Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms.

Activation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-1 (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM-1, recognized by a monoclonal antibody (MoAb) ENA1, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab')2 fragments of MoAb ENA1 up to 60%. An additive inhibition of the adhesion was established by pre-incubation of the PMN with anti-CD18 MoAb and/or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENA1, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD18 MoAb and/or LAI reduced the adhesion observed if intact ENA1 were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD18 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a role in graft rejection and in diseases where antibodies directed against endothelium are involved.     

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.     

Increased expression of IgG Fc receptor type I on neutrophils and monocytes from HIV-infected subjects.

Interferon-gamma (IFN-gamma) induces de novo expression of IgG Fc receptor type I (FcRI) on neutrophils and significantly raises the level of these receptors on monocytes. Since increased concentrations of IFN-gamma have been observed in sera from patients with HIV infection, FcRI expression might also be increased on these subjects' phagocytes. FcRI expression was assessed by indirect immunofluorescence staining of phagocytes in whole blood from 40 healthy controls and 55 HIV+ subjects, 24 belonging to CDC class III and 31 to CDC class IV; 42 were intravenous drug abusers (IVDA) and 13 were homosexual men. Plasma levels of IFN-gamma were measured using a modified immunoradiometric assay. The mean linear fluorescence intensity, used as a relative measure of receptor expression, was significantly higher on unseparated neutrophils from HIV+ subjects in CDC classes III (P < 0.001) and IV (P < 0.0001) than from controls. Similar changes in FcRI expression were observed on monocytes from HIV+ subjects. While no differences were observed between IVDA and homosexual HIV+ patients, there was a significant association between FcRI expression and the patients' CDC stage, those in class IV having the highest FcRI levels. Plasma IFN-gamma concentrations were significantly higher in HIV+ patients than in controls and a positive correlation with the stages of HIV infection was again observed. FcRI expression was also increased on freshly purified neutrophils from five HIV+ patients in CDC class IV but did not increase further after 18 h incubation with IFN-gamma, a treatment that up-regulated FcRI expression on control neutrophils. These data suggest that: (i) FcRI evaluation may be a sensitive marker for the biological activity of IFN-gamma in vivo; (ii) phagocytes from HIV+ subjects are activated in vivo by IFN-gamma, expressing increased levels of FcRI; (iii) these IFN-gamma-activated cells may play a role in the pathogenesis of AIDS.     

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.     

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.     

Identification of an Oolemmal IgG Fc Receptor: Its Role in Promoting Binding of Antibody-Labelled Human Sperm to Zona-Free Hamster Eggs

ABSTRACT: Antisperm antibodies (ASAs) present in sera of infertile men and women have been shown either to promote or inhibit penetration of zona-free hamster eggs by antibody-labelled human spermatozoa. Increased numbers of oolemmal-bound sperm have been noted in association with increased sperm penetration frequencies, following antibody labelling, when compared with antibody-free sperm. The promotion of adherence of ASA-labelled sperm to the oolemma could be mediated through the binding of antibodies to common epitopes present on the sperm and egg surfaces or through Fc-mediated binding to an oolemmal Fc receptor. In support of the latter hypothesis, we report that zona-free hamster eggs bind aggregated human IgG and IgG Fc fragments. The presence of an oolemmal IgG Fc receptor has been confirmed using a rat monoclonal antibody (2.4G2) directed against a murine IgG Fc receptor (Fc γ R_II) as judged both by indirect immunofluorescence and by immunobead binding. In addition, the pre-incubation of zona-free hamster eggs with IgG Fc diminished both adhesion to and penetration of the oolemma by human spermatozoa.     

Kupffer cell depletion in vivo results in preferential elimination of IgG aggregates and immune complexes via specific Fc receptors on rat liver endothelial cells.

