IgG Aggregates Research Articles

Effect of IgA Aggregates on Transforming Growth Factor-β1 Production in Human Mesangial Cells and the Intraglomerular Expression of Transforming Growth Factor-β1 in Patients with IgA Nephropathy

BackgroundTransforming growth factor-β (TGF-β) stimulates renal fibrosis in various renal diseases including IgA nephropathy.MethodsWe examined whether immunoglobulin A (IgA) stimulated TGF-β1 synthesis in human mesangial cells (MCs), and whether this effect was mediated through the protein kinase C (PKC) pathway. We measured the intraglomerular TGF-β1 mRNA expression by using competitive RT-PCR, and this was compared with various parameters in IgA nephropathy patients.ResultsThe IgA aggregate increased the TGF-β1 mRNA expression in MCs, while this expression was not affected by the culture media or IgG aggregate. Phorbol 12-myristate 13-acetate and calphostin C did not influence the TGF-β1 mRNA expression that was increased by the IgA aggregate. Intraglomerular TGF-β1 mRNA expression was significantly correlated with creatinine clearance (r=-0.764, p=0.027), daily proteinuria (r=0.781, p=0.022), serum creatinine (r=0.884, p=0.004), and tubulointerstitial changes (r=0.809, p=0.015). Glomerular TGF-β1 mRNA expression was associated with an increased tendency for glomerulosclerosis (r=0.646, p=0.084). After 4 years, patients with a high expression of intraglomerular TGF-β1 mRNA showed a tendency for an decrease of their renal function.ConclusionThe IgA aggregate increased TGF-β1 mRNA expression in MCs, and this was independent of the PKC pathway. The evaluation of intraglomerular TGF-β1 mRNA expression could be useful in predicting the progression of IgA nephropathy.

Responses of lymphocytes of patients with rheumatoid arthritis to IgG modified by oxygen radicals or peroxynitrite

We have previously demonstrated the presence of IgG aggregates modified by oxygen radicals (chlorinated IgG [Cl-IgG]) and peroxynitrite (nitrated IgG [N-IgG]) in synovial fluid of patients with rheumatoid arthritis (RA). A possible explanation for the longstanding chronic inflammatory process in RA is the establishment of an immune response to autoantigens. This study was undertaken to examine whether a T cell response to oxidatively modified IgG contributes to the inflammation in RA. We studied in vitro lymphocyte proliferation and interleukin 2 (IL-2) secretion in response to a common antigen (mumps), N-IgG, Cl-IgG, and heat-aggregated IgG (H-IgG) (control) in 15 normal blood donors and 16 RA patients not receiving immunosuppressive drugs. The responses of RA lymphocytes to mumps antigen were significantly lower that those in controls (mean +/- SEM 2,577 +/- 217 versus 6,367 +/- 365 counts per minute/well; P < 0.02). However, whereas in normal donors the cell responses to N-IgG and Cl-IgG were not significantly different than responses to H-IgG (N-IgG/H-IgG ratio 1.2 +/- 0.2, Cl-IgG/H-IgG 1.5 +/- 0.2), the RA lymphocyte responses to N-IgG and Cl-IgG were significantly higher than the responses to H-IgG (N-IgG/H-IgG 7.4 +/- 2.5, Cl-IgG/H-IgG 4.8 +/- 1.2). When ratios in RA cells were compared with normal cell responses as a group, there was a significant difference for both N-IgG (P < 0.017) and Cl-IgG (P < 0.014). When selected normal and RA lymphocyte culture supernatants were assayed for IL-2 secretion, the increase in IL-2 never exceeded 2-fold in normal cell cultures incubated with any of the IgG compared with unstimulated cultures, whereas responses of RA cells, particularly those incubated with N-IgG, were increased (range 2.6-15.7-fold) compared with unstimulated controls. These results suggest that in RA there are circulating T cells that are responsive to oxidatively modified IgG, a possible pathogenic mechanism contributing to the chronic inflammatory process within the inflamed joint.

