Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo.     

In vivo and in vitro effects of doxycycline on leucocyte membrane receptors.

Tetracyclines, particularly doxycycline, have adverse effects on granulocyte function in vitro. We have examined the effects of doxycycline on membrane receptors for IgG (Fc gamma-R) and C3b (C3b-R) on granulocytes and lymphocytes, as well as on the sheep erythrocyte receptor (E-R) on T lymphocytes. Acne patients given doxycycline orally had a lower percentage of Fc gamma-R positive granulocytes (57%) than before treatment (80%) or compared to healthy controls (81%). Following in vitro doxycycline incubation, normal granulocytes showed decreased levels of Fc gamma-R positive cells. This effect was counteracted by the addition of magnesium during incubation. The deleterious effect of doxycycline on granulocyte functions may be due to decreased levels of Fc gamma-R bearing granulocytes. Doxycycline in vivo or in vitro had no significant effect on the proportion of C3b-R bearing granulocytes or lymphocytes or the T lymphocyte percentage. After in vitro irradiation with light at 340-380 nm, however, both granulocytes and lymphocytes preincubated with doxycycline showed up to 50% decrease in Fc gamma-R bearing cells, while control cells without doxycycline were unaffected.     

Fc gamma receptor-dependent clearance is enhanced following lipopolysaccharide in vivo treatment.

Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram-negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)-specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythrocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fc gamma receptor (Fc gamma R)-dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody-dependent cellular cytotoxicity (ADCC), an in vitro indicator of Fc gamma R functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify Fc gamma R expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac-1), an adhesion molecule closely related to Fc gamma R activity. We conclude that LPS enhance Fc gamma R-dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules.     

Dynamics of mononuclear phagocyte system Fc receptor function in systemic lupus erythematosus. Relation to disease activity and circulating immune complexes

Seventeen pairs of longitudinal studies of mononuclear phagocyte system (MPS) Fc receptor function in 15 patients with systemic lupus were performed to explore the dynamic range of Fc receptor dysfunction in lupus and to establish the relationships between MPS function, clinical disease activity and circulating immune complexes (CIC). Fc receptor function was measured by the clearance of IgG sensitized autologous erythrocytes. At the time of first study the degree of MPS dysfunction was correlated with both clinical activity (P less than 0.05) and CIC (P less than 0.05). At follow-up patients with a change in clinical status show significantly larger changes in clearance function compared to clinically stable patients (206 min vs 7 min; P less than 0.001). MPS function changed concordantly with a change in clinical status in all cases (P = 0.002). Longitudinal assessments did not demonstrate concordance of changes in MPS function and CIC, measured by three different assays. The MPS Fc receptor defect in systemic lupus is dynamic and closely associated with disease activity. The lack of concordance of the defect with changes in CIC suggests that either CIC does not adequately reflect receptor site saturation or that other factors may also contribute to the magnitude of MPS dysfunction.     

Neuraminidase treatment of human T lymphocytes: effect on Fc receptor phenotype and function

Purified peripheral blood T cells or T mu cells from normal healthy donors were treated in vitro with neuraminidase and examined for the expression of IgM Fc and IgG Fc receptors. Increasing concentrations of neuraminidase selectively removed IgM Fc receptors, whereas the number of T cells expressing IgG Fc receptors was significantly increased. Following neuraminidase treatment, IgM Fc receptors could be regenerated by reincubation of T cells at 37 degrees C. The regeneration of IgM Fc receptors could be blocked by treatment with cycloheximide. Neuraminidase treatment of purified T mu cells resulted in the expression of IgG Fc receptors on a subpopulation of T mu lymphocytes. A small percentage of the neuraminidase-treated T cells expressed receptors for both IgG and IgM. Treatment of T cells with neuraminidase did not effect T cell-mediated spontaneous cytotoxicity (SLMC) or antibody-dependent cellular cytotoxicity (ADCC). Our results indicate that T cell Fc receptor phenotypes can be modulated in vitro without significantly altering their functional capacity.     

Evaluation of the possible role of B cell receptors in the tendency of B cells to migrate into follicles in mice and chickens.

