Fc fragment from human IgG induces PGE2 secretion. II. Negative regulation in B cell differentiation

In humans, in vitro, Fc fragment of IgG at a low concentration induces plasma cell generation. However, Fc fragment at a high concentration induces PGE2 release of monocyte activation capable of inhibiting this differentiation. The levels of PGE2 in the supernatant culture from mononuclear cells from normal donors were examined as a function of culture duration and concentration of Fc, Fab fragments and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin(s). PGE2 levels, from mononuclear cell cultures, were analyzed by the RIA test. The results indicated that the Fc fragments are able to induce PGE2 secretion. The maximal release of PGE2 occurs after 24 hr of culture; this level is proportionate to the quantity of Fc fragments introduced. The addition of indomethacin in the cell culture stimulated by a high concentration of Fc fragments reestablishes the percentage of plasma cells. These results suggest the regulatory role of Fc fragment by PGE2 secretion in B cell differentiation.     

Human B cell differentiation by Fc fragment. III. Effect of IL-1 and IL-2 on differentiation of human B lymphocytes induced by Fc fragments of human IgG

The human Fc fragment of IgG, when added to blood mononuclear cells in vitro, induces B cell differentiation after 6 days of culture. This activity requires the presence of T cells and monocytes. This work explores the roles of interleukin 1 (IL-1) and interleukin 2 (IL-2) in B cell differentiation induced by Fc fragments. Peripheral blood mononuclear cells (PBMC) from normal donors were examined for plasma cell differentiation following stimulation with Fc fragment (15 and 30 micrograms/ml) with or without IL-1 (6 U/ml) or IL-2 (2 U/ml). Results indicate that both IL-1 and IL-2 accelerated B cell differentiation by the Fc fragment to 3 days of culture, compared to 6 days required with the Fc fragment alone. The time required for differentiation was not further shortened when both IL-1 and IL-2 were present in culture; both IL-1 and IL-2 were able to partially induce B differentiation alone at 6 days of culture. The importance of IL-2 in B cell differentiation was further supported by the finding that antibodies specific for the IL-2 receptor blocked B cell differentiation induced by Fc fragments, with or without additional IL-1 or IL-2. The depletion of monocytes also blocked B cell differentiation and the requirement for monocytes could not be replaced by exogenous IL-1; however, Fc fragments were shown to induce monocytes to secrete IL-1 beta after 24 hr in culture. These results suggest that accelerated differentiation of B cells into plasma cells requires a double signal provided by Fc fragments and IL-1 or IL-2. Monocytes are necessary for Fc fragment-induced differentiation and cannot be replaced by either IL-1 or IL-2.     

抗人IgG Fc片段及Fab段单抗的鉴定及应用

本实验采用木瓜酶水解,SPA柱亲合层析等手段得到人IgGFc段及Fab段,以Sigma抗人IgGfFc段和抗人IgG Fab段单抗为标准品,鉴定了细胞库中抗人IgG系列的部分细胞株,得到特异性分泌抗人IgG Fc段和抗人IgG Fab段单抗的细胞各一株。 在上述实验基础上,用抗人IgG Fc及抗人IgG Fab单抗分别制备了Sepharose4B亲合层析柱,提纯了酶解人IgG Fc、Fab片段,经ELISA法鉴定,相互之间无交叉反应。同时用此方法制备了人抗HBe Fab片段,并将该片段进行了过氧化物酶标记,用来配制HBe ELISA诊断盒,证明其生物活性未受影响,而且消除了类风湿因子引起的HBe Ag假阳性现象。因抗HBe单抗来源困难,如采用HBe多抗制备ELISA试剂,本法将是提高质量的一个好方法。     

Polyclonal activation of murine B lymphocytes by Fc fragments. I. The requirement for two signals in the generation of the polyclonal antibody response induced by Fc fragments

