CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV.

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.

IgE directly affects eosinophil migration in chronic rhinosinusitis with nasal polyps through CCR3 and predicts the efficacy of omalizumab.

BACKGROUND: Whether IgE affects eosinophil migration in chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unclear. Moreover, our understanding of local IgE, eosinophils, and omalizumab efficacy in CRSwNP remains limited. OBJECTIVE: We investigated whether IgE acts directly on eosinophils and determined its role in omalizumab therapy. METHODS: Eosinophils and their surface receptors were detected by hematoxylin and eosin staining and flow cytometry. IgE and its receptors, eosinophil peroxidase (EPX), eosinophilic cationic protein, and CCR3 were detected by immunohistochemistry and immunofluorescence. Functional analyses were performed on blood eosinophils and polyp tissues. Logistic regression was performed to screen for risk factors. Receiver operating characteristic curve was generated to evaluate the accuracy. RESULTS: Both FcεRI and CD23 were expressed on eosinophils. The expression of FcεRI and CD23 on eosinophil in nasal polyp tissue was higher than in peripheral blood (both P < .001). IgE and EPX colocalized in CRSwNP. IgE directly promoted eosinophil migration by upregulating CCR3 in CRSwNP but not in healthy controls. Omalizumab and lumiliximab were found to be effective in restraining this migration, indicating CD23 was involved in IgE-induced eosinophil migration. Both IgE+ and EPX+ cells were significantly reduced after omalizumab treatment in those who experienced response (IgE+ cells, P = .001; EPX+ cells, P = .016) but not in those with no response (IgE+ cells, P = .060; EPX+ cells, P = .151). Baseline IgE+ cell levels were higher in those with response compared to those without response (P = .024). The baseline local IgE+ cell count predicted omalizumab efficacy with an accuracy of 0.811. CONCLUSIONS: IgE directly promotes eosinophil migration, and baseline local IgE+ cell counts are predictive of omalizumab efficacy in CRSwNP.

Analysis of the efficacy of azelastine nasal spray combined with mussel mucin in the treatment of allergic rhinitis and the influence of peripheral blood CCL26 and CCR3 levels.

OBJECTIVE: A retrospective study was conducted to investigate the efficacy of azelastine nasal spray combined with mussel mucin in the treatment of allergic rhinitis (AR) and the effects of CCL26 and CC chemokine receptor-3 (CCR3). PATIENTS AND METHODS: A total of 80 patients with AR admitted to our hospital from March 2020 to March 2022 were included as the research objects. All subjects were divided into two groups according to the different therapeutic strategies by reviewing the patient's treatment. The control group (n = 40) was given azelastine nasal spray, while the study group (n = 40) was treated with a combination of mussel mucin and azelastine nasal spray. The clinical efficacy, clinical symptoms, and sleep quality improvement of the two groups were calculated and compared retrospectively. The serological indexes were compared, and the incidence of adverse reactions between the two groups was calculated retrospectively based on the patient's medical records. RESULTS: In the study and control groups, the effective rate was 95.00% and 72.50%. After treatment, the symptom scores of nasal congestions, nasal itching, sneezing, and runny nose and the total score of Pittsburgh sleep quality index (PSQI) in the study group were remarkably less. After treatment, the serum levels of sVCAM-1, interleukin-4 (IL-4), and immunoglobulin E (IgE) were decreased, and the levels of IL-12 were upregulated. Following treatment, Minimum nasal cross-section (NMCA) and total nasal resistance (TNR) at 75Pa in the study group were reduced more noticeably (p < 0.05). After treatment, the expression levels of CCL26 and CCR3 in peripheral blood were significantly decreased. In the control and study groups, the incidence of adverse reactions was 7.50% and 10.00%. CONCLUSIONS: Azelastine nasal spray combined with mussel mucin is effective in the treatment of allergic rhinitis, which can effectively improve patients' clinical symptoms, alleviate nasal ventilation disorders, reduce inflammatory reactions, and improve sleep quality. This strategy of combined treatment is safe and, therefore, worth advocating.

