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Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics., (© 2019 Bersellini Farinotti et al.)

FcγRIIB functions to suppress the activation of immune cells. A single-nucleotide polymorphism in the transmembrane (TM) domain of FcγRIIB, FcγRIIB-T232, is associated with lupus. In this study, we investigated the pathogenic mechanism of FcγRIIB-T232 at both functional and structural levels. Our results showed that FcγRIIB-T232 exhibited significantly reduced lateral mobility compared with FcγRIIB-I232 and was significantly less enriched into the microclusters of immune complexes (ICs) after stimulation. However, if sufficient responding time is given for FcγRIIB-T232 to diffuse and interact with the ICs, FcγRIIB-T232 can restore its inhibitory function. Moreover, substituting the FcγRIIB-T232 TM domain with that of a fast floating CD86 molecule restored both the rapid mobility and the inhibitory function, which further corroborated the importance of fast mobility for FcγRIIB to function. Mechanistically, the crippled lateral mobility of FcγRIIB-T232 can be explained by the structural changes of the TM domain. Both atomistic simulations and nuclear magnetic resonance measurement indicated that the TM helix of FcγRIIB-T232 exhibited a more inclined orientation than that of FcγRIIB-I232, thus resulting in a longer region embedded in the membrane. Therefore, we conclude that the single-residue polymorphism T232 enforces the inclination of the TM domain and thereby reduces the lateral mobility and inhibitory functions of FcγRIIB., (© 2016 Xu et al.)

When studies on rheumatoid arthritis (RA) that were made many decades ago and could be considered "historical" in nature are analyzed in the context of recent observations, important insights on RA and on the function of rheumatoid factor (RF) become apparent. RF in the role of antibody to immune complexes (ICs) appears to be involved in activation of the complement system and in the production of chemotactic and inflammatory mediators, creating a condition that can be sustained and reinitiated. In the synovial cavity, a state of nonresolving inflammation is produced with the formation of citrullinated protein antigen-antibody complexes or other forms of ICs. This is followed by a second wave of IC production in the form of RF acting as antibody reactive with the initial ICs. Both of these processes are associated with complement consumption and production of inflammatory mediators. We present a model of an initiation phase of RA that might represent an example of repetitive formation of ICs and complement-mediated inflammation. Targeting therapy at this phase of RA to break the cycles of recurrent inflammation might be a novel approach to aid in further control of the disease., (© 2016 Tan and Smolen.)

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.

Inflammation induced by circulating immunoglobulin G-immune complexes (ICs) characterizes many immune-mediated diseases. In this work, the molecular requirements for the deposition of circulating ICs and subsequent acute leukocyte recruitment in mice were elucidated. We show that after intravenous injection, preformed soluble ICs are rapidly deposited in the postcapillary venules of the cremaster microcirculation, secondary to increased vascular permeability. This deposition is dependent on complement C1q. IC deposition is associated with leukocyte recruitment. Leukocyte rolling, which is mediated by P-selectin in the exteriorized cremaster muscle, is not further increased in response to ICs. In contrast, leukocyte rolling velocity is significantly decreased and leukocyte adhesion is significantly increased in the presence of ICs. The IC-mediated slow leukocyte rolling velocity and subsequent adhesion and emigration are dependent on Fcgamma receptors (FcgammaRs), particularly FcgammaRIII, with complement C3 and C5 having no detectable role. These studies suggest a regulatory mechanism of IC deposition and leukocyte trafficking in IC-mediated inflammation requiring C1q and FcgammaRs in sequential, noninteracting roles.