Fc fragments derived from human IgG1 induce murine splenic B lymphocytes to undergo proliferation and differentiation to antibody-secreting cells. The polyclonal antibody response was found to require both the presence of macrophages and T cells. Spleen cell cultures from nude mice or T cell-depleted normal mice proliferate to the level of untreated control mice but do not produce polyclonal antibody unless T cells are added. Regulation of the Fc fragment induced B cell differentiation to antibody synthesis apparently occurs through two distinct signals. One signal is provided by Fc fragments for proliferation and the other by T cells for differentiation. This suggestion is supported by the observation that spleen cell preparations, devoid of T cells, are capable of proliferation to the level of normal spleen cell cultures in response to Fc fragments, but are incapable of making a polyclonal antibody response. The cell population that responds to the differentiation signal also responds to the proliferative signal. "Hot pulse" experiments demonstrated that proliferation precedes polyclonal activation.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.     

Human monocyte chemiluminescence triggered by IgG aggregates. Requirement of phospholipase activation and modulation by Fc receptor ligands

Human mononuclear cells stimulated with soluble IgG aggregates generated chemiluminescence, a response attributable to monocytes. Some requirements of this reaction were examined by preincubation of the cells with a variety of inhibitors. The protease antagonists TPCK and TLCK, the phospholipase inhibitors quinacrine and BPB, and the calcium channel blocker verapamil were all inhibitory at micromolar concentrations. The oxygen metabolite scavengers SOD and catalase were less inhibitory. These findings are consistent with a major role for arachidonic acid metabolites in the generation of light. Modulation of monocyte Fc-mediated chemiluminescence also occurred by preincubation of the cells with Fc ligands. While IgG aggregates and monomeric IgG blocked Fc-dependent chemiluminescence, IgG Fc fragments were stimulatory of this response.     

Potentiation of the rat delayed-type hypersensitivity reaction by the Fc portion of human IgG1

Fc fragments derived from a human IgG1 myeloma protein potentiate the rat delayed-type hypersensitivity (DTH) reaction to antigen challenge. Lewis rats immunized with heat-killed tubercle bacilli give augmented DTH reactions to the purified protein derivative of tuberculin when Fc fragments are included in the challenge dose. Similar potentiation of DTH by pFc' fragments indicates that the active site is located in the CH3 domain of IgG1. Histologic evaluation of the augmented reaction sites revealed predominantly mononuclear cell infiltrates characteristic of DTH reactions. Skin tests of tubercle bacilli-sensitized rats with an unrelated antigen and/or Fc fragments fail to elicit significant reactions. Augmentation of the DTH reaction to purified protein derivative is restricted to the Fc or pFc' region fragments since intact monomeric IgG1, Fab fragments, and bovine serum albumin were all shown not to be active potentiators. The DTH reaction of ovalbumin-sensitized rats was similarly augmented when Fc fragments were included with a challenge dose of ovalbumin, thus supporting the general nature of the phenomenon. These results support the concept of Ig molecules as multifunctional proteins that can not only serve effector functions but also participate in the regulation of immune responses.     

Identification of a unique IgG Fc binding site in human intestinal epithelium

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.     

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.     

Interference with tolerance induction of primed B cells by the Fc fragment of immunoglobulin

The capacity to interfere with tolerance induction in primed B cells was examined. Previous work had shown that TNP-specific splenic B cells from mice primed and boosted with TNP-KLH are highly susceptible to in vitro tolerization upon a brief exposure to TNP on a carrier unrelated to KLH. In the present work it was found that tolerance induction in these primed B cells could be partially disrupted by addition of the Fc fragment of immunoglobulin, a B-cell mitogen, and adjuvant, during exposure of the B cells to tolerogen. Addition of Fc fragments prepared by papain digestion of human IgG interfered with tolerization routinely in approximately 30-60% of the spleen cells susceptible to tolerogen. Addition of whole IgG or Fab fragments had no effect on tolerance induction. As little as 5 micrograms/ml of the Fc fragment preparation significantly interfered with tolerization and 32-64 micrograms/ml was optimal. Disruption of tolerization was most effective when the Fc fragment was added to the spleen cells either 4 hr prior to tolerogen or simultaneously with tolerogen; addition of the Fc fragment 4 hr after exposure to tolerogen was significantly less effective. Disruption of tolerization by the Fc fragment was not through polyclonal activation of B cells, as antigen was required for generation of significant numbers of PFC to TNP. Also, disruption was not through expansion of low avidity clones of B cells insusceptible to tolerogen, as the avidity of the antibody produced with and without Fc fragments present was approximately the same. These results show that the Fc fragment of IgG can partially interfere with tolerization of primed B cells. The manner in which Fc fragments may function to prevent tolerization through its lymphoid cell stimulatory capacities is discussed.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

