大鼠自体移植脾组织再生的形态学研究

目的：探讨大鼠自体移植脾组织血管神经再生过程、VEGF及KDR表达的变化。方法：采用Wistar大鼠105只，行脾切除自体脾组织大网膜内移植术，分别于术后7、14、30、60、90、120、180d，采用免疫组化VEGF、KDR、NPY^ 神经纤维抗体染色方法，应用光镜、透射电镜、图像分析观测自体移植脾组织。结果：自体脾组织移植术后7d即有血管长入移植脾组织，180d再生血管接近正常；术后30d神经开始再生，180d趋于正常；术后7、14dVEGF、KDR阳性染色细胞密度迅速升高，60d达高峰，180d趋于正常；术后30d出现：NPY^ 神经纤维，120、180d NPY^ 神经纤维广泛分布。结论：移植脾组织再生血管神经来源于大网膜；移植脾组织内VEGF、KDR表达量早期升高，血管再生完成时恢复正常水平；移植脾组织血管再生早于神经再生。     

大鼠自体移植脾组织GAP-43+神经再生的实验研究

目的 研究大鼠自体脾组织移植术后移植脾组织GAP-43^ 神经纤维再生及分布规律。方法 健康Wistar大鼠105只，随机分为自体脾组织移植组和对照组，于术后7、15、30、60、90、120、180d取移植脾组织标本，应用免疫组化方法显示GAP-43^ 神经纤维，并用图像分析系统，对不同时相点免疫组化GAP-43^ 染色区域进行图像定量分析。结果 自体脾组织移植术后30d，移植脾组织周围大网膜内可见GAP-43^ 神经纤维，并向移植脾组织内伸展；自体脾组织移植术后60～120d，移植脾组织内再生神经纤维逐渐增多；术后180d，移植脾组织中GAP-43^ 神经纤维分布及密度接近正常。结论GAP-43^ 神经纤维于脾移植术后60d开始出现，术后180dGAP-43^ 神经纤维接近正常脾。     

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes.

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR-mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface-bound IgG displayed impaired phagocytosis of IgG-coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG (2-10 mg/ml). Monocytes pre-exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgG1 and with anti-D-treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR-mediated phagocytosis by monocytes. A flow cytometric analysis using anti-FcRI, FcRII and FcRII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.     

Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults

The role of specific IgG2 antibody in the protection against serious infection with Streptococcus pneumoniae is unclear. We therefore decided to investigate the relationship between serum antibody levels and opsonization and phagocytosis of this microorganism. We have measured serum IgM, IgA and IgG subclass antibody specific for pneumococcal capsular polysaccharide and in vitro phagocytosis of serotype 14 pneumococcus by polymorphs, in healthy adults before and after immunization with Pneumovax II. IgM and IgG2 were the predominant anti-pneumococcal antibodies seen, IgA and IgGl being present at low titre. No significant relationship of phagocytosis with specific IgM and IgA antibodies was found. However, both specific IgG 1 and IgG2 antibodies in post-immunization sera correlated significantly with phagocytosis of the pneumococcus in the presence of complement (r= 0.57, P= 0.029 and r= 0.59, P= 0.022 respectively). After heat-inactivation, the remaining opsonic activity of sera correlated only with levels of specific IgG2 antibody (r= 0.61, P = 0.0006). Whereas phagocytosis supported by specific IgG 1 and IgG2 antibody to serotype 14 pneumococcus after immunization is mediated by complement activation, IgG2-specific antibody in high titre may also be able to function by complement-independent interaction with Fcγ receptors on polymorphs.     

Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.     

Fcγ和Fcε受体在小鼠三叉神经节的表达

目的 检测正常小鼠三叉神经节(TG)是否表达免疫球蛋白G(IgG)和免疫球蛋白E(IgE) Fc段Fcγ受体和Fcε受体,及其在过敏小鼠TG中的变化.方法 通过腹腔注射OVA和铝剂,建立小鼠过敏性结膜炎(ACJ)模型.EHSA检测血清总IgE.用Westernblot和免疫荧光检测Fcγ受体和Fcε受体的表达.结果 小鼠TG表达Fcγ受体和Fcε受体.其中IgG激活型高亲和力受体FcγRI和抑制型低亲和力受体FcγRⅡ只表达在小鼠TG神经元上,而IgG激活型低亲和力受体FcγRⅢ表达在小鼠TG中的卫星胶质细胞上.IgE激活型高亲和力受体FcεR Ⅰ表达在小鼠TG神经元上,而IgE低亲和力受体FcεRⅡ同时表达在小鼠TG神经元和卫星胶质细胞上.与正常小鼠比较,ACJ小鼠的血清总IgE水平升高,TG的FcεR Ⅰ和FcγRⅡ表达增加(P＜0.05).而ACJ小鼠的FcεRⅡ和FcγRⅠ表达下降(P＜0.05).FcγRⅢ在正常和ACJ小鼠TG中的表达无显著差别.结论 小鼠TG表达的Fcγ和Fcε受体可能参与ACJ及其他过敏性疾病的发生和发展.     

