Soichiro Yoshikawa

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.

Inflammatory Monocytes Recruited to Allergic Skin Acquire an Anti-inflammatory M2 Phenotype via Basophil-Derived Interleukin-4

Monocytes and macrophages are important effectors and regulators of inflammation, and both can be divided into distinct subsets based on their phenotypes. The developmental and functional relationship between individual subsets of monocytes and those of macrophages has not been fully elucidated, although Ly6C(+)CCR2(+) inflammatory and Ly6C(-)CCR2(-) resident monocytes are generally thought to differentiate into M1 (classically activated) and M2 (alternatively activated) macrophages, respectively. Here we show that inflammatory monocytes recruited to allergic skin acquired an M2-like phenotype in response to basophil-derived interleukin-4 (IL-4) and exerted an anti-inflammatory function. CCR2-deficient mice unexpectedly displayed an exacerbation rather than alleviation of allergic inflammation, in spite of impaired recruitment of inflammatory monocytes to skin lesions. Adoptive transfer of inflammatory monocytes from wild-type but not IL-4 receptor-deficient mice dampened the exacerbated inflammation in CCR2-deficient mice. Thus, inflammatory monocytes can be converted from being proinflammatory to anti-inflammatory under the influence of basophils in allergic reactions.

The skin is an important bulwark of acquired immunity against intestinal helminths

IL-4–producing basophils promote the trapping of N. brasiliensis in the skin during secondary infection, a process critical for limiting the spread of infection to the lungs.

The Ectoenzyme E-NPP3 Negatively Regulates ATP-Dependent Chronic Allergic Responses by Basophils and Mast Cells

Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity.

Pre-B Cell Receptor Assesses the Quality of IgH Chains and Tunes the Pre-B Cell Repertoire by Delivering Differential Signals

It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that μH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of μH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their μH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their μH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of μH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of μH chains.

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.

Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils

Significance Recent studies have revealed that basophils, the rarest granulocytes, have crucial roles in various immune responses. Among their properties, the MHC class II (MHC-II) expression and their function as antigen-presenting cells are matters of considerable controversy. Here we show that basophils indeed express MHC-II on the cell surface, but with little transcription of corresponding genes. This could be achieved by the acquisition of peptide–MHC-II complexes from dendritic cells via cell contact-dependent trogocytosis in vitro and in vivo. The acquired complexes enabled basophils to stimulate and differentiate T cells toward Th2 cells. Thus, the present study clarified the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies. Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II–expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide–MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

The Basophil-specific Protease mMCP-8 Provokes an Inflammatory Response in the Skin with Microvascular Hyperpermeability and Leukocyte Infiltration

Basophils have often been erroneously considered to be minor relatives or blood-circulating precursors of tissue-resident mast cells because of some phenotypic similarity between them, including basophilic secretory granules in the cytoplasm. However, recent studies revealed that the repertoire of serine proteases stored in secretory granules is distinct in them. Particularly, mouse mast cell protease 8 (mMCP-8) is specifically expressed by basophils but not mast cells despite its name. Therefore, mMCP-8 is commonly used as a basophil-specific marker, but its functional property remains uncertain. Here we prepared recombinant mMCP-8 and examined its activity in vitro and in vivo. Purified recombinant mMCP-8 showed heat-sensitive proteolytic activity when α-tubulin was used as a substrate. One intradermal shot of mMCP-8, not heat-inactivated, induced cutaneous swelling with increased microvascular permeability in a cyclooxygenase-dependent manner. Moreover, repeated intradermal injection of mMCP-8 promoted skin infiltration of leukocytes, predominantly neutrophils and, to a lesser extent, monocytes and eosinophils, in conjunction with up-regulation of chemokine expression in the skin lesion. These results suggest that mMCP-8 is an important effector molecule in basophil-elicited inflammation, providing novel insights into how basophils exert a crucial and non-redundant role, distinct from that played by mast cells, in immune responses.

Basophil tryptase mMCP-11 plays a crucial role in IgE-mediated, delayed-onset allergic inflammation in mice.

Recent studies have identified nonredundant roles for basophils in immune responses including allergy and protective immunity. It is well known that activated basophils release granule contents such as histamine and proteases as do mast cells. However, the functional significance of basophil-derived proteases remains poorly understood in contrast to those released from mast cells. For this study we generated a line of knockout (KO) mice deficient for mouse mast cell protease-11 (mMCP-11) that is preferentially expressed by basophils rather than mast cells. In spite of normal development of basophils, the mMCP-11-deficient mice showed amelioration of immunoglobulin E-mediated chronic allergic inflammation (IgE-CAI), with reduction of cutaneous swelling, microvascular permeability, and leukocyte infiltration in the skin lesion, when KO mice were compared with wild-type mice. Repeated administration of recombinant mMCP-11 in the skin induced infiltration of leukocytes, including basophils, in a tryptase activity-dependent manner. The transwell migration assay in vitro suggested that mMCP-11-mediated proteolytic products of serum protein promoted migration of basophils, eosinophils, and macrophages via 1 or more G protein-coupled receptors. Thus, basophil tryptase mMCP-11 is a crucial effector molecule for the induction of IgE-CAI. This is the first demonstration that the basophil-derived protease plays a significant role in vivo.

CD41 is a reliable identification and activation marker for murine basophils in the steady state and during helminth and malarial infections

Basophils, a rare leukocyte population in peripheral circulation, are conventionally identified as CD45intCD49b+FcεRI+ cells. Here, we show that basophils from blood and several organs of naïve wild‐type mice express CD41, the α subunit of αIIbβ3 integrin. CD41 expression on basophils is upregulated after in vivo IL‐3 treatment and during infection with Nippostrongylus brasiliensis (Nb). Moreover, CD41 can be used as a reliable marker for basophils, circumventing technical difficulties associated with FcεRI for basophil identification in a Nb infection model. In vitro anti‐IgE cross‐linking and IL‐3 basophil stimulation showed that CD41 upregulation positively correlates with augmented surface expression of CD200R and increased production of IL‐4/IL‐13, indicating that CD41 is a basophil activation marker. Furthermore, we found that infection with Plasmodium yoelii 17X (Py17x) induced a profound basophilia and using Mcpt8DTR reporter mice as a basophil‐specific depletion model, we verified that CD41 can be used as a marker to track basophils in the steady state and during infection. During malarial infection, CD41 expression on basophils is negatively regulated by IFN‐γ and positively correlates with increased basophil IL‐4 production. In conclusion, we provide evidence that CD41 can be used as both an identification and activation marker for basophils during homeostasis and immune challenge.