敲除背根神经节Fcgr1减弱类风湿关节炎模型大鼠NF-κB/NLRP3通路活化

目的探讨外周神经元FcγRⅠ调控类风湿关节炎疼痛的机制。方法利用野生型SD大鼠和条件敲除Fcgr1大鼠建立类风湿关节炎模型。设野生型大鼠对照组(control组)、野生型大鼠类风湿关节炎(RA)组和条件敲除Fcgr1大鼠类风湿关节炎(CKO+RA)组,每组7只。用痛觉行为学检测各组大鼠建模前第3、1 d,建模后第3、5、7、9 d机械痛阈值和热痛阈值变化,用荧光原位杂交实验检测条件敲除Fcgr1大鼠背根神经节(DRG)中是否表达Fcgr1 mRNA,用免疫荧光实验检测大鼠DRG中pNF-κB(p65)、NLRP3、IL-1β和IL-18表达和脊髓背角(SDH)胶质细胞活化。结果与control组比较,RA组和(CKO+RA)组大鼠机械和热疼痛阈值均降低,(CKO+RA)组大鼠机械和热疼痛阈值比RA组大鼠明显提高(P<0.05);与RA组大鼠相比,(CKO+RA)组大鼠DRG中pNF-κB(p65)、NLRP3、IL-1β和IL-18表达明显下调(P<0.05);SDH中GFAP和Iba1表达降低(P<0.05)。结论DRG神经元FcγRⅠ可能通过NF-κB/NLRP3通路促进神经元炎性细胞因子IL-1β和IL-18合成释放,引起SDH胶质细胞活化,参与类风湿关节炎疼痛。

Endocytosis of FcαR is clathrin and dynamin dependent, but its cytoplasmic domain is not required

FcαR, the Fc receptor for IgA, is essential for IgA-mediated immune responses. Previous studies have shown that IgA and IgA immune complexes can be rapidly endocytosed by FcαR. However, the underlying mechanism remains unclear. Here, we investigated the endocytic pathway of FcαR in monocytic cell line, U937, that naturally express FcuR and in transfected Chinese hamster ovary （CHO）, COS-7 and Hela cells. By using selective chemical inhibitors of different endocytic pathways, overexpression of dominant-negative mutants of Eps15 and knockdown of clathrin heavy chain （CHC） via RNA interference, we demonstrated that endocytosis of FcaR was through a clathrin-mediated pathway. The endocytosed FcαR went into Rab5- and Rabll-positive endosomes. However, endocytosis of FcaR could not be blocked by a dominant-negative mutant of Rab5. We also demonstrated that endocytosis of FcαR was dynamin-dependent by overexpressing a dominant-negative mutant of dynamin. The potential endocytic motif for FcαR was also examined. Unexpectedly, we found that the entire cytoplasmic domain of FcaR was not required for the endocytic process of FcαR. We conclude that endocytosis of FcaR is clathrin- and dynamin-dependent, but is not regulated by RabS, and the endocytic motif is not located in the cytoplasmic domain of FcαR.

Distribution of Variant Genotypes of Fc gamma Receptor ⅢA in Healthy Chinese Population of Zhengzhou City

To investigate the distribution of variant genotypes of Fc gamma receptor Ⅲa (FcγRⅢa) in healthy Chinese population of Zhengzhou city, genomic DNA was extracted from peripheral blood of healthy donators The genotypes of FcγRⅢa-158 were determined by nested polymerase chain reaction (PCR) in 137 healthy people in Zhengzhou city The results showed that frequencies of variant genotypes FF, VV and VF were 42 3 %, 48 9 % and 8 8 % respectively The distribution of FcγRⅢa-158 in healthy Chinese population of Zhengzhou city was polymorphic and different from that of African Americans (AA) and Caucasian Americans (CA)

