Phagocytosis Of Immune Complexes Research Articles

The fate of immune complexes in membranous nephropathy.

The most characteristic feature of membranous nephropathy (MN) is the presence of subepithelial electron dense deposits and the consequential thickening of the glomerular basement membrane. There have been great advances in the understanding of the destiny of immune complexes in MN by the benefit of experimental models represented by Heymann nephritis. Subepithelial immune complexes are formed in situ by autoantibodies targeting native autoantigens or exogenous planted antigens such as the phospholipase A2 receptor (PLA2R) and cationic BSA respectively. The nascent immune complexes would not be pathogenic until they develop into immune deposits. Podocytes are the major source of autoantigens in idiopathic membranous nephropathy. They also participate in the modulation and removal of the immune complexes to a large extent. The balance between deposition and clearance is regulated by a wide range of factors such as the composition and physicochemical properties of the immune complexes and the complement system. Complement components such as C3 and C1q have been reported to be precipitated with the deposits whereas a complement regulatory protein CR1 expressed by podocytes is involved in the phagocytosis of immune complexes by podocytes. Podocytes regulate the dynamic change of immune complexes which is disturbed in membranous nephropathy. To elucidate the precise fate of the immune complexes is essential for developing more rational and novel therapies for membranous nephropathy.

50P Transcriptomic profiles of CD32b in breast cancer predict outcome and are associated with immune activation

50P Transcriptomic profiles of CD32b in breast cancer predict outcome and are associated with immune activation

TLR7/8 activation in neutrophils impairs immune complex phagocytosis through shedding of FcgRIIA.

Neutrophils play a crucial role in host defense. However, neutrophil activation is also linked to autoimmune diseases such as systemic lupus erythematosus (SLE), where nucleic acid-containing immune complexes (IC) drive inflammation. The role of Toll-like receptor (TLR) signaling in processing of SLE ICs and downstream inflammatory neutrophil effector functions is not known. We observed that TLR7/8 activation leads to a furin-dependent proteolytic cleavage of the N-terminal part of FcgRIIA, shifting neutrophils away from phagocytosis of ICs toward the programmed form of necrosis, NETosis. TLR7/8-activated neutrophils promoted cleavage of FcgRIIA on plasmacytoid dendritic cells and monocytes, resulting in impaired overall clearance of ICs and increased complement C5a generation. Importantly, ex vivo derived activated neutrophils from SLE patients demonstrated a similar cleavage of FcgRIIA that was correlated with markers of disease activity, as well as complement activation. Therapeutic approaches aimed at blocking TLR7/8 activation would be predicted to increase phagocytosis of circulating ICs, while disarming their inflammatory potential.

Neutrophil Function in Septic Dogs

Leukocytes appear to enter a hypo-inflammatory state in human patients with a severe bacterial infection, whereas, in swine, intra-abdominal sepsis produces an initial increase and subsequent decrease in neutrophil Fc receptor mediated phagocytosis. Impaired neutrophil function (hypo-inflammatory state) develops in dogs with sepsis. Thirteen adult client-owned dogs that developed clinical signs consistent with sepsis were assessed for evidence of neutrophil dysfunction. These results were compared with those of 12 healthy dogs. Flow cytometry combined with the appropriate fluorescent markers allowed for quantification of the phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis of immune complexes, neutrophil phagocytosis of opsonized Escherichia coli, and the intracellular concentration of reduced glutathione as a measure of oxidative stress. The phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis was significantly lower in neutrophils from septic dogs (mean fluorescence intensity +/- standard deviation; 118 +/- 13 and 165 +/- 27 for septic and control dogs, respectively, p = 0.001), although the phagocytosis of opsonized E. coli was significantly increased (155 +/- 74 and 77 +/- 44 for septic and control dogs, respectively, p = 0.004). Intracellular reduced glutathione was not significantly different in neutrophils from septic and healthy control dogs. An important component of neutrophil function is decreased in septic dogs. The diminished oxidative burst after Fc receptor-mediated phagocytosis in neutrophils from septic dogs could hinder the ability of the innate immune system to clear bacterial infections or it might help protect these patients from the systemic consequences of infection.