Profiling CCR3 target pathways for discovering novel antagonists from natural products using label-free cell phenotypic assays.

CC chemokine receptor 3 (CCR3) plays important roles in atopic dermatitis (AD) and other related allergic diseases. Activation of CCR3 receptor signaling pathways regulates the recruitment of eosinophils to related tissues, releasing inflammatory mediators and causing inflammatory responses. However, none of the known CCR3 antagonists exhibit promising efficacy in clinical trials. In this work, we sought new natural CCR3 antagonists for drug development. To construct a high-throughput screening model, we established a stably transfected CHO-K1-Gα15-CCR3 cell line, and receptor expression was demonstrated by real-time quantitative PCR, confocal detection and flow cytometry analysis. Then, we applied a label-free cell phenotyping technique to profile and deconvolute CCR3 target pathways in CHO-K1-Gα15-CCR3 cells and found that activation of CCR3 triggered the Gq-PLC-Ca2+ and MAPK-P38-ERK pathways. By in vitro and in silico experiments, we discovered a novel CCR3 antagonist emodin, with an IC50 value of 27.28 ± 1.71 µM out of 266 compounds that were identified in 15 traditional Chinese medicines used in the clinical treatment of skin diseases. Molecular docking graphically presented the binding mode of emodin on CCR3. This work reports a new approach for CCR3 antagonist screening and pathway detection and identifies a new antagonist that would benefit future drug development.

The Eotaxin-1/CCR3 Axis and Matrix Metalloproteinase-9 Are Critical in Anti-NC16A IgE-Induced Bullous Pemphigoid.

Bullous pemphigoid (BP) is the most common autoimmune bullous skin disease of humans and is characterized by eosinophilic inflammation and circulating and tissue-bound IgG and IgE autoantibodies directed against two hemidesmosomal proteins: BP180 and BP230. The noncollagenous 16A domain (NC16A) of BP180 has been found to contain major epitopes recognized by autoantibodies in BP. We recently established the pathogenicity of anti-NC16A IgE through passive transfer of patient-derived autoantibodies to double-humanized mice that express the human high-affinity IgE receptor, FcεRI, and human NC16A domain (FcεRI/NC16A). In this model, anti-NC16A IgEs recruit eosinophils to mediate tissue injury and clinical disease in FcεRI/NC16A mice. The objective of this study was to characterize the molecular and cellular events that underlie eosinophil recruitment and eosinophil-dependent tissue injury in anti-NC16A IgE-induced BP. We show that anti-NC16A IgEs significantly increase levels of key eosinophil chemoattractants, eotaxin-1 and eotaxin-2, as well as the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) in the lesional skin of FcεRI/NC16A mice. Importantly, neutralization of eotaxin-1, but not eotaxin-2, and blockade of the main eotaxin receptor, CCR3, drastically reduce anti-NC16A IgE-induced disease activity. We further show that anti-NC16A IgE/NC16A immune complexes induce the release of MMP-9 from eosinophils, and that MMP-9-deficient mice are resistant to anti-NC16A IgE-induced BP. Lastly, we find significantly increased levels of eotaxin-1, eotaxin-2, and MMP-9 in blister fluids of BP patients. Taken together, this study establishes the eotaxin-1/CCR3 axis and MMP-9 as key players in anti-NC16A IgE-induced BP and candidate therapeutic targets for future drug development and testing.

Rebalancing liver-infiltrating CCR3+ and CD206+ monocytes improves diet-induced NAFLD.

