anti-Fc Antibody Research Articles

Physical and functional association of p56lck with Fc gamma RIIIA (CD16) in natural killer cells.

The transmembrane receptor for immunoglobulin G immune complexes on natural killer (NK) cells and macrophages, Fc gamma RIIIA (CD16), mediates cellular activation through a tyrosine kinase-dependent pathway. We show that Fc gamma RIII crosslinking results in activation of the src-related kinase p56lck in NK cells and demonstrate a physical association of p56lck with Fc gamma RIIIA in immunoprecipitates from NK cells obtained using anti-Fc gamma RIII antibodies or immune complexes. Our studies show that the zeta chain, the signal transducing subunit of Fc gamma RIIIA and of T cell receptor, associates with p56lck and, in NK cells, is a substrate for this kinase. Such direct association of p56lck with the zeta subunit as confirmed by demonstrating the interaction in heterologous cells transfected with cDNA expressing p56lck and zeta. Our findings demonstrate both functional and physical association of p56lck with Fc gamma RIIIA, through direct interaction of the kinase with the zeta and/or the gamma signal transducer subunits of the receptor. These data suggest a possible mechanism by which activation via Fc gamma RIIIA occurs.

Coexpression of Fc gamma receptor IIIA and interleukin-2 receptor beta chain by a subset of human CD3+/CD8+/CD11b+ lymphocytes.

In this study we identify and characterize a subset of human peripheral blood T cells, present in all individuals, that has features previously described for T cells either separately or in special circumstances. These cells are found in purified suspensions of resting peripheral blood lymphocytes within the CD8+ T lymphocytes, express alpha beta T cell receptor (TCR), and can be identified and isolated because of high-density expression of surface CD11b (TCR alpha beta +/CD3+/CD8+/CD11b+ cells). They coexpress constitutively the IL-2 receptor beta chain, Fc gamma RIIIA, and CD56. Although they do not mediate spontaneous cytotoxicity, CD3+/CD8+/CD11b+ cells have cytotoxic potential, demonstrated in redirected cytotoxicity assays with P815 target cells in the presence of anti-Fc gamma RIII (CD16) or anti-CD3 monoclonal antibodies. Stimulation of CD3+/CD8+/CD11b+ cells with rIL-2 induces proliferation, cytotoxicity against NK-sensitive and NK-resistant target cells, and expression of surface activation antigens, including IL-2 receptor alpha chain (CD25). CD3+/CD8+/CD16+/CD56+ cell clones with cytotoxic functions including those mediated by engagement of surface CD16 were obtained by limiting-dilution cloning of purified CD3+/CD8+/CD11b+ cells in the presence of rIL-2 and autologous feeder cells. Our data support the hypothesis that the CD3+/CD8+/CD11b+/CD16+ cells represent a discrete peripheral blood lymphocyte subset that could be the physiological counterpart of that expanded in several pathological conditions and in large granular lymphocyte lymphocytosis.

Endomyocardial biopsies after heart transplantation. The presence of markers indicative of activation.

A series of 104 endomyocardial biopsies (EMB) from patients after heart transplantation was evaluated for the presence of immunological markers on graft component and infiltrating cells. This included markers for cells expressing alpha beta-T-cell receptors and gamma delta-T-cell receptors, and cytotoxic T cells with granules bearing the serine esterase Granzyme B; the presence of activation markers identified by CD25 (interleukin 2 receptor), CD30, CD69 (activation inducer molecule), CDw70; macrophages using antibody CD14 (WT14), and cells with Fc gamma-receptors type III (CD16). Almost all cells in T-cell infiltrates expressed the alpha beta-T cell receptor. Cells bearing the gamma delta-T cell receptor were scarcely found. The analysis with respect to the histopathologic diagnosis for rejection showed an absence of significance for T cell subsets, Granzyme B-positive cells, and activation markers except CD25. The numbers of macrophages labeled by CD14 and cells expressing Fc gamma RIII showed a significant relation to histopathology of rejection. Apart from leukocytes, also endothelium in EMB with rejection was labeled by the two anti-Fc gamma RIII antibodies used. In addition, in a small series of biopsies investigated, Fc gamma RI- and Fc gamma RII-positive cells were increased in EMB with rejection, and endothelium was labeled by Fc gamma RII antibodies. A cluster analysis on the basis of scores for CD25, CD14, and anti-Fc gamma RIII revealed three main clusters, one cluster comprising biopsies without abnormalities, one cluster containing EMB with the histopathology of rejection and high scores in immunophenotyping for lymphocytes and macrophages, and one cluster in between. The present data emphasize the importance of macrophage assessment in evaluating pathologic processes during rejection of heart allografts and diagnosing rejection.

