The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

Vaccination with the Omicron spike RBD boosts broadly neutralizing antibody levels and confers sustained protection even after acquiring immunity to the original antigen.

The immune evasion of SARS-CoV-2 Omicron variants caused by multiple amino acid replacements in the receptor-binding domain (RBD) of the spike protein wanes the effectiveness of antibodies elicited by current SARS-CoV-2 booster vaccination. The vaccines that target Omicron strains have been recently developed, however, there has been a concern yet to be addressed regarding the negative aspect of the immune response known as original antigenic sin. Here, we demonstrate that the breadth of neutralizing antibodies against SARS-CoV-2 variants is barely elicited by immunizing monovalent viral antigens via vaccination or natural infection in mice and human subjects. However, vaccination of Omicron BA.1 RBD to pre-immunized mice with the original RBD conferred sustained neutralizing activity to BA.1 and BA.2 not only original pseudoviruses. The acquisition of neutralizing antibody breadth was further confirmed in vaccinated-then-Omicron convalescent human sera in which neutralizing activity against BA.1 and BA.2 pseudoviruses was highly induced. Thus, our data suggest that Omicron-specific vaccines or the infection with Omicron viruses can boost potent neutralizing antibodies to the Omicron variants even in the host pre-vaccinated with the original antigen.

VSIG4/CRIg directly regulates early CD8+ T cell activation through its counter-receptor in a narrow window.

T-cell responses are fine-tuned by positive and negative co-signal molecules expressed on immune cells and adjacent tissues. VSIG4 is a newly identified member of the B7 family of ligands, which negatively regulates innate inflammatory and CD4+ T cell-mediated responses. However, little is known about the direct effects of VSIG4, which are exerted through an unidentified counter-receptor on CD8+ T cells. We investigated the binding of the VSIG4-Ig fusion protein during CD8+ T cell activation, and the functional involvement of VSIG4 pathway, using VSIG4-Ig and VSIG4-transfectants. VSIG4-Ig binding to CD8+ T cells was temporally observed in the CD44high phenotype during initial activation. VSIG4-Ig binding was observed earlier than the induction of PD-1, LAG3, and TIM-3, which are immune checkpoint receptors for exhausted CD8+ T cells. Immobilized VSIG4-Ig inhibited anti-CD3/CD28 mAb-induced CD8+ T cell activation, as indicated by proliferation and IFN-γ production, similar to the downregulation of T-bet and Eomesodermin transcription factors. VSIG4 on FcγR+ P815 or specific antigen-presenting E.G7 cells inhibited the generation of effector CD8+ T cells, as indicated by proliferation, IFN-γ and TNF-α expression, and granule degradation, compared to parental cells. However, the window for the regulatory function of VSIG4 was narrow and dependent on the strength of TCR (and CD28)-mediated signals. Our results suggested that VSIG4 directly delivers co-inhibitory signals via an as-yet unidentified counter-receptor on activated CD8+ T cells. VSIG4-mediated CD8+ T cell tolerance might contribute to the steady-state maintenance of homeostasis.

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.

Paliperidone-Induced Acute Hyperglycemia Is Caused by Adrenaline Secretion via the Activation of Hypothalamic AMP-Activated Protein Kinase.

Although paliperidone-related hyperglycemia has been extensively examined, the underlying mechanisms have not yet been elucidated. We investigated the effects of a single intravenous injection of paliperidone (0.2, 0.4, or 0.6 mg/kg) on serum concentrations of glucose and other endogenous metabolites in rats. We also examined the effects of a single intravenous injection of paliperidone (0.4 mg/kg) on AMP-activated protein kinase (AMPK) activity in the hypothalamus and liver. To clarify the relationship between AMPK activity and adrenaline secretion, the effects of berberine, which inhibits hypothalamic AMPK, on paliperidone-induced hyperglycemia were assessed. Significant increases were observed in serum glucose, adrenaline, and insulin concentrations following intravenous injections of paliperidone at doses of 0.4 and 0.6 mg/kg. A propranolol pretreatment attenuated paliperidone-induced increases in serum concentrations of glucose, but not adrenaline. Significant increases were also noted in phosphorylated AMPK concentrations in the hypothalamus following the administration of paliperidone at a dose of 0.4 mg/kg. A berberine pretreatment attenuated paliperidone-induced increases in blood concentrations of glucose, adrenaline, and insulin and phosphorylated AMPK concentrations in the hypothalamus. Collectively, the present results demonstrated that an acute treatment with paliperidone induced hyperglycemia, which was associated with the effects of hypothalamic AMPK activation on the secretion of adrenaline.

