Ig-binding receptors on human NK cells as effector and regulatory surface molecules.

The receptors on human natural killer 9NK cells which can specifically bind the Fc portion of immunoglobulin molecules (Fc receptors) have been extensively studied. The best known and studied Fc receptor on human NK cells is FcgammaRIIIa. Interactions of NK cells with IgG antibodies via this receptor are well known to induce a signal transduction cascade and lead to antibody-dependent cell-mediated cytotoxicity (ADCC) as well as release of various cytokines. In addition, interactions with monomeric IgG and FcgammaRIIIa have been demonstrated, which result in negative regulation of NK activity and other immunomodulatory effects. Over the past several years, it has also become increasingly appreciated that human NK cells express a variety of other Fc receptors, including FcmuR, which also can mediate effector and immunoregulatory functions. Also, a novel form of FcgammaR has been demonstrated on human NK cells, termed FcgammaRIIc. Recent molecular studies have shown considerable polymorphism in the genes for FcgammaIIc and the functional consequences are being dissected. This appears to include cross-talk between FcgammaRIIIa and at least some forms of FcgammaRIIc, which may have important functional consequences.

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.

Evaluation of apoptosis of tumor and of apparently normal cells in human renal carcinoma.

Apoptosis of tumor cells and of apparently normal renal cells (ANRC) isolated from the same kidney in 42 untreated patients with renal carcinomas (RC) was evaluated. Thirty five of the investigated tumors were of Grawitz type in different grades of differentiation. The intensity of the apoptotic process was routinely assessed by propidium iodide staining and flow-cytometry analysis. Similar results were obtained in the same cases by using TUNEL assay, by staining with annexin V and by DNA electrophoresis. In 85% of Grawitz carcinomas the proportion of apoptotic tumor cells was quite high, with mean% +/- SD of 57.7+/-27.3, whereas in transitional cell carcinoma of the bladder (TCC), the mean percentage of cells in apoptosis was of 22.3+/-13.9. Unexpectedly, in ANRC displaying normal morphology and normal DNA content (diploidy), the mean% +/- SD of apoptotic cells were found to exceed that of apoptotic tumor cells, 79.2+/-21.6. The percentages of cells expressing Fas receptor and/or Fas ligand varied between large ranges in both tumor and ANRC, thus suggesting that other mechanisms are also involved in the activation of apoptosis. Immunohistochemical studies showed that the intensity of apoptosis correlated well with high p-53 and low bcl-2 expression. The intensity of apoptosis was generally not correlated with the cell proliferation index (S phase fraction), suggesting that in RC apoptosis can be activated in any stage of the cell cycle. Further investigations are necessary to understand the peculiar behaviour of tumor cells as well as of ANRC in renal carcinomas as compared to other types of malignancies.

Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors.

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.

IgA monoclonal and polyclonal proteins as regulatory factors of the NK cytotoxic activity.

The presence of receptors for IgA (IgAR) on natural killer (NK) cells was only indirectly suggested yet. To elucidate the presence of IgAR on NK cells and its possible role in modulation of the NK activity, was initiated a preliminary study carried out in a homologous system, using human non adherent lymphocytes (NAL) and human seric or secretory IgA. A proportion of over 20% NK cells (CD16+ CD56+) was determined by flow-cytometry in the NAL-cells population. Dose-dependent IgA-binding to NAL cells was determined, showing a limitation of the IgA+ NAL proportion for a cell population of 32% and a ligand concentration of 4 mg/ml/10(7) cells. The binding parameters of the IgA/IgAR system, calculated by Scatchard procedure, revealed values of the affinity constant (K) and of the maximum number of ligand molecules bound/cell (n) depending on the aggregation degree of the ligands: K-values of 1.0 and 0.4 x 10(7) M-1 for dimeric and respectively, monomeric IgA, and n-values of 0.8 and 1.7 molecules/cell, respectively. A proportion of 3% IgA molecules endowed with cytophilic property was also calculated. The turnover rate of secretory IgA (sIgA) on the NAL cells surface showed values of the ligand half time (T 1/2) of 1 h. The effect of polyclonal IgA, (sIgA and normal seric IgA) and of monoclonal IgA myeloma (monomeric and dimeric IgA) on the NK cytotoxicity target K562 cell line was of inhibition, depended on the ligand doses and varied with the IgA type. The possible relevance of immunoglobulins A and of the activity of natural killer cells in processing of some mechanisms of the antiviral protection was discussed.

Optimization of immunochemical methods for intracellular protein detection.