Anti-inflammatory effects of nedocromil sodium: inhibition of alveolar macrophage function.

The IgE-dependent activation of mononuclear phagocytic cells through their capacity to express low affinity IgE receptors (Fc epsilon RII) has been proposed as a mechanism for the development of airways inflammation in allergic asthma. This Fc epsilon RII expression leads to the IgE-dependent production of the potent pro-inflammatory cytokines IL-1 beta and TNF-alpha. Expression by monocytes of Fc epsilon RII is regulated by several cytokines including interleukin-4, gamma- and alpha-interferons, and granulocyte-macrophage and macrophage colony stimulating factors. An anti-inflammatory effect of nedocromil on monocytes has been proposed as a possible mechanism for its anti-asthma activity. We therefore investigated the capacity of nedocromil to modulate mononuclear phagocyte Fc epsilon RII expression and cytokine production. We used an anti-Fc epsilon RII antibody and flow cytometric analysis to assess the capacity of nedocromil to modulate cytokine-induced Fc epsilon RII expression in normals and asthmatics. Monocytes, THP-1 monocyte leukaemia cells, and alveolar macrophages were exposed to varying concentrations of these cytokines for 48 hr at 37 degrees C with or without the additional presence of nedocromil (1-10 microM) and the per cent of monocytes expressing Fc epsilon RII was determined. No changes in Fc epsilon RII expression were observed. Subsequently, we investigated the capacity of nedocromil to affect the capacity of IgE plus anti-IgE complexes, allergen, and LPS (16 hr/37 degrees C) to stimulate IL-1 beta and IL-6 production. No changes were observed when nedocromil was applied concomitant with the stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells that present both specific ligand and costimulatory activity are the most efficient inducers of clonal expansion of normal CD4 T cells.

Clonal expansion of naive CD4 T cells is a necessary step in most adaptive immune responses. Two distinct signals are required for clonal expansion to occur, ligation of T-cell receptors by an antigenic peptide bound to self major histocompatibility complex-encoded class II molecules (signal 1) and a costimulatory signal derived from an antigen-presenting cell (signal 2). To study whether these two signals need to be delivered by a single cell in order to induce clonal expansion of normal CD4 T cells, we have used anti-CD3 bound to Fc receptors as a ligand for the T-cell receptor to deliver signal 1 to all CD4T cells, and we have inactivated signal 2 with a newly generated monoclonal antibody or by using Fc receptor-positive cells that lack the costimulator. Costimulation was delivered by cells whose Fc receptors were blocked with anti-Fc receptor monoclonal antibody. Our results indicate that delivery of ligand and costimulator on one cell is at least 30-fold more efficient than separate delivery. No significant clonal expansion was observed when signals 1 and 2 were delivered by different cells. We have also carried out experiments using fibroblast transfectants that can deliver either or both of these two signals. These studies show that separate delivery of these two signals is at least 80-fold less efficient than their combined delivery by one cell. These findings may explain why tissues can express autoantigens and contain active antigen-presenting cells without inducing autoimmunity.

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.

Granulocyte Fc gamma receptor recognition of cell bound and aggregated IgG: effect of gamma-interferon.

