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.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Human T cell activation induced by a monoclonal mouse IgG3 anti-CD3 antibody (RIV9) requires binding of the Fc part of the antibody to the monocytic 72-kDa high-affinity Fc receptor (FcRI)

The mitogenic activity of anti-CD3 mouse monoclonal antibodies (mAb) in cultures of human peripheral blood mononuclear cells (PBMC) depends on the ability of the mAb to interact with CD3 molecules on the T cells, and with Fc receptors (FcR) on monocytes. Two types of FcR with distinct specificity for murine (m) IgG subclasses are involved: a 72-kDa receptor (FcRI) binds mIgG2a and a 40-kDa receptor (FcRII) binds mIgG1. In this study we examined the mitogenic activity of mIgG3 anti-CD3 mAb RIV9. In cultures of human PBMC, the mAb induced T cell proliferation and interleukin 2 production. We found that subjects, unresponsive to mIgG2a anti-CD3 (e.g., OKT3), were also RIV9 nonresponders. In contrast, nonresponders to mIgG1 anti-CD3 (e.g., anti-Leu4) had a normal response to RIV9. Our results therefore suggested that anti-CD3 mAb of the mIgG2a and mIgG3 subclass bind to the same monocytic FcR. Human monomeric IgG, which has been shown to bind to FcRI only, blocked T cell proliferation induced by mIgG2a and mIgG3 anti-CD3, but had no effect on T cell proliferation induced by mIgG1 anti-CD3. In contrast, a mAb (IV.3) to FcRII, which blocks ligand binding of the receptor, blocked the mitogenic activity of mIgG1 anti-CD3 antibodies, but had no effect on T cell proliferation induced by mIgG3 anti-CD3 or by mIgG2a anti-CD3. Binding of RIV9 to FcR of responder monocytes could be demonstrated in immunofluorescence. Monocytes from the RIV9 nonresponder subjects however were unable to bind the Fc portion of this antibody. The binding of fluorescein (FITC)-conjugated mIgG3 or FITC-conjugated mIgG2a to responder monocytes could be inhibited by human monomeric IgG and by mIgG2a and mIgG3, but not by the mAb to FcRII. The results demonstrate that mIgG3 binds to FcRI on human monocytes and that this binding is needed for the mitogenic activity of mIgG3 anti-CD3.     

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.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

Role of Fc gamma RII in platelet activation by monoclonal antibodies.

mAb are being widely used to probe the function of cell-surface proteins. The cell stimulation that may be produced is often dependent on mAb interaction with both the target Ag and FcR. However, it remains unclear whether these interactions take place on the same cell or between adjacent cells and whether the FcR plays an anchoring or signaling role. Using the model of platelet activation, we demonstrate that two different Fc-dependent mAb, LeoA1 and ALB6, both activate the cell by forming intercellular links between Ag on one cell and FcR on the opposing platelet. We also show that the mAb differ with respect to the relative roles of target Ag vs FcR in provision of the stimulation signal. Thus Fc gamma RII played a mainly anchorage role in LeoA1 stimulation, whereas its role in ALB6 stimulation was mainly signaling. Therefore the precise contribution of each of these roles to the overall effect of a stimulatory antibody should be determined before the antibody is used as a specific functional probe.     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

Apparent sensitivity of human K lymphocytes to the spatial orientation and organization of target cell-bound antibodies as measured by the efficiency of antibody-dependent cellular cytotoxicity (ADCC)