Given the possibility that cell kinetics and p53 status may be important determinants of chemotherapeutical efficacy, the aim of the present study was to determine the optimal methods and conditions for qualitative and quantitative intracellular proteins detection. Bradford assay is the better choice for protein concentration detection because it is more sensitive and more rapid than Sheffield assay despite the fact that it utilizes a higher amount of samples. The direct staining method for flow-cytometrical detection of intracellular proteins is more rapid as compared to the indirect staining one, also providing information about protein expression during cell cycle phases, but it is low sensitive for protein expression estimation and is prohibitive for masked intracellular proteins like PCNA. More than that, it can be performed with both fixed and freshly isolated cells as compared to the indirect staining method, but the last one provides advantages by signal amplification and by its availability of using it for a large number of intracellular proteins detection.

Expression of functional CD32 molecules on human NK cells is determined by an allelic polymorphism of the FcgammaRIIC gene.

Human natural killer (NK) cells were thought to express only FcgammaRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcgammaR, ie, FcgammaRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcgammaRII from NK cells derived from several normal individuals that may represent four different products of the FcgammaRIIC gene. One transcript (IIc1) is identical with the already described FcgammaRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcgammaRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcgammaRIIc isoforms on their NK cells.

Cytophilic immunoglobulins revisited via natural killer cells.

Cytophilia is one of the biologic properties of the Fc region of immunoglobulins operationally defined as the ability of an antibody molecule to attach to certain cell types before combination with antigen. By attachment of cytophilic antibody to membrane Fc receptors, cells become "armed" with antibodies and able to interact specifically with soluble or particulate antigens. In contrast to this cytophilia, the so-called opsonic antibody binds to FcR of various cell types only after it has complexed with antigen. In spite of knowledge of cytophilic properties of immunoglobulins for more than 30 years and an impressive accumulation of facts regarding the structure and function of cell-surface FcR binding IgG, IgE, or IgA molecules, less progress has been made in understanding the molecular basis of cytophilia. Our extensive studies of the structural and functional aspects of the interaction of IgG in monomeric form with human natural killer cells, via the type IIIA IgG Fc receptor (Fc gamma RIIIA), provide a focal point for revisiting this major pathway of direct and continuous communication between the humoral and cellular compartments of the immune system.

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 effects of Fc gamma RIIIA ligands on the functional activities of human natural killer cells in vitro.

The Fc gamma receptor (R)IIIA (CD 16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via Fc gamma RIIIA, purified NK cells were cultured for 2-3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to Fc gamma RIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2R alpha, ICAM-1), cytotoxicity, cytokine production (IL-1 beta, IFN-gamma and TNF-alpha) and release of soluble IL-2R. Thus, mIgG binding to Fc gamma RIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2R alpha expression, cell proliferation and cytokine release.

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.

Natural killer (NK) activity in human responders and nonresponders to stimulation by anti-CD16 antibodies.

Various anti-Fc gamma RIII (CD16) monoclonal antibodies (mAbs) are shown here to have positive or negative modulatory effects on human NK cells. Thus, 3G8 mAb (IgG1) triggered a dose-dependent augmentation of NK activity in 67% (23/34) of individuals tested, who were designated as responders. All four IgG1 anti-CD16 mAb tested (BL-LGL/1, B73.1, Leu11c, and 3G8) were stimulatory for NK cells isolated from responders, whereas six non-IgG1 anti-CD16 mAbs were either inhibitory or had no significant effects on NK activity. The upregulation of NK activity in responders was not attributable to an increase in either the conjugate formation or the delivery of the lethal hit to target cells. This mAb-mediated up-regulation of NK activity was shown to be associated with a recycling capacity higher than that of controls and with enhanced release of cytokines by activated NK cells. Anti-CD16 mAb inhibited binding of either monomeric or polymeric IgG to Fc gamma RIIIA on NK cells. Also, mAb 3G8 or its F(ab')2 fragments decreased or reversed inhibition of NK activity induced by monomeric IgG (mIgG). Our data indicate that regulation of NK activity via the Fc gamma RIIIA is influenced by dose-dependent interactions between cytophilic mIgG and anti-CD16 mAb of IgG1 isotype.

Lymphocyte subset reference ranges in Romanian adult Caucasians.