Melatonin has been reported to improve nonalcoholic fatty liver disease (NAFLD), and exploring the underlying mechanisms will be beneficial for better treatment of NAFLD. Choline-deficient high-fat diet (CDHFD)- and methionine/choline-deficient diet (MCD)-fed mice with melatonin intervention exhibit significantly decreased liver steatosis, lobular inflammation, and focal liver necrosis. Single-cell RNA sequencing reveals that melatonin selectively inhibits pro-inflammatory CCR3+ monocyte-derived macrophages (MoMFs) and upregulates anti-inflammatory CD206+ MoMFs in NAFLD mice. Liver-infiltrating CCR3+CD14+ MoMFs are also significantly increased in patients with NAFLD. Mechanistically, melatonin receptor-independent BTG2-ATF4 signaling plays a role in the regulation of CCR3+ MoMF endoplasmic reticulum stress, survival, and inflammation. In contrast, melatonin upregulates CD206+ MoMF survival and polarization via MT1/2 receptors. Melatonin stimulation also regulates human CCR3+ MoMF and CD206+ MoMF survival and inflammation in vitro. Furthermore, CCR3 depletion antibody monotherapy inhibits liver inflammation and improves NAFLD in mice. Thus, therapies targeting CCR3+ MoMFs may have potential benefits in NAFLD treatment.

Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7).

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.

CD193 (CCR3) expression by B cells correlates with reduced IgE production in paediatric schistosomiasis.

We demonstrate that CD193, the eotaxin receptor, is highly expressed on circulating B cells in paediatric schistosomiasis mansoni. CD193 plays a role in directing granulocytes into sites of allergic-like inflammation in the mucosa, but little is known about its functional significance on human B cells. We sought to characterize CD193 expression and its relationship with S. mansoni infection. We found that CD193+ B cells increased with the intensity of schistosome infection. In addition, a significant negative association was observed between CD193 expression by B cells and IgE production. Decreased IgE levels are generally associated with susceptibility to re-infection. B cell stimulation with eotaxin-1 increased CD193 levels whereas IL-4 led to a reduction. This was supported by plasma levels of eotaxin-1 correlating with CD193 levels on B cells and other cells. In contrast, CD193 expression was induced on naive B cells with a combination of IL-10 and schistosome antigens. Whereas T cells had a modest increase in CD193 expression, only B cell CD193 appeared functionally chemotactic to eotaxin-1. Thus, CD193+ B cells, which co-express CXCR5, may be enroute to sites with allergic-like inflammation, such as gastrointestinal follicles, or even to Th2 granulomas, which develop around parasite eggs. Overall, our results suggest that schistosome infection may promote CD193 expression and suppress IgE via IL-10 and other undefined mechanisms related to B cell trafficking. This study adds to our understanding of why young children may have poor immunity. Nonetheless, praziquantel treatment was shown to reduce percentages of circulating CD193+ B cells lending hope for future vaccine efforts.

Chemokine CCL7 mediates trigeminal neuropathic pain via CCR2/CCR3-ERK pathway in the trigeminal ganglion of mice.

BACKGROUND: Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. METHODS: The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. RESULTS: Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CONCLUSION: CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

CCR3 plays a role in murine age-related cognitive changes and T-cell infiltration into the brain.

Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.

CCL24/CCR3 axis plays a central role in angiotensin II-induced heart failure by stimulating M2 macrophage polarization and fibroblast activation.