Granulocyte Fc gamma receptors are important components in the recognition of IgG-coated cells and immune complexes. Two proteins have been identified on resting human granulocytes which function as Fc gamma receptors, Fc gamma RII (CD32) and Fc gamma RIII (CD16). A third protein, Fc gamma RI (CD64), is not constitutively expressed on resting granulocytes, but can be induced by activation with gamma-interferon. We examined the role of these three Fc gamma receptors on human granulocytes in the binding of both IgG-sensitized erythrocytes and soluble oligomeric IgG. In these studies we employed anti-Fc gamma receptor antibodies which complete for the Fc gamma RII and Fc gamma RIII ligand binding sites. Preincubation of granulocytes with saturating concentrations of high-affinity anti-Fc gamma RII monoclonal antibody did not alter the recognition of IgG sensitized human cells by granulocytes. Furthermore, ligand binding studies demonstrated that anti-Fc gamma RII antibody altered neither the number nor the affinity of granulocyte binding sites for human trimeric IgG. In contrast, Fab anti-Fc gamma RIII inhibited the binding of both IgG (anti-D) sensitized human RBCs and IgG sensitized sheep RBCs. Similarly, a reduction in the expression of Fc gamma RIII by treatment with phosphatidyl-inositol specific phospholipase C reduced PMN recognition of IgG-sensitized cells. Also, anti-Fc gamma RIII decreased the number of granulocyte binding sites for human IgG trimer without a change in receptor affinity. Fc gamma RI, which was induced by gamma-IFN, increased granulocyte recognition of both IgG sensitized RBCs and IgG trimer. These data suggest that Fc gamma RIII is the primary Fc gamma receptor on granulocytes which recognizes IgG sensitized RBCs and low molecular weight complexes of IgG. With gamma-interferon activated granulocytes, Fc gamma RI appears to enhance this recognition process.

Functional consequences of monocyte/macrophage infection by HIV1

Monocyte/macrophage infection by human immunodeficiency virus type 1 (HIV1) was studied for its effects on the production of tumour necrosis factor alpha (TNF alpha) and the expression of the manganese superoxide dismutase (MnSOD) gene. For this purpose, human peripheral blood monocytes were obtained from healthy HIV1-seronegative donors by centrifugal elutriation and infected with either the HIV1/LAV1 strain or with the primary HIV1/DAS isolate. The results showed that (1) HIV1/LAV1-infected macrophages did not produce any biologically detectable TNF alpha during the few hours following lentiviral infection, despite rises in the TNF alpha mRNA level; (2) MnSOD gene transcription in the macrophages increased, as measured 2 and 4 h after infection; (3) the level of the MnSOD gene expression declined during the late phases of lentiviral infection, but TNF alpha synthesis and gene expression rose; and (4) bispecific antibody comprised of anti-Fc gamma RI (anti-CD64) and anti-gp41 monoclonal antibodies inhibited the in vitro infection of monocyte-derived macrophages by HIV1/DAS.

MHV S peplomer protein expressed by a recombinant vaccinia virus vector exhibits IgG Fc-receptor activity.

We have previously shown that cells infected with mouse hepatitis virus (MHV) bind rabbit, mouse, and rat IgG by the Fc portion of the IgG molecule. This Fc-binding activity appeared to be mediated by the MHV S protein. S protein could also be precipitated from MHV-infected cells by a monoclonal antibody directed against the murine Fc γ receptor (FcγR). To prove definitively that the S protein mediates Fc-binding activity, we have expressed the MHV S protein utilizing recombinant vaccinia viruses. The anti-FcγR monoclonal antibody, 2.4G2, precipitated recombinant S protein in cells of murine, human, and rabbit origin. Since the anti-Fc receptor monoclonal antibody does not react with human and rabbit Fc receptors these results demonstrate that the epitope recognized by this antibody is carried on the MHV S protein and is not murine in origin. Examination of various MHV isolates and escape mutants failed to identify the precise sequences in S responsible for the molecular mimicry of the murine FcγR. These data are consistent with the hypothesis that a previously identified region of similarity between the S protein and the FcγR mediates this activity. The Fc binding activity of S was expressed on the cell surface, since MHV-JHM-infected cells, but not uninfected cells, formed rosettes with anti-sheep red blood cell (SRBC) antibody-coated SRBC. The anti-FcγR monoclonal antibody neutralized MHV-JHM and inhibited syncytium formation induced by the MHV S protein.

Highly active enzyme preparations immobilized via matrix-conjugated anti-Fc antibodies

A novel method for enzyme immobilization to achieve highly active enzyme preparations is described. The enzyme is first reacted in solution with a specific monoclonal antibody (mAb) which does not interfere with its enzymic activity and which possesses a high affinity towards the enzyme, The immunocomplex is then reacted with immobilized anti-Fc antibodies which are coupled to the matrix by methods that preserve maximum immunological activity. Horseradish peroxidase (HRP) immobilization on epoxy-activated beads (CB6200) was chosen as a model system for this approach. Coupling of HRP via its mAbs to anti-Fc antibodies immobilized yielded the best results, corresponding to binding of 325 ng of fully active enzyme per bead. Binding of the enzyme to anti-HRP mAb directly immobilized on the beads was considerably lower.

