Relationship between murine macrophage Fc receptor-mediated phagocytic function and competency for activation for non-specific tumor cytotoxicity

The relationship between Fc receptor (FcR) function and activation of murine macrophage populations for non-specific tumor cytotoxicity was studied. Oil-elicited inflammatory peritoneal macrophages (PM phi) from C3HeB/FeJ mice had higher FcR function upon harvest than resident PM phi from the same strain or elicited PM phi from genetically deficient C3H/HeJ mice. C3HeB/FeJ inflammatory PM phi were uniformly responsive to activation by MAF and the complement activators: LPS, Poly I:C, cobra venom factor (CVF) and zymosan for tumoricidal activity. Resident cells from the same strain and C3H/HeJ-elicited PM phi were uniformly unresponsive to the same activators. In vitro culture of C3HeB/FeJ resident PM phi with fetal bovine serum for 24-48 h produced unregulation of FcR function which coincided with a conversion from an unresponsive to a responsive state for tumoricidal activity. Reconstitution of the FcR function of C3H/HeJ-elicited PM phi during 24-48 h culture with lymphokine or Poly I:C also coincided with the restoration of responsiveness to activation by LPS, CVF, and zymosan for tumor cytotoxicity. Thus, the consistent temporal relationship between upregulated FcR function and the capacity of macrophages to respond to activation for non-specific tumoricidal activity may be more than coincidental. Preincubation of responsive C3HeB/FeJ-elicited PM phi with insoluble immune complex or heat-aggregated IgG was shown to blockade FcR-mediated phagocytosis and to abrogate LPS-mediated tumoricidal activity. Interestingly, FcR blockade by IgG-opsonized sheep erythrocyte conjugates selectively inhibited activation by MAF, LPS, and Poly I:C, but had no inhibitory effect on activation by CVF or zymosan. Similar blockade of C3b receptors produced an identical pattern of selective inhibition of activation. This selective inhibition of non-specific tumoricidal activity by FcR/C3bR blockade suggests the existence of two pathways for antibody-independent activation of macrophages.     

Characterization of murine macrophage Fc receptor-dependent phagocytosis and antibody-dependent cellular cytotoxicity during in vitro culture with interferons-gamma, alpha/beta and/or fetal bovine serum

Resident and oil-elicited inflammatory peritoneal macrophages (PM phi) from competent C3HeB/FeJ and genetically deficient C3H/HeJ mice were characterized for their Fc receptor (FcR)-dependent binding, phagocytic and ADCC functions during in vitro differentiation under the influence of mouse recombinant interferon-gamma (rIFN-gamma), interferon-alpha/beta (IFN-alpha/beta) fetal bovine serum (FBS), or in serum-free medium. Freshly cultured resident PM phi from C3HeB/FeJ mice had low levels of FcR-mediated phagocytosis in response to mouse monoclonal IgG gamma 2a, IgG gamma 2b or IgG gamma 1, opsonized sheep erythrocytes as compared to oil-elicited inflammatory PM phi from the same strain. Resident PM phi were uniformly upregulated in their FcR-dependent phagocytosis after 24-48 h in vitro culture with FBS to levels approximating that of freshly cultured inflammatory PM phi which were also further upregulated after 24 h in vitro culture with FBS. Both resident and inflammatory PM phi were upregulated largely by an autostimulatory process in that they increased their FcR-mediated phagocytosis in serum-free RPMI-1640 medium without the addition of rIFN-gamma or IFN-alpha/beta, although FBS further augmented FcR upregulation. A synergistic effect of FBS and rIFN-gamma was required for total reconstitution of FcR-mediated phagocytosis of FcR-incompetent C3H/HeJ inflammatory PM phi in that FBS or rIFN-gamma alone only partially reconstituted FcR function, whereas in combination full reconstitution occurred. Thus, macrophages from competent C3HeB/FeJ mice were upregulated in their FcR-mediated functions largely by an autostimulatory process, presumably dependent on endogenous of IFN-beta, whereas, genetically-deficient C3H/HeJ macrophages required exogenous rIFN-gamma in combination with fetal bovine serum for synergistic reconstitution of FcR functions. The uniform upregulation of FcR-dependent effector functions in vitro appears to provide an efficient system for enhanced immune function during differentiation which may be applicable to in vivo situations.     