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Aggregated IgG Bind to Glomerular Epithelial Cells to Stimulate Urokinase Release through an Endocytosis-Independent Process

Background: In membranous nephropathy, the development of glomerular lesions is related to the formation of immune complexes at subepithelial sites. These deposits are associated with modifications in the fibrinolytic activity of glomerular cells leading to the appearance of fibrin degradation products in the deposits and the urine. A previous study has shown that immune complexes interact with glomerular epithelial cells (GEC) through the neonatal Fc receptor (FcRn). We therefore determined whether this binding could be responsible for a modification in the fibrinolytic activity of GEC. Methods: Endocytosis of heat-aggregated immunoglobulins (AgIgG) in cultured human GEC was studied by immunofluorescence and confocal microscopy. The release of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI) by GEC or whole glomeruli was assessed by ELISA, fibrin zymography and Northern blot. Results: Human GEC in culture bound AgIgG that possess characteristics similar to soluble immune complexes and internalized them by 10 min. This process was mediated by FcRn since chicken aggregated IgG (AgIgY), that do not bind FcRn, did not colocalize with AgIgG in GEC. AgIgG but not AgIgY induced a decrease of FcRn expression at the membrane and within the cells. The binding of AgIgG to GEC elicited a dose- and time-dependent increase in the release of uPA activity, as in the uPA protein and mRNA expression without modification in the release of PAI. This process was not abrogated by agents inhibiting endocytosis and/or transcytosis such as cytochalasin B, suggesting an endocytosis-independent uPA regulation. Conclusion: GEC response to AgIgG overload comprises at least two sequential steps: (1) a FcRn-mediated endocytosis; (2) an endocytosis-independent fibrinolytic imbalance leading to plasmin generation which could favor in vivo AgIgG clearance and matrix remodeling.

Atomic force bio-analytics of polymerization and aggregation of phycoerythrin-conjugated immunoglobulin G molecules

While bio-labeled immunoglobulin (IgG) or antibodies are extensively used in imaging studies and therapeutic modality, there have been no reports describing atomic force microscopy (AFM) micrographs of these labeled IgG molecules. Here, AFM studies of phycoerythrin (PE)-conjugated IgG were undertaken to examine whether PE conjugation induced conformational changes or molecular interaction, and subsequently resulted in an alteration in nano-structures of PE-conjugated IgG complex. Once immobilized on mica, single PE-conjugated IgG molecule exhibited globular shape with approximately 60 microns in diameter and 5 microns in height. PE-conjugated IgG were able to form monomers, spindle-like trimers, and hexamers that developed through an end–end connection of two trimers, after they were continuously immobilized on mica. Interestingly, these multimers could aggregate in different directions to form circular monolayers with a highly dense core of PE-conjugated IgG polymers. The formation of these well-organized polymers and aggregates of PE-conjugated IgG may be attributed to the PE conjugation as well as the air–liquid tension on subtract. These findings may help to understand the nano-structures of bio-labeled IgG or antibodies, and facilitate the potential use of PE-conjugated antibodies as markers or immunosensers for AFM bio-analytics of biomolecules in cells and membranes.

Activation of human mast cells by aggregated IgG through FcγRI: additive effects of C3a

Human mast cells (huMC) increase surface expression of FcγRI (CD64) in response to IFNγ. Subsequent receptor aggregation of FcγR1 using CD64-specific F(ab′) 2 or antibody directed against FcγR1-bound IgG results in cell activation. Human mast cells may be observed degranulating in inflammation associated with autoimmune disease and where IFNγ is produced. We sought to determine if human mast cells cultured in IFNγ would degranulate in response to aggregated IgG, what mediators might be generated (i.e., cytokines and eicosanoids), and whether C3a might enhance such activation. Activation of IFNγ-treated huMC sensitized with 1 μg/ml aggregated IgG 1 resulted in 15–30% degranulation (β-hexosaminidase release), which was half-maximal by 7.5 min; no degranulation was observed using heat-generated aggregates of IgG 2, IgG 3, or IgG 4. Activation using aggregated IgG 1 led to PGD 2 and LTC 4 generation as well as enhanced IL-3, IL-13, GM-CSF, and TNFα production. Preincubation of cells with F(ab′) 2 from CD64-specific clone 10.1 reduced aggregated IgG 1-mediated β-hexosaminidase release by 38% while degranulation was unaffected by blocking FcγRII with F(ab′) 2-specific antibody (clone 7.3). Simultaneous activation of huMC via aggregated IgG and C3a led to additive degranulation. These data support a mechanism by which mast cells may contribute to the inflammatory component in fibrosis, vasculitis, and arthritis.