In the present study we have investigated the clearance kinetics and tissue distribution of monomeric (m) IgG and soluble aggregates of IgG (AIgG) and immune complexes (IC) in normal and Kupffer cell (KC) depleted rats. In normal rats, clearance of mIgG occurred in a biphasic manner with a first half-life (T1/2) (T1) of 36.3 +/- 6.3 min and a second T1/2 (T2) of 168.4 +/- 4.7 min. AIgG composed of 20-27 IgG molecules per aggregate were cleared significantly faster than mIgG with a T1 of 2.5 +/- 0.1 min and a T2 of 32.5 +/- 5.6 min. KC depletion did not have a significant effect on the clearance rate of mIgG (T1: 33.4 +/- 8.9 min; T2; 159.5 +/- 12.5 min), while clearance of AIgG was delayed significantly with T1 4.8 +/- 0.7 min and T2 41.2 +/- 3.2 min. Eight minutes after injection, 77% of AIgG was found in the liver in normal rats while 62% was found in the liver of KC-depleted rats. Double immunofluorescence studies indicated that AIgG in the liver was associated with KC and endothelial cells (EC) in normal rats. In KC-depleted rats, AIgG was strongly associated with EC. A similar staining pattern was observed when IgG-immune IC were administered. The clearance of AIgG in KC-depleted rats was inhibited fully by pre-administration of high concentrations of IgG but not by pretreatment with IgA. asialofetuin (ASFe) or ovalbumin (OVA). Aggregated F(ab')2IgG was cleared with a comparable rate to mIgG from the circulation, again suggesting Fc gamma receptor-mediated elimination of AIgG by EC. There was a reduced degradation of AIgG in rats depleted of KC as compared with normal rats. These data suggest binding and degradation of AIgG by EC in vivo.     

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.     

抗人IgGFc和Fab单克隆抗体的鉴定及应用

本文将采用木瓜蛋白酶水解和ＳＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析等手段获得的人ＩｇＧＦｃ和Ｆａｂ，以抗人ＩｇＧＦｃ和抗人ＩｇＧＦａｂ单抗为参比品（Ｓｉｇｍａ）．鉴定了细胞库中抗人ＩｇＧ系列的部分细胞林，得到分泌特异性抗人ＩｇＧＦｅ和抗人ＩｇＧＦａｂ单抗的细胞各一株。在此基础上。应用抗人ＩｇＧＦｃ及抗人ＩｇＧＦａｂ单抗分别制备了Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱，纯化了酶解的人ＩｇＧＦｃ和Ｆａｂ。经ＥＬＩＳＡ法鉴定，相互间无交叉反应。同时用此方法还制备了人抗ＨＢｅＦａｂ，并将此Ｆａｂ标记过氧化物酶。配制ＨＢｅＥＬＩＳＡ诊断盒，证明其生物学活性未受影响，而且消除了类风湿因子引起的ＨＢｅＡｇ假阳性现象。     

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.     

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.     

An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta

During normal human pregnancy, maternal IgG crosses the placenta and provides passive immunity for the fetus. In so doing, IgG passes through two cellular barriers: the syncytiotrophoblast and the fetal capillary endothelium. The Fc region of IgG is required for its transport across the placenta, but the Fc receptors responsible have not been identified definitively. We recently reported the isolation from a placental cDNA library of clones encoding the α chain of a human homologue of the major histocompatibility complex class I-related Fc receptor, the neonatal Fc receptor (FcRn). In mice, FcRn is essential for the transport of maternal IgG to the fetus and the neonate. We report here the localization of human FcRn mRNA within the placenta by in situ hybridization, and of human FcRn protein by immunohistochemistry. Both methods show that human FcRn is expressed in syncytiotrophoblast, and is, thus, appropriately located to transport maternal IgG across the first barrier. We confirm previous findings that specific binding of IgG to placental membranes is greater at pH 6.0 than pH 7.5. This corresponds with the pH dependence of IgG binding to FcRn and is consistent with the presence of FcRn in syncytiotrophoblast. We propose a transport model in which maternal IgG binds FcRn at low pH in endosomes within the syncytiotrophoblast. FcRn is not expressed in fetal capillary endothelia, and the mechanism of IgG transport across the second barrier remains unknown.     