The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks ( Anas platyrhynchos)

Ducks produce a full length IgY and a truncated isoform (IgYΔFc). IgY and IgY(ΔFc) were isolated from ducks vaccinated against Escherichia coli and used to make E.coli-Ig immune complexes (IC). Phagocytosis of IC by duck monocytes decreased directly with the proportion of IgYΔFc ( p<0.001). IC containing IgY:IgY(ΔFc) at ratios of 100:0, 50:50, 0:100, and 0:0 ( E. coli alone) were injected intravenously into naı̈ve mallard ducks. At 24 h after injection, plasma hemopexin levels were higher in ducks given either the 0:100 ratio or the 100:0 ratio than those given 50:50 or E. coli alone ( p<0.05). Liver IL-1β mRNA levels followed a similar pattern. Splenic IL-1β mRNA decreased markedly as the proportion of IgY(ΔFc) increased ( p<0.01). Thus, IgY(ΔFc) may shift the response to IC from the spleen to the liver as infections progress from acute to chronic.

FcgammaRIIa polymorphism in Japanese patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an immune complex-mediated disease and organ damage is caused by the deposition of immune complex. Receptors which recognize the Fc portion of immunoglobulin G (FcgammaR) play a key role in the phagocytosis of immune complexes. As the gene encoding for FcgammaR of class IIa (FcgammaRIIa) has two allelic forms, H131 and R131, which differ in their affinity to IgG2, this polymorphism might have implications in handling immune complex. We studied the distribution of the FcgammaRIIa polymorphism in 90 Japanese patients with SLE. We also examined the association between FcgammaRIIa polymorphism and the disease activity of SLE and the histopathological findings of lupus nephritis. FcgammaRIIa polymorphism was determined by PCR and dot blot analysis. The allelic frequency of H131 in patients with SLE was significantly lower (H131/R131 = 0.44/0.56) than that of normal controls (H131/R131 = 0.62/0.38; P < 0.05). No significant association was observed between FcgammaRIIa polymorphism and the clinical parameters for the activity of SLE. There was no association between FcgammaRIIa polymorphism and the histological findings in lupus nephritis. The difference in the distribution of FcgammaRIIa alleles between patients with SLE and normal subjects indicates that this polymorphism is a candidate of susceptibility gene for SLE in Japanese.

Simultaneous Detection of Free Radical Release and Membrane Current during Phagocytosis

Stimulation of macrophages induces the "respiratory burst" response which is associated with the generation of superoxide (O2-), a drop in cytoplasmic pH, and a pronounced depolarization of the membrane potential. The purpose of the present studies was to determine whether an increase in O2- was temporally related to changes in membrane potential and transmembrane current. Release of O2- at the single cell level was photometrically monitored during phagocytosis of immune complexes while simultaneously measuring whole-cell current. Membrane depolarization and the generation of a non-selective current followed an increase in O2- production with a variable lag time which was correlated with the state of cellular maturation in culture. In the absence of phagocytosis, the exposure of macrophages to O2- generated by a xanthine-xanthine oxidase reaction activated a non-selective current similar to that seen after phagocytosis. These results provide the first demonstration of the relationship between free radical release and the ensuing electrophysiological signaling events which are linked to particle engulfment in phagocytic cells.

Antigen Distribution in Organs of Mink with Aleutian Disease Parvovirus Infection

On tissues from naturally infected non-Aleutian mink an immunohistological study was performed using monoclonal antibodies and the immunoperoxidase method. Structural proteins of ADV were demonstrated in cryosections and in ethanol-fixed and paraffin-embedded material which provide antigen detection in a similar amount together with good histological structure. In lymphoid organs viral antigen was restricted to B-cell areas, particularly lymphoid follicles. The pattern of antigen distribution was typical for follicular dendritic cells which are capable to retain immune complexes. Beside macrophages in the interior of lymphoid follicles most likely proliferating B-lymphoblasts reveal nuclear and cytoplasmatic presence of structural proteins indicating viral replication. Cells of the mononuclear phagocyte system such as cells of lymphatic sinuses and hepatic Kupffer cells harbor viral protein in the cytoplasm, probably resulting from phagocytosis of immune complexes. Renal glomeruli were consistently negative for virus antigen whereas in interstitial infiltrates cells resembling macrophages stained positive for ADV structural proteins.

Kupffer cell complement receptor clearance function after surgical injury and phagocytosis of immune complexes: effect of changes in plasma fibronectin.