Interleukin-10 (IL-10) is a potent deactivator of myeloid cells that limits the intensity and duration of immune and inflammatory responses. The activity of IL-10 can be suppressed during inflammation, infection, or after allogeneic tissue transplantation. We investigated whether inflammatory factors suppress IL-10 activity at the level of signal transduction. Out of many factors tested, only ligation of Fc receptors by immune complexes inhibited IL-10 activation of the Jak-Stat signaling pathway. IL-10 signaling was suppressed in rheumatoid arthritis joint macrophages that are exposed to immune complexes in vivo. Activation of macrophages with interferon-gamma was required for Fc receptor-mediated suppression of IL-10 signaling, which resulted in diminished activation of IL-10-inducible genes and reversal of IL-10-dependent suppression of cytokine production. The mechanism of inhibition involved decreased cell surface IL-10 receptor expression and Jak1 activation and was dependent on protein kinase C delta. These results establish that IL-10 signaling is regulated during inflammation and identify Fc receptors and interferon-gamma as important regulators of IL-10 activity. Generation of macrophages refractory to IL-10 can contribute to pathogenesis of inflammatory and infectious diseases characterized by production of interferon-gamma and immune complexes.

Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8alpha expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8(+) DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8(+) T cells. The use of immunoglobulin G Fc receptor (Fc(gamma)R) common gamma-chain-deficient mice revealed that the cross-presentation by CD8(-) DCs depended on the expression of gamma-chain-containing activating FcgammaRs, whereas cross-presentation by CD8(+) DCs was not reduced in gamma-chain-deficient mice. These results suggest that although CD8(+) DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8(-) DCs only do so after activation, such as via ligation of Fc(gamma)Rs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.

Systemic lupus erythematosus (SLE) is a potentially fatal non-organ-specific autoimmune disease that predominantly affects women. Features of the disease include inflammatory skin lesions and widespread organ damage caused by deposition of anti-dsDNA autoantibodies. The mechanism and site of production of these autoantibodies is unknown, but there is evidence that interferon (IFN) gamma plays a key role. We have used the involucrin promoter to overexpress IFN-gamma in the suprabasal layers of transgenic mouse epidermis. There was no evidence of organ-specific autoimmunity, but transgenic animals produced autoantibodies against dsDNA and histones. Autoantibody levels in female mice were significantly higher than in male transgenic mice. Furthermore, there was IgG deposition in the glomeruli of all female mice and histological evidence of severe proliferative glomerulonephritis in a proportion of these animals. Our findings are consistent with a central role for the skin immune system, acting under the influence of IFN-gamma, in the pathogenesis of SLE.

Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated

The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in situ immune complex formation by accumulated immunoglobulins and/or antigens bound to long-lived structural proteins that have undergone excessive nonenzymatic glycosylation. In this report, we have tested this hypothesis using nonenzymatically glycosylated collagen. Binding of both albumin and IgG averaged four times the amount bound to unmodified collagen. Both albumin and IgG (anti-BSA) bound to nonenzymatically glycosylated collagen retained their ability to form immune complexes in situ with free antibody and antigen.

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.

IgG1 immune complexes were identified as the humoral stimuli responsible for the synthesis of IgG1-specific IgM rheumatoid factor (RF), which occurs in the mouse during the early stages of secondary immune responses to protein antigens. The specificity of this phenomenon was illustrated by the fact that complexes made with IgG1 F(ab')2 fragments or with antibodies of a different isotype failed to induce significant anti-IgG1 RF synthesis. The importance of immune complexes in the induction of RF was further underscored by the substantial increase in the titers of isotype-specific RF observed in the serum of mice immunized with IgG1- or IgG2a-complexed antigen rather than with antigen alone. The RF-inducing capacity of the complexes varied with the antigen/antibody ratio: it was maximal in antibody excess or at equivalence, but dramatically reduced in large antigen excess. The importance of T cell priming in RF precursor cell activation by immune complexes was demonstrated by the failure of T cell-deprived spleen cells to reconstitute the capability of irradiated mice to produce RF, and by the optimal RF responses observed after reconstitution of irradiated recipients with primed T cells and naive B cells. The involvement of T cells in this process could not be explained by the release of nonspecific B cell activators, because antigenic stimulation of primed T cells failed to enhance the activation of RF precursor cells by immune complexes of unrelated antigen.

