The Placental Fcγ Receptors Studied Using Immune Complexes of Peroxidase

Immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP were used to study the Fcgamma receptors in normal human placenta. Cryostat sections of placental tissue were incubated with the complexes, and the peroxidase activity was revealed histochemically. The bound complexes were localized to the apical surface of the trophoblasts and endothelial cells of the fetal stem vessels. Binding also occurred within the wall of some fetal vessels, to stromal cells and occasionally to areas corresponding to the trophoblastic membrane. The strongest binding was obtained with immune complexes prepared at slight antigen excess. Eight- to sixteen-fold increased concentration of human and rabbit IgG was needed to block the binding of immune complexes. Bovine and porcine IgG did not block the binding. Treatment of tissue sections with neuraminidase enhanced the binding activity of the receptors. The technique is very convenient for studies of Fcgamma receptors in tissue. However, unfixed frozen placental tissue was not suitable for ultrastructural studies.     

Fc Receptors on Human T Lymphocytes: Loss of Fcμ and Expression of Fcγ Receptors by T Cells Stimulated in Mixed Lymphocyte Reaction

The consequences of allogeneic stimulation on the expression of Fc mu or Fc gamma receptors were analysed in T-cell populations responding in mixed lymphocyte reaction (MLR). Unfractionated T-cell populations were cultured with a pool or irradiated allogeneic T-depleted cells. The responder E-rosetting cells progressively lost Fc mu and acquired Fc gamma receptors. The change of the original Fc receptor phenotype is not the consequence of a preferential proliferative response of TG versus TM cells but is likely due to a de novo expression of Fc gamma receptors by T cells lacking detectable Fc receptors (T-null) and also to the loss of Fc mu and expression of Fc gamma receptors by TM cells. These data suggest that, after MLR, responder T cells can modify their Fc receptor phenotype.     

Activity of Two Types of Fc Receptors, FcγRI and FcγRII, in Human Monocyte Cytotoxicity to Sensitized Erythrocytes

We investigated the cytotoxicity of human monocytes mediated by two types of receptors for the Fc portion of IgG, Fc gamma RI and Fc gamma RII. Erythrocytes sensitized with human IgG (EA-human IgG) were used to assay Fc gamma RI function, and erythrocytes sensitized with mouse IgG1 (EA-mouse IgG1) were used to assay Fc gamma RII. Both types of Fc gamma R were observed to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which was further characterized by using different monoclonal anti-Fc gamma R antibodies (MoAb) and monomeric IgG. Lysis of EA-human IgG was inhibited by both monomeric human IgG and mouse IgG2a in a dose-dependent way, and also by anti-Fc gamma RI MoAb 10.1. Cytolysis of EA-mouse IgG1 was inhibited by monomeric mouse IgG1 and by two anti-Fc gamma RII MoAb, IV.3 and CIKM5. Antibodies of the mouse IgG2b isotype affected neither type of ADCC. The effectiveness of cytotoxicity mediated by either of the Fc gamma R was studied by means of targets sensitized with a calibrated number of IgG molecules. At least 20 times more IgG molecules per target cell were necessary to obtain half-maximal cytotoxicity mediated by Fc gamma RII than for Fc gamma RI-mediated cytolysis. Furthermore, the previously described polymorphism of Fc gamma RII was also reflected in Fc gamma RII-dependent cytotoxicity. These studies demonstrate that Fc gamma RII can mediate ADCC, although a higher degree of target cell sensitization is required than for Fc gamma RI-mediated ADCC.     

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.     

Fcγ Receptor Expression on Sheep Afferent Lymph Dendritic Cells and Rapid Modulation of Cell Surface Phenotype Following Fcγ Receptor Engagement In Vitro and In Vivo

Afferent lymph dendritic cells were analysed for the presence of Fcγ receptors by Western blotting and for modulation of surface markers following Fcγ receptor engagement in vitro and in vivo . The results showed thatunstimulated dendritic cells expressed FcγRII constitutively. When dendritic cells were incubated in vitro with antigen/antibody complexes in antibody excess, a marked reduction in surface staining was observed for MHC class II, CD1, CD44,and VLA-4 after 8 h in culture. These changes did not occur with antigen or antibody alone. DC expression of LFA-1 and LFA-3 were slightly reduced after 8 h in culture with Ova alone, but this was enhanced slightly when the cells werecultured with immune complexes. Even more marked reductions in surface staining for MHC class II, CD1, CD44 and VLA-4 were observed on dendritic cells 4–8 h following secondary antigen challenge in vivo . LFA-1 and LFA-3 expressionwas reduced only slightly. The level of expression of MHC class II, CD1, LFA-1 and LFA-3 was substantially increased over resting values 24 h after FcγR occupancy. The intensity of staining at this time was also significantly elevated for CD44, LFA-1, LFA-3 and VLA-4. These results show that engagement of Fcγ receptors cause a substantial modulation of the dendritic cell surface phenotype after immune complex uptake. The phenomenon may function to maximize subsequent presentation of thechallenge antigen to T cells.     