Chicken spleen and bursa cells were examined for the percentage of Fc receptor-bearing cells. Rosette formation was done with chicken 7S antibody-sensitized sheep erythrocytes and was inhibited by heat-aggregated chicken Ig. In the spleen, the percentage was found to increase with age to approximately 26% at 7 to 12 weeks. In contrast, only 3 to 5% of bursa cellss at this age demonstrated Fc receptors. Sleens from bursectomized chickens had 7--10% Fc receptor-bearing cells. In an attempt to determine a possible role of the C3 receptor on migration patterns, the effect of cobra venom factor (CVF) on the localization of transferred lymphoid cells was examined. Pretreatment of recipients with enough CVF to lower mean C3 levels to 11% of controls failed to affect follicular B cell localization in mice at either 24 or 48 h after transfer. Localization of thymus or bursa cells in chickens was similarly unaffected by CVF pretreatment. The possible roles of Fc and C3 receptors on migration of B lymphocytes into follicles and germinal centers were discussed.     

Surface markers on lymphocytes of multiple sclerosis patients.

Peripheral blood lymphocytes of twenty-two multiple sclerosis (MS) patients and thirty-five healthy controls were examined for the presence of surface markers characteristic for B lymphocytes (surface immunoglobulin, receptor for C3 (EAC), reporter for Fc (EA) and the spontaneous rosette-forming capacity characteristic of T cells. The results obtained indicate that the number of B and T cells in MS is similar to controls, as evaluated by the presence of surface immunoglobulin and E rosette-forming capacity. However, a statistically significant reduction in the percentage of lymphocytes bearing C3 receptors has been found in MS patients. It might have resulted from a reduction in the lymphocyte population bearing C3 receptor but no surface immunoglobulin. The EA rosette test revealed the greatest difference between the groups. The difference indicated a reduction in the density of the receptor for 7S Fc on lymphocytes from MS patients. The results obtained are consistent with the hypothesis of an immune deficit in multiple sclerosis.     

Shedding and synthesis de novo of Fc and C3b receptors by cultured guinea-pig macrophages.

Resident macrophages freshly obtained from the peritoneal cavity of guinea-pigs were demonstrated to form a higher percentage of Fc and C3b rosettes than elicited macrophages when low concentrations of IgG and IgM-C3b were used to sensitize ox red blood cells (ORBC) in rosette assays. Culture of the total resident and elicited macrophages for 6 h at 37 degrees C resulted in a decrease of Fc and C3b rosette-forming cells, the loss of Fc receptor-bearing cells by resident macrophages only being apparent when using a sub-optimal concentration of sensitizing IgG. After 24 h incubation the percentages of Fc and C3b rosettes returned to their initial values. In contrast, there was no decline in the percentage of Fc and C3b rosettes formed by the adherent population of resident and elicited macrophages cultured for 6 h. However, extending the incubation of the adherent macrophage to 24 h produced an increase of Fc receptor-positive cells and a dramatic decrease of C3b receptor-positive cells. Culture supernatants of the total macrophage population that had been incubated for 6 h inhibited Fc and C3b rosette formation by freshly obtained elicited macrophages. These results, together with the demonstration that treatment of the total macrophage population with cycloheximide led to an inhibition of Fc and C3b receptor expression after 24 h culture, suggest that the Fc and C3b receptors of guinea-pig macrophages are shed and synthesized de novo during short-term culture. This system could be applied to the study in vitro of soluble immunoregulatory mediators on macrophage functions which are dependent on the expression of Fc and C3b receptors.     

The isolation, long-term cultivation and characterization of bovine peripheral blood monocytes.