The mechanisms by which immune complexes deposit in the glomerular basement membrane have been the subject of much debate, with the relative importance of direct deposition of circulating immune complexes (IC) vs. formation of IC in situ from the binding of circulating antibody to structural or exogenous planted antigen being at issue. In order to determine whether intact IC can deposit as such, covalently linked IC were prepared by a two-step reaction involving the bifunctional reagent toluene-2,4-diisocyanate (TDI), the antigen bovine gamma globulin (BGG), and rabbit anti-BGG antibody. Antigen and antibody were covalently cross-linked, with little self-linkage of antigen or antibody, and IC were purified by gel filtration. The net charge of the complexes was varied by chemical means, either before or after IC formation. When cationic IC were injected intravenously into mice, there was codeposition of antigen and antibody diffusely in the glomerular basement membrane (GBM), and deposits were observed ultrastructurally in the laminae rarae, interna and externa, and the lamina densa. Thus, under conditions of restricted appropriate charge, intact IC can cross the glomerular basement membrane and deposit in subepithelial sites without being excluded by size alone.

Immune precipitates were solubilized by the alternative pathway of complement assembled from isolated proteins, i.e., C3, factor B, factor D, properdin, C3b inactivator (C3bINA), and beta 1H. The kinetic curves of solubilization in the isolated system and in EGTA-serum were virtually indistinguishable. No requirement of other factors was apparent. Deletion of C3bINA and beta 1H from the complete mixture caused total consumption of C3 in the fluid phase and resulted in neither C3 binding to the complexes nor solubilization. Thus, the presence of a regulated fluid-phase reaction is essential for efficient fixation of C3 and the consequent solubilization. In addition, properdin plays an essential role in the complement-mediated solubilization in the presence of the two regulators. A large amount of C3 was incorporated into the antigen-antibody lattice. Solubilization of immune complexes started after the binding of one C3 molecule to one antibody molecule in the complexes, and the molar ratio of C3:antibody in the solubilized complexes also is approximately 1.

Activation of the complement system by IgA was investigated with immune complexes containing a mouse IgA myeloma protein with specificity for phosphorylcholine linked to bovine serum albumin (PC-BSA). These IgA anti-PC-BSA immune complexes activated the alternative complement pathway in mouse and guinea pig serum, while human complement was not affected. The activation proceeded with consumption of C3 but little or no consumption of C5. C3 did not bind to the IgA immune complexes during complement activation although it did bind covalently to IgG immune complexes. It is suggested that IgA immune complexes do not supply a suitable surface for C3 binding and effective alternative pathway convertase assembly; therefore, cleavage is limited and occurs primarily in the fluid phase. Without C3 binding, C5 cleavage does not occur nor can the alternative pathway activation proceed to the amplification step.

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.

The structural heterogeneity of the human low affinity receptor for IgG, FcRII(CD32), has been elucidated through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. These clones predict amino acid sequences consistent with integral membrane glycoproteins with single membrane spanning domains. The extracellular domains display sequence homology to other Fc gamma Rs and members of the Ig supergene family. A minimum of three genes (Fc gamma RIIa, IIa', and Fc gamma RIIb) encode these transcripts, which demonstrate highly related extracellular and membrane spanning domains. IIa/IIa' differ substantially in the intracytoplasmic domain from IIb. Alternative splicing of the IIb gene generates further heterogeneity in both NH2- and COOH-terminal domains of the predicted proteins. Comparison to the murine homologues of these molecules reveals a high degree of conservation between the products of one of these genes, Fc gamma RIIb, and the murine beta gene in primary sequence, splicing pattern, and tissue distribution. In contrast, the sequence of IIa' indicates its relationship to the beta-like genes, with mutation giving rise to a novel cytoplasmic domain, while IIa is a chimera of both alpha- and beta-like genes. Expression of these cDNA molecules by transfection results in the appearance of IgG binding molecules that bear the epitopes defined by the FcRII(CD32) mAbs previously described.

