Partial characterization of different cell types found in the Xenopus laevis lymphoreticular tumor based on the presence or absence of surface immunoglobulins and Fc molecules

Cells of Xenopus laevis lymphoreticular tumor induced by tumor tissue transplantation were examined for surface Ig and Fc receptor molecules in order to evaluate the different cell types found in the tumor. Direct immunofluorescent technique, using fluorochrome conjugated rabbit antisera to Xenopus Ig's, detected Ig molecules on the surface of a mean of 31.7 +/- 11.3% of cells in tumor suspensions. Most of these molecules were of IgM isotype, reversibly bound to the cell membrane (cytophilic) and could be dissociated by acid pH or overnight cell culturing. In addition integral membrane IgM was detected on the surface of 10.2 +/- 5.9% of the cells. The serum origin of cytophilic Ig's and the cellular origin of integral membrane Ig's were confirmed by analysis of electrophoretic mobility of their heavy chains on SDS-polyacrylamide gels. The existence of Fc receptor molecules on the surface of 48.6 +/- 16.6% of the cells was demonstrated by fluorescent staining using heat aggregated FITC labelled IgM or FITC or TRITC labelled antigen-complexed IgY antibodies. 32.2 +/- 12.4% and 16.4 +/- 6.8% of the cells bore receptors for IgY or receptors for IgM respectively, while 6.3 +/- 3.1% carried receptors for both Ig's. Double fluorescent staining revealed that 28.9 +/- 4.5% of cells bearing IgM on their surface expressed also receptors for IgY. These results attest to the heterogeneity of the tumor cell population, in respect to the presence or absence of FcR-IgY, FcR-IgM, sIgM, and cytophilic IgM surface molecules.     

Tryptic digestion of Xenopus IgM and IgY molecules

Xenopus IgM and IgY molecules were digested by trypsin. Their respective fragments were separated by gel filtration and immunoadsorption. The purified fragments were characterized by SDS-PAGE and immunoblotting. Tryptic digestion of Xenopus IgM resulted in the release, at a low yield, of hexameric Fcmu, and of monovalent Fabmu fragments. The digestion of Xenopus IgY antibodies led to the recovery of divalent and monovalent Fab nu fragments. The antigen-binding property of these fragments was demonstrated. No Fc nu fragments of appreciable size could be detected.     

The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions.     

Receptors for the Fc portion of immunoglobulins (FcR) on murine oral mucosal T cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear Fc gamma R whereas less than 5% of T cells of any subset bear Fc alpha R or Fc mu R. In frozen tissue sections, Fc gamma R+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that Fc gamma R+ cells may be involved in the surveillance of the epithelium while the minor Fc alpha R+ L3T4+ T lymphocyte population may promote the expression of sIgA by resident sIgM-bearing B cells, and their differentiation into IgA plasma cells.     

Expression of Fc mu receptors on human natural killer cells

Fc receptors for IgG (CD16) have been described as the only type of immunoglobulin receptor on large granular lymphocytes (LGL). However, the ability of natural killer (NK) cells to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of monoclonal or polyclonal IgM and the inhibition of NK activity by highly purified IgM could not be explained on the basis of FcR for IgG. In order to directly assess the expression of Fc receptors for IgM (Fc mu R), NK cells were treated with human polyclonal IgM, and its binding was visualized by a direct anti-globulin rosette assay with identification of rosette-forming LGL on Giemsa-stained smears. The data indicated that a high proportion of LGL (up to 68%) were Fc mu R-positive cells. However, this percentage varied depending on the IgM preparation (polyclonal or monoclonal), the indicator reagent used for the rosette assays, and the cell preparations studied. Two-color flow cytometry of human nonadherent lymphocyte preparations confirmed the presence of CD56+IgM+ cells, which represented from 43 to 78% of CD56+ cells. Flow cytometry was also performed using highly enriched preparations of human NK cells (the mean percentage of CD3-CD56+ cells was 84%). Up to 88% of purified NK cells bound FITC-labeled monoclonal IgM at a saturating concentration. By indirect immunofluorescence, from 34 to 62% of NK cells purified from the peripheral blood of normal donors were able to bind polyclonal IgM. Similar results were obtained with LGL from a patient with NK lymphoproliferative disease. Thus the presence of Fc mu R on a majority of human NK cells was demonstrated by different techniques, using unseparated peripheral blood mononuclear leukocytes, purified normal NK cells, and also LGL from a patient with NK lymphoproliferative disease.     

