Functional interactions of aglycosylated monoclonal anti-D with Fc gamma RI+ and Fc gamma RIII+ cells.

Four human monoclonal antibodies (mAb) to the Rh antigen D were produced in aglycosylated forms by culture of B-cell lines in medium containing tunicamycin (Tm-mAb). Erythrocytes sensitized with these or control mAb were compared in U937 rosette and monocyte chemiluminescence assays to determine Fc gamma receptor I (Fc gamma RI)-mediated functional activity, and in lymphocyte rosette and lymphocyte antibody-dependent cell-mediated cytotoxicity (ADCC) assays to study Fc gamma RIII-mediated binding and lysis. Fc gamma RI-mediated interactions with Tm-mAb were greatly reduced compared with control mAb. All Tm-mAb failed to promote ADCC, although lymphocyte rosette formation was unaltered. The anti-D titre of Tm-mAb and their interaction with mAb JL512 (recognizing an epitope in the CH2 domain) were unchanged. These data suggest that glycosylation of IgG is required for CH2 domain interactions with both Fc gamma RI and Fc gamma RIII, but not for CH3 domain interactions with Fc gamma RIII.     

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.     

Monocyte-bound monoclonal antibodies inhibit the Fc gamma RI-mediated phagocytosis of sensitized red cells: the efficiency and mechanism of inhibition are determined by the nature of the antigen.

Monocyte-binding monoclonal antibodies (mAbs) inhibited the Fc gamma receptor I (Fc gamma RI)-mediated phagocytosis of red cells sensitized with human monoclonal immunoglobulin G (IgG) anti-D (E-IgG) via three distinct mechanisms depending on their specificity. First, all monocyte-binding mAbs tested inhibited the adherence (and hence the phagocytosis) of E-IgG. They also inhibited the binding of fluorescein isothiocyanate (FITC) conjugated IgG anti-D. This inhibition of ligand binding was more efficiently promoted by murine (m) IgG2a than mIgG1 mAbs and presumably involved receptor blockade via the formation of antigen (Ag)-mAb-Fc gamma RI complexes on the monocyte membrane. Monocytes passively sensitized with human monoclonal anti-D (M-IgG) were used in experiments to distinguish between inhibition of ligand binding and inhibition of phagocytosis. In this way, it was shown that mAbs to transmembrane molecules (CD11b/CD18, CD44, and HLA) inhibited the phagocytosis of red cells adherent to M-IgG. Under the same conditions, mAbs to glycosylphosphatidylinositol (GPI) linked molecules (CD14, CD55 and CD59) did not inhibit phagocytosis. These data suggested a second mechanism of inhibition of Fc gamma RI-mediated phagocytosis that involved the cross-linking of a proportion of Fc gamma RI (i.e. those not ligated with IgG anti-D) to molecules which are relatively constrained in the cell membrane. A third mechanism of inhibition was revealed by the use of F(ab')2 fragments of mAb to CD11b which inhibited Fc gamma RI-mediated interactions with E-IgG in a manner that did not involve IgG (Fc) crosslinking or blockade of Fc gamma RI. In this respect, Fc gamma RI-mediated phagocytosis was more susceptible to inhibition than receptor-mediated adherence.     

Soluble Fc gamma receptors II (Fc gamma RII) are generated by cleavage of membrane Fc gamma RII.

This study describes the production of soluble Fc gamma RII by a cell line, D1B1, obtained by transfection of mouse L cells with a murine beta 1 Fc gamma RII cDNA. Upon incubation at 37 degrees C, radioiodinated D1B1 cells release a 39-kDa soluble Fc gamma RII, reacting with the rat anti-mouse Fc gamma RII monoclonal antibody 2.4G2, and binding to mouse IgG2a, IgG2b and IgG1 but not IgG3. In contrast to the transmembrane 50- to 70-kDa receptor, this soluble Fc gamma RII does not react with antibodies directed against a peptide corresponding to the 15 carboxy-terminal intracytoplasmic amino acids of beta Fc gamma RIII. N-Glycosidase F treatment generates a 18-kDa polypeptide. A 32- to 40-kDa soluble Fc gamma RII, which resolves into 18.5- and 20-kDa polypeptides after deglycosylation, was also isolated from the culture medium of unlabeled D1B1 cells. Therefore, this study indicates that soluble Fc gamma RII corresponding to the two extracellular domains of Fc gamma RII are generated by cleavage of membrane Fc gamma RII. Proteolysis occurs most probably at the vicinity of the transmembrane region of the receptor, around amino acids 165 to 180.     

The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.     

The functional activity of human monocytes passively sensitized with monoclonal anti-D suggests a novel role for Fc gamma RI in the immune destruction of blood cells.