Dexamethasone attenuates oxidation of extracellular matrix proteins by human monocytes

In response to infection or in immune complex-mediated diseases, inflammatory cells may oxidatively damage extracellular matrix (ECM) proteins. In this study we evaluated whether human monocytes could oxidize ECM and whether this could be modulated by exposure to LPS, IgG complexes, and dexamethasone (DEX). Wells in tissue culture plates were coated with the ECM preparation Matrigel. Porous inserts with or without the human monocyte cell line THP-1 were placed into ECM-containing wells and cells were exposed to control conditions or to LPS (10 ng/ml), IgG complexes (200 and 500 μg/ml), or DEX (10 −7 and 10 −6 M). ECM was then subjected to Western blot analysis using an antibody to oxidized protein. In addition, Western blot analysis was carried out on DEX-treated cells to evaluate expression of the NADPH oxidase components p67- phox and gp91- phox. THP-1 cells enhanced ECM oxidation and this effect was augmented by LPS and by IgG aggregates. Preincubation of cells with DEX attenuated ECM oxidation and was also associated with decreased expression of p67- phox and gp91- phox. These findings suggest that human monocytes can oxidize ECM proteins and that this may be modulated by IgG complexes and LPS. Dexamethasone appears to attenuate ECM oxidation and a better understanding of this mechanism might allow for interventions to minimize oxidative damage to ECM proteins by monocytes in infectious and inflammatory states.

Differential Role of Actin, Clathrin, and Dynamin in Fcγ Receptor-mediated Endocytosis and Phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.

Effect of Preservatives on IgG Aggregation, Complement-activating Effect and Hypotensive Activity of Horse Polyvalent Antivenom Used in Snakebite Envenomation

Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4°C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

FcgammaRIIA exogenously expressed in HeLa cells activates the mitogen-activated protein kinase cascade by a mechanism dependent on the endogenous expression of the protein tyrosine kinase Syk.

HeLa cells transfected to express the human Fc receptor FcgammaRIIA were stimulated with aggregates of IgG, IgG-ovalbumin equivalence immune complexes and monoclonal antibody reacting with FcgammaRIIA. All of these stimuli activated the cells as judged from the band-shift characteristic of the activation of the p42-MAP/ERK kinase. Since this response is currently associated with the activation of the protein tyrosine kinase Syk, the expression of which is currently thought to be restricted to hemopoietic cells, the results were considered as an indirect evidence of the expression in HeLa cells of either Syk or another protein tyrosine kinase accounting for the same function. Transfection with a dominant negative Syk mutant abrogated the response to FcgammaRIIA cross-linking, whereas overexpression of Syk did not increase the extent of the response. Further evidence of the expression of syk was obtained by the reverse transcription PCR approach and sequencing of the DNA bands. Moreover, immunoprecipitation with anti-Syk antibody of the cell lysates obtained after cross-linking of FcgammaRIIA followed by immunoblotting with anti-phosphotyrosine antibody showed the phosphorylation of a protein band migrating as Syk. These data indicate that expression of FcgammaRIIA on epithelial HeLa cells conveys signals to the p42-MAP/ERK kinase by a mechanism involving the recruitment of Syk. In contrast, cross-linking of this receptor does not yield productive signals coupled to other responses associated to the FcgammaR system such as triggering of the arachidonic acid cascade, activation of the NF-kappaB system and production of chemotactic cytokines.