Although monoclonal antibodies (mAb) can elicit potent ADCC by human K lymphocytes, different mAb, even of the same antibody subclass or even of the same target antigen specificity, vary considerably as to their efficiency in eliciting ADCC. The extensive variability in ADCC efficiencies of murine IgG2a mAb is analyzed here. In cold-target inhibition experiments it was found that only cells coated with "ADCC-efficient" IgG2a mAb, and not "ADCC-inefficient" IgG2a mAb, inhibit K effector cell lysis of radiolabeled target cells by ADCC. This result indicates that the spatial orientation of the antibodies on the target cell membrane influences the net efficiency of ADCC reactions by affecting the efficiency of interaction between antibody and the Fc receptors (FcR) of K cells. It is proposed that a "favorable" orientation of antibodies on the target cell membrane is required for efficient ADCC reactions. This proposal is directly supported by the observation that one IgG2a mAb (20.8.4), which cross-reacts with several different H-2 alloantigens, was found to elicit efficient ADCC only when bound to certain of its possible target cell antigens. It was also observed in these studies that the organization of antibodies on a target cell membrane influences the net efficiency of ADCC reactions. It is proposed that a "favorable" antibody organization on the target cell membrane is also required for efficient ADCC reactions. This proposal is supported by the observation that certain antihuman beta 2m (anti-Hu beta 2m) IgG2a mAb, which elicit efficient ADCC lysis of human target cells, fail to elicit the lysis of murine cells having Hu beta 2m molecules coupled randomly to their external membrane surfaces. The differences in the way the Hu beta 2m was organized on the surfaces of the human cells and the murine-Hu beta 2m cell conjugates presumably caused differences in the way the bound antibodies were organized on the cell surfaces, which in turn resulted in the ADCC efficiency differences observed for the same mAb with the different target cell types. Because ADCC reactions appear to be sensitive to both the orientation and the organization of cell surface-bound antibodies, certain types of structural alterations or variations in the membrane molecules (relative to other neighboring structures on the target cell membrane) are potentially detectable by quantitative differences or variations in ADCC reactions.     

Differential regulation of murine B cell Fc gamma RII expression by CD4+ T helper subsets

The murine B cell FcR for IgG (Fc gamma RII) is a membrane glycoprotein reported to mediate inhibition of B cell activation and differentiation. We show that IL-4 inhibits the enhanced expression of Fc gamma RII by LPS-stimulated B cells. This activity is completely reversed by anti-IL-4 mAb and is specific, in that multiple other lymphokines tested do not exert a similar effect. This effect of IL-4 is apparent by day 1 of culture, although maximal inhibition occurs on day 4 at a concentration of 500 U/ml. The IL-4-induced inhibition of enhanced Fc gamma RII expression by LPS stimulation observed on day 4 of culture is associated with a significant reduction in the steady state level of Fc gamma RII beta gene-specific mRNA. IFN-gamma which inhibits many of the effects of IL-4 on B cells, does not reverse the IL-4-induced inhibition of Fc gamma RII membrane expression nor the levels of beta gene-specific mRNA. Fc gamma RII expression is significantly increased in B cells stimulated with antigen-specific, CD4+ T cell clones of the Th1 type (i.e., IL-2 and IFN-gamma-producing). By contrast, three different Th2 clones (i.e., IL-4-producing) fail to stimulate an increase in Fc gamma RII levels. Anti-IL-4 mAb significantly enhanced Fc gamma RII expression by Th2-stimulated B cells indicating that IL-4 was the active, inhibitory, substance produced by the Th2 cells. Supernatants from stimulated Th2 clones inhibited the enhanced expression of Fc gamma RII by LPS-stimulated B cells and this activity was completely reversed by anti-IL-4 mAb. By contrast, supernatants from stimulated Th1 clones further enhanced Fc gamma RII expression by LPS-stimulated B cells. The differential regulation of B cell Fc gamma RII expression by Th subsets may play an important role in the regulation of humoral immunity by altering the sensitivity of B cells to IgG immune complex-mediated inhibition of B cell activation and differentiation in vivo.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Activation of human platelets by a stimulatory monoclonal antibody

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.     

B cell activator. Effects on B cell expression of CD23, proliferation, and antibody secretion.

