The influence of tissue transglutaminase on the function of Fc receptors

In contrast to FcRII the soluble Fc receptor (FcRI) of human peripheral mononuclear blood cells (PMBC) is shed from PMBC following a 4-37 degrees C temperature shift and inhibits rosette formation of nonshed PMBC with antibody-coated erythrocytes (EA). Purified FcR, could be polymerized by tissue transglutaminase as was revealed by SDS-polyacrylamide gel electrophoresis. Comparing the Sephadex G-150 elution profile of the EA rosette inhibitory capacity of FcRI vs FcRI incubated in the presence of transglutaminase, the latter was found in a higher mol. wt region and could inhibit rosette formation by both FcRI and FcRII. Furthermore, the shedding of FcRI could be prevented by the addition of transglutaminase or Ca2+-ionophore A23187 (which leads to the activation of PMBC transglutaminase) to the cell suspension. The function of FcRII was not affected by either the addition of transglutaminase or Ca2+-ionophore to the cells. The results point to the involvement of transglutaminase in the determination of the functional state of the Fc receptor on the cell surface.     

Effect of lymphokine on the activation and Fc receptor expression of rat macrophages

Lymphocyte supernatant (LS) containing lymphokine (LK) activated rat peritoneal macrophages (PM) and at the same time enhanced their Fc receptor (FcR) activity. The signs of the early activation could not be observed on alveolar macrophages (AM) but the augmented FcR expression occurred under the effect of lymphokine. The LK-induced macrophage activation and the enhancement of erythrocyte—antibody (EA) rosette formation were found to be independent of each other. In the presence of soybean trypsin inhibitor (STI) the crude lymphokine-induced augmented EA rosette formation was only slightly diminished. However, it did not seem likely that the LK components of molecular weight over 15,000 could enhance the number of rosettes under the effect of protease inhibitor.These data indicate that the macrophage proteases play an important role in the generation of EA rosette formation enhancing breakbown product(s) during the macrophage—lymphokine interaction.     

Characterization of the Fc(IgG) receptor on the pluripotential leukaemia cell K-562.

K-562 cells express an Fc receptor that is murine isotype IgG specific. The receptor was defined by rosette formation using sheep erythrocytes sensitized with murine monoclonal haemagglutinin (EA) of known isotype. Optimal rosette formation occurred at 4 degrees C or ambient temperature; however, the number of rosettes formed at ambient temperature decreased after 8 h whereas formed rosettes were stable at 4 degrees C. EA in which IgG2b was the immunoglobulin isotype gave high numbers of rosettes while IgG2a gave lower but significant numbers. Aggregated human IgG inhibited rosette formation of EA(IgG2a) more easily than those of EA(IgG2b), indicating a higher affinity of the Fc receptor for IgG2b.     

Interaction between actomyosin complexes and Fc receptors on human peripheral mononuclear blood cells

The soluble FcRI shed from HPMBC following 4–37°C temperature shift interacts with AM, A-HMM and A-S1, respectively, but do not bind to A, M, HMM or S1. In contrast to the monovalent soluble FcRI the multivalent A-HMM-FcRI and A-S1-FcRI complexes agglutinate EA cells. Due to the interaction of soluble FcR with muscle proteins an alteration in the binding properties of the receptor was observed. FcRI in soluble form inhibits only the EA rosette formation of FcRI⁺ HPMBC in contrast to FcRI-A-HMM and FcRI-A-S1 complexes inhibiting the rosette formation of FcRI⁺ and FcRII⁺ cells as well. Based on these observations one can suppose that depending on their anchoring to cytoskeletal structures the FcRs possess one or two binding sites.     

Sequential changes of the T lymphocytes following ER rosette formation in guinea pigs. I. Expression of Fc and complement receptors

Changes in proportions of the Fc and complement receptor (FcR, CR) positive T lymphocytes from guinea pigs following their interaction with rabbit erythrocytes (ER) were studied using EA and EAC rosette forming assays. Significant increases in the percentages of EA and EAC rosette forming cell (RFC) were observed when thymocytes or lymph node cells were assayed after ER rosette formation. Furthermore, T-enriched fraction by the ER monolayer adherence technique also showed similar or somewhat higher increases in the proportions of both EA and EAC RFC than those of unfractionated cells after contact with ER. The double rosette assay by ER with EA or EAC showed that 50-80% of the Fc and/or complement receptor positive lymphocytes bound rabbit erythrocytes simultaneously. These findings strongly suggest that at least a subset of the guinea pig T cells is altered to express Fc and/or complement receptors on their surfaces following the interaction with ER.     