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.     

Neuraminidase treatment of human T lymphocytes: effect on Fc receptor phenotype and function

Purified peripheral blood T cells or T mu cells from normal healthy donors were treated in vitro with neuraminidase and examined for the expression of IgM Fc and IgG Fc receptors. Increasing concentrations of neuraminidase selectively removed IgM Fc receptors, whereas the number of T cells expressing IgG Fc receptors was significantly increased. Following neuraminidase treatment, IgM Fc receptors could be regenerated by reincubation of T cells at 37 degrees C. The regeneration of IgM Fc receptors could be blocked by treatment with cycloheximide. Neuraminidase treatment of purified T mu cells resulted in the expression of IgG Fc receptors on a subpopulation of T mu lymphocytes. A small percentage of the neuraminidase-treated T cells expressed receptors for both IgG and IgM. Treatment of T cells with neuraminidase did not effect T cell-mediated spontaneous cytotoxicity (SLMC) or antibody-dependent cellular cytotoxicity (ADCC). Our results indicate that T cell Fc receptor phenotypes can be modulated in vitro without significantly altering their functional capacity.     

Human Amnion as a Model for IgG Transport

ABSTRACT: The ability of fresh human amnion to bind and internalize horseradish peroxidase-Iabeled IgG (IgG-HRP) was examined in an in vitro Ussing chamber system. The amnion demonstrated unique cell membrane receptors for the Fc portion of IgG molecules (FeyR). The FcγR exhibit exquisite specificity and affinity for IgG monomers as demonstrated by staining with labeled IgG. Labeled IgA, IgM, F(ab')2 fragments of IgG, aggregated IgG, and antigen-antibody complexes all failed to bind to the amnionic epithelial cells. Binding was only minimally affected by changes in ionic strength or pH when viewed at the light microscopic level. The FcγR are located on both the apical and basal cell membranes. The binding of IgG-HRP to the amnion cell membrane was detectable within 1 min, and internalization of the ligand occurred within 5 min. No binding of IgG-HRP was observed following treatment of the membrane with 0.25% trypsin for 30 min at room temperature. Incubation of the amnion at 4°C or in the presence of colchicine or cytochalasin D prevented internalization of the IgG-HRP. These experiments demonstrate FcγR on human amnionic epithelial cells that both bind and internalize IgG, thus allowing the amnion to be used as a model system for studying IgG transport.     

Fc IgM receptors on human lymphoblastoid B cell lines.

Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis on the Fc IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having as cell origin due to the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity Fc IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited Fc IgM receptors (17–35%) while the T cell line Molt 4 had no detectable Fc IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation, IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect on EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was no correlation between the expression of Fc IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment to produce IgM or IgG.     

IgE Fc receptor positive T and B lymphocytes in patients with the hyper IgE syndrome.

