Enhanced phagocytic activity of blood leukocytes in athymic nude mice

After 30-min incubation, blood leukocytes of adult athymic BALB/c nude mice (nu/nu) have a distinctly higher methacrylate or yeast particle uptake than leukocytes of euthymic nu/+ or +/+ mice. This permanent enhancement is not due to humoral factors, since the percentage of phagocytosing nu/nu leukocytes increases further in nu/+ littermate's plasma. Also, chronic infection or intraperitoneal immunization causes an additional transitory increase of the percentage of phagocytosing leukocytes and numbers of particles ingested; the phagocytic performance of leukocytes of euthymic mice is raised under these conditions by a greater factor. In fetuses enhanced phagocytosis of nu/nu mice is found only in monocytes. The bulk of ingestion of methacrylate particles is mediated by Fc receptors and significantly more receptors for IgG2B, IgG1, and IgG2A are demonstrable on nu/nu neutrophils; ingestion of particles via these receptors is again higher in nu/nu neutrophils, whereas nu/nu monocytes display a higher uptake via Fc(IgG1) receptors.     

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.     

Fc receptor function on sheep alveolar macrophages

We have examined the binding to sheep alveolar macrophages (AM) and peripheral blood polymorphonuclear leukocytes (PMN) of sheep immunoglobulin G subclasses or rabbit IgG immune complexes formed between rabbit anti-DNP IgG and DNP-bovine serum albumin. Binding studies using 125I-rabbit IgG immune complexes demonstrated 6.6 +/- 3.5 X 10(4) receptors per alveolar macrophage; these receptors bound immune complexes with an average association constant of 3.3 X 10(7) M-1. Saturation binding was achieved by 90 minutes at 4 degrees C with 6 X 10(-8) M IgG. Binding of subclasses of sheep IgG was examined by immunofluorescence. Only 10% of alveolar macrophages bound monomeric IgG1 and no binding of sheep IgG2 monomer could be demonstrated. In contrast, most peripheral blood PMN (93.0 +/- 9.5%) bound IgG2, but not IgG1. No binding to adult peripheral blood PMN of rabbit IgG immune complexes could be demonstrated. To study further the development of pulmonary host defense, we examined the expression of receptors for IgG immune complexes (Fc gamma R) on alveolar macrophages obtained from animals aged 8 through 180 days. At 8 and 21 days of age, the number of Fc gamma R varied considerably (75,000-192,000 sites per cell) and equalled or even exceeded that of adult sheep. Fc gamma R number declined by 42 and 90 days of age, where a nadir was reached (37,000 +/- 6,000 and 25,000 +/- 6,000 sites, respectively). By 180 days of age, the number of receptors had approached those of normal adult sheep (70,000 +/- 20,000 sites per cell). These studies parallel previous observations that revealed age-related differences in the phagocytic capacity of ovine alveolar macrophages.     

Receptor associated defects of cultured monocytes in bronchial carcinoma

Using peripheral blood monocytes from individuals with bronchial carcinoma (BC) we have measured changes, over an 8 day culture period, in monocyte/macrophage complement (C3b) and IgG (Fc) rosettes, chemotactic factor (CF)-induced enhancement of C3b and IgG (Fc) rosettes, and the phagocytic index (PI) for yeast particles. The same measurements were made on monocyte/macrophages from individuals with non-malignant respiratory diseases (NMRD) and healthy controls (HC). Following 8 days of culture there was a significant increase in C3b rosettes, IgG (Fc) rosettes and the PI in NMRD and HC. In contrast there was no significant increase in C3b rosettes in BC. The PI increased in BC, but on day 8 was still significantly less than control monocytes/macrophages. IgG (Fc) rosettes increased in BC following culture to a similar degree as that observed with NMRD and HC. In addition, there were no differences between BC and HC in CF-induced enhancement of IgG (Fc) rosettes at either day 0 or day 8 of culture whereas complement receptor enhancement in BC was impaired both at the beginning and end of the 8 day culture period. These results indicate that in advanced BC there is an abnormality in the expression of monocyte/macrophage complement receptors (which is not readily demonstrable for IgG [Fc]receptors) and that the defect persists following an 8 day culture period.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

Bovine IgG1, but not IgG2, binds to human B cells and inhibits antibody secretion

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.     

