Similarities of Fcγ Receptors on Trophoblasts and Placental Endothelial Cells

The trophoblastic tissue and endothelium of fetal stem vessels in cryostat sections of first-trimester and term human placentae adsorbed erythrocytes sensitized with IgG antibodies of human, rabbit, and guinea pig origin. Native and heat-aggregated human and rabbit IgG, and human IgG subclasses except IgG2 inhibited the hemadsorption. However, the Fc fragment of IgG2 inhibited the hemadsorption similarly to the Fc of pooled IgG. No inhibition was obtained with o'ther immunoglobulin classes or fragments. The inhibitory activity of IgG and Fc fragments was abolished by partial reduction and alkylation, indicating that inter-heavy-chain disulfide bonds are required for the binding. Immune complexes prepared at slight antigen excess showed an eightfold stronger inhibition than antiserum alone. Both receptors were sensitive to formaldehyde, periodic acid, and heat. The Fcγ receptors on the trophoblasts and on the endothelium in human placentae are apparently similar.     

Immunological studies of human placentae. Binding of complexed immunoglobulin by stromal endothelial cells.

Endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae bind fluorescein-conjugated heat-aggregated human IgG. Soluble immune complexes of sheep anti-human albumin also bind in a pattern that is similar to that of aggregated human IgG, but native human IgG is not bound by placental endothelial cells. Aggregated IgG binding, unlike soluble immune complexes, is blocked by pretreatment of the sections with antiserum to human IgM. Cross-blocking experiments with aggregated IgG and immune complexes suggest that they may be bound by different receptors.     

Hassall's corpuscles in the thymus of fetuses, infants and children: immunological and histochemical aspects

Hassall's corpuscles (HC) were examined for immunological and histochemical markers in cryostat sections of thymus from fetuses, infants and children. HC could not be detected before 14 weeks of gestation. Receptors for the Fc part of IgG (Fc gamma R) were demonstrated by adherence of ox erythrocytes sensitized with anti-ox IgG using a closed chamber technique. Fc gamma R were also detected by immune complexes of horseradish peroxidase (HRP) and rabbit antibodies to HRP, and with an anti-Fc gamma R serum, using indirect immunofluorescence technique and indirect immunoperoxidase technique. The staining was seen along the outer cell membranes of the HC. The Fc gamma R activity was highest in early fetal life, and decreased with increasing age. Indicator cells which detect receptors for the Fc part of IgM and for the activated third component of complement did not adhere to HC. At 14 weeks of gestation, HC showed a weak alpha-naphthyl acetate esterase (ANAE) activity, while from 16 weeks the staining intensity and pattern was unchanged. In some HC, separate cells with strong ANAE activity were seen. These cells also showed endogenous peroxidase activity, and were stained by an antibody to HLA-DR antigens. Such cells were not seen until 16 weeks of gestation. HC were stained by antibodies to IgG in fetuses older than 16 weeks, and the intensity increased gradually up to 24 weeks. Antibodies to IgM weakly stained some HC in fetuses between 16 and 36 weeks of gestation, whereas antibodies to IgA stained a minority of HC in fetuses older than 24 weeks.     

Localization of IgGFc and Complement Receptors in Chicken Lymphoid Tissue

Chicken lymphoid organs were examined for IgGFc and complement receptors (FcR/CR) by immune adherence on frozen tissue sections. Indicator systems were sheep erythrocytes (E) coated with chicken anti-E IgG (Each). E coated with rabbit anti-E IgG (EArab) and normal chicken serum (EAC), and FITC-labelled zymosan particles coated with chicken serum (ZyC). In the spleen, FcR and CR activity was confined to B-dependent areas, i.e. the periellipsoidal sheaths and germinal centres and the red pulp. No FcR were found in the thymic or bursal lymphoid tissue, but CR activity was observed in the medulla of bursal follicles. Chicken and turkey IgG, chicken IgGFc, and bovine serum albumin (BSA)-chicken anti-BSA complexes inhibited binding of EAch. No inhibition was obtained with chicken IgGF(ab')₂. IgM or albumin, or with BSA-rabbit anti-BSA complexes and human or rabbit IgG. E did not adhere to the sections, nor did EArab, EArab incubated with heat-inactivated chicken serum, or EAC complexes prepared with EArab and guineapig complement. The data suggest that chicken B lymphocytes and macrophages have receptors for avian IgGFc and C which can be demonstrated in tissue sections.     

