Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes

Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease.     

Functional polymorphisms of Fc receptors in human monocyte-mediated cytotoxicity towards erythrocytes induced by murine isotype switch variants

Human monocytes can be triggered to antibody-dependent cell-mediated cytotoxicity (ADCC) by murine antibodies. In this study, a series of H chain isotype switch variant antibodies against glycophorin A on human RBC was used to study the influence of isotype on the induction of ADCC. Furthermore, it was studied whether the functional heterogeneity in responsiveness to IgG1 and IgG2b anti-CD3 antibodies, as found among different donors in T cell proliferation induction experiments, was reflected in ADCC. Whereas IgG2a induced ADCC to the same extent in monocytes from all donors, IgG1 showed a heterogeneous pattern, which corresponded to the heterogeneity in T cell proliferation studies. IgG1 anti-CD3 nonresponder monocytes could, however, be induced to ADCC by IgG1 antiglycophorin, although they needed a much higher antibody density on the target cell than did responder monocytes. IgG2b antiglycophorin at a high density induced ADCC in monocytes from all donors irrespective of responsiveness to IgG2b anti-CD3, whereas IgE and IgA antiglycophorin were barely effective in monocytes from all donors. By specific blocking with mAb, the FcR that were involved in ADCC directed by the various isotypes were characterized. ADCC by IgG2a was predominantly mediated by FcRI and could be specifically enhanced by culturing the monocytes with rIFN-gamma. ADCC by IgG1 was predominantly mediated through FcRII in both anti-CD3 responder and nonresponder monocytes. FcRII was also involved in ADCC by IgG2b, although other receptors seemed to contribute significantly to ADCC. When FcRII or FcRI were blocked, IgG1 and IgG2a could also functionally interact with FcRI and FcRII, respectively, provided that the target cells were sensitized to a high degree. These findings indicate that FcRI and both forms of FcRII can mediate cytotoxicity and that the specificity of human FcR for murine isotypes is relative.     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.     

Direct demonstration of binding of anti-Leu 4 antibody to the 40 kDa Fc receptor on monocytes as a prerequisite for anti-Leu 4-induced T cell mitogenesis

It has previously been demonstrated that about 30% of healthy Caucasian subjects are "nonresponders" in assays of the mitogenic activity of monoclonal mouse IgG1 (mIgG1) anti-CD3 antibodies (e.g., anti-Leu 4 and UCHT-1), and that this unresponsiveness is due to lack of monocyte helper function. In an immunofluorescence assay with fluorescence-activated cell sorter analysis, we studied the binding of phycoerythrin-conjugated anti-Leu 4 to monocytes from responders and nonresponders. Interaction was observed with monocytes from responders only, and was blocked by a murine monoclonal antibody (IV.3) directed to an epitope on the 40-kDa low affinity Fc receptor (FcRII). This indicates that the interaction represents binding of the Fc part of phycoerythrin-conjugated anti-Leu 4 to FcRII on responder monocytes. Indirect immunofluorescence with antibody IV.3 demonstrated, however, that monocytes from both responders and nonresponders express similar levels of FcRII. Thus, nonresponder monocytes apparently express a variant FcRII which is unable to bind the Fc part of mIgG1 antibodies. The anti-FcRII antibody completely blocked anti-Leu 4-induced (but not OKT3 (mIgG2a)-induced) T cell proliferation in cultures of peripheral blood mononuclear cells from responders. The results provide direct evidence that monocytes from anti-Leu 4 responders, but not monocytes from anti-Leu 4 non-responders, are able to bind the Fc part of mIgG1 to FcRII, and that this interaction with FcRII is essential for the mitogenic activity of mIgG1 anti-CD3 antibodies.     

