Human placentae membrane receptor for IgG—i. Studies on properties and solubilization of the receptor

Characterization of the human placental membrane receptor for human ¹²⁵I-IgG is described. The receptor bound specifically both monomers and aggregates of human IgG. Human colostral IgA, bovine, sheep, pig, and horse IgG were not bound. No effect of pH in the range 6.6–7.4, ionic strength in the range 0.1–0.5, and temperature between 4 and 45°C on the binding was found. A water-soluble fraction containing the active receptor (glycoprotein fraction-PGP) was obtained from the placental membranes using lithium diiodosalicylate. The solubilized receptor interacted with IgG better at 4°C than at 20°C or 37°C. The results on replacement of monomeric IgG by aggregated IgG, and vice versa, suggest that both monomers and aggregates of human IgG, were bound to the same receptor sites. The apparent association constant for monomeric human IgG was 0.86 ± 0.2 × 10⁷ mole^?1, and 2.0 ± 0.16 × 10¹⁵ IgG molecules were bound per l mg of the membrane protein. Formaldehyde (0.1%), 2-mercaptoethanol (50 mM), and periodate (4 mM) showed no effect on the binding properties of the membrane-bound and on the solubilized receptor, as well. Higher concentrations of periodate (10 mM or 20 mM) decreased the binding of IgG to membranes but showed no effect on the water-soluble receptor. Both the membrane-bound and the solubilized receptor were sensitive to papain. Pronose abolished the receptor activity after prolonged proteolysis only. Neuraminidase did not affect the activity of the receptor. The decrease of the binding activity of the membrane-bound receptor by trypsin and phospholipase C was due to a release of a material containing an active receptor. No effect of trypsin or phospholipase C on the activity of solubilized receptor was observed. The results obtained suggest a protein character of the placental Fc receptor. After electrophoresis of ¹²⁵I-labeled solubilized receptor in polyacrylamide gel in the presence of SDS, 2 major protein peaks with molecular weights of 74,000 and 104,000 and 3 minor peaks with molecular weights of 56,000, 144,000, and 163,000 were found.     

Size-dependent effect of IgA on the IgA Fc receptor (CD89)

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89⁺/γ⁺) enhance the IL-2 production 10–12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca²⁺]_i) in cells co-expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine-based activation motif of the FcR γ chain abolishes this increase in [Ca²⁺]_i, indicating association and involvement of the FcR γ chain in CD89-mediated signaling.     

An extended family of Fc receptor relatives

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.     

Macrophage function in high- and low-responder strains of mice

Unstimulated peritoneal macrophages of the high-responder A/J mice compared to the low-responder B10 strain have a lower number of cells with the different Fc receptor (FcR) subtypes and correspondingly the FcR mediated phagocytosis is also lower. The intraperitoneal immunization with SRBC leads to a prompt increase of FcR expression and of phagocytic and pinocytic activity in the A/J mice, while in the B10 strain the activity of macrophages remains unchanged.     

Co-localization of the neonatal Fcγ receptor and IgG in human placental term syncytiotrophoblasts

Transfer of maternal IgG through the human placenta furnishes the newborn with passive immunity to a number of infectious agents. The exact mechanism of this transfer is still unknown, but it is agreed that it involves active receptor-mediated transport. The neonatal Fc receptor is a major histocompatibility complex class I-like receptor originally identified in the intestines of newborn rodents. A similar receptor has recently been detected in human placental syncytiotrophoblasts. Using multilabeling fluorescence immunohistochemistry and confocal laser scanning microscopy, we found that the neonatal Fc receptor co-localizes with IgG and β2-microglobulin in granules of human placental syncytiotrophoblast. The Fc receptor is not detected on syncytiotrophoblast apical plasma membrane. Localization to the outermost cellular barrier between the fetal and maternal blood further strengthens the role of the Fc receptor in transplacental transport of IgG.     