Activated Human γδ T Lymphocytes Express Functional Lactoferrin Receptors

Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. αβ T cells, γδ T cells, CD8⁺ T cells, CD4⁺ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated γδ T cells is significantly larger than that of activated αβ T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for γδ T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.     

Properties of Fcγ Receptors in Normal and Malignant Human Tissues

Properties of the IgG receptors were studied by treating tissue sections or suspensions of peripheral mononuclear cells with various chemical reagents and determining changes in their ability to react with IgG-sensitized erythrocytes (EA). A heterogeneity of the Fc receptors was revealed. Iodoacetamide, 2-mercaptoethanol, sodium azide, EDTA, and a pH varying from 6.0 to 8.2 had no effect on Fc receptors in sections of normal lymphoreticular tissue and malignant tissue. Formaldehyde, low pH, and high salt concentrations affected receptor activity to various degrees. Receptors in liver sections and on monocytes were generally more resistant than receptors in spleen sections and on B lymphocytes. Receptors in malignant tissue behaved either like receptors in spleen or like receptors in liver. Although all tissues were sensitive to periodic acid, the Fc receptors in malignant tissue were always more resistant.     

Natural Cytotoxicity of Human Lymphocytes against Equine Target Cells in Vitro

Human lymphocytes displayed a frequent natural cytotoxicity (NK) in vitro against normal equine dermal fibroblasts (ED) and against equine tumour cells of a virus-containing cell line (Mc-1). Similarly, human normal sera contained antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) by normal human lymphocytes against the same target cells. Both NK and ADCC varied for different donors. For individual donors, however, cytotoxicity against the two target cells was significantly correlated both in NK and ADCC. For ED there was also a significant correlation between ADCC and NK activity. Both NK and ADCC showed some selectivity as assessed by cold target cell inhibition. Inhibition studies with Fab fragments of anti-human IgG established the involvement of immunoglobulins in the NK reaction. In this context, a marked and mainly immunoglobulin-dependent increase in both NK and ADCC activity against Mc-1 was observed in a laboratory worker frequently exposed to the target cells. The results indicate that variations of natural cytotoxicity in individual donors may sometimes be an indication of an ongoing spontaneous sensitization.     

Role of Infection in the Modulation of Mouse Circulating Soluble Cell-Free Fcγ2b/γ1 Receptor

We have recently demonstrated the occurrence of functional circulating soluble cell-free Fc gamma 2b/gamma 1 receptor in normal mouse serum by means of the 2.4G2 rat monoclonal antibody. With the solid phase radioimmunoassay described previously, we report here a dramatic increase in the level of circulating soluble cell-free Fc gamma 2b/gamma 1R in Schistosoma mansoni-infected mice individually tested before and on days 22, 41, 62, and 82 after infection. This increase was statistically highly significant when the five measurements from each mouse were compared, and also when the entire group of infected mice was compared with the stable level of Cf-Fc gamma 2b/gamma 1R measured in strain-, sex-, and age-matched control mice individually tested on the same days. This increase was commensurate with a rise in the amount of IgG detected in the sera of the S. mansoni-infected mice. Thus, infection seems to be one of the factors that modulate the expression of such soluble Cf-Fc gamma 2b/gamma 1R in mouse serum. Furthermore, the experimental device reported here for the production of high levels of Cf-Fc gamma 2b/gamma 1R by infection (in this case by parasitic infection) could serve as a model for obtaining large quantities of serum Cf-Fc gamma 2b/gamma 1R for further purification. Lastly, we point out that, by establishing a functional relationship with circulating IgG, such soluble Cf-Fc gamma 2b/gamma 1R may modulate some of the functions in which the Fc portion of immunoglobulins is involved.     

Effect of Human Anti-γ-Globulin Antibodies on Human Lymphocytes.

Addition of rheumatoid factor to cultures of human lymphocytes preincubated with substimulating amounts of rabbit anti-human lymphocyte antibody caused stimulation, while addition to lymphocytes preincubated with anti-HL-A or anti-human γ-chain antiserum had no effect. Neither was there any stimulatory effect on adding pepsin agglutinator, i.e. an antibody to the pepsin site of γG, to lymphocytes preincubated with pepsin-digested γG from anti-lymphocyte or anti-HL-A antiserum. The different results obtained in the various systems may partly be due to differences in the distribution of antigens on the lymphocyte membrane and partly to the lack of heterospecificity of pepsin agglutinator.     