The studies herein describe a B cell hybridoma-derived, low m.w. (less than 1000 Da), hydrophilic mediator denoted B cell activator (BCA). BCA stimulates B cell expression of IgE-specific FcR (Fc epsilon RII or CD23) in a manner similar to IL-4. However, BCA can be readily distinguished from IL-4 because it does not 1) enhance B cell Ia expression; 2) bind 11B11 anti-IL-4 mAb; or 3) elicit superinduction of Fc epsilon RII expression or IgE production in cultures of LPS-activated B cells. Moreover, BCA is considerably more mitogenic than IL-4 for LPS-activated B cells and, in contrast to IL-4, lacks mitogenicity for anti-mu-activated B cells. BCA can enhance IgG2b and IgG3 production by LPS-activated B cells, responses that are suppressed by IL-4. BCA alone did not stimulate IgE and IgG1 production by LPS-activated B cells, but exerted synergistic activity when combined with IL-4 in stimulating secretion of these antibody isotypes. Finally, secondary Ag-driven IgG1, IgE, and IgA antibody responses can be stimulated by BCA in vitro. Thus, BCA appears to be a novel mediator with broad B cell activation properties.     

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (Malayan pit viper), stimulates platelets by binding to α2β1 integrin and glycoprotein Ib, activating Syk and phospholipase Cγ 2, but does not involve the glycoprotein VI/Fc receptor γ chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.     

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein.

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.     

A novel function of the herpes simplex virus type 1 Fc receptor: participation in bipolar bridging of antiviral immunoglobulin G

We describe a novel function of the Fc receptor of herpes simplex virus type 1 (HSV-1), its ability to participate in antibody bipolar bridging. This refers to the binding of a single immunoglobulin G (IgG) molecule by its Fab end to its antigenic target and by its Fc end to an Fc receptor (FcR). We demonstrate that various immune IgG antibodies, including polyclonal rabbit antibodies to HSV-1 glycoproteins gC1 and gD1 and monoclonal human antibody to gD1 blocked rosetting of IgG-coated erythrocytes at IgG concentrations 100- to 2,000-fold lower than required for rosette inhibition with nonimmune IgG. Steric hindrance did not account for the observed differences between immune and nonimmune IgG since rabbit anti-gC1 F(ab')2 fragments did not block rosetting. Murine anti-gC1 or anti-gD1 IgG, a species of IgG incapable of binding by its Fc end to the HSV-1 FcR, also did not block rosetting. When cells were infected with a gC1-deficient mutant, anti-gC1 IgG inhibited rosetting to the same extent as nonimmune IgG. This indicates that binding by the Fab end of the IgG molecule was required for maximum inhibition of rosetting. Bipolar bridging was shown to occur even when small concentrations of immune IgG were present in physiologic concentrations of nonimmune IgG. The biologic relevance of antibody bipolar bridging was evaluated by comparing antibody- and complement-dependent virus neutralization of an FcR-negative mutant and its parent HSV-1 strain. By engaging the Fc end of antiviral IgG, the parent strain resisted neutralization mediated by the classical complement pathway. These observations provide insight into the role of the HSV-1 FcR in pathogenesis and may help explain the function of FcR detected on other microorganisms.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.     

Structural polymorphism of the human platelet Fc gamma receptor

A variable T lymphocyte proliferative response to murine IgG1 anti-T3 monoclonal antibodies, in which most North American Caucasians respond whereas a minority do not, is well established. This is most likely the result of a genetic polymorphism manifested by 1) the inability of the monocyte 40-kDa IgG FcR of some individuals to bind murine IgG1, and 2) a distinctive trimorphic pattern on IEF of the monocyte 40-kDa FcR, one form being seen in all individuals who do not respond and another form (or a combination of both forms) being seen in those who do respond. We have evaluated the IEF patterns of the platelet 40-kDa FcR and find that in every individual tested the pattern for platelet FcR correlates with that seen for the monocyte 40-kDa FcR pattern. Furthermore, the platelets of those individuals whose "nonresponder" monocyte 40-kDa FcR did not mediate a murine IgG1 anti-T3 response did not respond with an aggregation reaction to murine IgG1 immune complexes (opsonized E). In contrast, platelets from donors possessing "responder" monocytes displayed positive "aggregation" responses to E coated with murine IgG1 antibody. However, the platelet FcR structural polymorphism described earlier did not correlate with the donor-specific variability in capacity of platelets to respond functionally to aggregated human IgG described in an earlier paper. Rather, the variation in capacity of platelets from individual donors to respond functionally to aggregated human IgG was related to the quantitative expression of platelet FcR. These data indicate that the molecular mechanisms responsible for the platelet 40-kDa FcR structural polymorphism are quite different from the mechanisms governing the variation in quantitative expression of the receptor.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