Immune complexes in Takayasu''s arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.     

Shedding of Fc receptor from mononuclear cells and their ability of binding IgG1 and IgG3 anti-Rh/D antibodies

Fc receptors for IgG1 and IgG3 on peripheral blood lymphocytes and monocytes were studied before and after temperature shift from 4-37 degrees C. The investigations were performed in the EA test using human erythrocytes sensitized with anti-Rh/D/antibodies of IgG1 (EA IgG1) and IgG3 (EA IgG3) subclasses. It occurred that lymphocytes and monocytes were able to bind IgG1 and IgG3 antibodies before and after shedding, however, lower percentage of rosette was observed after temperature shift. This decrease was similar in the EAIgG1 and EAIgG3 tests. The supernatants obtained during shedding occurred to contain active Fc receptors since the inhibition of rosette formation was obtained after the incubation of sensitized erythrocytes with these supernatants. IgG1 as well as IgG3 myeloma proteins inhibited rosette formation in both EAIgG1 and EAIgG3 tests. Our data might suggest that IgG1 and IgG3 anti-D antibodies are able to bind to the same Fc receptor on lymphocytes as well as on monocytes.     

Mouse spleen cells and sensitized sheep red blood cells: An Fc-rosette-forming system allowing the detection of “activated” Fc structures

From a variety of Fc receptor-bearing cell/sensitized red blood cell combinations, mouse spleen cells, and sensitized SRBC were selected as an Fc-specific EA rosette assay system because only this mixture combined a high percentage (about 50%) of rosette-forming cells with complete absence of spontaneous rosettes and showed no influence of complement on the rosette formation. From studies on the minimal structural requirement of IgG both for mediation and inhibition of EA rosettes using IgG and several well-defined fragments, it appeared that both the C_H2 and the C_H3 domain of Fe are needed for optimal interaction with the lymphocyte Fc receptor. Finally, it was demonstrated that the assay system is able to detect “activated” Fc structures (here: heat-aggregated IgG) and to differentiate between varying amounts of such structures.     

Modulation of Fc and C3b receptor expression on guinea-pig macrophages by lymphokines.

The decrease in Fc-receptor-positive cells that occurred during a 6 h incubation of resident and elicited guinea-pig macrophages was partly abrogated when lymphokines were present in the culture. When the same lymphokine preparations were tested on C3b receptor-expression they preferentially sustained the percentage of C3b rosettes formed by resident rather than elicited macrophages. This lymphokine-induced maintenance of Fc and C3b rosettes by cultured macrophages may have been due to an inhibition of receptor release or an increase in receptor synthesis. Supernatants from cultured macrophages contain shed Fc and C3b receptors which inhibit rosette formation by other macrophages. From the demonstration that culture supernatants from both lymphokine-treated and untreated macrophages significantly inhibited Fc and C3b rosette formation by freshly obtained macrophages it seems that the shedding of Fc and C3b receptors is not modified by lymphokines. The maintenance of Fc and C3b rosettes by lymphokines was inhibited by treatment of the macrophages with cycloheximide, suggesting that the lymphokine effect was due to an increase in synthesis de novo of the Fc and C3b receptors. The lymphokine-inducing antigens, BGG and PPD, and control lymphokine preparations were devoid of receptor modifying activity. The reduction in the percentage of Fc rosettes after 6 h culture appears to be due to a loss of Fc receptors for IgG1. Although lymphokines partly inhibited this effect they could not prevent the loss of these receptors following 24 h culture, unlike their action in augmenting the expression of Fc receptors for IgG2. These findings suggest that a selective enhancement of Fc receptor synthesis by lymphokines may modify the functional activities of macrophages.     

Effect of angiotensin II on the Fc receptor activity of rat macrophages.