Immunogenetic studies of soluble Fc receptor (CD 16) antigens in human seminal plasma

Abstract: Two forms of CD 16 Fc receptors (FcRMIII) have been defined on human leukocytes. Type 1, present on polymorphonuclear neutrophils (PMN), expresses the NA alloantigen system and type 2, present on natural-killer (NK) cells and macrophages, has no known polymorphic variations. We have described the presence of soluble FcRMIII antigens in human seminal plasma (SP) (see Ref. 9). These SP antigens retain an affinity for IgG-Fc and are biochemically distinct from leukocytic FcRMIII. Their origin within the male reproductive tract and how they relate to FcRMIII types 1 and 2 were not known. By using monoclonal antibodies CLB/Gran 11 and Leu lie that differentially react with FcRMIII type 1 and type 2 from different individuals, we studied how SP FcRMIII antigens relate to leukocytic FcRMIII. CLB/Gran 11 and Leu 11c reactivities with PMN and NK cells from a selected panel of male donors were compared with enzyme-linked immunosorbent assay reactivities of CLB/Gran 11 and Leu lie with SP samples from these donors. The reactivity pattern with SP samples was shown to be different from findings with donors'PMN, but it corresponded with their NK cells. Our results indicate that SP FcRMIII antigens do not manifest the polymorphic variations that are detected by CLB/Gran 11 and Leu 11c on PMN. These findings suggest that SP FcRMIII antigens do not originate from PMN.     

Human IgG Fc promotes expression,secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice.Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.     

Characterization of Fc epsilon RI gamma in human natural killer cells.

We report the characterization of the Fc epsilon RI gamma chain which associates with the transmembrane form of CD16 to form the low affinity receptor for IgG (Fc gamma RIII) expressed on human natural killer (NK) cells. cDNA cloning and sequence analysis of Fc epsilon RI gamma from a polyclonal CD3-CD16+ NK line established that this molecule is identical to Fc epsilon RI gamma previously identified in human basophils as part of a high affinity receptor for IgE. Polymerase chain reaction analysis of Fc epsilon RI gamma gene expression in a series of CD3+CD16- and CD3-CD16+ NK clones reveals that Fc epsilon RI gamma is not directly linked to NK activity since clones of the CD3+CD16- phenotype lack Fc epsilon RI gamma RNA but nevertheless mediate cytotoxicity. Taken together, these results demonstrate that the Fc epsilon RI gamma molecule is expressed in various types within the hematopoietic system as part of multimeric surface receptors involved in different biological functions.     

Isolation of a rabbit IgG fraction with cytophilic properties

About 15% of rabbit IgG loaded on a column of Con A-Sepharose 4B was found to be specifically bound to the column due to a structural variation in its carbohydrate moiety. The Con A-retained rabbit IgG contained a higher amount of neutral hexoses than the initial IgG but its molecular weight, antigenic structure and half-life were identical or similar. The Con A-retained rabbit IgG has an affinity for the Fc receptor-bearing homologous macrophages which is 10 times higher than that of the initial IgG. The IgG fraction not retained on Con A-Sepharose is practically devoid of binding ability. These results suggest that the Con A-bound IgG may represent the cytophilic fraction of monomeric IgG responsible for the binding of IgG to Fc receptor-bearing cells.     

Function-blocking antibodies to human vascular adhesion protein-1: a potential anti-inflammatory therapy

Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.     

Etiology of leukocyte adhesion deficiency-associated periodontitis revisited: not a raging infection but a raging inflammatory response

In leukocyte adhesion deficiency type I, neutrophils fail to adhere to blood vessel walls and thus cannot transmigrate to peripheral tissues. Leukocyte adhesion deficiency type I patients invariably experience an aggressive form of generalized periodontitis, which has been historically attributed to defective neutrophil surveillance of the periodontal infection. This time-honored notion has now been challenged by a recent study, which showed that the underlying etiology involves a dysregulated host response that leads to overexpression of the proinflammatory and bone-resorptive cytokine IL-17.     

Phagocytic signaling strategies: Fc(gamma)receptor-mediated phagocytosis as a model system.

Phagocytosis is a phylogenetically ancient process by which eukaryotic cells engulf insoluble substances whose size exceeds approximately 0.5 microm. The engulfment process requires the concerted action of several fundamental cellular pathways and is governed by multiple transmembrane signaling events. Here we focus on phagocytosis mediated by a well-studied class of phagocytic receptors that recognize the Fc portion of IgG (Fc(gamma)Rs ).