THE EFFECT OF FCγ RECEPTOR ON THE PATHOGENESIS OF GRAVES' DISEASE

Objective To explore the roles of Fcγ recep tor in the pathogenesis of Graves' disease. Methods Fcγ receptor gene knockout mice(Fcγ R KO m ice) which were rooted in C57BL/6 mice and wild type C57BL/6 mice were immunized by hTSH receptor expressing cells (DAP3.WT). 1-2×107 DAP3.WT cells were peri toneally injected into mice every two weeks for a total of six times. Two weeks after final immunization, mice were killed for measurement of total thyroxine, T RAb and pathological examination. Results The thyroxine level of the immunized Fcγ recept or gene knockout mice was significantly lower than that of the immunized wild ty pe control mice (2.2±0.31 vs. 3.32± 0.59 g·dL -1, P< 0.05 ),but there was no significant difference between immunized Fcγ R KO mic e and non-immunized wild type control group. The TRAb levels of the immunized F γ R KO mice significantly increased compared to those of the immunized wild type mice (21.75±8.21 vs. 14.11±6.21, P< 0.05). The lymphocyte cel ls infiltration and destruction of thyroid follicles were found in the thyroid gland of the immunized Fcγ R KO mice. Conclusion These results suggest that Fcγ receptor may be involved in the pathogenesis of Graves' disease.

Effect of tumor necrosis factor-α on ventricular arrhythmias in rats with acute myocardial infarction in vivo

Acute myocardial infarction （AMI） is an acute cardiovascular emergency. This study was undertaken to assess the effect of tumor necrosis factor-a （TNF-a） on ventricular arrhythmias induced byAMI in rats in vivo. Two hundred and forty male Wistar rats were randomized into a sham- operation group, an AMI group, and a recombinant human tumor necrosis factor receptor：Fc fusion protein（rhTNFR：Fc） group. Acute anterior wall myocardial infarction was produced in the AMI group by ligating the left anterior descending coronary artery （LAD）, and there was no ligation but operation in the sham-operation group. The rhTNFR：Fc group was treated with rhTNFR：Fc（10 mg/kg）, a TNF-a antagonist, 24 hours before LAD ligation. The spontaneous and induced programmed electrical stimulation ventricular arrhythmias were recorded at baseline and 10 minutes, 20 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours and 12 hours after ligation. At the same time the protein and mRNA expression levels of TNF-a among different groups were detected by histochemistry and real-time fluorescent quantitative PCR. Expression of TNF-a increased markedly from 10 minutes after infarction, peaked at 20-30 minutes, and returned to baseline gradually in the AMI group and rhTNFR：Fc group. The time- windows of spontaneous and induced ventricular arrhythmias were similar. Compared with the AMI group, the rhTNFR：Fc group showed a lesser expression of TNF-a protein and a lower incidence of ventricular arrhythmias （P〈0.05）. There was no obvious change in the sham-operation group. The expression of TNF-a induced by AMI could contribute to the onset of ventricular arrhythmias.

Expression of endothelial cell IgG Fc receptors and markers on various cultures

Objective To determine and compare the expression of endothelial cell IgG Fc receptors (FcγR) and markers on various kinds of cultures.Methods Human breast microvascular endothelial cells (HMVEC), human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVEC) and canine aortic endothelial cells (CAEC) were stimulated with cytokines tumor necrosis factor α (TNF α) and interferon γ (IFN γ). The binding of anti Fcγ receptor (FcγR) type Ⅰ, Ⅱ and Ⅲ antibodies was measured using an enzyme linked immunosorbent assay (ELISA). The constitutive expression of endothelial cell markers was examined using anti von Willebrand factor antibodies, DiI low density lipoprotein (Dil Ac LDL) and fluorescein isothiocyanate (FITC) labeled ulex europaeus agglutinin 1. Results The binding of anti FcγRⅡ was significantly increased by the simultaneous stimulation with TNF α and IFN γ on all three types of human endothelial cells (ECs), but not on canine endothelial cells. Enhanced FcγRⅡ expression was most significant when human ECs were cultured in endothelial cell basal medium (ECBM). However, the expression of FcγRⅡ on CAECs could not be induced by human cytokines even after they were cultured in ECBM for 3 passages. Endothelial cells also showed diversity for the constitutive expression of classic markers. Conclusions This study demonstrates that cytokines TNF α and IFN γ enhance low affinity FcγR expression on human endothelial cells in vitro. The results indicate that heterogeneity of endothelial cells exists not only on constitutive expression but also on stimulative expression.