An improved anti-C3/IgG ELISA for quantification of soluble immune complexes

A semi-quantitative ELISA for complement-fixing, IgG-containing immune complexes (IC) is described. The assay is based on the insolubilization of IC by polyethyleneglycol, their capture by solid-phase anti-C3 antibodies, reaction with peroxidase-labeled anti-IgG antibodies and incubation with a chromogenic peroxidase substrate. It was markedly improved by the use of a single-step procedure which simultaneously washed and precipitated the insolubilized immune complexes. Intra-assay and inter-assay coefficients of variation were lower than 8.6 and 14.7%, respectively. As expected, higher levels of circulating immune complexes, in relation to healthy individuals, were found in patients with American visceral leishmaniasis (AVL), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), with prevalences comparable to those described in the literature. The ELISA can be quickly assembled from reagents and plasticware widely available commercially, detects immune complexes fulfilling three different criteria and is more sensitive than a previously published method based on the same principles (detection limit for complement-sensitized aggregated IgG of 2 μg ml −1 as compared with a detection limit above 16 μg ml −1).

Chimeric Human–Simian Anti-CD4 Antibodies Form Crystalline High Symmetry Particles

A chimeric human–simian IgG, antigen specific for CD4, when exposed to 0.5 M SO=4 containing 0.4% polyethylene glycol or Jeffamine, self-assembles into discreet, roughly spherical particles 23 nm in diameter. Increasing SO=4 to 1.55 M induces the IgG particles to crystallize in either a hexagonal or a monoclinic form. From X-ray diffraction, the former crystal is of space group P622, with one IgG particle in the unit cell; thus the particle itself must have 622 point group symmetry. Both crystal forms have been imaged using atomic force microscopy. Detailed features of the duodecamer were evident, including the symmetry and a large solvent channel along the sixfold axis. The particles in some ways resemble the hexameric IgG aggregates believed to activate compliment upon antigen binding and, therefore, may have physiological relevance. Investigation of seven other IgGs of diverse origins and subclasses indicates that many, if not most, IgGs form similar particles. To our knowledge, this is the first observation of the assembly of IgG into high symmetry aggregates in the absence of antigen or their crystallization.

Stimulation of Fc gamma R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I kappa B-alpha degradation and formation of p50/p65 NF-kappa B/Rel complexes.

THP-1 monocytic/macrophage cells were stimulated via their FcgammaR receptors with insoluble aggregates of human IgG and the production of the C-C chemokine monocyte chemoattractant protein (MCP)-1 assayed. A dose- and time-dependent production of MCP-1 comparable to that produced by the most potent agonists could be detected in the culture medium by a sensitive ELISA assay. This was accompanied by a parallel activation of the transcription factor NF-kappaB as judged from both the appearance of kappaB-binding activity containing p50/p65 NF-kappaB/Rel complexes in the nuclear extract and the disappearance of the NF-kappaB inhibitor IkappaB-alpha in the cell lysate. In contrast, IkappaB-beta and IkappaB-epsilon expression was not modified, thus pointing to the occurrence of a selective degradation of IkappaB-alpha under those conditions. Attempts to modulate MCP-1 production with compounds that display inhibitory effects on the activation of NF-kappaB such as the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal, the antioxidant pyrrolidine dithiocarbamate and the salicylate derivative 2-hydroxy-4-trifluoromethylbenzoic acid showed a parallel effect on both MCP-1 production and NF-kappaB activation, thus pointing to the involvement of kappaB-binding sites on the transcriptional regulation of MCP-1 production. Our findings suggest the existence in monocytic cells of a signaling mechanism initiated by cross-linking of low-affinity FcgammaR, most likely of the FcgammaRII family since THP-1 cells do not express FcgammaRIII receptors, that involves activation of NF-kappaB associated to the proteolytic degradation of IkappaB-alpha and leads to the transcriptional up-regulation of MCP-1.

Receptor mediated endocytosis by mesangial cells modulates transmigration of macrophages.