Angiotensin II (At II) in the concentration range of 10(-5) to 5 x 10(-7) M inhibited active and passive erythrocyte-antibody (EA) rosette formation on monolayers of rat peritoneal macrophages (PM) but stimulated it in the range of 10(-7) -10(08) M. Cytochalasin B (CB) and vinblastin (Vb) inhibited the dose-dependent biphasic effect of At II on the Fc receptor (FcR) expression of macrophages. The hormone acts on the last step of rosette formation, i.e. on the binding of sheep red blood cells (SRBC) but does not interfere with the binding of 125I-Ig to FcR. The influence of At II on macrophage rosette formation can be modified by changes in the density of FcR-Ig complexes on the surface of target cells, by the nature of the immunoglobulin (sub)classes involved in rosette formation, and by the biological properties of rosette-forming corpuscular antigen. Based upon the above results we suggest that At II exerts its effect on the contractile elements associated with the cytoskeleton of the macrophage.     

The functions of endogenous C1q, a subcomponent of the first component of complement, as a receptor on the membrane of macrophages

C1q, the Fc-recognizing subcomponent of the first component of complement is synthesized by peritoneal macrophages. During the secretion phase C1q serves as an Fc-binding protein in the membrane of macrophages. The Fc-mediated rosette formation was inhibited in a dose-dependent manner when macrophages were pretreated with anti-C1q F(ab')2, whereas C3b rosette formation was not affected. Furthermore, preincubation of peritoneal macrophages with anti-C1q F(ab')2 abolished, dose- and time-dependently, the polyanion-mediated stimulation of secretion of lysosomal enzymes. Polyanion-induced enzyme release was prevented after incubation of polyanions with highly purified C1q. The inhibition of Fc receptor activity by polyanions (i.e. dextran sulfate, liquoid, polyvinyl sulfate) is completely reversed upon treatment of these macrophages with protamine. These findings are compatible with the hypothesis that C1q produced by macrophages serves in the macrophage membrane as an endogenous receptor for Fc and polyanionic molecules. Thus, C1q mediates cell-bound biological receptor functions before it is released from these cells and is incorporated into the macromolecular C1 complex.     

Studies on the nature of EA binding by lymphocytes from rheumatoid arthritis patients

Investigation of the nature of the increased erythrocyte-antibody (EA) binding activity of peripheral blood lymphocytes (PBL) from rheumatoid arthritis (RA) patients reported in the preceding paper has revealed that IgG is the active class of antibody in this rosette formation. Some IgM binding also occurs. SRBC sensitized with F(ab)₂ preparations of IgG do not give rosette formation even at high concentrations. EA binding is inhibited by prior incubation of lymphocytes with heat-aggregated human IgG but antigen-antibody complexes did not give significant inhibition.The majority of these rosettes were found to be stable at 4°C and room temperature but labile at 37°C.Enzyme studies with pronase, trypsin, neuraminidase and treatment with sodium azide gave results strongly supporting the conclusion that the increased binding observed is increased Fc-receptor activity. This activity appears not to be a result of Fc binding by cell-bound rheumatoid factor.A range of titres of antibody and of IgG was used to sensitize erythrocytes to form EA and the enhanced EA-rosette formation by PBL from RA patients occurred throughout the range of concentrations of sensitizing antibody. Significantly more EA were bound by individual lymphocytes from RA patients than control subjects. This data suggest that the Fc receptors on RA lymphocytes are more avid for EA than receptors on lymphocytes from healthy people.     

Cellular requirements for the formation of EA rosettes by human monocytes.

The binding of sensitized red cells to Fc receptors in human monocytes was studied by evaluating the effects of various pharmacological reagents and other treatments on EA rosette formation. Cytochalasin B and 2-deoxyglucose inhibited rosette formation in a dose-dependent manner. Sodium azide and incubation at 4 degrees also inhibited rosette formation, while at 37 degrees increased numbers of RBCs bound to the monocytes. The microtubular poisons, vinblastine and colchicine at high concentrations resulted in decreased adherence of monocytes and inhibition of rosette formation, while at low concentrations of colchicine, enhanced rosette formation was sometimes observed. Contrary to the effects on rosette formation, binding of [125I] IgG to monocyte monolayers was not altered by treatment of the monocytes with drugs. Magnesium ions were required to promote monocyte adherence, but both magnesium and calcium were needed for the best rosette formation. We conclude that the formation of EA rosettes is dependent not merely on binding of IgG to the Fc receptor but requires metabolically active monocytes, an intact cytostructure and suitable environmental conditions (temperature and cation concentration).     

Re-examination of the EA rosette assay (Ripley) for Fc receptor leucocytes.