The present study evaluated the effect of changes in plasma fibronectin levels on the degree of depression of in vivo clearance function of Kupffer cell complement receptors after injury and the phagocytosis of immune complexes. In vitro studies have suggested that fibronectin may act as an opsonin for the clearance of immune complexes from the blood by binding to C1q and may influence the expression and activation of immune receptors on macrophages. Complement receptor clearance function was assessed in rats from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM. Increasing plasma fibronectin by the injection of purified fibronectin or decreasing fibronectin by the injection of gelatin had no effect on complement receptor function in otherwise normal animals. Surgical injury depressed both plasma fibronectin levels and complement receptor function. Injection of fibronectin at the time of injury prevented the depression of receptor function; however, when fibronectin was given 1 hour after injury, receptor function remained depressed even though fibronectin levels were increased. The phagocytosis of immune complexes (IgG-coated rat erythrocytes [EIgGs]) depressed complement receptor function but did not depress plasma fibronectin levels. The depression of receptor function caused by the phagocytosis of EIgGs was prevented by administering fibronectin and was potentiated by administering gelatin, which decreased plasma fibronectin levels. Therefore, plasma fibronectin concentrations can influence in vivo Kupffer cell complement receptor function under certain conditions that lead to the depression of complement receptor function.

Joint fluid leukocyte activation by preformed immune complexes.

Acute synovitis was induced in rabbit knee joints by intraarticular injection of preformed bovine serum albumin (BSA) -anti BSA immune complexes (ICs). Polymorphonuclear granulocytes (PMNGs) which had migrated into joints injected with ICs were degranulated and contained ICs as revealed by electron microscopy and were activated as revealed by the measurement of chemiluminescence (CL). In contrast, leukocytes from control joints injected with BSA and normal rabbit serum as well as glycogen-elicited peritoneal leukocytes did not display any morphological changes and did not show CL. Compared to cells from other sources, leukocytes from IC joints showed a decreased CL response when stimulated in vitro with ICs but not with opsonized zymosan, suggesting a stimulus-specific modification of the PMNG responsiveness. Inhibition experiments showed that oxygen radicals and formation of arachidonate metabolites, mainly of the lipoxygenase pathway, were involved in the CL response of the IC-stimulated joint fluid PMNGs. Our observations on morphology, activity, and responsiveness of emigrated cells from the various sources suggest, together with previous observations, that the reaction of leukocytes in IC-induced synovitis consists of an initial migration phase not related to an increased CL and a subsequent activation phase characterized by degranulation, phagocytosis of ICs and increased CL.

A specific antagonist of platelet-activating factor suppresses oedema formation in an Arthus reaction but not oedema induced by leukocyte chemoattractants in rabbit skin.

The properties of a novel platelet-activating factor (PAF) antagonist, L-652731, on oedema responses in rabbit skin induced by exogenous inflammatory mediators and by mediators generated endogenously in a reversed passive Arthus reaction have been investigated. Oedema responses in the skin were measured by using the local accumulation of i.v. injected 125I-albumin. The antagonist, mixed with mediators before intradermal injection, caused a dose-dependent suppression of oedema responses to PAF. In contrast, responses induced by other directly acting mediators (bradykinin and histamine) and responses induced by PMN leukocyte-dependent mediators (C5a des Arg, N-formyl-methionyl-leucyl-phenylalanine, and leukotriene B4) were not suppressed. Thus, a secondary release of PAF does not appear to be involved in mediating the actions of these agents. In a reversed passive Arthus reaction, intradermal injection of L-652731 together with antibody resulted in a significant inhibition of the oedema formation measured for 2 hr after i.v. antigen challenge. In contrast, oedema responses induced by intradermal injection of preformed immune complexes were not affected by the antagonist. These results suggest that the endogenous production of PAF, in close proximity to microvascular endothelial cells, appears to be an important step in the development of an Arthus reaction. The cellular source of PAF is unknown, but one possibility is the PMN leukocyte, which releases PAF during phagocytosis of immune complexes.

Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages.

The inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's), synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-alpha-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1 h at 37 degrees C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macrophages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.