Human epidermal Langerhans cells express only the 40-kilodalton Fc gamma receptor (FcRII)

In man, three distinct classes of receptors for the Fc fragments of IgG (FcRI, II, III) have been defined. The FcRI has a Mr of about 72 kDa, binds human IgG-coated E, and is recognized by mAb such as 32. The FcRII has a Mr of 40 kDa, binds murine IgG1-coated E, and reacts with the mAb IV.3 and CIKM5, which recognize CDw32 moieties. Lastly, the FcRIII has a Mr of 50 to 70 kDa and is recognized by anti-CD16 mAb. In the present study we have shown that i) only murine IgG1-coated E form rosettes with 49 +/- 1.5% (mean +/- SEM, n = 9) of CD1a+ epidermal cells (EC) (which represent Langerhans and indeterminate cells) and that ii) the mAb anti-FcRII CIKM5 prevents this rosette formation. Among the mAb reacting with the three different types of FcR, only those recognizing FcRII i) stain about 55 +/- 1.5% (mean +/- SEM, n = 9) of the CD1a+ EC and ii) reveal the presence of dendritic cells in epidermal sheets obtained by suction blister. Under the electron microscope i) apparently all the cells forming rosettes or reacting with the gold-labeled anti-FcRII mAb (CIKM5 or the F(ab) fragment of IV.3) contained Birbeck granules and ii) the gold-labeled mAb were internalized in unfixed Langerhans cells by receptor-mediated endocytosis and accumulated in lysosomes. Labeling by the anti-FcRII mAb of the CD1a+ cells in suspension disappears after 48 h of culture. All these observations strongly suggest that CD1a+ EC express only the FcRII. This conclusion was confirmed by immunoprecipitation experiments, whereas no specific immunoprecipitate was noted with the anti-FcRI or anti-FcRIII mAb, the anti-FcRII mAb immunoprecipitated a protein of Mr 40 kDa.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

Two distinct classes of IgG Fc receptors on a human monocyte line (U937) defined by differences in binding of murine IgG subclasses at low ionic strength

We have defined two distinct classes of IgG Fc receptors (FcR) on cells of a human monocytic line (U937) by analyzing the direct binding of murine IgG subclasses in medium of low ionic strength. Four lines of evidence support this contention. The binding of aggregated murine IgG2b (AggmIgG2b) to U937 and Daudi cells was enhanced at low ionic strength, whereas monomeric murine IgG2a (mIgG2a) did not bind to Daudi cells and its high affinity binding to U937 cells was unaffected by changes in ionic strength. Double reciprocal inhibition experiments with U937 cells indicated that the binding of both ligands was inhibited 30 to 135 times more efficiently by the homologous ligand than by the heterologous one. That is, the binding of 125I-AggmIgG2b was inhibited 50% by 3.5 micrograms/ml of AggmIgG2b and 100 micrograms/ml of mIgG2a. Similarly, the binding of 125I-mIgG2a was inhibited 50% by 2.5 micrograms/ml of mIgG2a and only 44% by 243 micrograms/ml of AggmIgG2b. A monoclonal antibody of the IgG2b subclass raised against an IgG FcR on K562 cells inhibited binding to U937 cells of AggmIgG2b but not of mIgG2a. Trypsinization of U937 cells abrogated by 32% the binding of mIgG2a but did not affect the binding of AggmIgG2b. Human IgG inhibited binding of both AggmIgG2b and mIgG2a to U937 cells. We propose that the newly recognized FcR that binds AggmIgG2b is the human homologue of the murine macrophage IgG2b/1 FcR (FcRII), and that the previously described 72,000 dalton high-affinity FcR on U937 cells that binds mIgG2a is the human equivalent of the murine macrophage IgG2a FcR (FcRI).     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.     