AIMS: We aimed to investigate the effect and mechanism of pleiotropic chemokine CCL24 in heart failure. METHODS AND RESULTS: Compared with normal donators, the expression of CCL24 and number of cardiac M2 macrophages in heart were higher in heart failure patients, the same as plasma CCL24. Treatment with CCL24 antibody hindered Ang II (1500 ng/kg/min)-induced cardiac adverse remodeling through preventing cardiac hypertrophy and fibrosis. RNA-seq showed that CCL24/CCR3 axis was involved in immune and inflammatory responses. Single-cell analysis of cytometry by time of flight (CyTOF) revealed that CCL24 antibody decreased the M2 macrophage and monocyte polarization during Ang II stimulation. Immunofluorescence co-localization analysis confirmed the expression of CCR3 in macrophage and fibroblasts. Then, in vitro experiments confirmed that CCL24/CCR3 axis was also involved in cardiac primary fibroblast activation through its G protein-coupled receptor function. CONCLUSION: CCL24/CCR3 axis plays a crucial part in cardiac remodeling by stimulating M2 macrophage polarization and cardiac fibroblast activation. Cardiac M2 macrophages, CCL24 and circulation CCL24 increased in heart failure patients. Treatment with CCL24 Ab hindered Ang II induced cardiac structural dysfunction and electrical remodeling. In CCL24 Ab group RNA-seq found that it was related to immune responses and hypertrophic cardiomyopathy, CytoF revealed M2 macrophages and monocytes decreased obviously. In vitro,CCL24 promoted activation and migration of cardiac fibroblast.

CCR3 blockage elicits polyploidization associated with the signatures of epithelial-mesenchymal transition in carcinoma cell lines.

Malignant features such as the acquisition of metastatic ability, stemness of cells, and therapeutic resistance of cancer cells are associated with epithelial-mesenchymal transition (EMT) accompanied by changes in motility and morphology. Recent reports implicated that the formation of polyploid giant cancer cells (PGCCs) in human malignancy correlated with the EMT processes. Chemokines are often involved in the regulation of cancer cell migration into tissues, and various types of human cancers exhibit enhanced expression of chemokine receptors, which could augment intrinsic potentials such as invasive activity, proliferating ability, and survival capacity in cancer cells. Nevertheless, the contribution of CCR3 in malignant cancer cells is controversial because it is a well-known primal receptor for the migration of eosinophils, one of the cells of the innate immune system. Here, we explored the blockage of chemokine receptor CCR3 in carcinoma cell lines and found that inhibition of CCR3 induced the formation of polyploid giant cells and stabilization of ß-catenin via the PI3K/Akt/GSK-3ß signaling pathway, which are processes associated with EMT. As a result of CCR3 inhibition, converted cells acquired enhanced mobile and proliferation abilities. In summary, these data indicate that modulation of the CCR3/PI3K/Akt/GSK-3ß signaling pathway regulates polyploidization associated with the EMT processes.

Knockdown of CCR3 gene inhibits Proliferation, migration and degranulation of eosinophils in mice by downregulating the PI3K/Akt pathway.

This study aimed to investigate the effects of CCR3 knockdown (CCR3-/-) on the proliferation, migration, and degranulation of the bone marrow eosinophils (EOS) in mice. Bone marrow cells from wild-type mice (WT) were harvested for primary culture and differentiated into mature EOS, which were then randomly divided into the control, 740Y-P, and LY294002 group. The effects of different concentrations of LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) on the proliferation viability of EOS, expressions of EPO, Akt, and p-Akt proteins, and migration changes of EOS were detected. CCR3-/- mice were identified. Then, bone marrow cells of WT and CCR3-/- mice were differentiated into mature EOS and grouped into WT EOS, WT EOS + eotaxin (100 ng/mL), CCR3-/- EOS, and CCR3-/- EOS + eotaxin (100 ng/mL) group. The changes in EOS proliferation, migration, as well as expressions of EPO, Akt, and p-Akt proteins were detected. The number of migrated cells (P < 0.01) and expression of EPO (p < 0.05) in the 740Y-P group were higher than those in the control group, while opposite trends were observed for the LY294002 group. Expression levels of p-Akt and Akt in the LY294002 group were significantly lower than in the control group (all P < 0.01). Also, the expression of p-Akt in the 740Y-P group was significantly higher than that in the control group (p < 0.05). The proliferative activity of EOS, expression of EPO and p-Akt, and the number of migrated cells in the WT EOS group were higher than those in CCR3-/- EOS group (all P < 0.05). After adding eotaxin, the WT EOS group was higher than the other three groups (all P < 0.05). Mechanistically, CCR3-/- inhibited EOS's proliferation, migration, and degranulation by downregulating PI3K/Akt pathway. This data suggests that the knockout of the CCR3 gene in bone marrow cells may inhibit the function of EOS by downregulating the PI3K/Akt pathway, thereby affecting AR; thus, the CCR3 gene may be a target gene for AR therapy.

