatically active, but not inactive, mMCP-11 induced migration of eosinophils, basophils and macrophages. Pre-treatment of these leukocytes with pertussis toxin (which prevents G-proteins from interacting with G-protein-coupled receptors) inhibited the mMCP-11-elicited leukocyte migration. Thus, mMCP-11 functioned as a protease and induced leukocyte migration through G-protein-coupled receptors. As expected, the addition of nafamostat, a protease inhibitor, efficiently blocked the leukocyte migration. Curiously, when culture medium was pre-incubated with mMCP-11 at 37°C for 2 h, followed by addition of nafamostat to inactivate the mMCP-11 activity, the treated culture medium was able to induce leukocyte migration. Moreover, serum depletion from culture medium abolished the mMCP-11-elicited leukocyte migration. These results suggested that mMCP-11 acts on a serum protein(s) present in culture medium, and its proteolytic product rather than mMCP-11 itself induces leukocyte migration. In the IgE-CAI setting in vivo, mMCP-11 released from activated basophils may cleave some as-yet-unidentified proteins present in the affected skin tissue, and their proteolytic proteins show chemotactic activity to recruit leukocytes including basophils (Fig. 2). Such recruited basophils can be activated to release more mMCP-11. This appears to drive a kind of amplification cycle that exacerbates and prolongs the allergic inflammation. Fig. 2. View largeDownload slide Basophil-derived mMCP-11 is an effector molecule contributing to the development of basophil-mediated allergic inflammation. (1) Basophils that are armed with IgE via the high-affinity IgE receptor (FcεRI) migrate into the skin where corresponding antigens have been administered. (2) Basophils are activated with antigens and release mMCP-11 molecules that are stored in the secretory granules. (3) Released mMCP-11 acts on a target protein(s) present in the skin tissue. (4) Proteolytic products of the target protein(s) show chemotactic activity and promote infiltration of leukocytes, leading to inflammation in the skin. Fig. 2. View largeDownload slide Basophil-derived mMCP-11 is an effector molecule contributing to the development of basophil-mediated allergic inflammation. (1) Basophils that are armed with IgE via the high-affinity IgE receptor (FcεRI) migrate into the skin where corresponding antigens have been administered. (2) Basophils are activated with antigens and release mMCP-11 molecules that are stored in the secretory granules. (3) Released mMCP-11 acts on a target protein(s) present in the skin tissue. (4) Proteolytic products of the target protein(s) show chemotactic activity and promote infiltration of leukocytes, leading to inflammation in the skin. As mentioned above, mMCP-8 is expressed specifically by basophils but not mast cells. It shows sequence similarity with mouse granzyme B in the region critical for substrate specificity, but its substrates remain to be identified (25, 30). To explore possible functions of mMCP-8, we prepared recombinant forms of mMCP-8 and found that mMCP-8 can cleave α-tubulin in vitro (31). This is the first demonstration that mMCP-8 indeed has a proteolytic activity, even though α-tubulin may not be a physiologically relevant substrate. A single intradermal injection of recombinant mMCP-8 induced cutaneous swelling with increased microvascular permeability through cyclo-oxygenase (COX)-mediated prostaglandin production. Multiple intradermal administrations of mMCP-8 resulted in infiltration and accumulation of leukocytes, predominantly neutrophils, and, to a lesser extent, monocytes/macrophages and eosinophils (31). Heat-treated mMCP-8 failed to do so, suggesting the importance of the protease activity in mMCP-8-mediated leukocyte recruitment. We then examined possible mechanisms underlying the action of mMCP-8 (31). In contrast to mMCP-11, mMCP-8 did not induce any significant migration of leukocytes when assessed with the transwell migration assay, implying that mMCP-8 evokes leukocyte migration through a mechanism distinct from that used by mMCP-11. We found that multiple intradermal administrations of mMCP-8 up-regulated the expression of genes encoding chemokines such as CXCL1, CCL2 and CCL24, which are known to attract neutrophils, monocytes/macrophages and eosinophils, respectively. On the basis of these observations, we assume that mMCP-8 may act on skin-resident cells such as fibroblasts to induce their production of chemokines, which in turn attract leukocytes (Fig. 3). Fig. 3. View largeDownload slide Basophil-derived mMCP-8 also induces inflammation with leukocyte infiltration, but through a mechanism distinct from that used by mMCP-11. (1, 2) Basophils migrate into the skin and are activated as described in Fig. 2. (3) Activated basophils release mMCP-8 that in turn acts on skin-resident cells such as fibroblasts. (4) Skin-resident cells activated with mMCP-8 produce chemokines that induce leukocyte infiltration to the skin. Fig. 3. View largeDownload slide Basophil-derived mMCP-8 also induces inflammation with leukocyte infiltration, but through a mechanism distinct from that used by mMCP-11. (1, 2) Basophils migrate into the skin and are activated as described in Fig. 2. (3) Activated basophils release mMCP-8 that in turn acts on skin-resident cells such as fibroblasts. (4) Skin-resident cells activated with mMCP-8 produce chemokines that induce leukocyte infiltration to the skin. Considering that basophil-selective expression of mMCP-8 and mMCP-11 and their pro-inflammatory activity observed in our studies, both proteases likely contribute to the non-redundant role of basophils, distinct from that played by mast cells, in various immune responses, including allergic inflammation and protective immunity against pathogens, particularly parasites. Our studies have cast new light on the functional significance of proteases produced by activated basophils. Conclusion We addressed the controversial issue of whether basophils indeed function as APCs for driving Th2 cell differentiation and defined the mechanism by which basophils display MHC-II on the cell surface at the protein but not transcriptional level. Trogocytosis-mediated acquisition of peptide–MHC-II complexes from DCs can confer the APC activity on basophils and, together with IL-4 production by basophils, make it possible for basophils to promote Th2 cell differentiation. This finding appears to reconcile some discrepancies observed in previous studies demonstrating that basophils expressed MHC-II on the cell surface and function as APCs under some but not other experimental conditions. We also demonstrated the important role of proteases, mMCP-8 and mMCP-11, that are expressed almost exclusively by basophils in contrast to other members of the mMCP family, in triggering allergic inflammation. Taken together, our studies have demonstrated that basophils are key players in Th2 immune responses even though they account for <1% of peripheral blood leukocytes. Basophils and their products could be therapeutic targets for the treatment of Th2 immune disorders including allergy, even though further studies are necessary to verify that the findings in mouse models are indeed relevant to human disease pathology. Funding This work is supported by a research grant, 15H05786, from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Conflicts of Interest statement The authors declared no conflicts of interest. References 1 Obata, K., Mukai, K., Tsujimura, Y., et al.   2007. Basophils are essential initiators of a novel type of chronic allergic inflammation. Blood  110: 913. Google Scholar CrossRef Search ADS PubMed  2 Wada, T., Ishiwata, K., Koseki, H., et al.   2010. 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Google Scholar CrossRef Search ADS PubMed  31 Tsutsui, H., Yamanishi, Y., Ohtsuka, H., Sato, S., Yoshikawa, S. and Karasuyama, H. 2017. The Basophil-specific protease mMCP-8 provokes an inflammatory response in the skin with microvascular hyperpermeability and leukocyte infiltration. J. Biol. Chem . 292: 1061. Google Scholar CrossRef Search ADS PubMed  © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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International ImmunologyOxford University Press

Published: Mar 21, 2018

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