Numerous investigations has utilized rosette formation with Ripley antibody-coated human erythrocytes (EA) to identify or deplete Fc receptor-bearing K lymphocytes in whole mononuclear cell preparations. This study examines the interaction between Ripley EA and purified preparations of human lymphocytes, monocytes and neutrophils and demonstrates that this technique is not specific for K lymphocytes. Indeed, 100% of blood monocytes rosette these EA target cells. Moreover, data from both rosetting studies and antibody-dependent cytotoxicity (ADCC) reactions suggest that the avidity of Ripley EA is actually greater for monocytes than for lymphocytes. In contrast to previous reports, 100% human neutrophils were found to possess Fc receptors, as determined by their ability to rosette Ripley EA. Thus, the extent of rosetting and ADCC by all three Fc receptor-bearing leucocytes depends significantly on the degree of antibody sensitization with neutrophils requiring the greatest, and monocytes the least, amount of target-bound antibody for Fc receptor-mediated interaction.     

Expression of Fc receptors is suppressed in alveolar macrophages from patients with sarcoidosis.

To study the expression of Fc receptors in human alveolar macrophages (AM), the cells were collected from 12 healthy controls and 22 patients with sarcoidosis and the activity involved in binding to 125I-soluble immune complexes (IC) was investigated. The binding of 125I-IC was significantly suppressed in cells from the patients. A Scatchard plot analysis revealed that this suppression was due to a reduction in the number of Fc receptors on the surface membrane and not the result of a decrease in the binding affinity of the receptors. Rosette formation was observed by incubating AM with antibody-coated ox red blood cells. The presence of rosette forming cells was about 70% in the AM from healthy controls, while, a rate of only 50% was seen in those with sarcoidosis. These results indicate that the decrease of the 125I-IC binding in the AM from patients with sarcoidosis is to some extent the result of an increase in the population of the Fc receptor negative macrophages.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

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.     

FcR blocking activity in serum of actively enhanced rat renal allograft recipients due to IgG anti-class II MHC alloantibody

In some rat strain combinations, pre-operative donor-specific blood transfusion produces long-term renal allograft survival, although the underlying mechanisms are unclear. This study has examined whether Fc receptor (FcR)-blocking activity could be detected in the serum of unmodified PVG strain recipients bearing a rejecting renal allograft and in recipients bearing an actively enhanced graft following pre-operative blood transfusion. Serum harvested on Day 5 from actively enhanced PVG recipients of DA rat renal allografts was shown to specifically inhibit erythrocyte-antibody (EA) rosette formation with donor strain, but not third-party, splenocytes, while the levels of EA rosette inhibition (EAI) in Day 5 serum from rejecting rats remained markedly lower. This FcR-blocking activity was present in enhanced serum fractions, prepared by discontinuous density gradient centrifugation, which corresponded to the 7 S peak. Purified IgG prepared from enhanced serum was also found to inhibit EA rosette formation with donor splenocytes, and absorption of the IgG preparations with donor strain erythrocytes failed to abrogate EA rosette inhibition. Further experiments, in which absorbed IgG from enhanced animals was tested for FcR blocking activity against splenocytes of defined major histocompatability complex (MHC) subregion specificities, established that FcR-blocking activity was mediated by IgG alloantibodies directed against donor MHC class II antigens. Whether the presence of such antibodies early after transplantation contributes to the beneficial effect of blood transfusion on graft survival remains to be determined.     

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.     

Quantitative contributions of IgG,IgM and C3 to erythrophagocytosisand rosette formation by peritoneal macrophages,and anti-opsoninactivity of dextran sulfate 500

In vitro phagocytosis by guinea pig peritoneal macrophages of immune complexes (EA) was shown to be dependent on IgG antibody in a dosedependent fashion. C3b enhanced phagocytosis of EA at limited IgG antibody concentrations only. When IgM antibody was used for sensitzation ofsheep red blood cells (SRBC), phagocytosis and rosette formation did notoccur in the absence of bound C3. The polyanion, dextran sulfate 500 (DS), was shown to depress both rosette formation and phagocytosis of EA_IgG, EA_IgGC1423 and EA_IgMC1423, as well as immune adherence of human group 0 erythrocytes and hemolytic activity of C3. This effect of DS was seen only when it was actually present in the incubation medium.