Differential tumor necrosis factor production by human monocyte subsets

The human monocyte (M phi subset rosetting with anti RH-coated human erythrocytes via high-affinity, 72 kD receptors (FcRI+), contains the PGE2-producing immunosuppressive subpopulation, while the non-rosetting M phi subset (FcRI-) is the major plasminogen activator-producing and antigen-presenting M phi. This study gives additional evidence for the functional disparity of the FcRI- and FcRI+ M phi subsets. We are demonstrating that the normal human M phi subset isolated by rosetting via the FcRI receptor (FcRI+) produces greater quantities of tumor necrosis factor (TNF) than the non-rosetting (FcRI-) M phi. TNF production by the FcRI+ M phi subset is greater than that of the FcRI- M phi subset whether secreted (P less than .001) or cell-associated (P less than .001) TNF is assessed. The rosetting M phi subset that expresses high densities of FcRI (FcRI+) produced the majority of normal human peripheral blood M phi TNF whether the stimulation was an interferon gamma (IFN gamma) prime followed by MDP or followed by interleukin-2 (IL-2). The Fc rosetting technique itself resulted in some TNF induction in the FcRI+ M phi subset accounting for some of the increased TNF production of this subset. However, increasing the stimulation level of the FcRI very-low-density (FcRI-) M phi subset did not induce it to produce TNF levels equivalent to the moderately stimulated FcRI+ M phi subset. These data, therefore, imply that only stimulation through the type I Fc gamma receptor can augment or induce TNF activity. The difference in the M phi subset's TNF response remained even after the FcRI- M phi subset received a 2.5-fold increase in stimulation with the classical M phi induction regimen of IFN gamma plus bacterial cell wall product. Although stimulation of the FcRI+ M phi subset via crosslinking of their FcRI receptors might represent a unique TNF stimulation pathway, this stimulation does not occur in the low-density FcRI (FcRI-) M phi subset, again indicating functional disparity between these subsets. Greater TNF production by the FcRI+ M phi subset was induced concomitant to elevation of its prostaglandin E2 production. Since both TNF and PGE2 are increased in some patient groups, a pathological shift in the FcRI+ versus FcRI- M phi ratio in these patients coupled to the functional differences in FcRI+ and FcRI- M phi subsets could be one mechanism for the development of immunoincompetence.     

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determinedin vitro by a classical rosette assay in 12 postmen-opausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, -mannosyl [IgG(Con A)] or -galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P<0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs. In addition, when both groups of women were compared, all three Fc-dependent functions measuredin vitro using IgG(PNA)-SRBCs were significantly lower (P<0.01 at least) in women without E than in women on therapy. In another series of experiments, we also found that the rosetting and phagocytosing capacities of MCs were dramatically and transiently reduced in three of three young women during the menstrual period, only when the IgG(PNA)-SRBCs were used as targets. Taken together, our data show that MC phagocytosis of SRBCs coated with IgG antibody exhibiting terminal, and/or accessible, -galactosyl residues in their Fc domain is selectively impaired by a physiological E deficiency and is restored when this deficiency is artificially or spontaneously corrected. They therefore suggest that these hormones are capable of affecting the PNA-like activity of IgG(Fc) receptors of human MCs.     

Interference of beta 2-microglobulin specific autoantibodies with EA-binding of human peripheral lymphocytes; inhibition of B-cell and enhancement of T-lymphocyte Fc-receptors

The effect of anti-beta 2 m-specific autoantibodies was investigated on the FcRs of human PBMCs. Anti-beta 2 m autoantibodies inhibited the FcRs of the lymphocyte subpopulation detectable by rosetting with EA(hu). On the contrary, when EA(ox) indicator system was used, in the majority of the cases an enhancement of EA rosette formation was detected. Using separated lymphocyte subpopulations we found that the binding of anti-beta 2 m autoantibodies increased the number of FcR+ non-B-cells and inhibited that of B-lymphocytes.     

Surface antigens of the murine cytostatic peritoneal macrophage.

