Functions of the various IgG Fc receptors in mediating killing of Toxoplasma gondii.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.     

Evaluation of the antibody-dependent cytotoxic capabilities of individual human monocytes. Role of Fc gamma RI and Fc gamma RII and the effects of cytokines at the single cell level.

In this report we present evidence that not all human peripheral blood monocytes mediate antibody-dependent cellular cytotoxicity (ADCC), and that this function may be determined on an individual cell by both the type and level of expression of FcR, and by the state of cellular activation and/or differentiation. Although the diverse range of effector and regulatory functions performed by human monocytes suggests the possibility of distinct subsets, it is not clear whether observed functional heterogeneity reflects the presence of true monocyte subpopulations, or whether this diversity represents a continuum of maturational states present in the peripheral circulation. In an attempt to address this question, we investigated the ability of human monocytes to carry out ADCC at the single cell level, with emphasis on the role of the three FcR for IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) in mediating cytotoxicity. Using a modified plaque assay, 58.3% +/- 4.9 of freshly isolated monocytes mediated ADCC, as evidenced by the formation of lytic plaques in monolayers of ox erythrocyte (oxE) target cells. Significant increases in the number of plaque-forming cells were observed after positive selection by flow microfluorimetry for those monocytes expressing high levels of Fc gamma RI and Rc gamma RII, but not Fc gamma RIII. Bispecific antibodies composed of Fab fragments of anti-oxE antibody covalently coupled to Fab fragments of anti-Fc gamma R antibodies were used to independently evaluate the ability of Fc gamma RI, Fc gamma RII, and Fc gamma RIII to mediate single cell cytotoxicity. Significant increases in the number of plaque-forming cells were observed in the presence of anti-Fc gamma RI x anti-oxE and anti-Fc gamma RII x anti-oxE bispecific antibodies, confirming the efficiency of Fc gamma RI and Fc gamma RII as cytotoxic trigger molecules on human monocytes. Incubation of monocytes with purified rIFN-gamma and granulocyte macrophage-CSF, but not IL-2, IL-3, IL-4, IL-6, or TNF-alpha, also resulted in significant increases in the number of monocytes mediating cytotoxicity, suggesting that cytotoxic ability at the single cell level may be influenced by factors which effect monocyte activation and differentiation, respectively. Overall, these studies demonstrate that freshly isolated human monocytes are heterogeneous in their ability to mediate ADCC, and suggest that this functional diversity arises not from discrete subpopulations of cells, but from a continuum of maturational/activational states present within the peripheral circulation.     

Modulation of human polymorphonuclear leukocyte IgG Fc receptors and Fc receptor-mediated functions by IFN-gamma and glucocorticoids

Human polymorphonuclear neutrophils (PMN) normally express two distinct types of IgG Fc gamma R, the 40-kDa Fc gamma R referred to as Fc gamma RII and the low affinity 50- to 70-kDa Fc gamma R designated Fc gamma RIII. A third type of Fc gamma R, the 72-kDa high affinity receptor known as Fc gamma RI, is also detectable on PMN that have been activated by IFN-gamma. Using mAb that discriminate among the three known types of Fc gamma R, we examined the effects of IFN-gamma and glucocorticoids on human PMN Fc gamma R expression. We also studied effects of IFN-gamma and the synthetic glucocorticoid dexamethasone (DEX) on antibody-dependent cytotoxicity (ADCC) of chicken erythrocytes and phagocytosis of IgG-coated ox RBC by human PMN. In 20 donors studied, we found that treatment of PMN with 400 U/ml IFN-gamma induced a 9- to 20-fold increase in the number of Fc gamma RI sites per cell, and DEX inhibited this induction of Fc gamma RI by 39 to 73%. Similarly, DEX significantly reduced the IFN-gamma stimulation of ADCC and phagocytosis. IFN-gamma had no effect on expression of Fc gamma RII or Fc gamma RIII. Fc gamma RI and Fc gamma RII expression was unaltered by 24 h of treatment with DEX alone, but Fc gamma RIII expression was sometimes increased by about 20% on PMN cultured with DEX. Nevertheless, we found a small but significant inhibition of ADCC and phagocytosis by 200 nM DEX. Our results indicate that Fc gamma RI plays a major but not exclusive role in the regulation of ADCC and phagocytosis by IFN-gamma and DEX.     