The percentages of peripheral blood lymphocytes (PBL), bearing Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) were determined in four patients with the hyper IgE syndrome by a rosette assay employing IgE and IgG coated fixed ox erythrocytes. The patients had 8 +/- 3% Fc epsilon R+ and 13 +/- 8% Fc gamma R+ PBL, compared to 1.2 +/- 1% Fc epsilon R+ and 17 +/- 4% Fc gamma R+ PBL for control donors. T cells were isolated by rosetting with neuraminidase treated sheep erythrocytes (EN). Indirect immunofluorescence with Lyt 3 monoclonal antibody (MoAb) to the sheep erythrocyte receptor, followed by rosetting for Fc epsilon R and Fc gamma R showed that the patients' T cells contained less than 0.1% Fc epsilon R+ and 1.4 +/- 0.2% Fc gamma R+ cells; T cells from the control subjects contained less than 0.1% Fc epsilon R+ and 11 +/- 4% Fc gamma R+ cells. The non-T (EN rosette depleted) cells of the patients included 56 +/- 18% sIgM+/sIgD+, 45 +/- 9% Fc epsilon R+ and 35 +/- 27% Fc gamma R+ cells. Indirect immunofluorescence with MoAb to IgM, IgD, and NK cells (antibody B73.1) followed by rosetting for Fc epsilon R and Fc gamma R, indicated that 92 +/- 2% of the Fc epsilon R+ cells and 9 +/- 7% of the Fc gamma R+ cells were B cells (mu+/delta+), while 3 +/- 4% of the Fc epsilon R+ and 30 +/- 23% of the Fc gamma R+ cells were NK cells (B73.1+). Thus, most of the Fc epsilon R+ non-T cells were B cells, and only a small fraction appeared to be NK cells. On the other hand, Fc gamma R+ B cells were outnumbered by Fc gamma R+ NK cells (B73.1+) by three to one. The data indicate that patients with the hyper IgE syndrome have increased numbers of Fc gamma R+ PBL, most of them being B cells, whereas their T cells contain less than 0.1% Fc epsilon R+ cells.     

Characterization of the receptor for IgM present on human B lymphocytes.

The presence of a receptor for the Fc of IgM (muFcR) was demonstrated on the pathological B cells of all of sixteen patients with hairy-cell leukaemia and most, but not all, of twenty-four cases of chronic lymphocytic leukaemia, by a rosette method employing ox erythrocytes sensitized with purified IgM (EAm). This muFcR was also demonstrated on a small population of normal human mononuclear cells from peripheral blood. Pathological B cells with this receptor (Bm) simultaneously expressed a different and distinct receptor for the Fc of IgG, and were detectable without preincubation in medium containing foetal calf serum (FCS). The muFcR on B cells was blocked by Fc5mu and IgM, but not by F(ab')2mu fragments, or by IgG, whether monomeric or aggregated. Monomeric IgM and IgM bound to its antigen blocked much more effectively than pentameric IgM. B cells also possessed surface immunoglobulin and the Ia-like P29, 34 antigen, and an antiserum to this antigen blocked the muFcR. The muFcR on B cells differs in a number of ways from the muFcR reported on T cells, and these differential characteristics are discussed in some detail. The muFcR was rapidly shed and resynthesized when washed Bm cells were maintained in medium not containing FCS and the general importance of this phenomenon in any study of muFcR is considered. It is suggested that Bm cells are memory cells and that the muFcR plays a part in the immune response.     

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.     

Effect of neuraminidase treatment on the expression of Fc IgG receptors on chicken embryonic bursa and thymus cells

Bursa and thymus cells from chicken embryos at different ages were analyzed for Fc IgG receptors by EA-rosette technique and by binding of heat aggregated chicken IgG (agg IgG) in the indirect immunofluorescence test. Neuraminidase treatment resulted in a substantial increase of agg IgG binding cells both in the embryonic bursa and thymus. The binding of agg IgG was shown to be specific for Fc IgG receptors, since IgM and F(ab')2 fragments were not bound to neuraminidase-treated embryonic cells. A far lower percentage of EA-rosette-forming cells than agg IgG binding cells were found both in untreated and neuraminidase-treated bursa and thymus cells. It was concluded that neuraminidase can reveal additional Fc IgG receptor sites mainly for agg IgG on embryonic thymus and bursa cells.     

A monoclonal antibody specific for immunoglobulin A receptor triggers polymorphonuclear neutrophil superoxide release.