Regulation of Fc receptors for IgG on cultured rat mesangial cells

Localization of immune complexes (IC) to the mesangium may contribute to glomerular disease. Recently, we and others characterized Fc receptors (Fc gamma R) for IgG-IC on mesangial cells (MC). This study examines regulation of Fc gamma R by cAMP, interferon gamma (IFN-gamma) and by macrophage colony stimulating factor (CSF-1), an agent controlling Fc gamma R in leukocytes and generated by MC. Preincubation of MC (3rd to 6th subculture) with CSF-1, db-cAMP or IFN-gamma for two to 48 hours resulted in a time dependent (maximal 24 to 48 hrs) two- to threefold increase of specific [125I] IgG-IC binding to MC at 4 degrees C. The increase of Fc receptors induced by CSF-1, db-cAMP or IFN-gamma was confirmed by enhanced binding of the monoclonal anti-Fc receptor antibody 2.4G2 to MC. Uptake of IgG-IC at 37 degrees C was also enhanced in MC pretreated with CSF-1, db-cAMP or IFN-gamma. This indicates that the increase in binding for IgG-IC is associated with functional receptors. Immunoprecipitation of extracts of [125I] surface labeled MC with polyclonal anti-Fc gamma R-Ab followed by SDS-PAGE also showed increased amounts of [125I] Fc gamma R protein after pretreatment with CSF-1, db-cAMP or IFN-gamma. The pretreatment also enhanced staining of MC with anti-Fc gamma R-Ab by immunogold-silver enhancement technique. We conclude that MC express Fc gamma R for IgG-IC that can be regulated by CSF-1, cAMP and IFN-gamma, factors that may be important in glomerular immune injury.

CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.

Augmentation of lymphokine-activated killer cell cytotoxicity by monoclonal antibodies against human small cell lung carcinoma : Tong AW, Lee JC, Wang R-M, Ordonez G, Stone MJ. Cancer Immunology Research Unit, Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX 75246. Cancer Res 1989;49:4103-8

This study examined the lymphokine-activated killer (LAK) cell cytotoxicity on monoclonal antibody (MoAb)-bound tumor cells from the human small cell lung carcinoma cell lines H69 and H128. LAK cells were generated from normal peripheral blood mononuclear cells by incubation with interleukin 2 for 3 or more days. Cells from the LAK culture were cytotoxic to natural killer-sensitive (K562, 84% cytotoxicity) and natural killer-resistant (Daudi, 85%; H69 and H128, 69% and 97%, respectively) cell lines, and to freshly excised human lung (49%) and breast (57%) tumors. LAK cytotoxicity to H69 or H128 cells was significantly augmented by target cell preincubation with the small cell lung carcinoma-reactive MoAbs 1096 (increases of up to 271%) or 5023 (up to 223%). SCLC 5023 or 1096 did not enhance LAK cytotoxicity to Daudi cells of lymphoblastoid origin. Pretreatment of LAK cells with an anti-Fc receptor antibody blocked MoAb augmentation by 1096 or 5023 (but not LAK cytotoxicity), suggesting that LAK-MoAb interaction may be mediated by Fc binding. LAK activity coincided with emergence of a large cell [interleukin 2-stimulated large mononuclear leukocyte (LML)] subset expressing the CD16 and NKH-1 surface determinants. Serial immunophenotyping of the LAK cell culture harvested at Days 3, 5, and 7 indicated that the level of LAK cytotoxicity, with or without MoAb augmentation, correlated with frequency of NKH-1-reactive LMLs. These observations support the hypothesis that LAK cytotoxicity is mediated by a NKH-1-reactive LML subpopulation. Antitumor cytotoxicity may be augmented by tumor-reactive MoAbs through Fc binding to this LML subset.