In vitro studies on the selective binding of IgG from different species to tissue sections of the bovine mammary gland

Tissue sections of the mammary gland of cattle provide a sensitive and reproducible model for studying the initial events of the selective transport of bovine IgGs by the acinar epithelium (AE). Mammary tissue was reacted with purified antibody to horseradish peroxidase (HRP) and exposed to HRP. The sites of peroxidase activity were revealed cytochemically. The highly selective binding of IgGs by AE of the colostrum-forming gland but never of the lactating gland was most notable, whereas IgG1 and IgG2 subclasses and IgM and IgA lacked any binding capacity. An inhibition assay showed that the inhibitory capacity of IgGs was abolished by either alanylation or acetylation, whereas binding of IgGs was blocked by both Fab/c and isolated H-chains, but not by F(ab')2, Fab and Fc. The inhibitory pattern suggests that the region on the IgGs molecule involved in binding to the AE receptor may be located within the CH2 domain. In addition, IgG preparations of human, sheep and rabbit origin were found to be equally efficient in inhibiting the binding of IgGs to AE, whereas no inhibition was obtained with nonmammalian (turtle and chicken) IgG. Similarly, human myeloma proteins of the subclasses IgG1 and IgG3 caused complete inhibition, while IgG2 and IgG4 were inefficient. It is suggested that molecular evolution of IgG within mammalian species is accompanied by the conservation of structures fitting to receptors on the cell surface of different species.     

Monocyte-dependent cell death of T lymphocyte subsets in chronic hepatitis C

Much evidence indicates that atherosclerotic lesions are largely of an inflammatory nature. Activated macrophages and macrophage-derived foam cells laden with cholesterol esters are a major constituent of these lesions and can influence lesion formation via several potential mechanisms. One such mechanism is Fcgamma receptor activation and/or Fcgamma receptor-mediated clearance of immune complexes containing cholesterol, such as lipoprotein immune complexes. That this mechanism contributes to lesion formation would be further supported if Fcgamma receptor expression in arterial lesions were demonstrated. We therefore used monoclonal antibodies and immunocytochemical methods to analyze frozen sections of human arterial lesions for expression of each of the three primary classes of mononuclear phagocyte Fcgamma receptors. Approximately 800 sections of aorta, carotid, and coronary arteries obtained from five elderly donors were analyzed. The presence of macrophages was determined by assaying reactivity of a monoclonal antibody specific to CD163, which is expressed only on cells of the human mononuclear phagocyte lineage. Results indicate that highly cellular preatheromatous lesions contained numerous macrophages in the zone of proliferation that expressed each class of Fcgamma receptor (FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA). Fcgamma receptor-positive cells were also present in medial and adventitial areas. Fcgamma receptor staining was both punctate and diffuse, the latter suggesting that soluble receptors were present in the extracellular matrix. These data further support that Fcgamma receptor-mediated clearance of immune complexes can occur in arterial lesions during atherogenesis. Expression of both the high affinity (FcgammaRIA) and lower affinity (FcgammaRIIA/FcgammaRIIIA) receptors indicates that mono- and multivalent IgG-containing immune complexes could engage Fcgamma receptors and influence lesion formation through several different inflammatory mechanisms triggered by receptor activation.     

Immunological studies of human placentae: subclass and fragment specificity of binding of aggregated IgG by placental endothelial cells.

Fluorescein-conjugated heat-aggregated human IgG binds to endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae. No binding was observed using native human IgG of heat-aggregated human albumin, IgM or IgA2. No inhibition of binding of heat-aggregated human IgG was observed by pre-treatment of placental tissue sections with native IgG or non-aggregated Fc fragments. The binding was blocked using heat-aggregated Fc fragments prepared from IgG1, IgG2, IgG3 and IgG4 myeloma proteins, but not with heat-aggregated human light chains, Fab and F(ab)2 fragments of human IgG, or with heat-aggregated human IgM and IgA2. It is suggested that the placental endothelial cell receptor for aggregated IgG may function to keep immune complexes from entering the foetal circulation.     

Studies of Fc receptors of heart valve and joint fibroblasts.

