Human IgG isotype-specific amino acid residues affecting complement-mediated cell lysis and phagocytosis

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophen-acetyl (NIP) mouse/human immunoglobulin (Ig)G4 chimeric molecules with altered amino acid residues in the C_H2 domain. Three mutants are described: Gln-268 is substituted by His in γ4 Q268H, Ser-331 is substituted by Pro in γ4 S331P, and in γ4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by FcγRII- and FcγRIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, γ4 Q268H was inactive, but both γ4 S331P and γ4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants γ4 S331P and γ4 Q268H/S331P were both active only when complement was introduced. γ4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by FcγRII and/or FcγRIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect FcγRII/FcγRIII-mediated phagocytosis. In this manner the mutants γ4 S331P and γ4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.     

Opsonophagocytic Activity Induced by Chimeric Antibodies of the Four Human IgG Subclasses With or Without Help from Complement

The Opsonophagocytic activity of the four human IgG subclasses was studied using chimeric mouse-human antibodies with specificity for the hapten NIP. As target cells we used haptenized sheep red blood cells and N. meningitidis , labelled with different amounts of hapten. We used polymorphonuclcar leucocytes (PMN) as effector cells to measure respiratory burst (RB), and U937 to measure phagocytosis/rosette formation. When the target cells were opsonized with antibody only, and PMN used as effector cells, lgG3 was highly efficient, while IgG1 revealed an intermediate activity and IgG2 and IgG4 were negative. The same pattern among the subclasses was obtained in the presence of complement source, when target cells with low hapten concentration were used. However, at high cpitope concentration on the target cells, in the presence of complement source, IgG2 was highly active. while IgG4 was still negative or only slightly positive. When U937 were used as effector cells and complement was omitted, IgG1, IgG3 and lgG4 all revealed high phagocylic/rosette-forming activity, while IgG2 was negative. When the target cells were opsonized with antibody and complement, the phagocytic/rosette-forming activity was often suppressed. Our results reveal that all four human IgG subclasses possess Opsonophagocytic capacity, but with different requirements concerning complement and Fc Rs. They also enlighten us as to how IgG2 might perform its protective effect against harmful bacteria displaying high density of carbohydrate epitopes on their outside surface.     

A standardized conversion of IgG antibody to bispecific form with inversely altered affinities for Fcγ-receptors II and III

This work aims to enhance killing of antibody-coated human tumor cells by altering the antibody's affinity for two effector-cell Fcγ-receptors (FcγR). Affinity for the activating FcγRIII is raised, affinity for the inhibitory FcγRIIB is reduced, with the ratio between the two association constants increasing >1000-fold. We use as a standard tool the Fab′γ from a monoclonal antibody specific for human FcγRIII. This Fab′γ module is bonded to an IgG antibody by a tandem thioether link running between cysteine residues in the hinge vicinity of each protein, thus forming a bispecific FabIgG construct. Simultaneously, effector function of the IgG module is adjusted by leaving its hinge open and adding negative charges. FabIgG constructs derived from the chimeric IgG1 antibody rituximab show the following properties. (1) The titer for antibody-dependent cellular cytotoxicity is enhanced by 12-100-fold, reflecting the affinity of the Fab′γ module for effector-cell FcγRIII. (2) Two functions of the construct's Fcγ, activation of complement and prolonged metabolic survival, are moderately reduced. (3) In contrast, affinities of the Fcγ for all FcγR are severely reduced, with two anticipated consequences. First, attacks by macrophages on antibody-coated cells are favored by reduced engagement of the inhibitory FcγRIIB. Second, reduced engagement of activating FcγR by the Fcγ lowers the probability of untoward crosslinkings of FcγR, which have been shown to provoke toxicity. If the Fab′γ module possesses human constant regions, the linkage strategy requires prior genetic deletion of at least one cysteine residue. With both Fab′γ and IgG modules available, FabIgG can be prepared in 35 h.     

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.     

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti‐CD25 monoclonal antibody is mediated by FcγRIII+ phagocytes

Depletion of CD4⁺CD25⁺FoxP3⁺ Treg using PC61 mAb (anti‐murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated ～70% of CD4⁺FoxP3⁺ cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcγ receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcγ receptor (FcγR)III^?/? mice were resistant to Treg depletion. FcγRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab‐dependent‐cellular‐cytotoxicity and Ab‐dependent‐cellular‐phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab‐dependent‐cellular‐phagocytosis of CD25⁺ cells by WT or FcγRIIB^?/?, but not FcγRIII^?/?, macrophages. Altogether these data demonstrate the critical role of FcγRIII⁺ phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.     

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.     

Activating and inhibitory Fcγ receptors in immunotherapy: being the actor or being the target

Membrane Fcγ receptors (FcγRs) can act either as potent activators of effector cell functions or as inhibitors of receptor-mediated cell activation following engagement by IgG antibodies bound to their target molecules. The remarkable ability of activating FcγRs to trigger antibody-dependent cellular cytotoxicity, cytokine release and phagocytosis/endocytosis followed by antigen presentation has stimulated the development of a number of therapeutic monoclonal antibodies whose Fc regions have been engineered to optimize their effector functions, mostly their killing activities. Conversely, the demonstration that inhibitory FcγRs can block or downmodulate effector functions has led to the concept that targeting these receptors is of interest in a number of pathologies. The use of bispecific antibodies leading to the crosslinking of FcγRIIB with activating receptors could induce immunomodulation in autoimmune or allergic diseases. Alternatively, the use of cytotoxic/antagonist anti-FcγRIIB antibodies could kill FcγRIIB-positive tumor cells or prevent the downmodulation of activating receptors. Thus, antibodies engineered to preferentially target activating or inhibitory FcγRs are currently being designed for therapeutic use.     

Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D‐related (HLA‐DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA‐DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2 weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over‐represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.     

Affinity of the interaction between Fcgamma receptor type III (FcγRIII) and monomeric human IgG subclasses. Role of FcγRIII glycosylation

Binding of the Fc region of IgG antibodies to low affinity Fcγ receptors (FcγR) triggers important effector functions in the immune system. The type IIIb FcγR (FcγRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of FcγRIIIb (sFcγRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore^TM instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab′)₂) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K_A) of 1.3 ± 0.6 × 10⁶ M^?1 and 2.6 ± 0.4 × 10⁵ M^?1, respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 ± 0.2 × 10⁶ M^?1), whereas that for human IgG3 was twofold higher (4.2 ± 0.4 × 10⁵ M^?1). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 ? IgG4 ? IgG2. Thus, the extracellular polypeptide of FcγRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.     

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.     

FcγRIa–γ-chain complexes trigger antibody-dependent cell-mediated cytotoxicity (ADCC) in CD5+ B cell/macrophage IIA1.6 cells

Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding α-chains, combined with accessory signalling (γ-, β-, or ζ-) chains. The myeloid class I receptor for IgG (FcγRIa) has been shown to be dependent on the FcR γ-chain for surface expression in vivo. In this study we assess the capacity of FcγRIa–γ-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcγRIa receptor complex. Both the FcR γ-chain and the FcγRIIa–ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcγRIa complex. The capacity of FcγRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered ‘professional phagocyte’ functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5⁺ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.     

Reactive oxygen product formation after Fcγ receptor-mediated neutrophil activation by monomeric mouse IgG2a: implications for the generation of first dose effects after OKT3 treatment

In the present study, we provide evidence that IgG2a monoclonal antibody (mAb) OKT3 is able to induce reactive oxygen intermediate (ROI) formation in polymorphonuclear leukocytes (PMN) when FcγRIIIB as well as FcγRII are bound concomitantly. Inhibition of binding to either FcγR by specific mAb (3G8 or IV. 3, respectively) resulted in complete abrogation of the OKT3-induced respiratory burst. The effect of OKT3 was independent from its specificity and thus also from its T cell-activating property, since nonbinding IgG2a isotype controls induced similar amounts of ROI. The IgG2b mAb BMA031 as well as the respective nonbinding isotype control were only minimally effective. With regard to the potential role of PMN activation in inflammation and tissue damage, our findings offer an extended explanation for the generation of initial adverse reactions to OKT3. Thus, one might speculate that the concerted action of cytokines liberated after its administration, what may lead to margination of leukocytes, and activation of PMN via FcγR might produce first-dose reactions to OKT3 by directing radical-mediated damage against the endothelium.     

The interaction of ruminant IgG with receptor type II for IgG on human phagocytes

The interaction of ruminant IgG with human phagocytes was assessed using Fc receptor (FcR)-mediated ingestion and the triggering of a respiratory burst as effector functions indicative of receptor-specific interaction. In monomeric form, ruminant IgG was three to five orders of magnitude less potent than homologous IgG in inhibiting FcR-specific phagocytosis by monocytes. However, when attached to tanned sheep erythrocytes (Es-T), ruminant IgG was opsonic, as it promoted enhanced phagocytosis of Es-T, comparable to ingestion of rabbit IgG-coated Es. This phagocytosis was inhibitable by high concentrations of human IgG in the fluid phase. Moreover, Es-T precoated with ferritin could be opsonized to a similar degree by anti-ferritin IgG from rabbit and cow. However, only bovine IgG1, but not IgG2, was opsonic. Bovine and goat IgG of some, but not other, suppliers were inactive. Similar results were obtained by measuring the respiratory burst triggered by heat-aggregated IgG, using a luminol-enhanced chemiluminescence assay. Thus, human IgG and ruminant IgG stimulated monocytes and, to a lesser extent, polymorphonuclear leucocytes (PMN), to generate CL. Depending on the manufacturer, some preparations of bovine and goat IgG were inactive, and bovine IgG2 failed to induce CL. These findings prove that certain ruminant IgG preparations, including bovine IgG1 interacting weakly with homologous PMN and monocytes, do interact with human PMN, monocytes and macrophages in a FcR-specific manner when offered in complexed form. Inhibition studies suggest that bovine IgG1 interacts mainly with human FcR type II. In contrast, bovine IgG2, regarded as cytophilic for homologous PMN, fails to interact with human PMN, monocytes and macrophages.     

Crystal structure of deglycosylated human IgG4-Fc

The Fc region of IgG antibodies, important for effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, contains an oligosaccharide moiety covalently attached to each C_H2 domain. The oligosaccharide not only orients the C_H2 domains but plays an important role in influencing IgG effector function, and engineering the IgG-Fc oligosaccharide moiety is an important aspect in the design of therapeutic monoclonal IgG antibodies. Recently we reported the crystal structure of glycosylated IgG4-Fc, revealing structural features that could explain the anti-inflammatory biological properties of IgG4 compared with IgG1. We now report the crystal structure of enzymatically deglycosylated IgG4-Fc, derived from human serum, at 2.7 Å resolution. Intermolecular C_H2-C_H2 domain interactions partially bury the C_H2 domain surface that would otherwise be exposed by the absence of oligosaccharide, and two Fc molecules are interlocked in a symmetric, open conformation. The conformation of the C_H2 domain DE loop, to which oligosaccharide is attached, is altered in the absence of carbohydrate. Furthermore, the C_H2 domain FG loop, important for Fcγ receptor and C1q binding, adopts two different conformations. One loop conformation is unique to IgG4 and would disrupt binding, consistent with IgG4's anti-inflammatory properties. The second is similar to the conserved conformation found in IgG1, suggesting that in contrast to IgG1, the IgG4 C_H2 FG loop is dynamic. Finally, crystal packing reveals a hexameric arrangement of IgG4-Fc molecules, providing further clues about the interaction between C1q and IgG.     

Insights into the effector functions of human IgG3 in the context of an antibody targeting transferrin receptor 1

The transferrin receptor 1 (TfR1) is involved in cellular iron uptake and regulation of cell proliferation. The increased expression of TfR1 observed in malignant cells, compared to normal cells, together with its extracellular accessibility, make this receptor an attractive target for antibody-mediated cancer therapy. We have developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1), which shows anti-tumor activity against certain malignant B cells in vitro through TfR1 degradation and iron deprivation, and in vivo through a mechanism yet to be defined. To further explore potential mechanisms of action of ch128.1, we examined its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). We now report that ch128.1 is capable of mediating ADCC and CDC against malignant B cells, which is consistent with its ability to bind FcγRI, FcγRIIIa, and the complement component C1q. To delineate the residues involved in these effector functions, we developed a panel of three constructs with mutations in the lower hinge region and C_H2 domain: 1) L234A/L235A, 2) P331S, and 3) L234A/L235A/P331S. The triple mutant consistently displayed a significant reduction in ADCC, while the L234A/L235A mutant exhibited less reduction in ADCC, and the P331S mutant did not show reduced ADCC. However, all three mutants exhibited impaired binding to FcγRI and FcγRIIIa. These results suggest that all three residues contribute to ADCC, although to different degrees. The P331S mutant showed drastically decreased C1q binding and abolished CDC, confirming the critical role of this residue in complement activation, while the other residues play a less important role in CDC. Our study provides insights into the effector functions of human IgG3 in the context of an antibody targeting TfR1.     

Role of FcγRIIIA (CD16) in IVIg-Mediated Anti-Inflammatory Function

The mechanism for anti-inflammatory action of intravenous immunoglobulin (IVIg) in the treatment of autoimmune and inflammatory diseases involves IgG Fc receptors (FcγR). Although the inhibitory FcγRIIB plays an important role in IVIg action, FcγRIIIA has recently been identified as another major anti-inflammatory actor. Interaction of FcγRIIIA with uncomplexed IgG1 or IVIg, or with bivalent anti-FcγRIII F(ab’)₂ dampened calcium responses, ROS production, endocytosis and phagocytosis, induced by heterologous activating receptors. This inhibitory action required the inhibitory configuration of the ITAM motif (ITAMi) present within the FcγRIII-associated FcRγ subunit. This allowed SHP-1 recruitment and formation of intracellular inhibisome clusters containing FcγRIII and the targeted activating receptor. Therefore, IVIg functionally interact with FcγRIIIA inducing ITAMi signaling which can prevent development of autoimmune and inflammatory disorders independently of FcγRIIB. This new mechanism of action for IVIg reveals a therapeutic potential for FcγRIIIA targeting in inflammatory diseases.     

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.