Proteolysis induces increased binding affinity of the monocyte type II FcR for human IgG

Human monocytes express two types of IgG FcR, Fc gamma RI and Fc gamma RII. These can be assayed by using indicator E sensitized by human IgG (EA-human IgG) or mouse IgG1, (EA-mouse IgG1), respectively. On mouse macrophages, Fc gamma RI is sensitive to trypsin, whereas Fc gamma RII is trypsin resistant. We studied the effects of the proteolytic enzymes pronase and trypsin on human monocyte Fc gamma R. Neither enzyme caused a decrease in rosetting mediated by monocyte Fc gamma RI. Human Fc gamma RII is polymorphic, and monocytes interact either strongly or weakly with mouse IgG1. The interaction of low responder monocytes with mouse IgG1 was dramatically increased (to the level exhibited by high responder monocytes) by protease treatment. The effects of proteases on Fc gamma RII were investigated in more detail by using monocytes from which Fc gamma RI was selectively modulated by using immobilized immune complexes. Proteolysis of such modulated monocytes induced an increased interaction with EA-human IgG. Fc gamma RII appears to mediate this interaction. This conclusion is supported by the observation that after proteolysis, the Fc gamma RII-mediated binding of EA-mouse IgG1 becomes susceptible to inhibition by (monomeric) human IgG. To quantify the effect of proteolytic enzymes on Fc gamma RII, we performed binding studies with cell line K562, that expresses only Fc gamma RII. A significant increase in Ka of Fc gamma RII for dimeric human IgG complexes was observed when K562 cells were treated with protease. To elucidate the mechanism of this enhancement of Ka by proteolysis, we performed immunoprecipitation studies. Neither m.w., nor IEF pattern of Fc gamma RII were influenced by proteolysis. Moreover, the expression of Fc gamma RII was not affected by proteolysis as evidenced by immunofluorescence studies and Scatchard analysis, and neither were Fc gamma RI or Fc gamma RIII induced. We conclude that proteolysis increases the affinity of Fc gamma RII for human IgG, and speculate that such a proteolysis-induced change may also occur in vivo, e.g., at inflammatory sites.     

IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.     

The mechanisms of antibody-dependent killing mediated by lymphoid and myeloid cells are distinct based on different divalent cation requirements

To further understand the mechanism(s) of antibody-dependent cell-mediated cytotoxicity (ADCC) by various effector populations, we have examined the extracellular Ca++ and Mg++ requirements for ADCC performed by lymphocytes, monocytes, polymorphonuclear leukocytes and peritoneal macrophages. We have used the anti-Fc gamma R-bearing hybridoma cell lines (HC) as self directed targets for ADCC to analyse the triggering ability of each of the three defined Fc gamma R; Fc gamma RI, Fc gamma RII, and Fc gamma RIII. Lymphocyte killing of the anti-Fc gamma RIII bearing HC (HC 3G8) was Ca++ dependent, but Mg++ independent. In contrast, monocytes and PMN killed the anti-Fc gamma RI- (HC 32) and the anti-Fc gamma RII- (HC IV.3) bearing HC in a Mg++-dependent, Ca++-independent fashion. In addition, freshly prepared monocytes were able to kill HC 3G8 in a Mg++-dependent, Ca++-independent fashion, indicating that low levels of Fc gamma RIII may be functionally detected on monocytes. Peritoneal macrophages were able to kill all three of the anti-Fc gamma R bearing HC in a Mg++-dependent, Ca++-independent fashion. Thus, the same target is lysed by myeloid cells in the presence of Mg++ without Ca++ and by lymphoid cells in the presence of Ca++ without Mg++. These results suggest that at least two distinct mechanisms of ADCC exist that depend on the type of effector cell mediating antibody-dependent killing and not necessarily on the Fc gamma R type triggered.     

IL-4 decreases Fc gamma R membrane expression and Fc gamma R-mediated cytotoxic activity of human monocytes

Monocytes can express three classes of FcR for IgG: Fc gamma RI, Fc gamma RII, and Fc gamma RIII (CD64, CD32, and CD16, respectively) of which the Fc gamma RIII is expressed after prolonged culture. Fc gamma R expression is regulated by IFN-gamma. Because IFN-gamma and IL-4 have antagonistic effects on the expression of the FcR for IgE on human monocytes, we studied the effect of IL-4 on Fc gamma R expression and function. We show that IL-4 down-regulates Fc gamma RI, Fc gamma RII, and Fc gamma RIII expression of cultured monocytes and inhibits IFN-gamma enhanced Fc gamma RI expression. Exposure of monocytes to IL-4 for 40 h resulted in a dose-dependent decrease of the expression of all three Fc gamma R that persisted throughout the whole culture period (7 days). Anti-IL-4 antibodies completely reversed the IL-4 effect. In addition the impaired Fc gamma R expression correlated directly with reduced Fc gamma R-mediated function because monocytes cultured in the presence of IL-4 have a reduced capacity to lyse human E opsonized with human IgG anti-D or mouse antiglycophorin A antibodies. These observations, together with the previous finding that IL-4 induces Fc epsilon RIIb expression on monocytes, indicate that IL-4 and IFN-gamma may control the Fc gamma R-mediated immune response by differentially regulating Fc gamma R expression.     

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.     