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.     

Toll-Like Receptor Expression and Function in Subsets of Human γδ T Lymphocytes

Two subsets of human γδ T cells can be identified by T cell receptor (TCR) V gene usage. Vδ2Vγ9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vδ1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-γ (IFN-γ) production in peripheral blood γδ T cells and in Vδ2Vγ9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated γδ T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vδ1 and Vδ2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vδ1 and Vδ2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-γ and chemokine secretion in TCR-activated Vδ1 and Vδ2 subsets, although the levels of IFN-γ secreted by Vδ1 T cells were much lower than those produced by Vδ2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vδ1 and Vδ2 T cells and underscore the intrinsically different capacity for IFN-γ secretion of Vδ1 versus Vδ2 T cells.     

Specificity of γδ Receptor-Bearing Cytotoxic T Lymphocytes Isolated from Human Peripheral Blood

T-cell receptor (TcR)-gamma delta-bearing lymphocytes were isolated from the peripheral blood of two healthy donors by immunomagnetic separation and subsequently cultured. The cell lines generated showed two distinct patterns of cytotoxicity. One TcR-gamma delta + cell line (HG.D) lysed K562 and U937 target cells, three TcR-gamma delta + cell lines lysed Daudi cells, and one TcR-gamma delta + cell line showed a shift from the former to the latter specificity during culture. Cold target inhibition experiments showed that the HG.D effector cells which were cytotoxic against U937 cells also lysed K562 cells. The cytotoxicity against Daudi cells was strongly inhibited by monoclonal antibodies (MoAb) against the CD3 complex, whereas the cytotoxicity of the HG.D cell line against K562 and U937 was unaffected by such antibodies. The cytotoxicity against Daudi cells was also strongly inhibited in the presence of anti-TcR-gamma delta MoAb. However, in two of the Daudi-specific cell lines, strong cytotoxicity against K562 cells was induced by anti-TcR-gamma delta MoAb. Anti-LFA-1 MoAb caused only a partial inhibition of cytotoxicity, while anti-CD2 and anti-TcR-alpha beta MoAb were found to have no effect. The results indicate that human gamma delta receptor-bearing T cells demonstrate a certain degree of target cell specificity, and that recognition of some target cells may be mediated through the TcR-gamma delta.     

Surface Markers on Human B and T Lymphocytes

A procedure to assess the cellular adherence displayed by lymphocytes, at the level of the single cell, is described. Adherence is defined as the capacity of a cell to hind acrylic acid polymer beads. 0.5 μm: in size, under standardized conditions These beads were found to adhere readily to lymphocytes that have a high density of surface immunoglobulin Such cells can be distinguished from other leukocytes with the same binding capacity by the extracellular localization of the beads on these cells Furthermore, adherence, as presently defined, is briefly characterized.     

Surface Markers on Human B and T Lymphocytes

The response of human peripheral lymphocytes to phytohemagglutinin-. concanavalin A-, pokeweed mitogen-, and mitomycin-treated allogeneic lymphocytes was characterized according to the surface markers of the responding cells Almost all blast cells derived from these cultures could be classified as either B or T cells Phytohemagglutinin, concanavalin A, and allogeneic lymphocytes triggered exclusively T-tell stimulation, whereas pokeweed mitogen gave rise to a dual response. No shift in the proportion of responding B and T cells was found If different concentrations of pokeweed mitogen under the present conditions Purified T cells, isolated by sheep erythrocyte (SRBC) rosette sedimentation, responded slightly more strongly than unseparated cells, whereas purified non-SRBC-binding; cells were essentially unresponsive to all three lectins. The presence of monocytes was found to increase the lectin responses as well as to make them more re producible. It is suggested that the classification of proliferating lymphocytes by surface markers may yield clinical information as well as serve as a useful tool in the evaluation of in vitro cellular cooperation     

Surface Markers on Human B and T Lymphocytes

An association has been found between the Epstein-Barr virus (EBV) and complement (C3d) receptors on human lymphoid cells. The evidence was four-fold: there was a correlation between the expression of these two receptors; inhibition experiments showed that the binding sites probably are close to each other in the cell membrane, although not identical; EBV and complement receptors have been found to co-cap in either order; and lymphoid cell lines lacking complement receptors could not be superinfected with EBV.