Gamma interferon augments functional and phenotypic characteristics of vitamin D3-induced monocytoid differentiation in the U937 human leukaemic cell line

We studied the effect of gamma interferon on 1,25-dihydroxyvitamin D3 (1,25(OH) 2D3)-induced differentiation of the human leukaemic cell line U937. Both phenotypic and functional aspects of 1,25(OH) 2D3-induced differentiation were significantly augmented by gamma interferon (IFN). Gamma interferon had little effect alone but increased butyrate esterase staining and expression of CD14 antigen and the 40 kD Fc receptor (FcRII) in response to 1,25(OH) 2D3. Ability to phagocytose IgG-opsonized bacteria, and superoxide burst in response to both IgG-opsonized bacteria and phorbol ester, was greater after incubation with both IFN and 1,25(OH) 2D3 than with either agent alone. The degree of functional activation of cells showed a positive correlation with FcRII expression. In addition, IgG-induced generation of superoxide by differentiated cells was considerably reduced by pre-incubation with the anti-Fc receptor antibody IV3. We conclude that gamma interferon augments 1,25(OH) 2D3-induced differentiation and functional activation of the U937 cell line. Increased functional activation may, in part, be due to up-regulation of surface FcR11.

Enhancement of the activity of thyroid-stimulating antibodies by anti-human IgG antibodies in vitro.

In an attempt to determine whether or not anti-human IgG antibodies could influence the activity of thyroid-stimulating antibodies (TSAb), we investigated the effects of anti-human Fc antibody (anti-Fc antibody) and IgG from rheumatoid arthritis patients (RA-IgG) on TSAb activity using FRTL-5 cells. It was found that these anti-human IgG antibodies enhanced the TSAb activity in vitro. FRTL-5 cells were first incubated with Graves' disease IgG for 30 min at 37 degrees C, then washed and incubated in Hanks' balanced salt solution with anti-human IgG antibodies for 60 min at 4 degrees C, and then for a further 120 min at 37 degrees C. The level of cAMP accumulated in the medium was determined by RIA. Anti-Fc antibody significantly augmented the cAMP formation stimulated by 16 out of 24 Graves' IgGs, whereas anti-F(ab')2 antibody did not potentiate cAMP accumulation. Three of five RA-IgGs, which are usually defined as specific antibodies for the Fc fragment of human IgG, mimicked these stimulatory effects. Protein A also potentiated the cAMP formation stimulated by Graves' IgGs. Furthermore, there was a significant correlation between the TSAb titres of these Graves' IgGs and the potentiating effects of anti-Fc antibody (r = 0.495, P less than 0.05, n = 21). These results suggest that the interaction of TSH receptor antibody with anti-human IgG antibodies might modulate thyroid function in Graves' disease.

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3).

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.

Inhibition of IgG-mediated immunosuppression by a monoclonal anti-Fc receptor antibody.

IgG-antibodies can efficiently suppress the antibody response against their specific antigen. The suppressive capacity is dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions are necessary for the induction of immunosuppression. The monoclonal antibody 24G2, which binds to murine Fc receptors on macrophages and B cells, was used in the present study to address the question of whether IgG-mediated suppression is in fact dependent on the binding of IgG antibodies to Fc receptors on splenocytes. In a murine in vitro immunization system, 24G2 is shown to reverse efficiently the suppression of the sheep erythrocyte-specific antibody response. The role of B cells or macrophages as effector cells is discussed.

The different roles of two distinct Fc gamma receptors on guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes.

The functional roles of two distinct types of Fc gamma receptors (Fc gamma 1/gamma 2R specific for both IgG1 and IgG2, and Fc gamma 2R specific for IgG2 alone) on the surface of guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes (EA) were investigated by the use of two Fab's of monoclonal anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibodies. The binding and subsequent ingestion of IgG1 antibody-sensitized erythrocytes (EA gamma 1) by macrophages were completely inhibited by anti-Fc gamma 1/gamma 2R Fab', indicating that the reactions are mediated only by Fc gamma 1/gamma 2R. On the other hand, the binding and subsequent ingestion of IgG2 antibody-sensitized erythrocytes (EA gamma 2) were substantially inhibited by anti-Fc gamma 2R Fab', but not by anti-Fc gamma 1/gamma 2R Fab'. The inhibitory activities of anti-Fc gamma 2R Fab' were dependent upon the amount of IgG2 antibody bound on erythrocytes; increasing the amount of bound IgG2 antibody from 0.15 to 0.91 micrograms/2 X 10(8) erythrocytes resulted in a decrease in the inhibition of binding of EA gamma 2 by anti-Fc gamma 2R Fab' from 50 to 0%, and also a decrease in the inhibition of ingestion of EA gamma 2 from 100 to 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow Cytometric Analysis of I-J Expression on Murine Bone Marrow-Derived Macrophages

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)