Universal chimeric Fcγreceptor T cells with appropriate affinity for IgG1 antibody exhibit optimal antitumor efficacy

Developing universal CARs with improved flexible targeting and controllable activities is urgently needed.While several studies have suggested the potential of CD16a in tandem with monoclonal antibodies to construct universal CAR-T cells,the weak affinity between them is one of the limiting factors for efficacy.Herein,we systematically investigated the impact of Fcγreceptor(FcγR)affinity on CAR-T cells properties by constructing universal CARs using Fcγreceptors with different affinities for IgG1 antibodies,namely CD16a,CD32a,and CD64.We demonstrated that the activities of these universal CAR-T cells on tumor cells could be redirected and regulated by IgG1 antibodies.In xenografted mice,64CAR chimeric Jurkat cells with the highest affinity showed significant antitumor effects in combination with herceptin in the HER2 low expression U251 MG model.However,in the CD20 high expression Raji model,64CAR caused excessive activation of CAR-T cells,which resulted in cytokine release syndrome(CRS)and the decline of antitumor activity,and 32CAR with a moderate affinity brought the best efficacy.Our work extended the knowledge about FcγR-based universal CAR-T cells and suggested that only the FcγRCAR with an appropriate affinity can offer the optimal antitumor advantages of CAR-T cells.

IgG Fc engineering to modulate antiboc affector functions

Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss pro- posed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.

Fcγ receptor IIIA polymorphisms and efficacy of rituximab therapy on Chinese diffuse large B-cell lymphoma

Background Rituximab is used extensively in combination with chemotherapy to cure non-Hodgkin＇s lymphoma （NHL）, and not only accelerates short-term improvement, but also prolongs patient survival and decreases relapse. The aim of this study was to evaluate the impact of Fcγ receptor IliA （FcyRIIIA） gene polymorphisms on the response to rituximab therapy for newly diagnosed B-cell lymphomas. Methods Patients with newly diagnosed histologically-proven CD20-positive B-cell lymphoma were eligible for the study. All of the patients received rituximab combined with chemotherapy （CHOP）. The FcyRIIIA type was analyzed by PCR. The initial efficacy was assessed after 6 cycles and the long-term survival was determined. Results Thirty-four patients were recruited between October 2005 and April 2006. The FcyRIIIA distribution was as follows： 11 patients were VV, 5 were FF, and 18 were VE After a median of 6 cycles （range 4-8） of rituximab combined chemotherapy, the overall response rate was 79% （82% in the VV group, 83% in the VF group, and 60% in the FF group; P=0.04）. After a median follow-up time of 37 months （range 34-41）, there were 12 relapses among 27 responders （44%）; 5 of 9 patients （5/9） in the VV group, 5 of 15 patients （33%） in the VF group, and 2 of 3 patients （2/3） in the FF group （P=0.21）. The 1-year overall survival in the VV, FF, and VF groups was 80%, 60%, and 80%, respectively, and the 3-year overall survival was 58%, 40%, and 69%, respectively （P=0.08）. After analysis by COX regression, only the international prognosis index and response to initial treatment were significantly related to overall survival. Conclusions The distribution of FcyRIIIA polymorphisms in this B-cell lymphoma population shows that VF is most frequently expressed, followed by VV and FE Patients with the FcyRIIIA VV and VF types are more sensitive to the initial treatment of rituximab combined with chemotherapy and have superior long-term survival compared with those with FF. Nevertheless, FcyRIIIA polymorphisms do not predict prognosis independently.