Quantitative assessment of Fc receptor expression and function during in vitro differentiation of human monocytes to macrophages.

The expression and function of IgG Fc receptors on peripheral blood monocytes and monocytes differentiating in vitro, within hydrophobic membranes, to macrophages have been determined. Three characteristic functional stages could be discerned, and these were reflected in the surface expression of specific IgG binding sites. Stage 1, represented by fresh monocytes, is characterized by efficient binding and phagocytosis of IgG-coated particles, although uptake was limited by the cell size. Within 1 day of culture, monocytes transform into Stage 2 cells. These bind and ingest particles with high IgG surface density only and these functions are exquisitely sensitive to inhibition by IgG in solution. The functional loss is paralleled by a decrease of the number of specific IgG binding sites, despite a gradual increase in cell size. Between Day 4 and Day 8, macrophages acquired the capacity to bind and ingest particles opsonized to a low degree, their phagocytic capacity is strongly enhanced, the number of IgG binding sites increases by a factor greater than 10, despite a moderate increase in cell size only. Mature human macrophages have about six times the Fc receptor number of blood monocytes, but the average binding affinity is decreased. It appears that the phagocytic capacity is related to the number of IgG binding sites, and the susceptibility to inhibition by IgG in solution is related to their surface density.     

T and B lymphocytes in the ontogenetic development of man.

Cord blood lymphocytes of premature and mature fetuses and peripheral blood of infants and adults were studied by rosette techniques and membrane immunofluorescence. Percentage of lymphocytes carrying surface immunoglobulins, receptors for complement and receptors for uncoated sheep erythrocytes or receptors for Fc IgG was determined. T lymphocyte receptors were found to appear later in ontogeny than the receptors typical of B lymphocytes. Receptors for Fc IgG were the earliest to appear: already in premature fetuses the percentage of lymphocytes resembled the one seen in healthy adults. However, the absolute number of T cells increased during development of fetuses and infants and slightly decreased in adults. On the other hand, the absolute number of B lymphocytes were highest in mature fetuses and decreased in subsequent age groups. In the course of human ontogeny a progressive increase was found in lymphocytes carrying full sets of T or B lymphocyte receptors, paralleled by a corresponding decrease in mean values of "null" lymphocytes free of detectable receptors.     

The binding of human and guinea-pig IgG subclasses to homologous macrophage and monocyte Fc receptors

Guinea-pig IgG2 and IgT1 bind to contiguous Fc receptors on homologous peritoneal macrophages. Equilibrium association constants determined for the binding of human IgG subclasses to homologous peripheral blood monocytes show that the order of binding is IgG1 greater than IgG3 greater than IgG4 greater than IgG2. Direct binding and rosette assay techniques independently established that both guinea-pig IgG2 and human IgG bind to homologous macrophage-monocyte Fc receptors through a site present in whole Fc (CH2. CH3)2, but absent in pFc' subfragments (CH3)2.     

Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents

To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed.     

Interferon-gamma restores T lymphocyte proliferation of nonresponders to IgG1 anti-CD3 via the induction of Fc gamma 1 receptors on monocytes

Human peripheral blood T lymphocytes are stimulated to grow and divide by some mouse anti-CD3 monoclonal antibodies. This polyclonal mitogenesis is dependent on both their immunoglobulin subclass and the presence of monocytes. The unresponsiveness of T lymphocytes from certain individuals to mouse IgG1 (or IgG2a) antibodies is due to a failure of their monocytes to bind these IgG isotypes. In this study, we have selected such nonresponder subjects to IgG1 anti-CD3 (UCHT 1) in order to study their monocytes. Two assays were used: IgG1 and IgG2 EA rosettes to evaluate their Fc receptor-binding capacity, and IgG-mediated monocyte chemiluminescence to test their receptor-related activation since mouse anti-T cell antibodies binding to lymphocytes trigger monocyte chemiluminescence via their Fc receptor. We have observed that in all nonresponder subjects the absence of IgG1 anti-CD3 monocyte chemiluminescence strictly correlates with the absence of IgG1 EA rosettes. Thus, the failure to respond to UCHT 1, in all nonresponders tested to date, is due to the absence of Fc gamma 1 receptors on their monocytes. Treatment of nonresponder monocytes by recombinant interferon-gamma was shown to restore T cell proliferation and monocyte chemiluminescence in nonresponders. This effect of interferon-gamma correlates with the appearance of Fc gamma 1 receptors on monocytes from these individuals. This work strongly suggests that nonresponder monocytes possess functional genes for Fc gamma 1 receptors which are not expressed normally at a detectable level but can be induced by interferon-gamma.     