Bovine peripheral blood monocytes were isolated from buffy coats of ACD-anticoagulated whole blood. Cells cultivated on plastic surfaces for 20 h were judged to be 95% monocytes based on nonspecific esterase-1 staining, uptake of latex particles and surface receptor characteristics. Bovine monocytes were maintained up to 80 days in vitro in Dulbecco's modified Eagle medium with 15% horse serum and 15% foetal bovine serum. Several morphological and physiological features of bovine monocytes were examined during the course of culture. Cell size and cytoplasmic spreading, granulation and vacuolization increased progressively. Multinucleated giant cells predominated during the latter stages of in vitro culture. A high percentage of bovine monocytes possessed C3 and IgG Fc receptors, whereas IgM Fc and sheep erythrocyte receptors were not detected. Phagocytosis was mediated by the IgG receptor, but not by the C3 receptor. Peroxidase activity declined in a linear fashion, with cells essentially negative after 8 days of culture. Total cell protein and acid phosphatase increased during cultivation. Lysozyme activity was undetectable in both lysates and supernatants of bovine monocyte. These findings are consistent with the concept of maturation of mononuclear phagocytes. The procedures for isolation and cultivation described in this paper will provide methodology for detailed study of bovine mononuclear phagocytes.     

Expression of HLA DR antigens, Fc IgG and C3 receptors on lung macrophages in sarcoidosis and idiopathic lung fibrosis

The studies concerned an expression of HLA DR antigens, Fc IgG receptors and C3 complement on lung macrophages in patients with active sarcoidosis and idiopathic lung fibrosis as well as in the control group of smokers and nonsmokers. Macrophages were isolated by broncho-alveolar lavage with the aid of bronchofiberoscope. The expression of HLA DR antigens, Fc IgG and C3 receptors was tested before during and after the treatment. The percentage of macrophages with HLA DR antigens and Fc IgG receptors was observed to be high before the treatment in the examined as well as in the control groups. During steride treatment, the percentage of macrophages with HLA DR antigens and Fc IgG receptors diminished in relation to the control (p less than 0.05). After termination of the treatment, the percentage of macrophages with HLA DR antigens and Fc IgG receptors was lower than before the treatment. The differences in percentages of macrophages with C3 receptors did not achieve statistical significance in neither of the groups examined in the period of treatment.     

Modulation of Fc and C3b receptor expression on guinea-pig macrophages by lymphokines.

The decrease in Fc-receptor-positive cells that occurred during a 6 h incubation of resident and elicited guinea-pig macrophages was partly abrogated when lymphokines were present in the culture. When the same lymphokine preparations were tested on C3b receptor-expression they preferentially sustained the percentage of C3b rosettes formed by resident rather than elicited macrophages. This lymphokine-induced maintenance of Fc and C3b rosettes by cultured macrophages may have been due to an inhibition of receptor release or an increase in receptor synthesis. Supernatants from cultured macrophages contain shed Fc and C3b receptors which inhibit rosette formation by other macrophages. From the demonstration that culture supernatants from both lymphokine-treated and untreated macrophages significantly inhibited Fc and C3b rosette formation by freshly obtained macrophages it seems that the shedding of Fc and C3b receptors is not modified by lymphokines. The maintenance of Fc and C3b rosettes by lymphokines was inhibited by treatment of the macrophages with cycloheximide, suggesting that the lymphokine effect was due to an increase in synthesis de novo of the Fc and C3b receptors. The lymphokine-inducing antigens, BGG and PPD, and control lymphokine preparations were devoid of receptor modifying activity. The reduction in the percentage of Fc rosettes after 6 h culture appears to be due to a loss of Fc receptors for IgG1. Although lymphokines partly inhibited this effect they could not prevent the loss of these receptors following 24 h culture, unlike their action in augmenting the expression of Fc receptors for IgG2. These findings suggest that a selective enhancement of Fc receptor synthesis by lymphokines may modify the functional activities of macrophages.     

Leukocyte membrane receptors in meningitis and other bacterial infections

Blood leukocytes from 37 patients with acute bacterial infections, and cerebrospinal fluid (CSF) granulocytes from 12 patients with bacterial meningitis, were examined for the distribution of membrane receptors (R) for (1) untreated sheep erythrocytes (E), (2) the Fc portion of IgG (Fc gamma), and (3) complement component C3b. We found a decreased percentage of granulocytes bearing Fc gamma-R in the CSF from patients with meningitis, and in blood from patients with respiratory tract infections. This group also had a decreased percentage of C3b-R bearing granulocytes on admission, whereas meningitis patients had lower levels of C3b-R and Fc gamma-R bearing granulocytes in the 2nd and 3rd week and even later. Several patients with meningitis and gastroenteritis had granulocytes bearing the E-R, previously considered specific for T lymphocytes. Such cells were also found in the CSF. Meningitis and respiratory tract infections were associated with a decreased percentage of 'active' T lymphocytes. The total percentage of T lymphocytes was also decreased in meningitis. Conversely the proportion of Fc gamma-R bearing lymphocytes (consisting mostly of B lymphocytes) was increased in most infections. During the first 3 weeks of bacterial meningitis, the percentages of Fc gamma- and C3b-R bearing granulocytes, and of Fc gamma-R bearing lymphocytes, gradually decreased, while the T lymphocyte percentage increased from the initial low values.     