Accumulation of immune complexes in the mesangium is a common finding. Since migration of macrophages (Mphi) in the mesangium has been demonstrated to be an important event in the development of glomerular lesions, we studied the role of immune complexes and mesangial cell (MC) interaction in the transmigration (Tm) of Mphi. To determine the effect of MC and immune complexes (aggregated IgG, IgGAg) on transmigration of Mphi. MC were incubated with or without IgGAg in the lower compartment of a modified Boyden Chamber. To determine the effects of the secretory products (as a result of endocytosis of IgGAg by mesangial cells), MC-IgAg conditioned media was prepared and placed in the lower compartment of the Boyden chamber. We evaluated the effects of MC alone, MC + IgGAg, or MC-IgGAg conditioned media on the transmigration of macrophages across a filter. To determine the effect of free radicals on MC-IgAg endocytosis-induced Mphi migration we evaluated the effect of free radical scavengers such as dimethyl thiourea (DMTU) and tetramethylthiourea (TMTU) in MC-IgAg endocytosis-induced Mphi migration. To determine the role of chemokines in MC-IgAs endocytosis-induced Mphi migration we evaluated the effect of ani-MCP-1 antibodies on MC-IgAg endocytosis-induced Mphi migration, and also studied the effects of IgAg on MC mRNA expression of MCP-1 and RANTES. In addition, we evaluated the role of Fc receptors and actin cytoskeleton of MC in transmigration of Mphi. Mesangial cell endocytosis of IgG aggregates (IgGAg) is associated with enhanced (P < 0.001) transmigration of Mphi (control, 11.2 +/- 0.2 vs. MC + IgGAg, 22.1 +/- 0.9 migrated Mphi/field). IgGAg also induced MC mRNA expression for RANTES and MCP-1 on MC. DMTU and TMTU attenuated (P < 0.001) the MC + IgGAg-induced migration of Mphi as well as IgGAg-induced mRNA expression for RANTES and MCP-1. MC and IgGAg interaction products (MC-IgGAg conditioned media) also increased (P < 0.01) transmigration of Mphi (control, 18.3 +/- 1.7 vs. MC-IgGAg conditioned media, 30.7 +/- 0.6 Mphi/field). This effect of MC-IgGAg conditioned media on the migration of macrophages was dose dependent. Anti-MCP-1 antibody partially inhibited MC-IgGAg-induced migration of macrophages. MC and monomeric IgG (MIgG) interaction (MC-MIgG conditioned media) showed a lower (P < 0.05) migration of Mphi, when compared to the MC-IgGAg conditioned media. MC-IgGAg conditioned media prepared from cytochalasin B pretreated MCs also showed a lower (P < 0.001) migration of Mphi when compared with MC-IgGAg conditioned media-induced migration. These results indicate that MC-IgGAg conditioned media-induced transmigration of macrophages may be mediated through the generation of RANTES and MCP-1 by MC.

Evidence ofPasteurella haemolyticalinked immune complex disease in natural and experimental models

The pathogenesis of bovine pneumonic pasteurellosis is not completely understood, and studies have not established thatPasteurella haemolyticaA1 (Ph1) virulence is exclusively responsible for the development of acute pulmonary lesions. The purpose of this investigation was to determine if immune complex disease is involved in the pathogenesis of bovine pneumonic pasteurellosis. A retrospective immunohistologic study of lung tissue from natural cases of bovine pneumonic pasteurellosis (44) as performed, and immune complexes were observed in alveloar spaces and walls in 88% of these cases. To study this pathologic mechanism experimentally, groups of mice were immunized with purified Ph1 outer membranes (OMs) or sham immunized on days 0 and 14. Mice were challenged intratracheally on day 24 with either live Ph1 or Ph1 OMs, and pulmonary lesions were assessed 24 h after challenge. Placebo immunized mice developed focal infiltrates of neutrophils and macrophages centered around large caliber bronchi. Mice immunized with Ph1 OMs and challenged with live Ph1 or OMs developed severe bronchointerstitial pneumonia with diffuse neutrophilic infiltration, focal necrosis, hemorrhage and edema, that is histologically similar to bovine pneumonic pasteurellosis. Immunohistology revealed flocculent aggregates of IgG and complement positive material within alveolar spaces and walls from mice challenged with live Ph1, and fine granular deposits of IgG and complement positive material were observed lining the alveolar walls from mice challenged with Ph1 OMs. Immunized mice exhibited high serum IgG antibody titers to Ph1 outer membrane proteins (OMPs). Results of this study suggest that immune complex disease plays a role in the pathogenesis of bovine pneumonic pasteurellosis.