Unusual expression of IgG Fc receptors on peripheral granulocytes from patients with leukocyte adhesion deficiency (CD11/CD18 deficiency)

Leukocyte adhesion deficiency (LAD) is a hereditary disease characterized by defective expression of leukocyte adhesion glycoproteins; lymphocyte function-associated Ag-1 (CD11a/CD18), CR3 (CD11b/CD18) and p150,95 (CD11c/CD18). Granulocytes, monocytes, and lymphocytes of patients with LAD show profoundly defective in vivo and in vitro adherence-dependent immune functions. We investigated the expression of FcR for IgG on polymorphonuclear cells (PMN) and monocytes from patients with LAD, and their luminol- and lucigenin-enhanced chemiluminescence production in response to SRBC sensitized with murine (m) IgG2a and IgG2b. Unstimulated patient PMN showed an enhanced chemiluminescence in response to mIgG2a-SRBC and an increased phagocytosis of mIgG2a-SRBC. The up-regulated functions were inhibited by monomeric human IgG in a dose-dependent manner, which was attributed to an increase in expression of FcRI on patient PMN, as shown by flow cytometry using monoclonal antibody, 32.2, specific for human FcRI. In contrast, neither the expression of FcR on the monocytes of LAD patients nor their FcR-mediated functions were different from those of controls.     

Phagocytosis of serum-opsonized zymosan down-regulates the expression of CR3 and FcRI in the membrane of human monocytes

The effect of iC3b receptor (CR3)-mediated phagocytosis on the expression of CR (C3b receptor, CR3) and IgG FcR (FcRI, FcRII) has been investigated by using serum-opsonized zymosan as a multivalent ligand for CR3. Sixteen hours after a short (1-h) pretreatment of human monocyte monolayers with zymosan opsonized with human AB serum (250 micrograms/ml), CR3 expression (as assessed by flow cytometric analysis with mAb Mo1) was significantly reduced by 59 +/- 3% (mean +/- SEM, n = 15, p less than 0.001). Concomitant with CR3 down modulation, FcR binding activity (as assessed by binding of IgG-coated E) was also found to be decreased to 41 +/- 4% of control (n = 7, p less than 0.001). Reduced FcR function was paralleled by a decrease in the expression of FcRI (as assessed with mAb 32.2). This FcRI modulation was not caused by zymosan-bound IgG because zymosan opsonized with agammaglobulinemic serum equally down regulated CR3 and FcRI expression. Pretreatment with zymosan opsonized with human AB serum, however, did not change the expression of other IgG and C-binding sites such as FcRII (examined with mAb IV.3 and 2E1) and CR1 (assessed with mAb 57F) as well as of unrelated cell membrane structures (beta 2m, MHC class II). In contrast, co-modulation for FcR function and CR3 expression induced by polymeric IgG is accompanied by a decreased expression of FcRII. These data indicate that interaction of a specific receptor with its ligand not only changes the expression of the receptor triggered, but has also a modulating effect on other receptor systems on the same cell.     

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.     

Expression of IgG Fc receptors in myeloid leukemic cell lines. Effect of colony-stimulating factors and cytokines

Three classes of FcR have been defined on human myeloid cells by their reactivity with mAb; FcRI (mAb 32); FcRII (mAb IV3); and FcRIII (mAb 3G8). We have quantitated the expression of each FcR on human myeloid leukemia cells and cell lines (KG-1, HL-60, U937, and K562). Detailed analysis of FcR surface expression is provided for the U937 cell line after exposure to CSF and cytokines. Increased expression of FcRI and FcRII occurred at 72 h in cells exposed to GCT or Mo cell line-conditioned medium as well as to medium from PHA-treated mononuclear cells. The augmentation of FcRII required protein synthesis and was diminished by a neutralizing antibody to granulocyte-macrophage CSF. We also show that fractions containing natural granulocyte CSF or granulocyte-macrophage CSF as well as r-granulocyte and r-granulocyte-macrophage CSF are capable of inducing FcRII on these cells, whereas other cytokines such as IL-1 and IL-2, TNF-alpha, INF-gamma and macrophages CSF failed to do so.     

The monoclonal antibody 41H16 detects the Leu 4 responder form of human Fc gamma RII.