The role of Fc gamma RI in the immune destruction of blood cells is uncertain as serum IgG levels are sufficient to competitively inhibit interactions between this high-affinity receptor and sensitized red cells. In the current study, it is proposed that, rather than functioning as a receptor for opsonized red cells, Fc gamma RI might, under appropriate conditions, mediate the passive sensitization (or 'arming') of human macrophages with IgG antibodies resulting in the in vivo destruction of unsensitized cells expressing the corresponding antigen. To examine this hypothesis, Fc gamma RI-bearing human monocytes and U937 cells were first passively sensitized by incubation in vitro with human monoclonal anti-D, and then incubated with D-positive red cells. The uptake of monoclonal anti-D by U937 cells was rapid and, in the presence of 2.5 micrograms/ml IgG1 or IgG3 anti-D, was almost complete after 5 min at 37 degrees. Subsequent incubation of passively sensitized U937 cells in an IgG-free medium for 1 hr at 37 degrees resulted in the loss from the cell surface of approximately 50% cell-bound IgG; the remaining cell-bound IgG was lost more slowly despite repeated washing. In functional assays, passively sensitized monocytes (M-IgG) mediated adherent, phagocytic and chemiluminescent (CL) responses to D-positive red cells. After incubation of M-IgG in 50% v/v fresh normal human serum (FNHS) for 2 hr, sufficient anti-D remained bound to monocytes to promote the adherence of red cells. The adherence and phagocytosis of red cells by M-IgG was enhanced by the simultaneous addition of 50% FNHS, probably owing to the binding of low levels of C3bi to red cells. In contrast, phagocytic and CL responses of unsensitized monocytes to anti-D-sensitized red cells (E-IgG) were abrogated in the presence of 0.25% v/v FNHS, presumably owing to blocking of Fc gamma RI by IgG. It is considered that in vivo, Fc gamma RI may mediate the passive sensitization of macrophages in close proximity with antibody-secreting cells in the reticular network of the splenic cords. Once 'armed' in this way, macrophages may destroy cells expressing the appropriate antigen.     

Reactivity of cloned, expressed human Fc gamma RIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of Fc gamma RIII.

We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.     

Characterization of Fc epsilon RI gamma in human natural killer cells.

We report the characterization of the Fc epsilon RI gamma chain which associates with the transmembrane form of CD16 to form the low affinity receptor for IgG (Fc gamma RIII) expressed on human natural killer (NK) cells. cDNA cloning and sequence analysis of Fc epsilon RI gamma from a polyclonal CD3-CD16+ NK line established that this molecule is identical to Fc epsilon RI gamma previously identified in human basophils as part of a high affinity receptor for IgE. Polymerase chain reaction analysis of Fc epsilon RI gamma gene expression in a series of CD3+CD16- and CD3-CD16+ NK clones reveals that Fc epsilon RI gamma is not directly linked to NK activity since clones of the CD3+CD16- phenotype lack Fc epsilon RI gamma RNA but nevertheless mediate cytotoxicity. Taken together, these results demonstrate that the Fc epsilon RI gamma molecule is expressed in various types within the hematopoietic system as part of multimeric surface receptors involved in different biological functions.     

Functional interactions of red cells sensitized by IgG1 and IgG3 human monoclonal anti-D with enzyme-modified human monocytes and FcR-bearing cell lines

Human monoclonal IgG1 and IgG3 antibodies specific for the Rh antigen D (anti-D) were tested for their ability to promote the binding of D-positive red cells to peripheral blood monocytes and Fc receptor (FcR)-bearing cell lines (U937, K562 and Daudi). Monocyte-mediated antibody-dependent cell-mediated cytotoxicity and metabolic (chemiluminescent) responses were also determined. By comparing the activity of different cell lines in rosette assays, and by using murine myeloma IgG2a and IgG1 to block FcRI and FcRII respectively, these functional interactions of sensitized red cells (E-IgG1 and E-IgG3) with monocytes or cell lines were shown to be mediated predominantly and perhaps solely by FcRI. E-IgG3 bound to human monocytes and cell lines to a greater extent than E-IgG1. Rosette formation by E-IgG3 was relatively less susceptible to inhibition by fluid-phase murine IgG2a than was rosette formation by E-IgG1. These findings may be due to the long hinge region of IgG3 which enables it to bridge the gap between two negatively charged cells more efficiently than IgG1. Consistent with this hypothesis was the greatly increased rosette formation achieved by treating monocytes or U937 cells with neuraminidase or bromelain, procedures shown to reduce the zeta potential of these cells. The lytic and metabolic activities of untreated human monocytes were also greater towards E-IgG3 than E-IgG1, red cell binding being a prerequisite for these responses. However, after pretreatment of monocytes with neuraminidase, these responses were greater with E-IgG1 than with E-IgG3. Further, the addition of polybrene to non-specifically enhance cell to cell binding also resulted in greater lysis and chemiluminescence with E-IgG1 than with E-IgG3. These results indicate that, although E-IgG3 are more effective than E-IgG1 in promoting red cell binding to monocytes, E-IgG1 are more efficient at activating the lytic and metabolic processes providing the steric disadvantages of the shorter hinge region of cell-bound IgG1 are circumvented.     