CEACAM1 specifically suppresses B cell receptor signaling-mediated activation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igµ F(ab')2 fragment. Overexpression of CEACAM1 in a murine B cell line, A20, resulted in reduced expressions of activation surface markers with decreased Ca2+ influx after BCR signal activation. Overexpression of CEACAM1 suppressed BCR signal cascade in A20 cells in association with decreased spontaneous proliferation. Our results suggest that CEACAM1 can regulate BCR-mediated mature B cell activation in lymphoid tissues. Therefore, further studies of this molecule may lead to greater insights into the mechanisms of immune responses within peripheral tissues and the potential treatment of inflammatory diseases.

Tolerogenic properties of CD206+ macrophages appeared in the sublingual mucosa after repeated antigen-painting.

The sublingual mucosa (SLM) in the oral cavity is utilized as the site for sublingual immunotherapy to induce tolerance against allergens. We previously reported that CD206+ round-type macrophage-like cells were induced in the SLM after repeated antigen (e.g. cedar pollen or fluorescein isothiocyanate (FITC))-painting. In this study, we examined the phenotypic and functional properties of CD206+ cells induced by repeated FITC-painting on the SLM. CD206+ cells after the repeated FITC-painting possessed a macrophage-like CD11b+Ly6C+ F4/80+CD64+ phenotype and expressed TIM-4, which was expressed in tolerogenic tissue-resident macrophages, at a high level. SLM CD206+ cells preferentially expressed molecules related to endocytosis and homeostatic processes, including the novel B7 family of immune checkpoint molecules, as assessed by microarray analyses. SLM CD206+ cells showed preferential expression of M2-related genes such as Fizz1, Aldh1a1 and Aldh1a2 but not Ym-1 and Arginase-1. A CD206+ cell-rich status inhibited OVA-specific CD4+ T-cell responses but reciprocally enhanced the proportion of both IL-10+CD4+ cells and Foxp3+ regulatory T-cells in regional lymph nodes. Co-culture of CD206+ cells with dendritic cells (DCs) showed that IL-12 production was suppressed in DCs concurrent with the decline of the MHC class IIhiCD86+ population, which was restored by neutralization of IL-10. These results demonstrate SLM CD206+ cells show the feature of tolerogenic macrophages and down-regulate the antigen-presenting cell function of mature DCs resulting in the inhibition of CD4+ T-cell responses.

Comparison of the Safety and Effectiveness of Four Direct Oral Anticoagulants in Japanese Patients with Nonvalvular Atrial Fibrillation Using Real-World Data.

Direct oral anticoagulants (DOACs) are widely used for the prevention of ischemic stroke and systemic embolism in patients with nonvalvular atrial fibrillation (NVAF). However, the differences in safety and effectiveness among four DOACs, dabigatran, rivaroxaban, apixaban, and edoxaban, in Japanese patients have not been clarified. Therefore, we conducted a retrospective cohort study to directly compare the safety and effectiveness among the four DOACs using the Japan Medical Data Center (JMDC) claims database. We identified 3823 patients with NVAF who started receiving a DOAC between March 2011 and June 2017. The safety outcome was major bleeding (a composite outcome of intracranial, gastrointestinal, respiratory, or renal/urinary tract bleeding) and the effectiveness outcome was the composite of ischemic stroke including transient ischemic attack (TIA) or systemic embolism. We constructed a Cox proportional hazard model to calculate the hazard ratio (HR) for all four DOAC combinations. The risk of major bleeding was significantly lower in the dabigatran group than in the apixaban group (HR, 0.55; 95% confidence interval (CI), 0.31-0.93; p = 0.03). In contrast, there was no significant difference in the risk of major bleeding among the other DOACs. In the composite risk of ischemic stroke including TIA or systemic embolism, there was no significant difference among the four DOACs. This study suggested that in the current use of DOACs in Japanese patients with NVAF, dabigatran had a significantly lower risk of major bleeding than apixaban, but there was no significant difference in effectiveness among the four DOACs.