By rosetting techniques, peritoneal exudate cells (PEC) from mice stimulated intraperitoneally with starch have been shown to be a mixed population of cells consisting of the following subpopulations of cells: 75% FcR, 26% C3R, 18% Ig+, 29% Ia+, Ig- and 5% Thy-1+, Ig-. By separating rosetting from non-rosetting cells, it was possible to establish the phenotype of the PEC which was cytostatic for tumour cells. This cell possessed receptors for Fc and C3 but lacked surface Ig, the Thy-1 antigen and I-region controlled antigens and was NSE positive. Thus by presently available criteria, the cytostatic PEC can be identified as a macrophage. The lack of Ia distinguishes this type of macrophage from the antigen-presenting macrophage which bears Ia.     

Human blood B lymphocytes with receptors for sheep erythrocytes--their relevance in techniques to obtain T cells depleted of mature B cells

An autoradiographic technique for the determination of viable bacteria in individual cells is described, based on the incorporation of [3H]thymidine into the DNA of viable Escherichia coli X43, following phagocytosis by resident mouse peritoneal macrophages. The results of the autoradiographic technique were in overall agreement with viable colony counts. Investigation of the killing of E. coli X43 with the autoradiographic technique showed that the percentage viable bacteria tended to be the same irrespective of the number of bacteria ingested per macrophage, although there was a definite correlation between the numbers phagocytosed and the percentage killed in some of the experiments.     

Size-dependent effect of IgA on the IgA Fc receptor (CD89)

The IgA Fc receptor (FcR; CD89) is expressed on several types of cells of the myeloid cell lineage. We investigated whether different sizes of heat-aggregated IgA (aIgA) bind to CD89 and subsequently induce cellular activation. As a model we used the murine B cell line IIA1.6 transfected with CD89 or IIA1.6 cells transfected with CD89 as well as with the FcR γ chain to study the binding of IgA to CD89. When these cells expressing CD89 were incubated with monomeric IgA, no significant binding of IgA to the cells was detectable by fluorescence-activated cell sorter analysis; however, incubation of the cells with aggregated IgA resulted in 93 ± 2% positive cells. Incubation of the cells with different sizes of IgA-containing aggregates revealed optimal binding with aggregates containing five to six molecules of IgA per aggregate. No difference was observed between the binding to CD89 of both IgA1- or IgA2-containing aggregates. Furthermore, the binding of aIgA was found to be CD89-specific, since the binding of IgA was completely inhibited by the CD89-specific monoclonal antibody My43 and no detectable binding occurred to the IIA1.6 parent cell line. Activation studies using interleukin-2 (IL-2) production as a marker, showed that the FcR γ chain is necessary to induce cellular activation. Only cells transfected with both CD89 and the FcR γ chain (CD89⁺/γ⁺) enhance the IL-2 production 10–12-fold upon stimulation with aggregates of IgA. Furthermore, triggering of CD89 only results in increase of intracellular calcium concentration ([Ca²⁺]_i) in cells co-expressing FcR γ chain. Mutation of the tyrosine residues in the FcR γ chain immunoreceptor tyrosine-based activation motif of the FcR γ chain abolishes this increase in [Ca²⁺]_i, indicating association and involvement of the FcR γ chain in CD89-mediated signaling.     

An extended family of Fc receptor relatives

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.     

Infection enhancement of influenza A NWS virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody.

Antibody-dependent enhancement (ADE) of influenza A NWS virus infection was investigated in primary murine macrophages (M phi) using anti-hemagglutinin(HA) monoclonal antibody (mAB). Contrary to previous reports of abortive influenza virus infection in primary M phi, this study demonstrated that the NWS virus replicated productively in both resident peritoneal M phi and thioglycolate-elicited peritoneal M phi providing cleavage of the HA was achieved by trypsin; 5 micrograms/ml of trypsin was the optimum concentration for the induction of infectivity. Under multiple-cycle growth conditions in the presence of mAB at various concentrations in trypsin-containing media, ADE was demonstrated in both M phi in the presence of subneutralizing concentrations of mAB. Flow cytometric analysis showed that the mechanism of virus entry into M phi could be through HA to specific virus receptors, or HA plus antibody to Fc receptors. These results indicate that ADE of the NWS virus infection actually occurs on Fc receptor-bearing primary murine M phi depending on the concentration of antibody in the presence of the appropriate protease for cleavage of viral HA.     