An immunoglobulin M (IgM) monoclonal antibody, My43, specific for IgA Fc receptor (Fc alpha R) on human monocytes, bound to human polymorphonuclear neutrophils (PMNs) and inhibited their ability to bind IgA but not IgG. It was observed that the PMN oxidative burst was induced by both polymeric IgA and aggregated IgG, whereas IgM was without effect. The IgG-mediated oxidative burst was inhibited by anti-Fc gamma RII Fab and anti-Fc gamma RIII F(ab')2 but not by My43. Conversely, the IgA-mediated oxidative burst was inhibited by My43 but not by anti-Fc gamma RII or anti-Fc gamma RIII. When anti-Fc receptor monoclonal antibodies (mAbs) were used directly as ligands, it was observed that both anti-Fc gamma RII Fab and anti-Fc gamma RII F(ab')2 promoted the oxidative burst when cross-linked. Moreover, My43, when cross-linked with F(ab')2 antimouse IgM, also triggered the oxidative burst, whereas an IgM anti-CD15 mAb, PM81, did not stimulate function. This demonstrates that IgA receptors on PMNs are function-triggering molecules and that an anti-IgA receptor mAb may be substituted as a ligand.     

The specificity of the Fc receptor on murine lymphocytes for immunoglobulins of the IgG and IgM classes.

It is shown that the Fc receptor for IgG on lymph node cells recognizes IgG molecules of mouse IgG1, IgG2a and IgG2b subclasses. The presence of an intact CH3 domain is required to bind IgG to the Fc receptors. While monomeric IgG molecules are shown to bind to Fc receptors, heat aggregated IgG was found to bind more efficiently. The various forms of IgM molecules investigated show only weak or no binding in comparison. The significance of these results is discussed with relevance to the possible biological function of the Fc receptors on lymphocytes.     

Receptors for immunoglobulin isotype on T and B lymphocytes from untreated patients with chronic lymphocytic leukaemia

Peripheral blood lymphocytes from eighteen untreated patients with chronic lymphocytic leukaemia (CLL) were analysed for the proportions of T and B lymphocytes with receptors for IgM, IgG or IgA. T lymphocytes with Fc receptors for IgM (T mu cells) or IgA (T alpha) cells were found in proportions comparable to those found in the controls. However, the proportion of T lymphocytes with receptors for IgG (T gamma cells) was significantly increased (P < 0.001) resulting in an abnormally low ratio of T mu/T gamma (P < 0.001), when compared with normal controls. The proportion of B cells bearing Fc receptors for IgM, IgG or IgA was determined simultaneously. No significant differences were found between the normal controls and the patients with CLL. In vitro treatment of the purified T and B lymphocyte preparations with human leucocyte interferon, did not alter the proportions of the lymphocytes expressing Fc receptors for various immunoglobulin isotypes. The significance of these findings is discussed.     

Allotypic and isotypic specificities of rabbit IgM: localization to the Fab mu or Fc mu fragments

IgM from trypanosome-infected rabbits was digested with trypsin under different conditions to obtain Fab mu or Fc5 mu fragments suitable for analysis with anti-allotype and anti-isotype antibodies. The Fab mu but not the Fc5 mu fragment was shown to have the n-locus allotypic specificities, n80, n81, n82, n83 and n87, characteristic of the IgM class of immunoglobulins. Thus, the n82 and n83 allotypic specificities, conformationally dependent on the a VH locus for expression, and the n80, n81 and n87 allotypic specificities, independent of the a VH locus for expression, are in either the CH1 or CH2 domain of IgM heavy chains. In addition, two high-affinity mouse monoclonal antibodies (MoAbs) specific for IgM and able to bind IgM in direct-binding radioimmunoassays were produced and characterized. One MoAb (3C1) was specific for an isotypic determinant (epitope) in the Fab mu fragment, presumably in the CH1 or CH2 domain, whereas another MoAb (8C2) was specific for an isotypic epitope in the Fc5 mu fragment, presumably in the CH3 or CH4 domain. The proximity of the n-locus allotypic specificities (CH1 or CH2 domain) to the VH domain is consistent with the finding that some IgM allotypic specificities are expressed only in conjunction with certain a VH locus allotypic specificities.     