On the interaction between agalactosyl IgG and Fcγ receptors

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (FcγR). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity FcγRII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for FcγR-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.     

Measurement of the binding activity of defined IgG aggregates to macrophage Fc receptors

Small soluble IgG aggregates of defined size were prepared from pooled human IgG by gel filtration chromatography, and examined by analytical ultracentrifugation. Three such fractions, dimer-rich, trimer-rich and 25S aggregate were used to inhibit IgG monomer binding in a study of the influence of aggregation in the binding of human IgG1 to mouse macrophage Fc receptors. Of the polymers tested, IgG in the trimeric form was found to bind with the greatest avidity, being 158 times more active than monomeric IgG, whereas IgG as a larger 25S aggregate had an increased binding activity of 80 times; the avidity of IgG as dimer was increased by a factor of 2 over monomeric IgG. The possible mechanisms involved in achieving enhanced binding are discussed.     

Localization of the site of the IgG molecule that regulates maternofetal transmission in mice

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the mouse IgG1 (mIgG1) molecule that is involved in the intestinal transfer of recombinant Fc hinge fragments in neonatal mice. This site encompasses Ile-253, His-310, Gln-311, His-433 and Asn-434, localized at the CH2-CH3 domain interface and overlapping with the staphylococcal protein A-binding and catabolic sites. In the present study, the effect of these mutations on the maternofetal transfer of Fc hinge fragments has been studied. Experiments to analyze transfer of radiolabeled Fc hinge fragments from the circulation of 15–18 day pregnant mice to fetuses in utero demonstrate that the mutations affect the maternofetal transmission in a way that correlates closely with the effects of the mutations on intestinal transfer and catabolism. The studies indicate that the neonatal Fc receptor, FcRn, is involved in transcytosis across both yolk sac and neonatal intestine in addition to the regulation of IgG catabolism.     

Critical mass of splenic autotransplant needed for the development of phagocytic activity in rats

When total splenectomy is inevitable, heterotopic splenic autotransplantation seems to be the only alternative to maintain the functions of the spleen. The present study was carried out to analyse the critical mass of splenic autotransplant (SAT) for the development of phagocytic activity in rats. Wistar rats were submitted to total splenectomy (TS) alone or in combination with slices of SAT ranging from an average rate of 21·9% (one slice) to 100% (five slices) of the total splenic mass implanted into the greater omentum. Sixteen weeks after the beginning of the experiment, the animals were inoculated intravenously with a suspension of Escherichia coli labelled with Tc‐99m. After 20 min, the rats were killed and the liver, lung and spleen or SAT, as well as blood samples were removed to determine the percentage of labelled bacteria uptake in these tissues. As the percentage of the total splenic mass contained in the SAT increased, the bacteria remaining in the blood decreased. From the implant of 26% up to the implant of the total splenic mass (100%) there was no difference in the bacteria remaining in the blood between the healthy animals of the control group and those submitted to TS combined with SAT. This finding shows that the critical mass needed for the development of phagocytic activity of macrophages in splenic autotransplants in adult rats is 26% of the total splenic mass.     

Photometric microassay for quantitation of macrophage Fc and C3b receptor function

A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37 degrees C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4 degrees C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay.     