FcR cross-linking on monocytes results in impaired T cell stimulatory capacity

Presentation of antigen to T lymphocytes without the appropriateco-stlmulatory signals results in a state of antigen-specificunresponsiveness. Despite the presumed importance of the B7-CD28interaction for the initiation and maintenance of T cell-mediatedimmune responses, relatively few studies have addressed theregulation of B7 expression. We have studied the expressionof the CD80 (B7-1) and B7-2 molecules on peripheral blood monocytesfollowing different activation signals, and it was demonstratedthat not only IFN-, but also granulocyte macrophage colony stimulatingfactor can induce CD80 expression on monocytes. In addition,we found that cross linking of FcR on monocytes strongly inhibitsthe up-regulation of CD80 and B7-2, with as a functional consequencethat the capacity to function as antigen presenting cells (APC)and to.stimulate T cell activation is severely impaired. Whencultures were prepared In 96-well plates coated with human IgG,stimulation of T cells with allogenelc monocytes resulted onlyin modest T cell proliferation and no detectable IL-2 secretionas compared with untreated culture plates or plates coated withFab fragments of human IgG. Under these conditions cross-linkingof CD28 on the T cells with specific mAb completely revertedthe inhibitory effect observed after culture on IgG-coated plates.Furthermore, FcR cross-linking on monocytes strongly Inhibitedthe capacity of monocytes to Induce a specific memory T cellresponse to viral, bacterial and fungal antigens, whereas thetreatment did not impair the capacity of the T cells to respondto pokeweed mitogen, phytohemagglutinin and concanavalin A.We conclude that after FcR cross-linking, the impaired APC functionis most likely due to the inability of monocytes to providethe essential costimulatory signals to the T cells via the B7–CD28/CTLA-4interaction.     

Effect of Fc receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The effect of Fc receptor (FcR) blocking by aggregated human gamma-globulin (AGG) was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of [³H]TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 μg/ml concanavalin A (Con A). Under these conditions but without Con A considerable NCMC was not elicited by normal lymphocytes. FcR blocking by AGG treatment of effector cells resulted in a significant NCMC activity to HEp-2 targets. In contrast, AGG treatment profoundly depressed LDCC. Monocyte depletion of effector cells had no major influence on the effect of AGG on NCMC and LDCC activities. An interference of FcR blocking by AGG and LDCC in response to Con A is suggested.     

An IgG-Fc Binding Protein in Seminal Fluid*

ABSTRACT: Human seminal fluid, at low dilutions, prevented the binding of aggregated human IgG (AHG) to bull spermatozoa. Seminal fluids from vasectomized men were also inhibitory. Preincubation of the seminal fluid with the spermatozoa prior to washing and addition to AHG had no inhibitory effect, indicating that the fluid component was reacting directly with AHG. Human seminal fluid was fractionated by gel exclusion chromatography on Ultrogel AcA-34, and AHG inhibitory activity was found in fractions corresponding to a molecular weight of 94,000. The activity in this fraction was stable to boiling for 10 min. It was sensitive to pronase but resistant to glycosidase, phospholipase C, neuraminidase, ribonuclease, and deoxyribonuclease, indicating that it was a protein. The gel filtration fraction readily bound recrystallized Fc and AHG; IgG was bound to a lesser extent, and no reactivity was observed with F(ab')₂, IgA, or IgM. Thus, the seminal fluid fraction appeared to specifically react with the Fc portion of IgG. The seminal fluid Fc-binding protein was isolated by affinity chromatography on Fc coupled to CNBr-activated Sepharose 4B. Scatchard analysis revealed that the binding of the seminal fluid Fc-binding protein to recrystallized Fc is reversible and had a Kd of approximately 3 × 10^?6 M.     

Specific alteration of Sendai virus glycoprotein subunits.