Normal human and rabbit sera, and the IgG isolated from them, have been shown by immunofluorescence to react with bovine and human heart valve fibroblasts. Analogous results were obtained with sera of children under the age of 2. Positive reactions were observed also with fibroblasts, chondrocytes and osteocytes of human fetal joint tissues. The reactions are mostly due to monomeric immunoglobulins, since soluble immune complexes give much weaker reactions with the fibroblasts. The reactions are apparently dependent on the presence of Fc receptors on these cells. This conclusion is confirmed by positive reactions with IgG Fc fragments, with pure antibodies to ovalbumin and with human monoclonal IgG. The monoclonal IgG1 possesses the strongest ability to bind with fibroblast Fc receptors. No Fc-IgG receptors have been revealed on the fibroblasts and other structures of the interstitial connective tissue of human and bovine myocardium.     

De Novo Formation of Immune Complexes in Toman Kidney Allografts

Cryostat sections of 11 rejected human renal grafts were selected for positive results in immunofluorescence tests. Immune complexes were detected in glomerular basement membrane (9 cases), tubular basement membrane (3 cases), and vessels (5 cases). Preincubation of the sections with FII of pooled human serum, but not of rabbit serum, prevented the staining of immune complexes for human IgG. These findings were interpreted by the assumption that most complexes under study were produced by reaction of the patient's altered IgG with the rheumatoid-like factor of IgG variety.     

Immune receptors (IgG-Fc and complement receptors) in normal human organs.

The presence of immune receptors (IgG-Fc and complement receptors) was examined in normal human tissues from various organs. Sheep erythrocytes sensitized with rabbit IgG antibody (IgG-EA) or with rabbit IgM antibody and human complement (IgM-EAC) were used for the detection of IgG-Fc receptors and complement receptors, respectively. IgG-Fc receptors were detected on sinuses of the lymph node, splenic red pulps, hepatic lobules, renal glomeruli, alveolar wall of the lung, intestinal villi, superifical layer of the synovium, and subcutaneous tissue. The presence of complement receptors was demonstrated in the follicles and the sinuses of lymph nodes, white pulp of the spleen, renal glomeruli, alveolar wall of the lung, and lamina propria of the intestine. The specific binding of IgG-EA was consistently inhibited by heat-aggregated human IgG or by a high concentration of native human IgG. The detection of immune receptors in these various tissues might be helpful for understanding why the immune complexes are often detected when immunologically mediated disease processes involve these tissues.     

Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fcgamma RIIIa (CD16)

Human red cells (RBC) coated with IgG anti-D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcgamma R). Polymorphisms of Fcgamma RIIa and Fcgamma RIIIa affect IgG binding in vitro, and may alter the efficiency of clearance of immune complexes in vivo. In a RBC clearance study, 22 Rh D-negative subjects were given 100-400 micro g human monoclonal or polyclonal IgG anti-D i.m. followed 48 h later by 51Cr-labelled D+ RBC. The half lives of the infused D+ RBC were determined, together with the coating levels of anti-D on the D+ RBC. Fcgamma RIIA and FcgammaRIIIA genotyping was performed. Large ranges of phagocytosis and extracellular lysis of RBC in vitro, and of half lives of RBC in vivo, were observed. Clearance of RBC coated with monoclonal IgG3 anti-D (BRAD-3) was more rapid in five subjects homozygous for Fcgamma RIIIa-F/F158 than in three subjects expressing the Fcgamma RIIIa-V158 allele (P = 0.024). This effect was not observed, however, for those individuals given polyclonal anti-D. There was also no significant difference in the efficiency of RBC destruction in vitro or of RBC clearance in vivo between the subjects analysed for individual genotypes or alleles or combinations of alleles. In conclusion, the presence of the Fcgamma RIIIa-V158 allele compromised the efficiency of removal of RBC coated with IgG3 anti-D.     

Tissue distribution of IgG Fc receptors.

The mechanism causing deposition of circulating immune complexes is largely unknown. The possible role of tissue IgG Fc receptors in immune complex localization has been evaluated using IgG coated ox RBC (ox erythrocyte antisera [EA]) as indicator particles. Cryostat tissue sections of normal human synovium, skin, kidney, choroid plexus, lung and uveal tract were examined for the presence of IgG Fc receptors, with human spleen used as a positive control. Ox EA were shown to bind to splenic red pulp. This binding could be almost completely blocked by heat aggregated human IgG. In none of the other normal tissues examined were IgG Fc receptors demonstrated. To investigate the possibility that inflamed tissues express Fc receptors, biopsy specimens of rheumatoid synovium and skin demonstrating vasculitis were studied. No ox EA binding to these tissues was noted. We concluded that IgG Fc receptors are probably not present in tissues that are targets for immune complex deposition and are therefore unlikely to play a role in this process.     