Antiplatelet Effect of Mirtazapine via Co-blocking of the 5-HT2A and α2-Adrenergic Receptors on Platelets.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT2A and α2-adrenergic receptors on platelets and suppresses platelet aggregation mediated by ADP, increased by either 5-HT or adrenaline. Thus, a detailed monitoring of bleeding is recommended for patients taking MTZ.

Comprehensive Exploration of Medications That Affect the Bleeding Risk of Oral Anticoagulant Users.

Oral anticoagulants (OACs) pose a major bleeding risk, which may be increased or decreased by concomitant medications. To explore medications that affect the bleeding risk of OACs, we conducted a nested case-control study including 554 bleeding cases (warfarin, n = 327; direct OACs [DOACs], n = 227) and 1337 non-bleeding controls (warfarin, n = 814; DOACs, n = 523), using a Japanese health insurance database from January 2005 to June 2017. Major bleeding risk associated with exposure to concomitant medications within 30 d of the event/index date was evaluated, and adjusted odds ratios (aORs) were calculated using logistic regression analysis. Several antihypertensive drugs, such as amlodipine and bisoprolol, were associated with a decreased risk of bleeding (warfarin + amlodipine [aOR, 0.64; 95% confidence interval (CI): 0.41-0.98], DOACs + bisoprolol [aOR, 0.51; 95% CI, 0.33-0.80]). As hypertension is considered a significant risk factor for intracranial bleeding in antithrombotic therapy, antihypertensive drugs may suppress intracranial bleeding. In contrast, telmisartan, a widely used antihypertensive drug, was associated with an increased risk of bleeding [DOACs + telmisartan (aOR, 4.87; 95% CI, 1.84-12.91)]. Since telmisartan is an inhibitor of P-glycoprotein (P-gp), the elimination of rivaroxaban and apixaban, which are substrates of P-gp, is hindered, resulting in increased blood levels of both drugs, thereby increasing the risk of hemorrhage. In conclusion, antihypertensive drugs may improve the safety of OACs, and the pharmacokinetic-based drug interactions of DOACs must be considered.

Stable lines and clones of long-term proliferating normal, genetically unmodified murine common lymphoid progenitors.

Common lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution.

Aggregation makes a protein allergenic at the challenge phase of basophil-mediated allergy in mice.

A hapten is a small molecule that is not immunogenic on its own but can stimulate the production of antibodies at the sensitization phase when conjugated to carrier proteins. The hapten then reacts specifically with the antibodies generated against it to elicit an immune or allergic response at the challenge phase. Here, we compared various carrier proteins conjugated with the same hapten in their ability to induce hapten-specific IgE-mediated allergic responses in vitro and in vivo, and characterized the nature of carrier proteins that determines the magnitude of response at the challenge phase of allergic reactions. Hapten 2,4,6-trinitrophenol (TNP)-conjugated ovalbumin (TNP-OVA) and bovine serum albumin (TNP-BSA) elicited TNP-specific, mast cell-dependent, immediate-type allergic reactions at a comparable level in mice that had been passively sensitized with TNP-specific IgE. In contrast, TNP-OVA but not TNP-BSA efficiently induced a basophil-dependent, IgE-mediated chronic allergic inflammation (IgE-CAI), even though both proteins could stimulate basophils in vitro at a comparable level. By comparing different carrier proteins and structurally modifying them, we found that the formation of large aggregates is crucial for TNP-conjugated carrier proteins to efficiently elicit IgE-CAI, regardless of the type of protein. Thus, the aggregation status of carrier proteins appears to determine the magnitude of allergic response at the challenge phase of hapten-specific IgE-CAI. Our findings suggest that the allergenicity of substances is a matter of importance not only at the sensitization but also at the challenge phase in a certain type of allergy including a basophil-mediated allergic inflammation.

MiR221 promotes precursor B-cell retention in the bone marrow by amplifying the PI3K-signaling pathway in mice.

Hematopoietic stem cells and lineage-uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B-lineage committed preBI-cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221-controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B-cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221-enhanced PI3K signaling leads to increased expression of the anti-apoptotic protein Bcl2 and VLA4 integrin-mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI-cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221-expressing hematopoietic cells.

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

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.

IL-7 and immobilized Kit-ligand stimulate serum- and stromal cell-free cultures of precursor B-cell lines and clones.