Human FcR for IgG can be divided into three classes (Fc gamma RI, II, and III) based on their structure and reactivity with mAb. Fc gamma RII can be further subdivided into two categories based on functional and biochemical assays. These two Fc gamma RII subtypes were initially recognized by the failure of T cells from 40% of individuals to proliferate in response to mAb Leu 4 (mouse IgG1, anti-CD3), a response that requires the binding of the Fc region of the Leu 4 mAb to Fc gamma RII on monocyte accessory cells. Inas-much as mouse IgG1, does not bind efficiently to the nonresponder form of Fc gamma RII, mAb Leu 4 is unable to induce proliferation in these individuals. IEF data on Fc gamma RII from Leu 4 responder and nonresponder individuals suggested that the structural gene for Fc gamma RII consisted of two allelic forms R (responder) and N (nonresponder) producing the phenotypes RR, RN, and NN. Thus, exclusive expression of the nonresponder allele in monocytes of "nonresponder" individuals, appeared to be responsible for the lack of proliferation observed. In cooperation with the IVth International Conference on Human Leukocyte Differentiation Antigens, we analyzed CDw32 mAb to determine if they could distinguish the responder and nonresponder forms of Fc gamma RII. We report that mAb 41H16 binds preferentially to the responder allotypic form of Fc gamma RII expressed on human monocytes. When quantitative flow cytometry is used to measure the binding of both mAb 41H16 (responder Fc gamma RII) and mAb IV.3 (all myeloid cell Fc gamma RII), we are able to subdivide the responder population into homozygous and heterozygous responders. In addition, mAb 41H16 blocks the binding of mAb IV.3 to monocytes and inhibits proliferation when added to cells before addition of mAb Leu 4. We also show that polymorphonuclear leukocytes and platelets have the same allotypic differences in the binding of 41H16 as do monocytes. However, a subset of lymphocytes (previously shown to be B cells) expresses the 41H16 epitope with no evidence for donor to donor variability.     

Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG

It is shown that a mAb specific for the human 40-kDa FcR (FcRII) leads to activation of human monocytic cells but that extensive cross-linking of the receptor is required. Calcium mobilization can be induced in immature monocytic cells (undifferentiated U937 cells) and peripheral blood monocytes with an intact IgG1 anti-FcRII antibody (CIKM5) but not by F(ab')2 fragments of this antibody. The intact antibody can bind in a tripartite manner by its two F(ab') sites and its Fc-binding site whereas the F(ab')2 fragments of this antibody can only bind in a divalent fashion. A rise in intracellular free calcium ion concentration occurs when F(ab')2 fragments are cross-linked with F(ab')2 anti-mouse Ig indicating that more extensive cross-linking of FcRII is required rather than an obligatory requirement for an Fc-FcRII interaction. Calcium mobilization in response to intact or cross-linked F(ab')2 fragments of CIKM5 is associated with superoxide production only in IFN-gamma-primed peripheral blood monocytes and IFN-gamma differentiated U937 cells indicating that the activation signal produced via FcRII is inadequate to fully stimulate non-"primed" cells. A second mAb reactive with FcRII (2E1) does not cause calcium mobilization in monocytes or U937 cells, and partially blocks the effects of CIKM5. 2E1 also blocks CIKM5 superoxide production in IFN-gamma-primed monocytes and differentiated U937 cells. This may be explained in part by the fact that 2E1 is an IgG2a antibody and can only participate in bipartite binding with FcRII. When 2E1 is cross-linked with F(ab')2 anti-mouse Ig there is a small calcium response. This does not cause superoxide generation in IFN-primed monocytes but does do so in IFN-gamma differentiated U937 cells. FcRII is also expressed on granulocytes and some B cells but the effects of cross-linking the receptor on these cells differ from those seen in monocytes.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.     