Potent CCR3 Receptor Antagonist, SB328437, Suppresses Colonic Eosinophil Chemotaxis and Inflammation in the Winnie Murine Model of Spontaneous Chronic Colitis.

Eosinophils and their regulatory molecules have been associated with chronic intestinal inflammation and gastrointestinal dysfunctions; eosinophil accumulation in the gut is prominent in inflammatory bowel disease (IBD). The chemokine receptor CCR3 plays a pivotal role in local and systemic recruitment and activation of eosinophils. In this study, we targeted CCR3-ligand interactions with a potent CCR3 receptor antagonist, SB328437, to alleviate eosinophil-associated immunological responses in the Winnie model of spontaneous chronic colitis. Winnie and C57BL/6 mice were treated with SB328437 or vehicle. Clinical and histopathological parameters of chronic colitis were assessed. Flow cytometry was performed to discern changes in colonic, splenic, circulatory, and bone marrow-derived leukocytes. Changes to the serum levels of eosinophil-associated chemokines and cytokines were measured using BioPlex. Inhibition of CCR3 receptors with SB328437 attenuated disease activity and gross morphological damage to the inflamed intestines and reduced eosinophils and their regulatory molecules in the inflamed colon and circulation. SB328437 had no effect on eosinophils and their progenitor cells in the spleen and bone marrow. This study demonstrates that targeting eosinophils via the CCR3 axis has anti-inflammatory effects in the inflamed intestine, and also contributes to understanding the role of eosinophils as potential end-point targets for IBD treatment.

Lack of CCR3 leads to a skeletal phenotype only in male mice.

We recently showed that adult male mice that lacked the C-C-chemokine receptor 3 (CCR3) exhibited disturbed bone remodeling, which resulted in a cortical bone phenotype of thin femoral cortical bone. However, it remains unknown whether this phenotype would be present during bone modeling, or it affects female mice. Here, we analyzed juvenile and adolescent CCR3-deficient mice to determine when bone modeling was affected in the absence of CCR3 signaling. To investigate whether the CCR3 bone phenotype was sex-related, we analyzed both young female and male mice, and adult females. Micro-computed tomography (µCT) and histomorphometric analyses in adolescent CCR3-deficient male mice revealed reduced cortical bone volume and thickness, and an increase in periosteal mineralization. Interestingly, no skeletal phenotype was observed in adolescent or adult female CCR3-deficient mice. Among juvenile CCR3-deficient mice, neither males nor females showed a skeletal phenotype, which indicated that bone modeling was not affected by the CCR3 deficiency. In summary, adolescent and adult male mice that lacked CCR3 receptors exhibited a cortical phenotype that was not present in female mice, probably due to an estrogen protective mechanism. Based on these and our previous results, we suggest that the importance of CCR3 in cortical bone turnover is related to sex hormones. Because only a few molecules are known to control cortical bone turnover, our novel finding that CCR3 regulated cortical bone thickness only in males suggested that CCR3 is a novel target for controlling cortical bone morphology in male individuals, and perhaps, in post-menopausal women.

The rs13075270 and rs13092160 polymorphisms of CCR1 and CCR3 genes on oral aphthous-like lesions in PFAPA syndrome.