The alteration produced by chemical and physical agents in the structure and neuraminidase activity of Sendai virus glycoproteins was studied. While dissociation of glycoproteins with 1% sodium dodecyl sulphate (SDS), 2% beta-mercaptoethanol and 5 M urea for 2 min at 100 degrees C yielded the known HN and F subunits (mol. wts. 60,000 and 53,000), in the presence of 1% SDS the glycoproteins were converted into components with approximate mol. wts. of 60,000, 120, 000 and higher. Treatment of the glycoproteins with 2% beta-mercaptoethanol and 0.1% SDS favoured the formation of a single component with a mol. wt. of 75,000. The alterations in the glycoprotein structure were very likely caused by their free -SH groups content. An average value of 6 free -SH groups per glycoprotein subunit was estimated. Glycoproteins stored at 4 degrees C contained only 53,000 mol. wt. subunits. During storage a kind of conversion of 67,000 to 53,000 mol. wt. components took place, preserving about 60% of the initial neuraminidase activity.     

A striking example of convergent evolution observed for the ggFcR:IgY interaction closely resembling that of mammalian FcR:IgG

We have recently identified a novel IgY specific chicken FcR (ggFcR) on chromosome 20, a region where no FcR gene is present in mammals. Serially deleted IgY fusion proteins were tested in a reporter assay to identify C_H domains involved in ggFcR binding. Single C_H domains did not bind to ggFcR, whereas Fcυ2 to Fcυ4 induced good and the Fcυ3 to Fcυ4 domains moderate activity. When IgY from diverse birds were assayed, only IgY from gallinaceous birds showed binding, which enabled us to pinpoint several potential contact sites by a sequence comparison and molecular modelling. Point mutations of critical residues at these sites revealed the Fcυ2 and Fcυ3 domains as major ggFcR:IgY binding sites similar to mammalian IgG. These results demonstrate that ggFcR has a contact site to IgY which closely resembles that of human IgG bound to FcR.     

The role of Fc receptors in HIV prevention and therapy

Over the past decade, a wealth of experimental evidence has accumulated supporting the importance of Fc receptor (FcR) ligation in antibody-mediated pathology and protection in many disease states. Here we present the diverse evidence base that has accumulated as to the importance of antibody effector functions in the setting of HIV prevention and therapy, including clinical correlates, genetic associations, viral evasion strategies, and a rapidly growing number of compelling animal model experiments. Collectively, this work identifies antibody interactions with FcR as important to both therapeutic and prophylactic strategies involving both passive and active immunity. These findings mirror those in other fields as investigators continue to work toward identifying the right antibodies and the right effectors to be present at the right sites at the right time.     

Fcγ-Receptor Activity of Placental Annexin II

We have previously produced a MoAb, B1D6, against a plaeental FcR. The antigen isolated using F(ab')₂-fragments of B1D6 exhibits Fc-binding properties with low affinity for IgG. The antigen is a single-chained glycoprotein with a molecular weight of approximately 37 kDa and a pi of about 7.0-8.5. Amino acid sequences from enzymatic digests of the antigen indicated that it is annexin II. Immunoreactivity using anti-annexin antisera and purified placental annexin II have further established the specificity of BID6 to annexin II. The B1D6 epitope appears to be intramembraneous and intracellular on placental syncytiotrophoblasts, monoeytes and other cells investigated.     

The effect of corticosteroids on monocyte and neutrophil activation in bronchial asthma

The expression of IgG (Fc) receptor (FcR) and complement receptor (CR) on peripheral blood monocytes and neutrophils was determined by the rosette technique in patients with asthma receiving different forms of treatment. In 31 patients taking inhaled therapy (i.e., bronchodilators alone or in combination with inhaled corticosteroids), monocyte FcR (48.19 +/- 1.24%, mean +/- SEM) and complement (66.54 +/- 1.09%) rosettes were significantly higher (FcR p less than 0.001, CR p less than 0.001) than in the 17 healthy, normal control subjects (FcR 37.94 +/- 0.82%, CR 59.7 +/- 0.98%). These increases in the percent rosettes between the two groups were observed even when a wide concentration range of IgG or complement was used to coat the red cells. No significant differences in monocyte receptor expression were observed between those patients being treated with bronchodilators alone or patients being treated in combination with inhaled corticosteroids. In 19 patients with asthma receiving oral corticosteroids, the mean monocyte FcR (38.21 +/- 1.73%) and CR (52.78 +/- 2.09%) were significantly reduced when these patients were compared with those patients receiving inhaled therapy alone (FcR p less than 0.001, CR p less than 0.001), and there was a significant inverse correlation between the percent rosettes and the dose of prednisolone. Neutrophil CR (51.32 +/- 1.30%, p less than 0.05) but not FcR expression (24.7 +/- 0.80%) was significantly increased when these were compared with those of control subjects (FcR 24.7 +/- 0.60%, CR 47.11 +/- 0.86%), and both neutrophil FcR and CR expression was significantly reduced (FcR p less than 0.01, CR p less than 0.001) in those patients with asthma receiving oral corticosteroids. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isolation of a glycoprotein from Mycoplasma pneumoniae membranes.