Activation of lymphocytes alters Fc receptor-beta 2-microglobulin interrelationship on the lymphocyte surface

The effect of anti-beta 2-microglobulin (anti-B2Mi) on the expression of Fc receptors (FcR) of human lymphocytes was compared on resting and activated cells. Previously we reported that anti-B2Mi induces a "co-shedding" of FcR with the beta 2-microglobulin (B2Mi)-anti-B2Mi complexes when used under the conditions where the redistribution of membrane molecules is allowed (Sármay et al., Cell. Immunol. 56, 452, 1980; Sármay et al. Immunology 36, 339, 1979). Furthermore our group also described two types of FcR-bearing cells, one which shed their FcR during a temperature shift from 4 to 37 degrees C (FcRI+ cells) and the other which has an immobile type FcR under the same circumstances (FcRI+ cells) (Sándor et al., Immunology 38, 553, 1979; Sármay et al., Immunology 34, 315, 1978). In this work we have characterized the FcR released from the membrane as a consequence of anti-B2Mi treatment. We have found that they are the mobile, FcRI type. It was proved that the shedding of this FcRI is a consequence of the anti-B2MI-induced transformation of FcRII into the FcRI form on the membrane of the antibody-treated lymphocytes. On the activated T cells, however, anti-B2Mi is incapable of inducing the same phenomenon in the early phase of activation. In contrast, FcR expression is blocked by anti-B2Mi treatment similarly to that on resting lymphocytes, on the surface of activated B cells, or on activated T cells in the later phases of activation.     

Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937

Human C-reactive protein (CRP) is an acute phase reactant that is opsonic and an activator of macrophage tumoricidal function. CRP also activates the classical C cascade. These activities suggest that CRP might interact with monocytes/macrophages via specific receptors in a manner analogous to the interaction of IgG with FcR. With the use of radio-labeled human CRP, we have observed specific binding of CRP to human blood monocytes and the human monocytic cell line U-937. Binding was saturable at a pathophysiologic concentration of CRP, with an estimated KD of 9.5 x 10(-8) M and 3.6 x 10(5) binding sites/cell. Specific binding was inhibited by polyclonal human IgG as well as an IgG1 myeloma. In the converse experiment, CRP failed to inhibit specific [125I]IgG binding. The mAb IV.3, which inhibits binding of IgG immune complexes to FcRII, did not inhibit CRP binding. A 100-fold excess of phosphorylcholine or the phosphorylcholine binding peptide of CRP (residues 47-63) failed to inhibit binding. Although human rIFN-gamma and PMA increased FcRI expression, these reagents had no affect on CRP receptor expression. A single membrane protein of 38 to 41 kDa from U-937 cells was chemically cross-linked to [125I]CRP; the cross-linking was inhibited by human IgG1 but not the IV.3 mAb. Furthermore, two membrane proteins with a Mr of 38 to 40 kDa and 58 to 60 kDa were isolated by CRP ligand-affinity chromatography. These proteins were of a distinct size from those isolated for FcRI from an IgG ligand matrix. These studies demonstrate specific binding of human CRP to a human monocytic cell line via receptors that are distinct from the IgG FcR and implicate CRP in nonspecific, preimmune host defense reaction mediated by cells of the monocytic lineage.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

Effects of inhibitors of C1q biosynthesis on macrophage Fc receptor subclass-mediated antibody-dependent cellular cytotoxicity and phagocytosis

We examined the effects of the inhibitors of C1q or collagen biosynthesis, 2,2'-dipyridyl (DP), and 3,4-dehydro-DL-proline (DHP) on murine macrophage (M phi) FcR subclass-mediated antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of sheep erythrocyte targets. Oil-elicited peritoneal M phi from C3HeB/FeJ mice which were cultured for 24 hr with DP (0.08 or 0.10 mM) or DHP (0.8 or 1.0 mM) showed a significant decrease in FcR subclass-mediated ADCC for murine monoclonal IgG2a (FcRI) and IgG2b/IgG1 (FcRII) as well as for heterologous polyclonal IgG. These collagen inhibitors also blocked phagocytosis mediated by both IgG2a- and IgG2b-opsonized erythrocytes. DP was more potent than DHP in blocking FcR effector functions in a reversible fashion and neither inhibitor affected M phi C3b receptor function. Pretreatment of M phi with collagenase resulted in significant reduction in FcR-mediated ADCC and phagocytosis. The inhibition of M phi FcR subclass-mediated ADCC and phagocytosis by collagen C1q synthetic inhibitors or by collagenase treatment further confirms a functional relationship between cell-associated C1q and FcR-dependent functions.