Anti-CD16 autoantibodies and delayed phagocytosis of apoptotic cells in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by chronic hepatic inflammation and immune mediated apoptosis of bile duct epithelial cells. Delayed macrophage phagocytosis of opsonized apoptotic cells, noted in other autoimmune diseases, may promote inflammation. Recent studies suggest serum anti-CD16 autoantibodies contribute to impaired macrophage phagocytosis by blocking complement receptor 3 (CR3) signaling via CD16. Therefore, serum anti-CD16 levels and the ability of monocyte derived macrophages from individuals with PBC to phagocytosis apoptotic cells were compared to controls. The mean level of anti-CD16 IgM autoantibodies (0.86+/-0.62 v. 0.35+/-0.22, respectively, p=0.031) was increased in PBC compared to control sera, and mean PBC phagocytosis of opsonized apoptotic cells was significantly decreased compared to controls (23.9+/-12.2% v. 43.9+/-14.4%, respectively, p=0.020). However, PBC phagocytosis of opsonized apoptotic cells was not significantly affected by the presence or absence of autologous serum (20.8+/-13.5% v. 23.9+/-12.2%, respectively, p=0.560). PBC phagocytosis of opsonized apoptotic cells inversely correlated with CD16 (and CR3) expression levels on Day 5 after culture in the presence or absence of autologous serum (r=-0.546, p=0.033 and r=-0.519, p=0.042, respectively). Phagocytosis of non-opsonized apoptotic cells did not correlate with CD16 or CR3 expression (p>0.050). In conclusion, PBC macrophage phagocytosis of opsonized apoptotic cells is impaired, irrespective of serum factors and may increase hepatic inflammation.     

Human IgG4: a structural perspective

IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs.     

Development of a new in vivo anaphylactic histamine release assay in rats

A new method has been developed to detect antigen-induced histamine release in the blood of allergic rats. This in vivo model of systemic anaphylaxis has been utilized to evaluate drugs for histamine release inhibition activity. Compounds were administered by various routes at appropriate times prior to or concomitant with antigen challenge. One minute after challenge the rats were bled into tubes containing heparin and aminoguanidine. Histamine concentrations were determined by a radioenzyme technique after separation of the plasma. Experiments demonstrated that: (a) rats passively sensitized with reaginic serum exhibited elevated blood histamine upon i.v. antigen challenge in a reproducible and dose-dependent manner; (b) heat-treatment of the reaginic serum abolishes antigen-induced histamine release; and (c) antigen-induced histamine release is inhibited by disodium cromoglycate, in a dose-dependent manner and it is also inhibited by isoproterenol or isobutyl-methylxanthine.     

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determinedin vitro by a classical rosette assay in 12 postmen-opausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, -mannosyl [IgG(Con A)] or -galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P<0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs. In addition, when both groups of women were compared, all three Fc-dependent functions measuredin vitro using IgG(PNA)-SRBCs were significantly lower (P<0.01 at least) in women without E than in women on therapy. In another series of experiments, we also found that the rosetting and phagocytosing capacities of MCs were dramatically and transiently reduced in three of three young women during the menstrual period, only when the IgG(PNA)-SRBCs were used as targets. Taken together, our data show that MC phagocytosis of SRBCs coated with IgG antibody exhibiting terminal, and/or accessible, -galactosyl residues in their Fc domain is selectively impaired by a physiological E deficiency and is restored when this deficiency is artificially or spontaneously corrected. They therefore suggest that these hormones are capable of affecting the PNA-like activity of IgG(Fc) receptors of human MCs.     

The multiple facets of FcRn in immunity

The neonatal Fc receptor, FcRn, is best known for its role in transporting IgG in various tissues, providing newborns with humoral immunity, and for prolonging the half-life of IgG. Recent findings implicate the involvement of FcRn in a far wider range of biological and immunological processes, as FcRn has been found to bind and extend the half-life of albumin; to be involved in IgG transport and antigen sampling at mucosal surfaces; and to be crucial for efficient IgG-mediated phagocytosis. Herein, the function of FcRn will be reviewed, with emphasis on its recently documented significance for IgG polymorphisms affecting the half-life and biodistribution of IgG3, on its role in phagocyte biology, and the subsequent role for the presentation of antigens to lymphocytes.     

大网膜内植入自体脾组织与原位脾组织的结构比较

目的 :为临床应用自体脾组织植入术提供实验研究资料。方法 :大鼠分为实验组和对照组 ,前者切取 1 /2脾脏去包膜后切成 1mm× 1mm× 1mm大小均匀组织块 ,植入大网膜囊袋内。饲养 6个月后取 2组脾组织制片 ,光镜和电镜定性观察组织结构变化 ,计算机图像分析系统比较血管、红髓、白髓及胶原纤维的面密度 ;免疫组化法结合计算机图像分析测定神经肽 (NPY)阳性神经纤维密度。结果 :神经和边缘窦内皮细胞结构恢复较好 ,血管 ,白髓的面密度值较对照组减少 ,红髓与对照组相当 ,胶原纤维面密度增加。结论 :大网膜内植入的自体脾组织通过再生能恢复脾脏的主要组织结构 ,但不能完全恢复正常。