Functional cooperation of C3b-acceptors, Fcγ-receptors and cell-surface proteases on macrophages

Macrophages are FcR-positive cells, synthetize complement components and express proteolytic enzymes on their surface. In this paper a functional cooperation of C3b acceptor (C3bA) sites, which bind covalently nascent C3b molecules via their metastable binding site, IgG FcRs and cell surface proteases are described and the possible importance of this cooperation in regulation of immune response is discussed. It was found that isolated monocytes did not express C3bA in contrast to cultured macrophages which showed immune adherence positivity. Stimulation of macrophages resulted in enhanced expression of C3bA. C3 synthetized by macrophages was shown to be cleaved by cellular proteases which resulted in the binding of nascent C3b to C3bA. C3bA-nascent C3b interaction inhibited FcR-dependent effector functions, such as immune complex phagocytosis and antibody-dependent cellular cytotoxicity.

Superoxide radical production by human leukocytes exposed to immune complexes: inhibitory action of gold compounds.

Superoxide radicals produced by phagocytic cells are considered to be important mediators in the rheumatoid inflammation. The effect of the gold compounds auranofin (AF) and gold sodium thiomalate (GST) on superoxide production by human leukocytes was investigated in two models of immunologic injury: immune-complex phagocytosis and frustrated phagocytosis. In both systems, AF (0.5-1.0 micrograms Au/ml) showed a potent inhibitory activity on superoxide generation, quantitated by ferricytochrome c and NBT reduction. GST showed only modest inhibition at higher concentrations (100 microM). The thiol protecting agent dithiothreitol, 1 mM, completely blocks the inhibitory effect of AF. The inhibition of the oxy radical generation by AF may play an important role in the control of rheumatoid inflammation; it is suggested that this action might be mediated through sulfhydryl-AF interaction at the cellular membrane level.

Modulation of human mononuclear cell responses by neutrophil-derived factors. II. Partial characterization of a neutrophil-derived lymphocyte-enhancing factor (N-LEF).

We previously reported that neutrophil (PMN)-derived factors modulated human mononuclear cell (MNC) responses. These interactions are important in understanding MNC localization, growth, and activation in inflammatory events such as occur in the rheumatoid synovium. We have partially identified and characterized a human neutrophil-derived, lymphocyte-enhancing factor (N-LEF). Highly purified PMN, devoid of MNC or platelets, were obtained by density gradient sedimentation. Supernatants (spt) were prepared from PMN phagocytosis of complement-coated zymosan particles. N-LEF was determined by [3H]TdR uptake or Ig secretion by stimulated MNC of cell lines. Neutral protease activity was quantitated by digestion of 125I-labeled Hb. Sodium dodecylsulfate (SDS) polyacrylamide gel electropheresis (PAGE) was carried out in 15% gels. We found (1) N-LEF biologic activity was cold stable, heat labile, nondialyzable, and diminished at acid pH (less than 4) or alkaline pH (greater than 10); (2) N-LEF biologic activity was abrogated by human plasma; (3) spt contained neutral protease and granule-associated but not cytoplasmic enzyme activities; (4) protease activities (for Hb, fibrin, ATEE, and Ac-Phe-BNa) were recovered from aprotinin-sepharose affinity columns and contained approximately 25,000 and approximately 28,000 molecular weight material on SDS-PAGE; (5) absorbtion of N-LEF spt on aprotinin-sepharose diminished both protease and biologic activities; and (6) N-LEF biologic activity and nonelastase neutral protease activity were found in approximately 13,000 mol wt fractions of Sephadex G100 (where cathepsin G chromatographs). Our observations suggest that phagocytosis of immune complexes by PMN induces release of a mononuclear cell enhancing factor, N-LEF, which may be an important mediator of inflammation, promoting transition from an acute to a chronic inflammatory response. Biologic properties of N-LEF are those of cathepsin G.

Evidence for the presence of double-stranded polynucleotides in the neutrophil inclusions of circulating neutrophils and those which form in the presence of SLE sera