Ligand binding specificities of two cloned murine Fc gamma Rs (Fc gamma R-alpha, Fc gamma R-beta [9]) were determined by gene transfer into Fc gamma R negative cell lines. Both receptors were expressed as full-length molecules capable of IgG immune complex binding that was inhibitable by the mAb 2.4G2. The ligand binding profiles of these receptors were indistinguishable whereby both bound immune-complexed mouse IgG1, IgG2a, and IgG2b, but not IgG3. Neither receptor could bind monomeric IgG2a, indicating these receptors to be low-affinity IgG Fc receptors. Accumulation of the Fc gamma R-alpha mRNA can be induced with murine IFN-gamma at a concentration of 200 U/ml in the macrophage-like cell lines RAW 264.7 and J774a. The time course for induction indicates that the mRNA accumulation is transient but does not return to the uninduced level even after 50 h of treatment. Fc gamma R-beta mRNA was not induced by IFN-gamma, rather its expression was down modulated in mouse peritoneal macrophages. Both RAW and J774a cells lines exhibited increased receptor levels after IFN-gamma stimulation as measured by 125I-2.4G2 and ligand binding. In the absence of IFN-gamma, the RAW and J774a cell lines were minimally phagocytic, while P388D1 cells were actively phagocytic. In the presence of IFN-gamma, however, RAW 264.7 and J774a cells were induced to become actively phagocytic. Induction of Fc gamma R-alpha mRNA and protein by IFN-gamma may be part of the process by which macrophages become activated to engulf antibody-coated particles.

50% of individuals of selective IgA deficiency have high serum titers of antibody to bovine proteins, and high levels of circulating immune complexes that contain bovine antigens. Because in animal studies, immunization with antigen-antibody complexes is a very effective means of producing anti-idiotypic antibodies, we sought such autoantibodies in two sera known to have large amounts of anticasein. After IgG isolation and two-stage affinity chromatography, IgG-like material (molecular weights of H and L chains on SDS-PAGE), with binding activity for the F(ab')2 of anticasein were isolated from both sera. Pooled human gamma globulin or IgG myeloma proteins did not inhibit binding of specific anti-anticaseins to the corresponding anticasein, but sodium caseinate did block this binding (by 80 and 95%) indicating that most of these autoantibodies have affinity for the casein-binding site. Naturally occurring anti-idiotypic antibodies have been difficult to conclusively demonstrate in human sera; consequently, these experiments provide evidence of a unique model which may be used to explore the network theory of immunoglobulin regulation in humans.

An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns (4), have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of 125I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary. Two further findings of great relevance for the concept of induction of immune complex glomerulonephritis by histones were: (a) glomerular-bound histone was accessible for specific antibody given intravenously; and (b) prior binding of histones promoted glomerular deposition of anionic antigens, as could be shown with ssDNA fragments. These data justify the proposal that glomerular deposition of histones can induce immune complex formation, start an inflammatory process, and produce tissue damage.

Either of two immunostimulating factors (lpr, lipopolysaccharide) enhanced the pathogenic autoimmune responses of MRL/n mice, but the serologic and immunopathologic characteristics differed. In contrast, either factor acting alone, caused minimal immunopathology in normal mice, despite autoantibody induction. Combined immunostimulation, however, caused fatal glomerulonephritis in normal-background C57BL/6 mice. These results show the profound influence of the background genome on the effects of immunostimulating agents, and show that resistance to autoimmune disease in immunologically normal mice is not absolute.

DNA-anti-DNA immune complexes, produced from single-stranded DNA and IgG from a patient with systemic lupus erythematosus were cleared from the circulation of normal mice extremely rapidly, at a rate similar to the clearance of DNA alone. The initial clearance of these complexes was more rapid than the clearance of aggregated IgG, a surrogate immune complex containing a comparable number of IgG molecules, suggesting that the antigen (DNA) in the complexes significantly altered the clearance kinetics of the complexes. Analysis of the late clearance component of these complexes showed that anti-DNA is released back into the circulation after initial removal, and is then cleared at a rate similar to monomeric IgG. Whether this anti-DNA represents free antibody, or antibody bound to small nuclease digested DNA fragments, awaits further study.