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.     

Synergism of 3-deazaadenosine with some Fc receptor ligands in the inhibition of antibody-dependent phagocytosis

Inhibition of phagocytosis by 3-deazaadenosine, present in vitro during the assay, is unique in that it is dependent on the concentration of IgG on Ig-coated erythrocyte target cells. This regulatory interaction is now further examined utilizing the capacity of other Fc receptor ligands, like soluble immune complexes and Ig preparations, to modulate the effect of 3-deazaadenosine on antibody-dependent phagocytosis (AD phi) in mouse resident peritoneal macrophages. Non-inhibitory concentrations of soluble immune complexes formed in mixtures of ovalbumin (OVA) antigen and rabbit anti-OVA (RAOVA) antibodies enhanced the inhibition by 3-deazaadenosine of AD phi. The effect was reversed by using F(ab')2 antibodies, that lack the Fc portion of IgG, in OVA/RAOVA mixtures. Similar synergistic effects were achieved with human gammaglobulin and IgG preparations, and were further enhanced by aggregated IgG. Another source of Fc receptor ligands, autoimmune MRL/Mp-lpr/lpr mouse sera, unlike MRL/Mp-+/+ sera, also showed synergism with 3-deazaadenosine in the inhibition of AD phi. This effect was reversed by treatment of MRL sera with F(ab')2 fragments of an anti-mouse Fc antibody. It has been pointed out that the regulatory interaction between Fc receptor ligands, like Ig-coated cells, soluble immune complexes, Ig aggregates, on one hand, and the effects of 3-deazaadenosine, on the other hand, may represent a novel mechanism by which an agent inhibits phagocytosis indirectly, by manipulating the competition among these various ligands for the macrophage Fc receptor.     

Eosinophils of human colonic mucosa: C3b and Fc gamma receptor expression and phagocytic capabilities

Because little is known about eosinophils of the human intestine, we measured their C3b and Fc gamma receptor expression and phagocytic activity in mucosal suspensions from colon resections for large bowel neoplasms. Enzymatically dissociated suspensions were enriched for eosinophils by countercurrent centrifugation. C3b and Fc gamma receptors were measured by immunofluorescent assays with flow cytometry. Phagocytosis of Escherichia coli ON2 was determined by an in vitro microscopic method. Suspensions of normal tissue from neoplasm resections yielded 1.8 X 10(6) eosinophils/g mucosa, and these cells were more numerous than either macrophages or neutrophils. Fivefold enrichment was achieved by countercurrent centrifugation, and 75% of these cells expressed C3b receptors and 90% expressed Fc gamma receptors. Sixty-seven percent of mucosal eosinophils were phagocytic for E. coli ON2 and ingested a mean of 4.7 bacteria per cell. Eosinophils accounted for more overall phagocytic activity than either neutrophils or macrophages.     

Difference in the Effect of Immobilized Ligands on the Fc and C3 Receptors of Mouse Peritoneal Macrophages in Vitro

The function of the Fc and C3 receptors on the free surface of normal and endotoxin activated mouse peritoneal macrophages seeded on glass bound antibody--antigen (AbAg) complexes or complement was examined. We found that the glass bound AbAg complexes interfered with attachment and internalization of particles recognized by the Fc receptor. The C3 receptor function in these cells was not affected. On the other hand, normal and activated macrophages seeded on glass bound complement showed no change in the function of the C3 and the Fc receptors.     