Intracytoplasmic inclusions containing immunoglobulin (Ig) and complement (C3) are found in normal neutrophils (PMN) after incubation with SLE sera (indirect method). These inclusions are believed to be immune complexes removed by phagocytosis from the SLE patients' sera in vitro. Similar inclusions have also been noted in the circulating PMN from patients with SLE (direct method). By both direct and indirect methods, IgG-staining intracytoplasmic inclusions were present in preparations from patients with SLE, chronic liver disease, and Felty's syndrome while none of the normal controls had any inclusions. Using ethidium bromide (EB), a fluorescent stain for double-stranded polynucleotides, red fluorescence was noted in the inclusions of some circulating SLE PMN (36% of patients) and in normal PMN exposed to SLE sera in 67% of SLE patients, suggesting that polynucleotides were part of these complexes. Such staining was not present in control inclusions produced with heat aggregated IgG, nor in any of the patient control groups. Addition of DNA to some SLE sera increased the intensity of the red fluorescence suggesting the presence of antibodies to polynucleotides which can combine with the added DNA. Some originally negative sera were converted to positive by addition of DNA. Purified IgG fractions from SLE sera did not produce inclusions or EB positively unless DNA was added. IgG fractions from normal sera did not produce inclusions with or without added DNA. These findings suggest (1) that normal PMN on exposure to SLE sera develop intracytoplasmic inclusions by phagocytosis of immune complexes containing double-stranded polynucleotides and antibodies to polynucleotides and (2) such complexes may be phagocytosed in vivo as indicated by the presence of inclusions in vivo and contribute to the number of granulocyte disturbances seen in SLE patients. These abnormalities in granulocyte function may be important predisposing factors for infection in patients with active SLE.

Evidence for two mechanisms of dengue virus infection of adherent human monocytes: trypsin-sensitive virus receptors and trypsin-resistant immune complex receptors.

Trypsin treatment of adherent human monocytes greatly reduced or eliminated the ability of these cells to support dengue virus replication. However, addition of dilute (nonneutralizing) antibody to the inoculum and the culture medium resulted in viral yields similar to those from monocytes not treated with trypsin. These results suggested that viral entry was facilitated by phagocytosis of immune complexes via Fc receptors on the monocytes. This concept was tested by (i) pretreating monocytes with aggregated gamma globulin, which resulted in a 40-fold reduction of viral yields after infection with dilute antibody-virus complexes and (ii) forming an immune complex with virus, antivirus F(ab')2 fragments, and rabbit anti-human Fab. Whereas F(ab')2 fragments alone would not enhance virus replication in trypsin-treated monocytes, the immune complex containing a rabbit Fc piece did increase the yield of dengue virus. These results suggest that dengue virus can infect a cultured monocyte in two ways: (i) through a viral receptor that is trypsin sensitive or (ii) through an Fc receptor that is not trypsin sensitive.

Association of severe rheumatoid arthritis with heterozygosity for alpha 1-antitrypsin deficiency.

Genetic types of alpha 1-antitrypsin (protease inhibitor types, or Pi types) were determined in 108 patients with rheumatoid arthritis. These patients were selected for severely destructive disease and had classical rheumatoid arthritis according to ARA criteria, were seropositive, and had joint erosions shown by X-ray. Heterozygotes for the deficiency Z allele (Pi types MZ, SZ, etc.) were found among 9.2% of patients and 3.5% of a control adult population. The increased frequency in patients was statistically significant. Heterozygotes were most frequent among female patients with an early onset of disease. Heterozygosity for alpha 1-antitrypsin deficiency may be a factor in familial recurrence of rheumatoid arthritis. Among 98 patients with juvenile rheumatoid arthritis not selected for severity, 4.1% were Z heterozygotes compared with 1.3% of control children, not a statistically significant difference. Reduced concentrations of alpha 1-antitrypsin in Z heterozygotes may be inadequate to inhibit the proteolytic enzymes released into the joints of adults with rheumatoid arthritis during phagocytosis of immune complexes. This may be a factor promoting severe joint destruction.

Exogenous immunoglobulin and the macrophage origin of Reed-Sternberg cells in Hodgkin's disease.

We studied Reed-Sternberg cells from 14 patients with Hodgkin's disease to learn whether they had monoclonal immunoglobulin synthesized by the cell or polyclonal immunoglobulin of external origin. Double-label immunofluorescence with F(ab')2 anti-serums to human light chains showed that cytoplasmic immunoglobulin of individual Reed-Sternberg cells is always polyclonal and usually associated with membrane-bound immunoglobulin of the same type. The predominant immunoglobulin was IgG; in one case IgM was also present. In vitro studies confirmed the internalization of exogenous IgG and phagocytosis of immune complexes by viable Reed-Sternberg cells. Their exclusion of trypan blue dye and lack of albumin and fibrinogen suggests relatively specific uptake of immunoglobulin, mediated by the Fc receptor or antigen (or antigens) associated with Hodgkin's disease at the cell membrane. Our studies support other recent evidence that the Reed-Sternberg cell is derived from a macrophage.

80. Phagocytosis of immune complexes by human eosinophils

80. Phagocytosis of immune complexes by human eosinophils