The paper reports the distribution of lymphocyte subpopulations in the Caucasian population of Romania. Investigations were carried out in cells bearing the following antigens: CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic and some NK cells), and CD16 CD56 (NK cells). Reference values for the lymphocyte subpopulation were obtained from over 100 healthy Caucasian adult volunteers. Blood from these donors was analyzed using FACScan flow cytometer, Leuco-GATE, Simultest and FACS Lysing Solution, and SimulSET software. As an internal quality control, it was verified that %T+%B+%NK approximates 100% in all samples. The results presented here, obtained on healthy donors (51 males and 49 females), showed that there are no statistically significant variations of CD4/CD8 ratio related to sex or age, the mean value of this ratio (2.0 +/- 0.02) being similar to that reported by the West-European countries. Additional similarities were found when the relative percentage, mean +/- standard deviation (CD3 = 74.2 +/- 2.8; CD19 = 10.8 +/- 1.6; CD4 = 42.0 +/- 2.5; CD8 = 28.9 +/- 5.7; CD56 = 15.2 +/- 0.4) and the absolute cell number of the major peripheral blood mononuclear subsets established in our study were compared with other published results. This study was entirely supported by Becton Dickinson--Europe (Division Heidelberg, Germany).

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.

Expression and function of Fc gamma RII on human natural killer cells.

In this report, we present data on the expression and function of Fc gamma RII (CD32) by natural killer (NK) cells. Highly enriched NK cell populations were isolated from peripheral blood lymphocytes by negative selection and consisted of > or = 95% CD3-/CD56+ cells. Flow cytometric analyses with anti-CD32 monoclonal antibodies (mAbs) demonstrated that a small proportion of NK cells were recognized by mAbs IV.3 and 41H16. Two-color flow cytometric analysis indicated coexpression of the epitope on NK cells recognized by both these mAbs. Verification of expression of CD32 on NK cells was obtained by demonstrating coexpression of CD32 on either CD16+ or CD56+ cells. The CD32+/CD16+ and CD32+/CD56+ cells represented approximately 7 and 3% of the total, respectively. CD32 transcripts were identified from highly purified NK cells using reverse transcription-polymerase chain reaction with CD32-specific primers, followed by Southern blotting. Enhanced chemiluminescence-Western blot (ECL-WB) analysis of lysates of purified NK cells indicated that mAb IV.3 recognized a molecule of approximately 40 kD. The Fc gamma RII on NK cells was able to transduce intracellular signals in several types of assay. Cross-linking of anti-CD32 resulted in a mobilization of intracellular Ca2+, although to a lesser extent than that induced by cross-linking CD16. Both mAbs IV.3 and 41H16 were found to be capable of inducing reverse antibody-dependent cellular cytotoxicity against FcR+ target cells (e.g. P815). These data represent the first direct description of the expression and function of Fc gamma RII on human NK cells.

Characterization of the Fc mu receptor on human natural killer cells. Interaction with its physiologic ligand, human normal IgM, specificity of binding, and functional effects.

After treatment with human normal IgM, 78 +/- 8% of purified CD3-CD56+ resting human NK cells and 93 +/- 6% of IL-2-activated NK cells selected by adherence to plastic reacted with FITC-goat anti-human IgM. Binding of IgM to the FcR for IgM (Fc mu R) on human NK cells was not species specific because mouse myeloma IgM also bound to these cells. The percentage of CD56+ cells binding IgM after incubation with anti-CD16 mAb was similar to that of cells incubated with medium alone (95 +/- 1% vs 93 +/- 4%). Binding of anti-CD16 mAb to Fc gamma RIII on NK cells was unaffected by pretreatment with IgM (65 +/- 12% vs 69 +/- 4%). The CD7 molecule has been reported to be the Fc mu R on the surface of T cells. Two-color flow cytometry showed that 94 +/- 3% of CD3-CD56+ resting NK cells and 71 +/- 16% of activated NK cells were CD7+. Preincubation of NK cells with three anti-CD7 mAb (Leu-9, 8H8-1, and LAU-A1) failed to block the binding of IgM to the Fc mu R. Modulation of the CD7 molecule off the cell surface (CD7+ = 1.5% +/- 0.3) did not reduce IgM binding, thus excluding the possibility that IgM anti-CD7 might bind to different epitopes of the same molecule. These data indicate that the Fc mu R is a specific Ig-binding structure, distinct from the Fc gamma RIII (CD16) or CD7. The Fc mu R on NK cells functions as a signal-transducing molecule because the addition of 0.2 mg/ml IgM to R-NK cells caused a rapid increase in [Ca2+]i (delta = 40 nM). One of the early events that followed signaling through the Fc mu R was the down-modulation of IFN-gamma gene expression and IFN gamma production in NK cells. The presence of IgM during culture of NK cells consistently decreased the expression of HLA-DR (16% vs 40% in control). Thus, the Fc mu R, a constitutively-expressed receptor on human NK cells, seems to be an important functional molecule, which delivers negative regulatory signals to NK cells.

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.