Long-term proliferating, DH JH -rearranged mouse precursor B-cell lines have previously been established in serum- and IL-7-containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre-B cells to undefined factors, hampering accurate analyses of ligand-dependent signaling, which controls pre-B cell proliferation, survival, residence and migration. Here, we describe a novel serum-free, stromal cell-free culture system, which allows us to establish and maintain pre-B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit-ligand, also called stem cell factor, produced as a kit-ligand-Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre-B cells in an optimal density in plate-bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre-B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.

Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150+ long-term repopulating murine hematopoietic stem cells.

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin- Sca1+ c-Kit+ CD34- Flt3- CD150+ CD48- cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporterlow cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporterhigh cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporterlow cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporterhigh HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporterlow cells develop to Flt3L-eGFP-reporterhigh cells in vitro, although Flt3L-eGFP-reporterhigh cells remain Flt3L-eGFP-reporterhigh . CD150+ Flt3L-eGFP-reporterlow cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporterhigh cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin- CD150+ CD48- EPCR+ CD41+ HSCs allows a further 5-fold enrichment of functional LT-HSCs.

Erk kinases link pre-B cell receptor signaling to transcriptional events required for early B cell expansion.

The pre-B cell receptor (pre-BCR) plays a crucial role in the development of immature B cells. Although certain aspects of proximal pre-BCR signaling have been studied, the intermediate signal transducers and the distal transcription modulators are poorly characterized. Here, we demonstrate that deletion of both Erk1 and Erk2 kinases was associated with defective pre-BCR-mediated cell expansion as well as a block in the transition of pro-B to pre-B cells. Phosphorylation of transcription factors Elk1 and CREB was mediated by Erk, and a dominant-negative mutation in the Erk-mediated phosphorylation sites of Elk1 or CREB suppressed pre-BCR-mediated cell expansion as well as expression of genes including Myc, which is involved in the cell-cycle progression. Together, our results identify a crucial role for Erk kinases in regulating B cell development by initiating transcriptional regulatory network and thereby pre-BCR-mediated cell expansion.

Basophils play a pivotal role in immunoglobulin-G-mediated but not immunoglobulin-E-mediated systemic anaphylaxis.

Anaphylaxis is an acute, severe, and potentially fatal systemic allergic reaction. Immunoglobulin E (IgE), mast cells, and histamine have long been associated with anaphylaxis, but an alternative pathway mediated by IgG has been suggested to be more important in the elicitation of anaphylaxis. Here, we showed that basophils, the least common blood cells, were dispensable for IgE-mediated anaphylaxis but played a critical role in IgG-mediated, passive and active systemic anaphylaxis in mice. In vivo depletion of basophils but not macrophages, neutrophils, or NK cells ameliorated IgG-mediated passive anaphylaxis and rescued mice from death in active anaphylaxis. Upon capture of IgG-allergen complexes, basophils released platelet-activating factor (PAF), leading to increased vascular permeability. These results highlight a pivotal role for basophils in vivo and contrast two major, distinct pathways leading to allergen-induced systemic anaphylaxis: one mediated by basophils, IgG, and PAF and the other "classical" pathway mediated by mast cells, IgE, and histamine.

Large particulate allergens can elicit mast cell-mediated anaphylaxis without exit from blood vessels as efficiently as do small soluble allergens.

Anaphylaxis is a rapid-onset, life-threatening allergic reaction in that IgE, mast cells and histamine are commonly involved. It can be experimentally induced in IgE-sensitized animals by intravenous injection of corresponding allergens, and the sign of anaphylactic reaction can be detected within minutes after allergen challenge. However, it remains puzzling why the anaphylactic reaction can be initiated in vivo so quickly, considering that allergens are delivered into the blood circulation while mast cells reside within peripheral tissues but not in the blood circulation. To address this issue, we compared two different forms of the same allergen, small soluble and large particulate ones, in their ability to induce anaphylaxis in IgE-sensitized mice. In contrast to our expectation, particulate allergens could induce anaphylaxis as quickly and efficiently as did soluble allergens, even though they remained inside of blood vessels. In vivo imaging analysis suggested the direct interaction of intravascular particulate allergens and perivascular mast cells across the capillary wall. Taken together with previous report that perivascular mast cells can capture IgE in the blood circulation by extending cell processes across the vessel wall, our findings imply that blood-circulating allergens, regardless of their size, can stimulate mast cells without exit from blood vessels, by means of cross-linking IgE on mast cell processes inserted into the vessel lumen, and hence initiate anaphylactic reaction so quickly.