The Fc Receptors of Normal and Cancer Patients Monocytes

The ability of human monocytes from normal donors and gastric-cancer patients to form rosettes with ?0? Rh⁺(D) human erythrocytes coated with hyperimmune IgG anti-D antibody (EA_hu) and to kill the same target in antibody-dependent cellular cytotoxicity (ADCC) were assessed. Trypsin pretreatment of normal monocytes decreased their ability to form rosettes with EA_hu complexes, but their ADCC activity was unaffected. The Fc receptor (FcR) expression and ADCC activity of monocytes of cancer patients were elevated, and trypsin-treatment led to their further increase. The elevated values were related to the presence of the tumour. These results may suggest that human monocytes possess trypsin-sensitive and trypsin-resistant Fc receptors. The trypsin-resistant FcR seems to be involved in ADCC phenomenon and to be preferentially expressed on monocytes of some cancer patients.     

Effect of Fc receptor blocking on human natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The effect of Fc receptor (FcR) blocking by aggregated human gamma-globulin (AGG) was studied on natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. Cytotoxicity was measured by detachment from the monolayer of [³H]TdR-prelabelled HEp-2 cells. LDCC was evaluated in a 24 h assay at 50:1 effector-target cell ratio in the presence of 25 μg/ml concanavalin A (Con A). Under these conditions but without Con A considerable NCMC was not elicited by normal lymphocytes. FcR blocking by AGG treatment of effector cells resulted in a significant NCMC activity to HEp-2 targets. In contrast, AGG treatment profoundly depressed LDCC. Monocyte depletion of effector cells had no major influence on the effect of AGG on NCMC and LDCC activities. An interference of FcR blocking by AGG and LDCC in response to Con A is suggested.     

The effect of corticosteroids on monocyte and neutrophil activation in bronchial asthma

The expression of IgG (Fc) receptor (FcR) and complement receptor (CR) on peripheral blood monocytes and neutrophils was determined by the rosette technique in patients with asthma receiving different forms of treatment. In 31 patients taking inhaled therapy (i.e., bronchodilators alone or in combination with inhaled corticosteroids), monocyte FcR (48.19 +/- 1.24%, mean +/- SEM) and complement (66.54 +/- 1.09%) rosettes were significantly higher (FcR p less than 0.001, CR p less than 0.001) than in the 17 healthy, normal control subjects (FcR 37.94 +/- 0.82%, CR 59.7 +/- 0.98%). These increases in the percent rosettes between the two groups were observed even when a wide concentration range of IgG or complement was used to coat the red cells. No significant differences in monocyte receptor expression were observed between those patients being treated with bronchodilators alone or patients being treated in combination with inhaled corticosteroids. In 19 patients with asthma receiving oral corticosteroids, the mean monocyte FcR (38.21 +/- 1.73%) and CR (52.78 +/- 2.09%) were significantly reduced when these patients were compared with those patients receiving inhaled therapy alone (FcR p less than 0.001, CR p less than 0.001), and there was a significant inverse correlation between the percent rosettes and the dose of prednisolone. Neutrophil CR (51.32 +/- 1.30%, p less than 0.05) but not FcR expression (24.7 +/- 0.80%) was significantly increased when these were compared with those of control subjects (FcR 24.7 +/- 0.60%, CR 47.11 +/- 0.86%), and both neutrophil FcR and CR expression was significantly reduced (FcR p less than 0.01, CR p less than 0.001) in those patients with asthma receiving oral corticosteroids. (ABSTRACT TRUNCATED AT 250 WORDS)     

The role of Fc receptors in HIV prevention and therapy

Over the past decade, a wealth of experimental evidence has accumulated supporting the importance of Fc receptor (FcR) ligation in antibody-mediated pathology and protection in many disease states. Here we present the diverse evidence base that has accumulated as to the importance of antibody effector functions in the setting of HIV prevention and therapy, including clinical correlates, genetic associations, viral evasion strategies, and a rapidly growing number of compelling animal model experiments. Collectively, this work identifies antibody interactions with FcR as important to both therapeutic and prophylactic strategies involving both passive and active immunity. These findings mirror those in other fields as investigators continue to work toward identifying the right antibodies and the right effectors to be present at the right sites at the right time.     