Biological Activities of Murine Low-Affinity Fc Receptors for IgG

Murine low-affinity Fc receptors for IgG (FcγRIIbl, FcγRIIb2, and FcγRIII) bind the same IgG subclasses and are not distinguished by available anti-FcγRII/III mAbs (2.4G2). They trigger various biological activities, among which are the internalization of soluble and particulate immune complexes, cell activation, and its regulation. To determine the biological properties of the three murine receptors, each was expressed by stable transfection of corresponding cDNAs in two model cells: the murine lymphoma B cell IIA1.6 and the rat basophilic leukemia cell RBL-2H3. Biological activities of recombinant receptors were triggered with soluble immune complexes or 2.4G2 IgG in IIA1.6 cells, which express no FcγR, and with 2.4G2 Fab or F(ab′)₂, cross-linked with mouse anti-rat F(ab′)₂ in RBL, which express rat FcγR. Conditions for studying cell activation and endocytosis in both cell models are described, as are conditions for studying phagocytosis in RBL cells and antigen presentation or regulation of cell activation in IIA1.6 cells. Internalization of immune complexes was triggered by FcγRIIb2 and FcγRIII, but not by FcγRIIb1. Intracytoplasmic sequences required for phagocytosis and endocytosis could be distinguished in FcγRIIb2, but not in FcγRIII. Cell activation was restricted to FcγRIII. FcγRIII-mediated endocytosis, phagocytosis, and cell activation involved the consensus tyrosine-containing activation motif found in the intracytoplasmic domain of the γ subunit. Regulation of cell activation was induced by both FcγRII isoforms and depended on the same sequence as endocytosis. As a consequence, a single motif can determine more than one biological response of the cell, and a given response may be triggered by several motifs, borne by different FcγR.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.     

Monoclonal antibody to human IgG Fc receptors. Cross-linking of receptors induces lysosomal enzyme release and superoxide generation by neutrophils

The monoclonal antibody KuFc79 binds to a determinant on the Fc receptors (Fc gamma R) of human leukocytes. We examined the biologic effects of the interaction of this antibody with Fc gamma R on human neutrophils (PMNL). The univalent Fab fragment of KuFc79 inhibits the formation of rosettes with IgG-sensitized sheep erythrocytes by as much as 91.7%. In other experiments in which PMNL were washed after exposure to Fab of KuFc79, phagocytosis of IgG-sensitized sheep erythrocytes was inhibited by 36%. Fab fragments of other mouse IgG2b monoclonal proteins did not have these effects. When PMNL are exposed to coverslips coated with univalent Fab fragments of this antibody, the Fc gamma R are removed from the surface of the PMNL. Under these conditions, rosetting could be inhibited by 85.4%. We examined cross-linking of receptor bound monoclonal antibody or its Fab fragment by either Protein A or F(ab')2 of an anti-mouse Ig. As much as 31.7% of beta-glucuronidase, a marker for lysosomal enzymes, is specifically released by cross-linking the Fc gamma R on PMNL. The generation of O2- is also induced by specifically cross-linking Fc gamma R with Fab and anti-Fab. The data constitute the first formal demonstration that cross-linking of Fc gamma R on PMNL leads to enzyme release and superoxide generation.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

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

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

Protein tyrosine phosphorylation induced via the IgG receptors Fc gamma Ri and Fc gamma RII in the human monocytic cell line THP-1.

We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes.     

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.