Murine epidermal gamma/delta T cells express Fc gamma receptor II encoded by the Fc gamma R alpha gene.

Short-term bulk cultures and some long-term clones and lines of murine T cell receptor (TcR) gamma/delta-bearing epidermal T cells (dEC) were found to express an Fc gamma receptor II (Fc gamma RII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the Fc gamma RII expressed on dEC is encoded solely by the Fc gamma R alpha gene. While all the various cultured dEC cell populations analyzed exhibit lectin-dependent cellular cytotoxicity, only those which expressed Fc gamma R alpha were also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of Fc gamma R alpha on mouse natural killer cells support an essential role for Fc gamma R alpha in ADCC and extend an analogy with surface CD16 (Fc gamma RIII) expression and ADCC in human natural killer cells and peripheral TcR gamma/delta T cells.     

A polymorphic Fc receptor for mouse IgG2b on human B cells and monocytes.

With respect to murine (m)IgG1 anti-CD3 monoclonal antibody (mAb), a polymorphic mitogenic response of peripheral blood mononuclear cells (PBMC) has been described which is caused by polymorphism of monocyte Fc gamma RII, and which defines high responders to mIgG1 (mIgG1-HR, approximately 70% of normal individuals) and low responders (mIgG1-LR). PBMC also exhibit a polymorphic mitogenic response to mIgG2b anti-CD3 mAb. In the present study 18 out of 550 individuals (3%) were mIgG2b-HR. Purified monocytes from mIgG2b-HR were able to support the mitogenic response to mIgG2b anti-CD3 mAb of purified T cells from mIgG2b-LR. Surprisingly, a significant mitogenic response to mIgG2b anti-CD3 mAb remained after vigorous depletion of monocytes from mIgG2b-HR PBMC. Apparently B cells are responsible for this accessory function since Epstein-Barr virus (EBV)-transformed B cells from mIgG2b-HR (but not from mIgG2b-LR) were able to support T-cell proliferation induced by mIgG2b anti-CD3 mAb. Only EBV B cells from mIgG2b-HR were able to form rosettes with human red blood cells (RBC) that had been sensitized with mIgG2b anti-glycophorin A mAb (EA-mIgG2b). These EBV B cells did not express Fc gamma RI or Fc gamma RIII, and could bind some but not all anti-Fc gamma RII mAb. The mitogenic response to mIgG2b anti-CD3 was not inhibited by any of the anti-Fc gamma RII mAb. From these studies we conclude that a polymorphic Fc receptor is expressed on human B cells and monocytes, which cross-reacts with mIgG2b. This receptor is different from Fc gamma RI and Fc gamma RIII, and apparently also from Fc gamma RII.     

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.     

Role of neutrophil Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes.

The four subclasses of IgG have different biological activities associated with their Fc regions. Fc gamma receptors on leucocytes (Fc gamma R) mediate binding and phagocytosis of opsonized particles. Two structurally and functionally distinct allelic polymorphisms of the Fc gamma R have been defined: the H/R131 forms of Fc gamma RIIa (CD32), and the neutrophil antigen 1 (NA1)/NA2 forms of Fc gamma RIIIb (CD16). In this study the activities of allotypes of CD16 are analysed with antibacterial IgG subclass antibodies and with IgG1 and IgG3 anti-Rhesus D, and the activities of CD32 with IgG1 and IgG3 anti-Rhesus D. With respect to the allotypes of CD16, polymorphonuclear leucocytes (PMN) homozygous for Fc gamma RIIb-NA2 exhibited a lower (21-25%) IgG1-mediated phagocytosis of Staphylococcus aureus strain Wood (STAW), Haemophilus influenzae type b (Hib), and Neisseria meningitidis group B (NMen) than IIIb-NA1 PMN. The difference was apparent only when the micro-organisms were opsonized in the absence of complement, and was furthermore enhanced (34-52%) upon blockade of Fc gamma RIIa. In addition, monoclonal IgG3 anti-D-mediated rosette formation and phagocytosis was consistently found to be lower (16%) with Fc gamma RIIIb-NA2 than with IIIb-NA1 PMN. For the allotypes of CD32 we now show that IgG3 anti-D sensitized erythrocytes formed more (50%) rosettes and were phagocytosed at a higher rate with PMN carrying Fc gamma RIIa-H131 than with PMN carrying IIa-R131. Heterozygous Fc gamma RIIa-H/R131 PMN exhibited intermediate phagocytic activity in this respect. This study illustrates a critical role of Fc gamma R allotypes in functional interactions with biologically relevant IgG subclass antibodies.     

Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.     

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.     

Receptor specific probes for the study of Fc gamma receptor specific function.

Human leukocytes express three distinct families of receptors for the Fc region of IgG (Fc gamma R). We have prepared erythrocytes (E) coated with monoclonal anti-Fc gamma R antibodies for the study of receptor specific phagocytosis using biotin and streptavidin. In this technique, both the E and the monoclonal antibody (mAb) are biotinylated and coupling of the mAb to the E occurs through the use of streptavidin. The same biotin/streptavidin principle was used to prepare E coated with human IgG. Using this technique, receptor specific probes or probes coated with natural ligand (IgG) can be prepared rapidly with the use of small amounts of mAb or IgG. Finally, we have used these receptor specific probes to demonstrate that all three families of Fc gamma R (Fc gamma RI, Fc gamma RII and Fc gamma RIII) expressed on human monocytes and human macrophages are phagocytic receptors.     

T-cell hybridoma co-expressing Fc receptors for different isotypes. I. Reciprocal regulation of Fc alpha R and Fc gamma R expression by IgA and interferon.

To clarify the co-expression phenomenon of T-cell Fc receptors (FcR) specific for different isotypes on the clonal level, a murine hybridoma clone T2D4 was studied. T2D4 cells originally reported to bear FcR for IgG (Fc gamma R) and to release a Fc gamma R-related T-cell factor binding to IgG (immunoglobulin binding factor; IBF) proved to have also the receptor for IgA. The binding of IgA was detected by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) after preincubation of T2D4 cells with MOPC 315 IgA having anti-TNP activity, or directly with TNP-ORBC sensitized with MOPC 315 IgA. While the binding of MOPC 315 IgA was competed for by IgA but not by IgG2A nor IgG2B, IgA failed to inhibit the rosette formation of the cells with ORBC sensitized with rabbit IgG antibody (EA ox gamma), proving that T2D4 cells express FcR specific for IgA (Fc alpha R) in addition to Fc gamma R. Co-expression of both receptors on the same cell surface was demonstrated by a double rosette technique using TNP-quail red blood cells (TNP-QRBC) and EAox gamma. Fc alpha R activity of the cells was completely abrogated by 15 min. incubation with 0.1 mg/ml trypsin, whereas Fc gamma R was resistant even to 1 mg/ml trypsin. The expression of Fc alpha R was augmented (up-regulation) by IgA at the concentration above 300 micrograms/ml and inhibited (down-regulation) by 1000 u./ml of murine beta-interferon (beta-IFN). Conversely, the expression of Fc gamma R was down-regulated by IgA and up-regulated by alpha-IFN. Thus, Fc gamma R and Fc alpha R are co-expressed and reciprocally regulated on these cell lines. The possible co-production of IBF and the Fc alpha R-related binding factor specific for IgA is discussed.     

Role of activatory Fc gamma RI and Fc gamma RIII and inhibitory Fc gamma RII in inflammation and cartilage destruction during experimental antigen-induced arthritis.

IgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(Fc gamma RI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR gamma-chain-deficient mice that lack functional Fc gamma RI and Fc gamma RIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory Fc gamma RI and Fc gamma RIII and inhibitory Fc gamma RII in inflammation and functional cartilage damage in knee joints with AIA using Fc gamma RI-, Fc gamma RII-, and Fc gamma RIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in Fc gamma RI-/- and Fc gamma RIII-/- but higher in Fc gamma RII-/- if compared to controls. Joint swelling as measured by (99m)Tc uptake at days 1, 3, and 7 was similar in Fc gamma RI-/- and Fc gamma RIII-/- mice and significantly higher in Fc gamma RII-/-. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in Fc gamma RI-/- and Fc gamma RIII-/- and significantly higher (230% and 340%) in Fc gamma RII-/- mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in Fc gamma RI-/- (79 to 87% and 87 to 88%, respectively) and comparable in Fc gamma RIII-/-. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in Fc gamma RII-/- mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in Fc gamma RII-/- (222% and 186%, respectively) but not in Fc gamma RI-/- or Fc gamma RIII-/- mice. These results suggest that during T-cell-mediated AIA, Fc gamma RI and Fc gamma RIII act in concert in acute and chronic inflammation whereas Fc gamma RI is the dominant FcR involved in severe cartilage destruction. Fc gamma RII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion.