Ligand binding specificities and signal transduction pathways of Fc gamma receptor IIc isoforms: the CD32 isoforms expressed by human NK cells.

We recently reported that human NK cells express, in addition to CD16 [Fcgamma receptor (FcgammaR) IIIA], a second type of FcgammaR, namely CD32 (FcgammaRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcgammaRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcgammaRIIc-specific isoforms isolated from NK cells, namely FcgammaRIIc1 and FcgammaRIIc3, which differ in their cytoplasmic tails. The ligand binding specificity for both murine and human IgG isotypes was found to be similar to that observed for FcgammaRIIb isoforms. Immunoprecipitation studies of FcgammaRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcgammaRIIc1, and a protein of around 32 kDa for FcgammaRIIc3. Signal transduction studies performed on FcgammaRIIc1-expressing Jurkat cells indicated that this molecule is functional, i. e. capable of Ca2+ mobilization and activation of Lck, Zap-70 and Syk protein tyrosine kinases, although the CD3 zeta chain was not found to functionally associate with FcgammaRIIc1. In contrast, FcgammaRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcgammaRIIc3. These studies demonstrate the functional activity of FcgammaRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.

Beta1 integrin-mediated activation of focal adhesion kinase and its association with Fyn and Zap-70 in human NK cells.

Adhesive interactions mediated by cell surface receptors have been shown to induce signal transduction pathways that regulate changes in cellular function. We have reported recently that fibronectin (FN) receptors, alpha4beta1 and alpha5beta1 integrins, on NK cells transduce transmembrane signals leading to tyrosine phosphorylation of 60-, 70-, and 120-kDa proteins. In the current study, we have identified a 120-kDa phosphoprotein as the focal adhesion kinase (p125FAK), a structurally unique nonreceptor protein tyrosine kinase that localizes to focal adhesions. Activity of p125FAK was induced by adhesion of NK cells to plastic-immobilized FN, by cross-linking of cell surface-bound FN or FN fragments, FN120 or FN40, with anti-FN mAb, or by cross-linking of alpha4beta1 or alpha5beta1 integrins with alpha-chain-specific Abs. We also observed that enhanced in vitro kinase activity was associated with immunoprecipitates of alpha4beta1 or alpha5beta1 integrins from lysates of FN-adherent NK cells as compared with BSA-treated NK cells. In addition to p125FAK activity, FN-induced kinase activity was also found to be mediated by Fyn, Lyn, and Zap-70, as assessed by in vitro phosphorylation of the immunoprecipitated kinases in the presence of [gamma-32P]ATP. Clustering of FN receptors on NK cells by agonists such as immobilized FN or alpha4- or alpha5-specific Abs also induced association of Fyn and Zap-70 with p125FAK. Our observations indicate that activation and phosphorylation of p125FAK as well as Zap-70 and certain kinases of the src family play an important role in formation of active signaling complexes in response to triggering via beta1 integrins on NK cells. These results also suggest the existence of cross-talk or points of convergence between the beta1 integrin-mediated and other receptor-signaling pathways.

Physical and functional association of Fc mu receptor on human natural killer cells with the zeta- and Fc epsilon RI gamma-chains and with src family protein tyrosine kinases.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

Divergent phosphotyrosine signaling via Fc gamma RIIIA on human NK cells.

Previous studies have indicated that interaction of Fc gamma RIIIA on natural killer (NK) cells with various immunoglobulin ligands or monoclonal antibodies (mAbs) can have either stimulatory or inhibitory effects on cytotoxic activity, but the basis for such divergent functional effects has been unclear. We report here that stimulation of NK cells via Fc gamma RIIIA by monoclonal anti-human CD16 (3G8), monomeric IgG (mIgG), or dimeric IgG (dIgG), used either alone or cross-linked by secondary Ab (goat anti-mouse IgG or goat anti-human IgG), resulted in different phosphotyrosine protein patterns. These results suggest that distinct substrates are involved in signaling pathways activated via various agonists of the same triggering surface molecule. Three protein tyrosine kinases, i.e., LCK, LYN, and SYK, were activated by occupancy of the Fc gamma RIIIA, and only LCK activity showed a divergence in effects induced by the various ligands, with strong autophosphorylation induced by mIgG upon cross-linking. We observed no ligand-induced activation of p59fyn, p60c-src, or p62c-yes, src-related protein tyrosine kinases which are expressed in NK cells. Activity of phosphatidylinositol 3-kinase (PI 3-kinase) induced by receptor-specific antibodies or IgG ligands had different kinetics while the level of cytoplasmic free calcium was greatest upon 3G8-induced stimulation. Although the changes in kinase activities associated with Fc gamma RIIIA-mediated regulation of NK cells are complex, it appears that the patterns induced varied with the nature of the ligand and the direction of the regulation of NK activity.

Induction of protein tyrosine phosphorylation in human natural killer cells by triggering via alpha 4 beta 1 or alpha 5 beta 1 integrins.

Recent studies have shown that cell-surface integrins expressed on platelets, fibroblasts, or carcinoma cell lines serve not only as adhesion receptors that connect the extracellular matrix to the cytoskeleton, but also as signal-transducing molecules involved in altering cellular patterns of tyrosine phosphorylation. In this present report we provide evidence that adhesion of freshly purified human natural killer (NK) cells to fibronectin (FN) induces tyrosine phosphorylation of intracellular proteins of approximate molecular mass of 60, 70, and 120 kD. Increases in phosphorylation induced by NK cell binding to immobilized FN were partially blocked by EILDV- (CS-1) or RGD-containing peptides, which compete specifically for a distinct binding site for either alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, within the FN molecule. The presence of either one of the inhibitory peptides alone inhibited tyrosine phosphorylation primarily during short-term (30 minutes) and, to a lesser extent, during long-term (2 to 3 hours) periods of adhesion. These observations indicate that triggering either via alpha 4 beta 1 or alpha 5 beta 1 integrins, which are constitutively expressed on NK cells, induces protein tyrosine phosphorylation. Moreover, FN fragments of 40 or 120 kD, known to contain the binding sites for alpha 4 beta 1 or alpha 5 beta 1 integrins, respectively, used as immobilized substrates for NK cell adhesion, were able to initiate tyrosine kinase activity. The induced tyrosine phosphorylation was observed mainly on intracellular proteins of greater than 50 kD molecular weight. We have identified a 70-kD tyrosine phosphoprotein as paxillin, a cytoskeletal-associated tyrosine kinase substrate previously identified in fibroblasts and shown to localize to focal adhesions. Thus, interaction of NK cells with immobilized extracellular matrix glycoproteins required for migration and extravasation of these cells involves activation of intracellular protein tyrosine kinases and tyrosine phosphorylation of cytoskeleton-associated protein, paxillin, which may play a role in signaling between beta 1 integrins and the underlying cytoskeleton.

Divergent signal transduction pathways and effects on natural killer cell functions induced by interaction of Fc receptors with physiologic ligands or antireceptor antibodies.

Natural killer (NK) cells express receptors that bind to the Fc portion of immunoglobulin molecules (FcR), and their function can potentially be modulated positively or negatively by such receptor:ligand interactions. This review discusses recent developments in the understanding of expression of various forms and isoforms of FcRs by NK cells, and the sequelae of binding of physiologic ligands. Particular attention is paid to the significance of FcR:Ig interactions in both activating and inactivating NK cells under various conditions.

Regulation of human natural cytotoxicity by IgG. IV. Association between binding of monomeric IgG to the Fc receptors on large granular lymphocytes and inhibition of natural killer (NK) cell activity.

The regulation of human natural killer (NK) activity by IgG described previously by us depends on the ability of cytophilic molecules of monomeric IgG (mIgG) to inhibit the subsequent killing of NK-sensitive targets. Highly purified NK cells obtained from human peripheral blood are able to directly bind mIgG as well as antigen-complexed IgG through its Fc region. The demonstration that NK cells bear labile cytophilic IgG, a property which usually has been attributed to L cells, indicates that mIgG-induced inhibition of NK activity is mediated by direct interactions between the inhibitory ligand and cytotoxic effector cells. The Fc receptor (FcR) mediating downregulation of NK cytotoxicity appeared to be FcR gamma III, previously found to be selectively expressed on NK cells and granulocytes. In studies of unidirectional cross-inhibition of mIgG binding to NK cells by various anti-CD16 monoclonal antibodies, binding characteristics of mIgG or complexed IgG were similar. Thus, the FcR gamma III for mIgG appear to be indistinguishable from receptors responsible for binding of polymeric IgG on human NK cells. The negative regulation of NK activity by mIgG was not attributable to inhibition of conjugate formation between effector cells and K532 targets, but rather to inhibition of a post-binding event involved in killing of conjugated targets. The data presented suggest that the Fc gamma RIII on human NK cells can either mediate killing against IgG antibody-coated target cells or, upon interaction with cytophilic monomeric ligand in soluble form, induce inhibition of NK activity.

Not only quantitative but also qualitative changes of serum immunoglobulins in diabetes mellitus.

To extend previous observations on the quantitative changes of IgA and other serum Ig in diabetics, additional immunochemical investigations were carried out in 96 patients, 63 males and 33 females, mean age 43.5 +/- 15.7 years, 51 with type 1 (insulin-dependent) and 45 with type 2 (non-insulin-dependent) diabetes. The immunological data were correlated with the clinical-metabolic aspects. In the whole group, the IgA level was increased (144.1 +/- 57.2 I.U.). Significant differences were recorded with respect to age for IgG, to age and diabetes type for IgA, to sex for IgM. Qualitative Ig changes, reflecting disturbances of molecular structure, mainly for IgG, seldom for IgM, but never for IgA, were observed in 20% of the patients with both types of diabetes, more seldom in cases with long disease duration. The IgG with qualitative changes were purified and their functional capacity of inhibiting the natural cytotoxic activity (NK) was tested in comparison with that induced by pretreatment of the effectory cells with normal IgG. Some of these modified IgG showed a reduced capacity of inhibiting the NK activity. These data confirm the existence of certain quantitative changes of the main serum Ig in diabetics and reveal the presence of qualitative disorders of the IgG molecules, with consequences on their functionality.

Expression of Fc mu receptors on human natural killer cells.

Fc receptors for IgG (CD16) have been described as the only type of immunoglobulin receptor on large granular lymphocytes (LGL). However, the ability of natural killer (NK) cells to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of monoclonal or polyclonal IgM and the inhibition of NK activity by highly purified IgM could not be explained on the basis of FcR for IgG. In order to directly assess the expression of Fc receptors for IgM (Fc mu R), NK cells were treated with human polyclonal IgM, and its binding was visualized by a direct anti-globulin rosette assay with identification of rosette-forming LGL on Giemsa-stained smears. The data indicated that a high proportion of LGL (up to 68%) were Fc mu R-positive cells. However, this percentage varied depending on the IgM preparation (polyclonal or monoclonal), the indicator reagent used for the rosette assays, and the cell preparations studied. Two-color flow cytometry of human nonadherent lymphocyte preparations confirmed the presence of CD56+IgM+ cells, which represented from 43 to 78% of CD56+ cells. Flow cytometry was also performed using highly enriched preparations of human NK cells (the mean percentage of CD3-CD56+ cells was 84%). Up to 88% of purified NK cells bound FITC-labeled monoclonal IgM at a saturating concentration. By indirect immunofluorescence, from 34 to 62% of NK cells purified from the peripheral blood of normal donors were able to bind polyclonal IgM. Similar results were obtained with LGL from a patient with NK lymphoproliferative disease. Thus the presence of Fc mu R on a majority of human NK cells was demonstrated by different techniques, using unseparated peripheral blood mononuclear leukocytes, purified normal NK cells, and also LGL from a patient with NK lymphoproliferative disease.

Inhibition of human natural killer cell activity by polyclonal IgM.

Pretreatment of effector cells with normal human IgM induced strong dose-dependent inhibition of NK activity. The degree of inhibition by normal IgM was stronger than that induced by monomeric IgG, which has previously been reported to be a negative regulator of NK activity. For 100% inhibition, 1.1 x 10(-6) M of IgM was required, whereas 66.6 x 10(-6) M of IgG was needed to abolish NK activity. This inhibitory property of polyclonal IgM appeared to be localized in the Fc region of the molecule, and also was significantly reduced upon mild reduction of disulfide bonds. Monoclonal IgM purified from sera of five patients with Waldenström's macroglobulinemia and tested in parallel with normal IgM lacked or had a decreased capacity to inhibit the cytotoxic reaction. As with IgG, IgM interfered mainly with the lytic event, after binding of effector cells to target cells. The inhibition by IgM appeared to be a direct effect on NK cells, since similar effects were observed with purified large granular lymphocytes as with non-adherent lymphocytes. These results indicate a new mechanism for negative regulation of NK cells and suggest the presence of Fcmu receptors on these effector cells.

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon.

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab')2 fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A2 and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoyl-phorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.

Regulation of human natural cytotoxicity by IgG. III. Interaction between negative regulation by monomeric IgG and positive regulation by interferons of different types.

Our prior reported results have demonstrated the dose-dependent inhibition of human natural killer (NK) cell activity upon treatment of peripheral blood mononuclear cells (PBMC) with monomeric IgG (mIgG) prior to the cytotoxic assay. In the present study, the combined effects on NK activity of human interferon (IFN) of each of the three types and mIgG, respectively, were determined. NK cells incubated with IFN alpha or IFN beta had augmented cytotoxicity against K562 target cells but remained responsive to negative regulation by mIgG. PBMC treated with human recombinant IFN gamma had unchanged cytotoxic activity but became partially resistant to suppression by mIgG. This ability of IFN gamma to interfere with the negative regulation of NK activity by cytophilic mIgG was seen when the cytokine was preincubated with effector cells prior to, simultaneously with, or after their exposure to inhibitor protein. These data provide some clues regarding the possible biological significance of the mIgG-induced down-regulation of NK cells which, when required for host protection, might be appreciably reversed or blocked by IFN gamma produced by NK cells or T cells in response to various agents.

Anti-phospholipase C antibodies inhibit the lectin-induced proliferation of human lymphocytes.

A novel approach was used to assess the role of phosphoinositide hydrolysis in the mitogenic action of phytohemagglutinin (PHA) or concanavalin A (ConA). The treatment of human peripheral blood leukocytes (PBL) with monospecific antibodies against phospholipase C (PLC) produced a dose-dependent inhibition (up to 100%) of PHA (10 micrograms/ml) or ConA (25 micrograms/ml) proliferative effects. Thus, the activation of membrane-bound PLC is a sine-qua-non condition for lectin-induced proliferation of T lymphocytes. The key-role of PLC versus protein kinase C (PKC) is stressed by the fact that the inhibition of PKC with Hidaka's compound H-7 (40 microM) produced only a partial blockade (about 25%) of lectin mitogenic effect.

Regulation of human natural cytotoxicity by IgG--I. Characterization of the structural site on monomeric IgG responsible for inhibiting natural killer cell activity.

Inhibition of human natural killer (NK) cell activity upon exposure of peripheral blood lymphocytes (PBL) to IgG in monomeric form (mIgG) was found to be dose-, time- and temp-dependent. PBL incubated for 2 hr at 37 degrees C in the presence of myeloma protein of a certain class or subclass had a significant reduction of their NK activity when exposed to IgG, but not to IgM or IgD, and the IgG-induced inhibition of NK cells was observed only when IgG1 or IgG3 paraproteins were used. IgG3 isolated from normal serum had a higher inhibitory property than that of total mIgG. The cytophilic activity of the IgG molecules was confined entirely to the Fc region and seemed to be localized in the CH3 domain, since human and rabbit Facb fragments had a reduced ability to inhibit NK activity. When synthetic peptides representing various sequences of the human gamma-chain were tested for inhibition of NK activity, only treatment of effector cells with a peptide comprising the sequence Tyr407-Arg416 of the CH3 domain showed a reduction of NK cell function comparable to the inhibition obtained following incubation of cells in the presence of mIgG. However, on a molar basis, this peptide was 20 times less active than mIgG. In contrast, peptides derived from sequences in the CH2 domain lacked this inhibitory capacity. Our data indicate that the structural site responsible for inhibiting NK cell activity is located in the C-terminal domain of the IgG molecule.

Polymorphism of C3 components and the B factor in patients with chronic renal failure, dialyzed iteratively.

The value of C3, of the cleavage C3 components and of the B factor were determined in 25 normal controls and in 22 patients with chronic renal failure (CRF), iteratively dialyzed. Low values of C3 and progressive hypomorphism of the cleavage components of C3 were observed in the patients at the three moments of determination. The activity of the B factor was also found significantly decreased in the patients. Our results indicated an activation of the complement alternative pathway in the dialyzed patients but the activation through the classic pathway, could not be excluded either. It is concluded that this activation might be due to the initial disease which generated CRF but also to some factors transported by hemodialysis.

Immunogenicity and effector functions of glutaraldehyde-treated rabbit and mouse immunoglobulin G.

Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.

Quantitative determination of in vitro immunoglobulin secretion with protein A from Staphylococcus aureus.

A micromethod for the quantitative determination of Ig secreted in vitro by mice lymphocytes isolated from the spleen of normal animals is described. The indicator system consists in sheep erythrocytes radiolabelled with sodium chromate (51Cr) and coated with protein A of Staphylococcus aureus (51Cr-labelled ES). When splenocytes were incubated in fluid phase at 37 degrees C for 3 1/2 h with rabbit antisera to mouse Ig (IgM and IgG) and with guinea pig complement, the immune complexes formed between the secreted Ig and its specific IgG antibody are bound to protein A on the erythrocyte surface allowing the complement-mediated lysis of 51Cr-labelled ES. The degree of haemolysis produced in this experimental system, which reflects the amount of in vitro secreted Ig, was quantitatively measured by radioactive determination of 51Cr release. In combination with the ES plaque assay the method also gives information as immunoglobulin secretion per plaque forming cell.

Antibodies against glutaraldehyde-modified albumin in normal mouse sera.

An antigen-coated plate radioimmunoassay was used to detect antibodies against homologous glutaraldehyde-modified albumin (in monomeric or polymeric form) in normal mouse sera. Mouse antibodies reacted also with heterologous glutaraldehyde-treated albumin; for instance human albumin. Immunoelectrophoresis of purified anti-albumin antibodies and adsorption experiments on protein A-Sepharose 4B gel indicated that mouse antibodies belong mainly to the IgG class. The ability of mouse anti-albumin antibodies to react also with pyridinium compounds suggested that such structures are probably part of the new antigenic determinants induced in mouse albumin by glutaraldehyde treatment. It was assumed that anti-albumin antibodies have a physiologic role in the recognition and removal from the circulation of in vivo 'aged' mouse albumin.

Inhibition of human natural killer cell activity by cytophilic immunoglobulin G.

The natural killer (NK) cell activity of human peripheral blood mononuclear cells (PBL) was found to be increased after incubation at 37 degrees C for 2 hr. The observed increase was shown to be associated with release from inhibition by human serum factors, because incubation in autologous serum interfered with augmentation. The serum-mediated effect appeared attributable to the degree of binding of labile IgG to PBL and could be reduced by selective depletion of IgG from the serum. Human monomeric IgG was found to efficiently inhibit the culture-induced augmentation of NK activity; the inhibitory IgG had properties consistent with those described for cytophilic IgG and was mediated through the Fc region of IgG. The inhibition by monomeric IgG occurred at 0 degrees C as well as at 37 degrees C and this could be induced even after culture-induced augmentation of NK activity. Thus, binding of monomeric IgG to human PBL appears to reversibly inhibit their NK activity. These results provide evidence for a novel mechanism for negative regulation of NK activity.