Properties of visceral primary afferent neurons in the nodose ganglion of the rabbit

The active and passive membrane properties of rabbit nodose ganglion cells and their responsiveness to depolarizing agents have been examined in vitro. Neurons with an axonal conduction velocity of less than 3 m/s were classified as C-cells and the remainder as A-cells. Mean axonal conduction velocities of A- and C-cells were 16.4 m/s and 0.99 m/s, respectively. A-cells had action potentials of brief duration (1.16 ms), high rate of rise (385 V/s), an overshoot of 23 mV, and relatively high spike following frequency (SFF). C-cells typically had action potentials with a "humped" configuration (duration 2.51 ms), lower rate of rise (255 V/s), an overshoot of 28.6 mV, an after potential of longer duration than A-cells, and relatively low SFF. Eight of 15 A-cells whose axons conducted at less than 10 m/s had action potentials of longer duration with a humped configuration; these were termed Ah-cells. They formed about 10% of cells whose axons conducted above 2.5 m/s. The soma action potential of A-cells was blocked by tetrodotoxin (TTX), but that of 6/11 C-cells was unaffected by TTX. Typically, A-cells showed strong delayed (outward) rectification on passage of depolarizing current through the soma membrane and time-dependent (inward) rectification on inward current passage. Input resistance was thus highly sensitive to membrane potential close to rest. In C-cells, delayed rectification was not marked, and slight time-dependent rectification occurred in only 3 of 25 cells; I/V curves were normally linear over the range: resting potential to 40 mV more negative. Data on Ah-cells were incomplete, but in our sample of eight cells time-dependent rectification was absent or mild. C-cells had a higher input resistance and a higher neuronal capacitance than A-cells. In a proportion of A-cells, RN was low at resting potential (5 M omega) but increased as the membrane was hyperpolarized by a few millivolts. A-cells were depolarized by GABA but were normally unaffected by 5-HT or DMPP. C-cells were depolarized by GABA in a similar manner to A-cells but also responded strongly to 5-HT; 53/66 gave a depolarizing response, and 3/66, a hyperpolarizing response. Of C-cells, 75% gave a depolarizing response to DMPP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

Activation of complement by soluble IgG aggregates and their subsequent interaction with macrophages

The mononuclear phagocyte system (MPS) plays an important role in the removal of both particulate and soluble immunogenic material from the circulation. E.IgG adhere to mononuclear phagocytes if the Fc portion of the IgG can interact with the phagocytes' Fc receptors. Simultaneous sensitization with IgG and C3b enhances the effectiveness of binding and ingestion. If soluble material cannot adhere to the surface of macrophages, it will be endocytosed in vitro via fluid-phase pinocytosis at the concentration that is present in the medium. If the material adheres to the cell's surface via its chemical properties or via specific receptors, it will be selectively concentrated at the cell's surface and endocytosed by an adsorptive pinocytosis. Ingestion of IC via Fc gamma R and C3b depends on the ability of the antibodies to interact with Fc gamma R and their capacity to activate the complement system. IC-bound C3b enhances the adsorptive pinocytosis of IC. Soluble AIgG are also pinocytosed more efficiently when C3b is bound to AIgG. The degree of endocytosis varies with the level of C3b sensitization. The highly effective C3b-mediated pinocytosis can be abolished by treating the macrophages with trypsin to inactivate C3bR. This observation illustrates that C3-mediated pinocytosis can replace Fc-mediated pinocytosis in unstimulated macrophages. Soluble IC and AIgG are removed from the circulation mainly by hepatic Kupffer cells. It seems that the size, the Ag/Ab ratio, the capacity of the IC to bind C1, activate C1, and allow deposition of C3b together with the degree of phagocyte activation determine the degree of binding and subsequent degradation of soluble IC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface T- and B-cell markers on murine lymphomas and plasmacytomas

Seven murine lymphomas and three plasmacytomas were examined for the distribution of the theta antigen, immunoglobulin determinants, the receptor for Fc portion of antigen—antibody complexes and the receptor for the third component of complement. The theta-bearing tumours lacked C3 and Fc receptors and easily detectable surface immunoglobulin. The theta-negative lymphomas, while being morphologically lymphocytic, lacked all but the Fc receptors. One of the plasmacytomas possessed clearly detectable surface immunoglobulin. All three lacked the receptors for Fc and C3.