Identification of a mycoplasmal protein which binds immunoglobulins nonimmunologically.

Immunoblotted protein samples from several strains of Mycoplasma hominis and from one strain of Mycoplasma arginini each contain a polypeptide of a molecular mass of 95,000 to 105,000 Da which binds immunoglobulin nonimmunologically. Immunoblots from these organisms were probed with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin, conjugated goat immunoglobulin G (IgG) Fab fragments, and conjugated goat IgG Fc fragments. The polypeptide bound the goat anti-rabbit molecules and the Fab fragments but not the Fc fragments. These reactions could be blocked with nonimmune unconjugated goat IgG and unconjugated human IgM. Controls probed with alkaline phosphatase alone did not stain. Binding of the conjugated preparations to whole mycoplasmal cells was dependent on concentrations of both conjugate and cells for the goat anti-rabbit preparation and for Fab. The mycoplasmal polypeptide may be a light-chain-specific reactant.     

Aggregated IgG inhibits the differentiation of human fibrocytes

Fibrocytes are fibroblast-like cells, which appear to participate in wound healing and are present in pathological lesions associated with asthma, pulmonary fibrosis, and scleroderma. Fibrocytes differentiate from CD14+ peripheral blood monocytes, and the presence of serum delays this process dramatically. We previously purified the factor in serum, which inhibits fibrocyte differentiation, and identified it as serum amyloid P (SAP). As SAP binds to Fc receptors for immunoglobulin G (IgG; Fc gammaRs), Fc gammaR activation may be an inhibitory signal for fibrocyte differentiation. Fc gammaR are activated by aggregated IgG, and we find aggregated but not monomeric, human IgG inhibits human fibrocyte differentiation. Monoclonal antibodies that bind to Fc gammaRI (CD64) or Fc gammaRII (CD32) also inhibit fibrocyte differentiation. Aggregated IgG lacking Fc domains or aggregated IgA, IgE, or IgM do not inhibit fibrocyte differentiation. Incubation of monocytes with SAP or aggregated IgG inhibited fibrocyte differentiation. Using inhibitors of protein kinase enzymes, we show that Syk- and Src-related tyrosine kinases participate in the inhibition of fibrocyte differentiation. These observations suggest that fibrocyte differentiation can occur in situations where SAP and aggregated IgG levels are low, such as the resolution phase of inflammation.     

Duck lymphoid organs: their contribution to the ontogeny of IgM and IgY.

The occurrence of mRNAs encoding mu, nu and nu(delta Fc) immunoglobulin heavy chains and lambda light chains in organs of duck embryos from 16 days of incubation and ducklings up to 74 days of age was assessed by Northern hybridization. The mu message was first detected in bursa of Fabricius and spleen at 16 days of incubation and in cervical lymph nodes at 23 days of incubation, but in other organs (bone marrow, buffy coat, Harderian gland, liver) not until 7 17 days after hatching; in general, the appearance of the lambda message paralleled that of mu. Messenger RNAs encoding one or both of the nu isoforms were first detected in cervical lymph nodes at 25 days of incubation, in spleen and bursa in 1-day-old ducklings, in Harderian gland, bone marrow and liver from 10 to 17 days post-hatching and in buffy coat from 46 days. In most organs, the nu(delta Fc) message was detected prior to the nu message and predominated during the experiment; Harderian gland expressed the nu(delta Fc) message exclusively. These results indicate that bursa of Fabricius, spleen and cervical lymph nodes play early roles in the development of B cells and the ontogeny of duck immunoglobulins while other lymphoid organs support the later differentiation of plasma cells, and that IgY and IgY(delta Fc) are probably not simultaneous products of the same plasma cells.