Activation of human monocytes occurs on cross-linking monocytic antigens to an Fc receptor

Murine mAb to CD13, CD14, and class II MHC, are able to mobilize calcium in normal human monocytes and enhance superoxide production in primed cells. Antibodies to CD35 (CR1) also cause a minor calcium response in some individuals. Antibodies to CD11a, CD11b, CD11c, CD15, CD17, CD18, and CD45 do not activate monocytes. The ability of mAb to cause monocyte activation is not only dependent on the Ag with which they react but also on the isotype of the antibodies and the individual from whom the monocytes were obtained. It is shown that this is because the mAb that activate monocytes do so by formation of Ag-antibody-FcR complexes. F(ab')2 fragments of mAb to CD13 and CD14 do not therefore activate monocytes even when cross-linked with F(ab')2 anti-mouse Ig but do so when cross-linked with intact anti-mouse Ig. These data indicate that activation via the FcR requires perturbation of this receptor but does not necessarily require cross-linking of one FcR to another. Antibody-coated particles or cells able to bind to cell surface receptors on monocytes other than the FcR would thus augment FcR-mediated activation.     

Platelet activation induces increased Fc gamma receptor expression

Platelet contain Fc gamma RII. However, little is known about how the expression of these receptors is regulated. Inasmuch as platelet activation by a variety of agonists increases the expression of several proteins on the platelet surface, we used flow cytometry to study the effect of platelet activation on the expression of platelet Fc gamma R by measuring the binding of fluorescein-labeled oligomeric IgG (FITC-IgG oligomer) and fluorescein-labeled mAb IV.3 (FITC-IV.3), a mAb that recognizes Fc gamma RII, to platelets. The number of Fc gamma R per platelet was determined by relating the binding to platelets of FITC-IV.3, measured by flow cytometry, to the binding of 125I-labeled IV.3, measured using a standard filtration assay. Nonactivated, gel-filtered platelets from nine healthy donors expressed a mean of 891 Fc gamma R per platelet, whereas platelets activated at 25 degrees C by thrombin or PMA expressed a mean of 1382 Fc gamma R an average increase of 55% (p less than 0.001). Binding of FITC-IgG oligomer increased to a similar extent when platelets were stimulated by these agonists. A smaller increase in the number of Fc gamma R expressed on the platelet surface was measured when platelets were stimulated with ADP, though no increase was observed with epinephrine. The agonist-dependent increase in Fc gamma R expression did not occur when platelets were studied at 4 degrees C or in the presence of agents that elevate intracellular levels of cAMP, suggesting that platelet activation was required for this process. Agonist-stimulated Fc gamma R expression did not depend on dense-granule secretion, because it was observed at low agonist concentrations in the absence of 14C-serotonin release. These studies demonstrate that the number of Fc gamma R expressed on the platelet surface increases when platelets are activated by several agonists, perhaps as a result of the exposure of Fc gamma R located along the surface-connected open canalicular system, or the fusion of platelet alpha-granule and plasma membranes during the activation process. Increased Fc gamma R expression may promote the clearance of IgG-containing immune complexes from the circulation, and contribute to the development of immune complex-mediated thrombocytopenia.