Fever is a common symptom of infection in children. Periodic fever syndromes are less common but more complex. One of these Periodic fever syndromes is PFAPA (periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis) syndrome which is known as the most benign syndromes. The cause of this disease is unknown. Various factors, including environmental and genetic factors, are involved in the development of this disease. In this study, the association of rs13075270 and rs13092160 polymorphisms were investigated in CCR1 and CCR3 genes with susceptibility to this syndrome in the Chinese population. In this regard, 38 patients with PFAPA syndrome and 100 healthy individuals were selected. After DNA sampling and extraction, polymorphisms of CCR1 and CCR3 receptor genes were examined by the PCR-RFLP method. Findings were analyzed using SPSS software version 22 with a significant level of P <0.05. The frequency of T/T genotype rs13092160 polymorphism in the patient and control groups was 78.95% and 83%, respectively, C/T genotype was 21.05% and 17% (P = 0.421). The frequency of the C/C genotype was 0 in both groups. Regarding rs13075270 polymorphism, the frequency of T/T genotype in patient and control groups was 15.79% and 81%, C/T genotype was 78.95% and 18% and C/C genotype was 5.26% and 1%, respectively (P<0.05). Thus, in rs13075270 polymorphism, the C/T genotype was associated with the risk of PFAPA syndrome (P<0.05), but rs13092160 polymorphism did not show a significant difference between individuals with PFAPA syndrome and controls.

C3Mab-3: A Monoclonal Antibody for Mouse CC Chemokine Receptor 3 for Flow Cytometry.

CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed in eosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 was found in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be a useful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the Cell-Based Immunization and Screening method. The established anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry. C3Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells, which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that KDs of C3Mab-3 for CHO/mCCR3, P388, and J774-1 cells were 4.3 × 10-8 M, 2.6 × 10-7 M, and 2.4 × 10-7 M, respectively. C3Mab-3 could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.

Establishment of Novel Anti-Mouse CCR3 Monoclonal Antibodies (C3Mab-6 and C3Mab-7) by N-terminal Peptide Immunization.

The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 × 10-9 M, 1.4 × 10-7 M, and 1.7 × 10-7 M, respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 × 10-9 M, 5.1 × 10-7 M, and 3.1 × 10-7 M, respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3 through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.

Gene knockdown of CCR3 reduces eosinophilic inflammation and the Th2 immune response by inhibiting the PI3K/AKT pathway in allergic rhinitis mice.

The CCR3 gene plays a critical role in allergic airway inflammation, such as allergic rhinitis (AR), and there is an inflammatory signal link between the nasal cavity and the CCR3 gene in bone marrow. However, the effects of the CCR3 gene in bone marrow cells on AR are not clear. The present study investigated the roles and underlying mechanisms of the bone marrow CCR3 gene in AR mice. Conditional knockout of the bone marrow CCR3 gene (CKO) in mice was generated using the Cre-LoxP recombination system, and offspring genotypes were identified using polymerase chain reaction (PCR). An ovalbumin-induced AR model was established in CKO and wild-type mice to measure eosinophilic inflammation and the Th2 immune response. The following mechanisms were explored using a specific PI3K/AKT pathway inhibitor (Ly294002). We successfully constructed and bred homozygous CKO mice and confirmed a significant increase in CCR3 expression and PI3K/AKT pathway activity in AR mice. Deficiency of the bone marrow CCR3 gene caused a remarkable reduction of CCR3 expression and the PI3K/AKT signaling pathway activity, inhibited histopathological lesions and eosinophil infiltration of the nasal cavity, and reduced the production of Th2 cytokines in serum, which led to the remission of allergic symptoms in AR mice. Ly294002 treatment also decreased these inflammatory indexes in a concentration-dependent manner and blocked inflammatory signals from CCR3, but it did not affect the high expression of CCR3 in AR mice. Collectively, our results suggest that conditional knockout of the bone marrow CCR3 gene can reduce eosinophilic inflammation and the Th2 immune response, which may be due to inhibition of the PI3K/AKT pathway.

C3Mab-2: An Anti-Mouse CCR3 Monoclonal Antibody for Immunocytochemistry.

The C-C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4, and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressed in eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic and therapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognized endogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cell line) cells and are usable in immunocytochemistry.