Enhanced antigen presentation using human Fc gamma receptor (monocyte/macrophage)-specific immunogens.

A major new challenge for vaccine development is to target APC such as monocytes and macrophages for efficient Ag processing and presentation. It has been shown that Fc gamma R-mediated uptake of Ag-antibody complexes can enhance Ag presentation by myeloid cells at least 100-fold, and directing Ag to Fc gamma R in mice brings about a substantial increase in the effectiveness of immunization while eliminating the requirement for adjuvant. It has not been determined which of the three subclasses of human Fc gamma R on myeloid cells (Fc gamma RI, Fc gamma RII, or Fc gamma RIII) function to enhance Ag presentation. We have targeted our Ag (TT) to each of the three subclasses of human Fc gamma R on monocytes using Fc gamma R subclass-specific mAb-TT conjugates, and have measured TT presentation by monitoring T cell proliferation in response to TT. In addition, we have examined enhanced Ag presentation mediated by a human IgG1 (HIgG1) anti-TT mAb. All anti-Fc gamma R-TT conjugates enhanced Ag presentation. HIgG1 anti-TT, in monomeric form, enhanced Ag presentation through Fc gamma RI only. Anti-Fc gamma RI-Ag conjugates appear to be optimal for application as vaccines. They are monocyte/macrophage-specific, are very efficiently processed and presented, and enhance Ag presentation despite occupation of Fc gamma RI with HIgG.     

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.     

Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells

As part of an effort to define the cytotoxic trigger molecules on human myeloid cells, the ability of the different Fc receptors for IgG (Fc gamma R) to mediate killing of tumor cell lines by monocytes and granulocytes was examined. This was accomplished by studying cytolysis of hybridoma cell (HC) targets bearing surface antibody directed toward the different Fc gamma R. The HC line, HC IV.3A, which bears Ig directed to the low affinity Fc gamma R (Fc gamma RII) on monocytes and neutrophils was lysed by human monocytes. The extent of lysis of HC IV.3A was approximately equal to that of anti-Fc gamma RI (the high affinity Fc gamma R on human monocytes) bearing HC lines (HC 32.2A and HC 62A) and was not augmented by treatment of the monocytes with interferon-gamma (IFN-gamma). In contrast, neutrophils lysed HC IV.3A and HC 32.2A only after activation with IFN-gamma. Since Fc gamma RI is not detectable on untreated neutrophils and is induced by IFN-gamma on these cells, lysis of HC 32.2A by IFN-gamma-activated neutrophils correlated with receptor induction. On the other hand, Fc gamma RII was present at equal levels on untreated and IFN-gamma-treated neutrophils, but only IFN-gamma-treated neutrophils mediated cytotoxicity via Fc gamma RII. In this case, enhanced killing appeared to be due to events other than an increase in Fc gamma RII number. Neither untreated nor IFN-gamma-treated neutrophils mediated the lysis of the anti-Fc gamma RIII bearing HC 3G8A. Thus, binding to the tumor target via this Fc receptor does not lead to lysis and may initiate signals distinct from those triggered through Fc gamma RI or Fc gamma RII. Surprisingly, HC bearing high amounts of mouse IgG1 antibody of irrelevant specificity were also lysed by monocytes. This lysis was blocked by soluble IV.3 antibody and thus appeared to be due to binding of the Fc portion of the surface Ig to Fc gamma RII on monocytes. Furthermore, monocytes from donors with a form of Fc gamma RII incapable of binding aggregated mouse IgG1 did not lyse these HC, but displayed normal lysis of HC IV.3, demonstrating that this structurally different Fc gamma RII remained a functional trigger molecule. Overall, these studies have demonstrated the specificity of Fc receptors in triggering monocyte- and granulocyte-mediated antibody-dependent tumor cell killing and have begun to dissect functional similarities and differences among the three defined Fc gamma R on human myeloid cells.     

Alveolar and peritoneal macrophages bear three distinct classes of Fc receptors for IgG

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.     

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (Fc gamma RI) and the IgA FcR (Fc alpha RI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-gamma, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to Fc alpha RI or Fc gamma RI on MDM. Although Fc alpha RI and Fc gamma RI share a common signaling pathway contingent on association with the gamma-chain (FcR gamma subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by Fc alpha RI and Fc gamma RI; however, IFN-gamma-treated MDM phagocytosed tumor cells only with the Fc gamma RI-directed bispecific Abs. Similarly, IFN-gamma-cultured MDM lysed tumor cells more efficiently via Fc gamma RI then by Fc alpha RI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via Fc alpha RI than Fc gamma RI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-gamma-mediated enhancement of Fc gamma RI expression and Fc gamma RI gamma-chain complexes, the regulation of Fc gamma RI- or Fc alpha RI-mediated activity occurred without significant change in either receptor expression or total complexes with gamma subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.     

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.     

Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.     

Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.