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.

A novel polymorphism in the extracellular domain 2 (EC2) of FcgammaRIIIA affects ligand binding by natural killer (NK) cells and monocytes from genotyped homozygous normal donors independently of receptor expression. The nonconservative T to G substitution at nucleotide 559 predicts a change of phenylalanine (F) to valine (V) at amino acid position 176. Compared with F/F homozygotes, FcgammaRIIIa expressed on NK cells and monocytes in V/V homozygotes bound more IgG1 and IgG3 despite identical levels of receptor expression. In response to a standard aggregated human IgG stimulus, FcgammaRIIIa engagement on NK cells from V/V (high-binding) homozygotes led to a larger rise in [Ca2+]i, a greater level of NK cell activation, and a more rapid induction of activation-induced cell death (by apoptosis). Investigation of an independently phenotyped normal cohort revealed that all donors with a low binding phenotype are F/F homozygotes, while all phenotypic high binding donors have at least one V allele. Initial analysis of 200 patients with SLE indicates a strong association of the low binding phenotype with disease, especially in patients with nephritis who have an underrepresentation of the homozygous high binding phenotype. Thus, the FcgammaRIIIa polymorphism at residue 176 appears to impact directly on human biology, an effect which may extend beyond autoimmune disease characterized by immune complexes to host defense mechanisms.

Potentiation of C1 inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma.

Activation of the complement system may contribute to the pathogenesis of many diseases. Hence, an effective inhibitor of complement might be useful to reduce tissue damage. Some glycosaminoglycans (GAG), such as heparin, are known to inhibit the interaction of C1q with activators and the assembly of the classical and the alternative pathway C3 convertases. Furthermore, they may potentiate C1 inhibitor-mediated inactivation of C1s. To search for potential complement inhibitors, we systematically investigated the complement inhibitory properties of various synthetic and naturally occurring GAG (dextran sulfates 500,000 and 5,000, heparin, N-acetylheparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates A and C). First, we assessed the effect of GAG on the second-order rate constant of the inactivation of C1s by C1 inhibitor. This rate constant increased 6- to 130-fold in the presence of the GAG, dextran sulfate being the most effective. Second, all tested GAG were found to reduce deposition of C4 and C3 on immobilized aggregated human IgG (AHG) and to reduce fluid phase formation of C4b/c and C3b/c in recalcified plasma upon incubation with AHG. Dextran sulfate again was found to be most effective. We conclude that GAG modulate complement activation in vitro and that the low molecular weight dextran sulfate (m.w. 5000) may be a candidate for pharmacologic manipulation of complement activation via potentiation of C1 inhibitor.