A glycoprotein was detected in Mycoplasma pneumoniae membranes. Its apparent molecular weight was about 60,000, as observed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It corresponded to the single band that was detected on the gels by the carbohydrate stain, periodic acid-Schiff reagent. The intensity of this stained band varied for membranes derived from cells harvested between 4 and 10 days, with maximal intensity found for cells grown for 6 days. The carbohydrate-containing polypeptide was extracted with lithium diiodosalicylate. The extracted fraction consisted of about 80 to 90% amino acids (mainly glycine and histidine) and about 7% carbohydrates (mainly glucose, galactose, and glucosamine). The fraction was immunologically active, as indicated by the complement fixation and precipitin tests with antisera against whole cells, membranes, and membrane proteins.     

Human Peripheral Eosinophils with Receptors for IgM: Demonstration and Ultrastructural Morphology

Receptors for IgM were detected on peripheral blood human eosinophils by a rosette technique with ox red blood cells coated with the IgM fraction of the specific immunserum. Between 14 % and 43 % (mean 27 %) FcµR positive cells were found after an overnight incubation period at 37°C by using this technique. The specificity of the receptors for IgM was assessed by studying the inhibitory capacity of purified human IgM in the rosette assay. From an ultrastructural point of view, the EA^M rosette-forming cells are mature eosinophlic granulocytes characterized by a nucleus with a variable number of lobes and a certain number of «first type» granules partially or totally devoid of their content.     

Non-specific normal immunosuppressive protein (Nip): purification and further biochemical characterization

The non-specific normal immunosuppressive protein (Nip) has been described in our laboratory and its biological activity was extensively studied. In the present study, further purification analyses of Nip were conducted. Fractionation of Nip by Ultrogel AcA 34 column resulted in peak (I) that displayed Nip activity in that it exhibited marked inhibition of in-vitro blastogenic responses of human lymphocytes to mitogenic stimulation, in vitro it inhibited EL4 tumour-cell proliferation. Partial dissociation of Ultrogel peak I on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) resulted chiefly in three bands: one of high molecular weight, which is considered to be the carrier, an intermediate band of 50,000-60,000 (under non-reducing conditions) and a band of high mobility and low molecular weight approximately 6,000-14,000. Further fractionation of peak I on Sepharose-6B in 6 M guanidine-HCl resulted in two main peaks. The biological activity resided in the second peak, which corresponded to the low molecular weight fraction of 6,000-14,000. Nip is suggested to be a complex molecule comprised of a low molecular weight peptide or glycopeptide, which displays the biological activity, and a macromolecular glycoprotein carrier, which conserves its stability.     

Heterogeneity of envelope polypeptides among strains of venezuelan encephalitis virus.

Comparative virion polypeptide analysis of 29 strains of purified Venezuelan encephalitis virus by electrophoresis side by side on discontinuous, slab polyacrylamide gels revealed heterogeneity in electrophoretic mobility and apparent numbers of polypeptides. All strains had two common proteins: a capsid protein of 36,000 molecular weight, and an envelope glycoprotein of 50,000–51,000 molecular weight. Another glycoprotein ranged in molecular weight between 51,000 and 55,000. Eight strains yielded an additional glycoprotein peak in the range of 56,000–58,000; two others showed a small glycoprotein peak at 45,000–46,000. These polypeptide patterns were not influenced by the cell species (chick, hamster, monkey) in which the virus was cultured.