Characterization and structural analysis of Fc gamma receptors of human monocytes, a monoblast cell line (U937) and a myeloblast cell line (HL-60) by a monoclonal antibody

A monoclonal antibody, FR51, raised against the IgG Fc receptor (Fc gamma R) of the human monoblast cell line U937 was used to analyze the distribution of this antigen on various human cells. This antibody inhibited the binding of human IgG to the Fc gamma R on U937 cells, HL-60 cells and human peripheral blood monocytes. In contrast, the Fc gamma R on human granulocytes (neutrophil cells) and on an Epstein-Barr virus-transformed human lymphoblastoid cell line (Raji) were not recognized, indicated by the failure of blocking the binding of human IgG ligand to the Fc gamma R on these cells. By affinity chromatography of detergent-containing cell free lysates of surface-iodinated U937 cells, HL-60 cells and monocytes, a protein of 70-kDa was isolated. This protein was identified as the Fc gamma R by rebinding the isolated protein to immobilized human IgG. Removal of the carbohydrate moiety with endo-beta-N-acetylglucosaminidase F demonstrated that the receptors consist of a 40-kDa polypeptide. Analysis of the polypeptide patterns obtained by proteolytic digestion of either mature (70-kDa) or deglycosylated (40-kDa) receptors isolated from monocytes, U937 cells and HL-60 cells strongly suggests that the Fc gamma R are identical. The monoclonal antibody FR51 specifically reacts with Fc gamma R on human monocytes, a myeloblast and a monoblast cell line but not with the receptors on a B cell line and neutrophil cells.     

Effect of polymeric IgG on human monocyte functions

Fc and iC3b receptors are involved in various biological functions of phagocytic cells, such as immune adherence and phagocytosis of opsonized particles, degranulation and superoxide generation. In the present study we examined the expression of specific receptors for the Fc portion of IgG (FcR) and for iC3b (CR3), a cleavage product of the third complement component, on human monocytes following in vitro treatment with polymeric and monomeric IgG. Interaction of polymeric IgG (fluid phase) with the monocyte membrane led to a concomitant modulation of both Fc and iC3b receptors. Monomeric IgG, however, down modulated Fc receptor expression only if surface bound. Under these conditions, no concomitant modulation of the iC3b receptor could be observed. The down modulation of Fc and iC3b receptors induced by fluid-phase IgG polymers was also accompanied by a decrease in monocyte functions as expressed by reduced Fc receptor-mediated phagocytosis, decreased release of oxygen metabolites following stimulation by aggregated IgG and opsonized zymosan, as well as in impaired killing of bacteria. These data suggest that a down modulation of Fc and iC3b receptors might have important implications for host defense mechanisms, since interaction with these receptors is required for the proper elimination of many pathogens.     

IgG on Infants'' B Lymphocytes: Enhanced Binding of IgG by IgM-Bearing Lymphoid Cells in Early Childhood

IgM/IgD-bearing lymphocytes (B cells) from children in the first few weeks of life were found to also have surface IgG, unlike most normal adult B cells. The IgG was loosely bound to the lymphocyte surface and was partially or completely removed by incubation at 37 degrees C or by trypsinization. When F(ab')2 antisera were employed, very few infant B cells had surface IgG, although the IgM staining was similar to that obtained with the native antisera. IgM/IgD-positive cells bound IgG anti-Rh-coated (Ripley) erythrocytes, unlike most adult B lymphocytes. Capping experiments suggested that an Fc receptor on the cells could be redistributed by the anti-IgM-surface IgM complex. These data indicate that, during infancy, B-lymphocyte receptors for IgG are of altered affinity, frequency, or availability compared with adult B-lymphocyte Fc receptors and resemble the Fc receptors found on "third population" (Fc + Ig-) mononuclear cells and monocytes.     

Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation.     

Transformation of monocytes into "fat" cells

Human peripheral blood leukocytes, when cultured in soft agar give rise to giant (100 to 500 micrometer.) "foam cells." Investigation of the origin and properties of the cells proved that they were derived from monocytes in that the cells adherent to glass after 24 hours in culture were phagocytic, elaborated lysozyme and bore receptors for complement and immunoglobulin. The increment in size was accounted for primarily by large inclusions which on histochemical and biochemical analyses were shown to consist predominantly fo neutral fat. Transformation to fat cells took place in the absence of mitosis. Fc receptors were retained but complement receptors were lost. These observations suggest a role for monocytes in the replacement of hematopoietic tissue by fat in certain hypoplastic states. The cultured monocytes may also serve to facilitate the study of fat synthesis and metabolism in vitro.     

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.     

Alpha-fetoprotein and phagocytosis in athymic nude mice

Alpha-fetoprotein (AFP) was found to suppress the phagocytic activity of the blood monocytes and neutrophils in vitro. The amounts of AFP detectable by immunofluorescence in the livers of nu/nu, nu/+ and +/+ mice were quite comparable, and thus could not have been responsible for the alterations in phagocytosis found in leukocytes of athymic nude mice during their ontogenetic development.     

Shedding of Fc receptor from mononuclear cells and their ability of binding IgG1 and IgG3 anti-Rh/D antibodies

Fc receptors for IgG1 and IgG3 on peripheral blood lymphocytes and monocytes were studied before and after temperature shift from 4-37 degrees C. The investigations were performed in the EA test using human erythrocytes sensitized with anti-Rh/D/antibodies of IgG1 (EA IgG1) and IgG3 (EA IgG3) subclasses. It occurred that lymphocytes and monocytes were able to bind IgG1 and IgG3 antibodies before and after shedding, however, lower percentage of rosette was observed after temperature shift. This decrease was similar in the EAIgG1 and EAIgG3 tests. The supernatants obtained during shedding occurred to contain active Fc receptors since the inhibition of rosette formation was obtained after the incubation of sensitized erythrocytes with these supernatants. IgG1 as well as IgG3 myeloma proteins inhibited rosette formation in both EAIgG1 and EAIgG3 tests. Our data might suggest that IgG1 and IgG3 anti-D antibodies are able to bind to the same Fc receptor on lymphocytes as well as on monocytes.     

Bovine IgG can aggregate at conditions simulating pasteurization and binds to some human Fc gamma receptors

The ability of bovine IgG preparations to bind to the various distinct human leukocyte Fc gamma receptors was studied. In experiments using intact cells and isolated Fc gamma receptors, it was demonstrated that bovine IgG can bind to Fc gamma receptors of four human cell types but not to Fc gamma receptors of human neutrophils. 125I-labeled Fc gamma receptors purified on human IgG-Sepharose columns from human B and T lymphocytes, monocytes and eosinophils were able to rebind specifically to insolubilized bovine IgG. In contrast, radioiodinated human neutrophil Fc gamma receptors did not rebind to bovine IgG-Sepharose. A similar pattern of specificity was demonstrated in studies of the binding of 125I-labeled heat-aggregated bovine IgG to various human leukocyte populations. The labeled aggregated bovine IgG bound to peripheral blood mononuclear cells, to B cells from chronic lymphocytic leukemia patients and to macrophage-like U-937 cells, but bound poorly to normal human granulocytes. Labeled non-aggregated bovine IgG was not appreciably bound to any of the cell populations. Since bovine IgG in dietary sources is frequently exposed to heat, the effect of heating on the physical state and Fc-binding properties of bovine IgG was examined. The data show that heating bovine IgG at concns of 0.9-3.6 mg/ml at 63 degrees C for 30 min in neutral buffer causes aggregation of bovine immunoglobulin (10-16% aggregation) and increases the ability of bovine IgG preparations to bind to human Fc gamma receptors of intact cells. Gel filtration studies suggest the possibility that bovine IgG may also be aggregated during the pasteurization of raw milk.