Processing of IgA Aggregates in a Rat Model of Chronic Liver Disease

Heavy alcohol intake and/or lipotrope-deficient diet induced hepatocellular injury and mesangial deposition of IgA and often IgG in Lewis rats. The experimental animals showing more severe urinary abnormalities and histologic damage in the glomeruli had increased levels of IgA antibodies to dietary antigens and altered intestinal permeability. Based on human studies, the prolonged circulation of IgA-containing complexes associated with the liver disease could be envisaged as important for the development of mesangial IgA deposits. In order to verify this hypothesis, four groups (G) of Lewis rats were studied: G1 received thrice a week an intragastric infusion of 1.5 ml/100 g body wt of whiskey; G2 rats were nourished with lipotrope-deficient diet; G3 rats were given both whiskey and LD diet; G4 rats were nourished with regular chow. After 12 weeks, heat-aggregated rat monomeric IgA was labeled with123I and intravenously injected. Three control subgroups of rats, one given whiskey, one nourished with LD diet, and one with regular chow, were injected with radiolabeled heat-aggregated rat IgG. A large field-of-view digital gamma camera, equipped with an ultra-high-resolution collimator and interfaced to a dedicated computer, was used to analyze tracer kinetics and fate. The liver was the main organ involved in clearance of both test probes. The hepatic mean transit (MTT) was 114 ± 11 min in G1 (proteinuria of 6.9 ± 1.41 mg/day and hematuria +/++), 221 ± 19 min in G2 (proteinuria 9.1 ± 0.64 mg/day and hematuria ++/+++), and 230 ± 15 min in G3 (proteinuria 9.5 ± 0.58 mg/day and hematuria ++/+++). In each case MTT value was found to be significantly prolonged compared to G4 (85 ± 4 min). The multiple regression analysis showed that MTT values, proteinuria, and hematuria were significantly correlated (P< 0.01). Controls had trace amount proteinuria (0.82 ± 0.17 mg/day, significantly lower than for each study group,P< 0.03) and undetectable hematuria. Similar results were obtained in control rats injected with aggregated IgG; i.e., MTT values were more prolonged in rats given whiskey or LD diet than normally nourished rats (P< 0.01). The lipotrope-deficient diet and the chronic alcohol abuse per se seem to lead to critical changes in hepatic uptake and catabolism of both an IgA and an IgG aggregate, which could account in turn for the reported appearance of renal immunoglobulin deposits in this experimental model. Due to the comparable delay in removal of IgA and IgG probes in equally nourished animals, additional factors are likely to be involved in the prominent deposition of IgA.

Regulation of RANTES and ICAM-1 expression in murine mesangial cells.

Chemokines and adhesion molecules play a pivotal role in leukocyte infiltration during tissue injury. RANTES (regulated upon activation, normal T cell expressed and secreted) is a monocyte chemoattractant that induces the expression of CD11/CD18 integrins on leukocytes for which intercellular adhesion molecule-1 (ICAM-1) is the ligand. Both RANTES and ICAM-1 can be expressed by mesangial cells (MC) in culture and in glomeruli during immune injury. In this study, the role of reactive oxygen species (ROS) in the activation of RANTES and ICAM-1 in murine MC was examined. Tumor necrosis factor alpha (TNF-alpha) and aggregated immunoglobulin (aggr. Ig) G, which enhance ROS formation in MC, increased mRNA transcripts of both RANTES and ICAM-1. Thiol-containing free-radical scavengers N-acetyl cysteine, dimethyl- and tetramethylthiourea, or pyrrolidinedithiocarbamate abrogated the increase in mRNA for RANTES and ICAM-1 in response to TNF-alpha or IgG. Hydroxy-methoxy acetophenone, an inhibitor of NADPH-dependent oxidase, also attenuated RANTES and ICAM-1 in response to TNF-alpha or IgG. ROS generated by addition of xanthine oxidase and hypoxanthine induced RANTES and ICAM-1 expression, whereas hydrogen peroxide caused no response. Because cAMP can interfere with gene activation in MC, the effects of 8-Br-cAMP, forskolin, and prostaglandin E2 on mRNA levels were examined for RANTES and ICAM-1. These agents attenuated the response to IgG aggregates and also to superoxide generation. Finally, the effect of glucocorticoids, which are frequently used in glomerular immune injury, was examined. Dexamethasone decreased mRNA for both RANTES and ICAM-1 after stimulation with aggr. IgG or TNF-alpha. Both forskolin and dexamethasone also reduced the amount of RANTES protein secreted by MC in response to aggr. IgG. Only dexamethasone decreased RANTES secretion in response to TNF-alpha stimulation. The inhibitory effects of cAMP and dexamethasone may explain the beneficial effects of cAMP mimetics, such as prostaglandin E2 and glucocorticoid administration on glomerular inflammatory processes.