Immunization with HBsAg-Fc fusion protein induces a predominant production of Thl cytokines and reduces HBsAg level in transgenic mice

Background The Fc receptor associated pathway might improve the immune responses against hepatitis B virus （HBV） as previously described by us. In addition, the Fit3 ligand （FL） has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice. Methods The fusion proteins composed of HBsAg and the Fc domain of murine IgG1 （HBsAg-Fc） and/or the Fit3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase （ALT）/AST were investigated after immunization. Results After six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Thl cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Fit3 ligand stimulated an alternative Thl-type immune response featured with high level productions of tumor necrosis factor a （TNF-a） and monocyte chemoabstractant protein 1 （MCP-1）, causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level. Conclusion HBsAg-Fc is effective in eliciting both the humeral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

Transferrin receptor and Fc α/μ receptor may not be the major IgA_1 receptor on human mesangial cells

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. 1 The histopathology of IgAN is characterized by abundance of mesangial matrix and proliferation of mesangial cells. IgA_1 deposition in the mesangium plays an important role in the inflammatory process in this disease.

Binding of Divalent H-2K^(d)/IgG2aFc Fusion Protein to Murine Macrophage via Fc-FcR Interaction

Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal Protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.

Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.

Understanding SARS-CoV-2-Mediated Inflammatory Responses: From Mechanisms to Potential Therapeutic Tools

Currently there is no effective antiviral therapy for SARS-CoV-2 infection, which frequently leads to fatal inflammatory responses and acute lung injury. Here, we discuss the various mechanisms of SARS-CoV-mediated inflammation. We also assume that SARS-CoV-2 likely shares similar inflammatory responses. Potential therapeutic tools to reduce SARS-CoV-2-induced inflammatory responses include various methods to block FcR activation. In the absence of a proven clinical FcR blocker, the use of intravenous immunoglobulin to block FcR activation may be a viable option for the urgent treatment of pulmonary inflammation to prevent severe lung injury. Such treatment may also be combined with systemic anti-inflammatory drugs or corticosteroids. However, these strategies, as proposed here, remain to be clinically tested for effectiveness.

Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis

Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis （CIA）, a mouse model of rheumatoid arthritis. We show that splenic marginal zone （MZ）, but not B- 1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in na＇=＇ve mice. Upon immunization with heterologous collagen II in complete Freund＇s adjuvant the collagen-reactive MZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor （TLR） 4 and 9-1igands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitroand induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors I and 2 demonstrated increased proliferation and cytokine production, while Fcy receptor lib deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.

ADAP restraint of STAT1 signaling regulates macrophage phagocytosis in immune thrombocytopenia

Heightened platelet phagocytosis by macrophages accompanied by an increase in IFN-γplay key roles in the etiology of immune thrombocytopenia(ITP);however,it remains elusive how macrophage-mediated platelet clearance is regulated in ITP.Here,we report that adhesion and degranulation-protein adaptor protein(ADAP)restrains platelet phagocytosis by macrophages in ITP via modulation of signal transducer and activator of transcription 1(STAT1)-FcγR signaling.We show that ITP was associated with the underexpression of ADAP in splenic macrophages.Furthermore,macrophages from Adap^(−/−)mice exhibited elevated platelet phagocytosis and upregulated proinflammatory signaling,and thrombocytopenia in Adap^(−/−)mice was mitigated by the depletion of macrophages.Mechanistically,ADAP interacted and competed with STAT1 binding to importinα5.ADAP deficiency potentiated STAT1 nuclear entry,leading to a selective enhancement of FcγRI/IV transcription in macrophages.Moreover,pharmacological inhibition of STAT1 or disruption of the STAT1-importinα5 interaction relieved thrombocytopenia in Adap^(−/−)mice.Thus,our findings not only reveal a critical role for ADAP as an intracellular immune checkpoint for shaping macrophage phagocytosis in ITP but also identify the ADAP-STAT1-importinα5 module as a promising therapeutic target in the treatment of ITP.