Fc epsilon receptors on human cell lines and peripheral blood lymphocytes detected by binding of IgE immune complexes

To identify Fc epsilon receptors on human cell lines and peripheral blood lymphocytes, we developed a new method which relies on the binding of constructed immune complexes to Fc epsilon receptor-positive cells. Cell suspensions from either cell lines or peripheral blood lymphocytes were incubated with complexes of human myeloma IgE and murine monoclonal anti-human IgE at various ratios prior to cytocentrifugation. The complexes bound to the cells were subsequently visualized by immunoperoxidase staining. The specificity of this assay to detect cell surface Fc epsilon receptors was shown by the ability of human myeloma IgE to block the binding of the IgE complexes, resulting in unstained cells, whereas IgM, IgG, and IgA were unable to block the binding of the complexes (stained cells). This method is reproducible, allows quantification of a single sample at different times, and provides a record of the results. It can also be adapted to identify any cell surface receptor for which the ligand is known.     

Chlamydophila pneumoniae infection of human aortic endothelial cells induces the expression of FC gamma receptor II (FcgammaRII)

Chronic endothelial infection is believed to be one of the factors able to cause endothelial cell damage and trigger the onset of human atherosclerosis. Chlamydophila pneumoniae infects endothelial cells and has received special attention because of both epidemiological and experimental evidence supporting its role as a risk factor for atherosclerosis. It is also possible that otherwise independent risk factors for atherosclerosis may have synergistic effects. Immune phenomena, such as the formation of circulating immune complexes (IC) containing modified LDL and corresponding antibodies, have been linked to the development of coronary artery disease. The antibodies involved in the immune response to modified lipoproteins are predominantly of the pro-inflammatory IgG1 and IgG3 subclasses. However, it is difficult to understand how circulating IC could cause endothelial damage and initiate the atherosclerotic process, unless they were formed in the subendothelial space or immobilized by endothelial cells. The last hypothesis would be possible if endothelial cells expressed Fcgamma receptors. Healthy endothelial cells do not express Fcgamma receptors, but endothelial cells infected by a variety of infectious agents do. Thus we decided to investigate whether infection of endothelial cells with C. pneumoniae is also able to cause the expression of Fcgamma receptors. The expression of Fcgamma receptors (CD64, 32, and 16) on human aortic endothelial cells infected with C. pneumoniae for 4, 24, 36, and 48 h was studied by flow cytometry. Twenty-four hours after infection 30-40% of the endothelial cells had detectable inclusion bodies, 8-9% of the total number of cells (approximately 25% of the infected cells) expressed FcgammaRII, and about 1.5-2% (5% of infected cells) expressed FcgammaRI and FcgammaRIII. Double-staining studies confirmed that the expression of FcgammaRII was limited to C. pneumoniae-infected endothelial cells. We conclude that C. pneumoniae infection induces primarily the expression of FcgammaRII by endothelial cells and this may be a significant link between two proposed pathogenic mechanisms involved in the pathogenesis of human atherosclerosis.     

Fc receptor function on sheep alveolar macrophages

We have examined the binding to sheep alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN) of sheep immunoglobulin G subclasses or rabbit IgG immune complexes formed between rabbit anti-DNP IgG and DNP-bovine serum albumin. Binding studies using 125I-rabbit IgG immune complexes demonstrated 6.6 +/- 3.5 X 10(4) receptors per alveolar macrophage; these receptors bound immune complexes with an average association constant of 3.3 X 10(7) M-1. Saturation binding was achieved by 90 minutes at 4 degrees C with 6 X 10(-8) M IgG. Binding of subclasses of sheep IgG was examined by immunofluorescence. Only 10% of alveolar macrophages bound monomeric IgG1 and no binding of sheep IgG2 monomer could be demonstrated. In contrast, most peripheral blood PMN (93.0 +/- 9.5%) bound IgG2, but not IgG1. No binding to adult peripheral blood PMN of rabbit IgG immune complexes could be demonstrated. To study further the development of pulmonary host defense, we examined the expression of receptors for IgG immune complexes (Fc gamma R) on alveolar macrophages obtained from animals aged 8 through 180 days. At 8 and 21 days of age, the number of Fc gamma R varied considerably (75,000-192,000 sites per cell) and equalled or even exceeded that of adult sheep. Fc gamma R number declined by 42 and 90 days of age, where a nadir was reached (37,000 +/- 6,000 and 25,000 +/- 6,000 sites, respectively). By 180 days of age, the number of receptors had approached those of normal adult sheep (70,000 +/- 20,000 sites per cell). These studies parallel previous observations that revealed age-related differences in the phagocytic capacity of ovine alveolar macrophages.     