Antimicrobial activities of dialysate-elicited and resident human peritoneal macrophages.

Recent studies of the antimicrobial capacity of peritoneal macrophages (PM phi) isolated from patients undergoing chronic peritoneal dialysis have raised the question of whether these cells might be analogous to stimulated or activated murine PM phi. To explore this possibility, we compared PM phi from these patients (dialysate-elicited PM phi) with PM phi obtained from women undergoing laparoscopy (resident PM phi) in several in vitro assays of phagocyte function. Although bacterial phagocytosis by cells from both groups of donors was similar, significant differences were found in their chemiluminescence responses to opsonized zymosan. Although the mean peak luminol-enhanced chemiluminescence response of dialysate-elicited PM phi was 4.7 X 10(5) cpm, that of resident PM phi was only 1.3 X 10(5) cpm (P less than 0.05). In a lucigenin-enhanced chemiluminescence assay, dialysate-elicited PM phi again generated significantly greater chemiluminescence than did resident PM phi, suggesting that dialysate-elicited PM phi have a relatively increased capacity for O2- production. Using a fluorochrome microassay to assess the intracellular candidicidal activities of these cells, we found that dialysate-elicited PM phi killed 17% of cell-associated blastospores compared with only 1.5% killing by resident PM phi (P less than 0.05). These investigations led us to conclude that results of studies of the functional activity of dialysate-elicited PM phi cannot necessarily be extrapolated to resident PM phi and that dialysate-elicited PM phi do in some respects behave as stimulated or activated cells.     

抗Fc受体γ链单克隆抗体的研制及生物学特性鉴定

目的:制备Fc受体γ链的单克隆抗体(mAb),并对其生物学特性进行鉴定,为研究受体γ链相关的疾病提供实验材料。方法:人工合成的蛋白多肽偶联后免疫BALB/c小鼠,用间接ELISA法筛选阳性克隆,Western blot、流式细胞术(FCM)检测鉴定抗体的特性及抗原识别位点。结果:通过细胞融合和亚克隆,共筛选出3株能稳定分泌抗体并且反应性好的杂交瘤细胞株5B6、7D3和8D4。其中7D3抗体反应性最强,2.5μg/mL与体内体外的γ链都能特异性反应。结论:获得了抗Fc受体γ链特异性的mAb,为进一步研究Fc受体γ链在相关疾病发生发展过程中所发挥的作用提供了良好的研究手段。     

Human placental membrane receptor for IgG. Purification of the receptor and its subunit structure

The method of purification of the human placental Fc receptor to an active form is described. The FcR was purified from the glycoprotein fraction of the placental membranes by immunoprecipitation and chromatography on DEAE-cellulose. The purifield FcR corresponded to 1.5–2% of the protein present in the crude glycoprotein fraction (PGP) and showed the tendency to aggregate. In the presence of 1% SDS, 4 M urea or 5 M guanidine-HCl the placental FcR dissociated into subunits of molecular weight of 60,000–65,000. The 60,000–65,000 dalton glycoprotein subunits regarded as monomers of FcR are composed of two chains of molecular weight 25,000–30,000, linked by disulphide bonds. The subunits, after removal of dissociating agents, displayed IgG binding activity.     

Spontaneous cell-mediated cytotoxicity in humans. Distribution and characterization of the effector cell.

When lymphocytes from healthy donors were tested as effector cells, the cytotoxic activities observed in spontaneous and in antibody-dependent cell-mediated cytotoxicity were positively correlated. However, with lymphocyte preparations obtained from renal patients, a dissociation between the two activities was occasionally observed. Human natural killer cells are lymphocytes, with receptors for the Fc fragment of IgG molecules, but with no surface immunoglobulin. Their cytotoxicity is reduced by the presence of granulocytes or monocytes. After separation of rosetting and non-rosetting cells with AET- (2-aminoethylisothiouronium bromide hydrobromide) or neuraminidase-treated sheep erythrocytes, the majority of the activity was recovered in the non-rosetting fraction, but a portion of it was present consistently in the rosetting cell fraction. Cells in the latter fraction also displayed receptors for the Fc fragment of immunoglobulin G.     

Inhibition by monoclonal anticomplement receptor type 1 on interactions between senescent human red blood cells and monocytic-macrophagic cells

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.