Ureaplasma urealyticum binds mannose-binding lectin

Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.     

Fcγ Receptor Heterogeneity in the Human Placenta

Fc receptors for IgG, FcRI (CD64), FcRII (CD32), and FcRIII (CD16) in human placenta were studied by indirect immunohistochemistry using avidin-biotin peroxidase complexes for the staining of cryostat sections. The MoAb 32.2 against FcRI stained cells in the loose connective tissue of the placental villi. The MoAb IV3 (FcRII) and C1KM5 (FcRII) also stained stromal cells and in addition stained the endothelium of the placental villi. The MoAb anti-Leu-11b against FcRIII and B1D6 against a 40-kDa FcR from placenta stained both stromal cells and endothelium as well as the fetal trophoblasts lining the villi. The MoAb 3G8 (FcRIII) also stained trophoblasts and stromal cells but did not stain the endothelium. The heterogeneity of FcR expression on human placenta is established. The function of the different receptors is still unclear.     

Direct Binding of Monomeric Anti-DNA Antibodies to Raji Cells

The Raji-cell test is one of the most widely used methods for the detection and quantitation of immune complexes. Immune complexes and not 7 S IgG bind via C3 to complement receptors on the cell membrane of the Raji cell. During sucrose gradient fractionation of human and murine systemic lupus erythematosus sera, with a high Raji cell-binding activity, we could not demonstrate immune complexes in these sera. Subsequent analysis showed that the major part of the Raji cell binding was used by 7 S IgG with an anti-DNA specificity. Blocking experiments wilh complement-bearing aggregated IgG revealed that complement and Fc receptors were not involved in the binding of these anti-DNA antibodies to Raji cells. We conclude that the Raji cell test is not suitable for the detection and quantitation of immune complexes in sera containing anti-DNA antibodies.     

Noncognate binding to histones of IgG from patients with idiopathic systemic lupus erythematosus

The mechanism of attachment of circulating immune complexes (CIC) to glomerular basement membranes (GBM) has not yet been elucidated. Since it has been proposed that histone may be the ligand between GBM and DNA/anti-DNA CIC, we explored by ELISA and Western blots the nature of the interaction of IgG with histone on solid phase. Cognate binding of IgG anti-histone antibody was characteristically dependent on in its F(ab')(2) fragment and was inhibited by free histone. On the other hand, heat-aggregated IgG, a model for CIC, and IgG from most patients with idiopathic SLE had a characteristic noncognate binding behavior to histone: it was dependent on Fcgamma rather than on the F(ab')(2) fragment and was not effectively inhibited by free histones. Also, binding to histone of in vitro generated DNA/anti-DNA immune complexes was not dependent on DNA as a histone ligand, but on Fcgamma. Finally, there was good agreement between the binding of this IgG to histone and to C1q. We concluded that: (1) altered IgG and/or CIC bind to solid-phase-attached histone primarily through their Fcgamma and (2) CIC may mimic IgG antihistone antibodies in solid-phase immunoassays.     

Specificity of Fcγ Receptors in Human Malignant Tissue and Normal Lymphoreticular Tissue

The specificity of the Fcγ receptors in normal spleen and liver and in malignant tissues was studied using hemadsorption to cryostat sections. Indicator cells (EA) were sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The binding of EA to sections of normal and malignant tissues was inhibited by pooled IgG of human, rabbit, and guinea pig origin and by human IgG1, and IgG3, and IgG4 myeloma proteins. Heat-aggregated IgG inhibited the binding to sections of liver and some malignant tissues more effectively than monomeric IgG. The Fc fragments of IgG were also inhibitory, but not the F(ab')₂, Fab', and Facb fragments. The inhibition obtained increased with decreasing amounts of A used for sensitization of E. The inhibitory activity of IgG was abolished after partial reduction and alkylation. No inhibition was obtained with IgG2, IgM, IgA, or albumin. E sensitized with Facb or F(ab')₂ fragments of A did not bind to normal or malignant tissues. The specificity of the Fc receptors in normal spleen and liver and in malignant tissues is apparently very similar.