The independent B lymphocyte surface membrane receptors IgM and Fc IgG receptors were evaluated for interactions using immunoflourescence. Ligand [F(ab')2 anti-mu]-induced capping of surface IgM resulted in capping of Fc IgG receptors only if the latter were occupied during the capping process by: (a) soluble antigen-antibody complexes that themselves provided insufficient cross-linking to result in capping; or (b) monomeric IgG at physiologic concentrations (or less) either purified or as normal serum. Ligand-induced capping of Fc IgG receptors did not result in capping of surface IgM occupied by monomeric F(ab') anti-mu. Control experiments showed that ligand binding to or capping of only one of these two receptors has no effect on the other, and that there were no cross-reactions. The interaction appears specific in that ligand-induced capping of surface IgM did not induce capping of ligand-occupied surface IgD or I-A antigens. Thus, there appears to be a specific interaction between ligand-bound surface IgM and ligand-bound Fc IgG receptors on the B lymphocyte surface. The results also indicate that binding of monomeric IgG produces a reversible alteration in the Fc IgG receptor leading to association with ligand-bound surface IgM. Because Fc IgG receptors are continuously exposed to monomeric IgG in vivo, these results suggest that whenever surface IgM is involved in a B lymphocyte response to an immunologic stimulus, the Fc IgG receptor is also involved.

It is known that polycations bind to and neutralize glomerular polyanions. Here we examine the effect of the polycation polyethyleneimine (PEI) on glomerular deposition of preformed immune complexes. Bovine serum albumin (BSA)-anti-BSA immune complexes made in 40 times antigen excess were administered following intravenous injection of PEI. Glomerular localization of immune deposits was assessed by quantitative immunofluorescence and electron microscopy and compared to controls receiving diluent without PEI followed by the same dose in immune complexes. In rats receiving PEI, deposits were localized within the glomerular basement membrane (GBM) of all peripheral capillary walls and in the mesangium. In controls, deposits localized exclusively within the mesangium in smaller amounts than after PEI. Thus, neutralization of glomerular polyanion by a circulating polycation enhances the deposition and alters the distribution of immune complexes in glomeruli.

To characterize the physiology of circulating IgA immune complexes (IgA-IC), the dynamics of IgA-IC removal by the liver were examined. After intravenous injection, covalently cross-linked IgA antibodies to the dinitrophenyl determinant were rapidly removed from the circulation by the liver. Immunofluorescence microscopy and light and electron microscope autoradiography showed that the IgA-IC were associated with Kupffer cells. With increasing doses of injected IgA-IC the clearance velocity approached a maximum, thus prolonging the circulation of IgA-IC. All these observations indicated a receptor-mediated process. Saturating doses of various potential receptor-blocking agents, heat-aggregated mouse IgG, microaggregated human serum albumin, and purified dimeric IgA did not influence the clearance pattern and hepatic uptake of radiolabeled IgA-IC. Mouse livers were also perfused via the portal vein with 1 microgram of IgA-IC. In the presence or absence of serum proteins, 43% of the perfused IgA-IC were removed in a single passage. This liver uptake was not reduced with simultaneous perfusion of large doses of aggregated mouse IgG, aggregated human serum albumin, or purified free dimeric mouse IgA. In contrast, the liver uptake of radiolabeled IgA-IC was decreased by 88% with the addition of 1 mg unlabeled IgA-IC. These observations support the conclusion that removal of IgA-IC from circulation is mediated by a specific IgA receptor on Kupffer cells.

We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.

Mouse macrophage Fc receptors specific for IgG1/IgG2b mediate the binding and pinocytic uptake of soluble IgG-containing antibody-antigen complexes. Internalization of these multivalent IgG complexes is accompanied not only by the intracellular degradation of the ligand, but also by a net decrease in the number of plasma membrane Fc receptors and an accelerated rate of receptor turnover. In contrast, internalized receptors bound to a monovalent ligand, the high affinity Fab fragment of the antireceptor mAb 2.4G2, escape degradation by rapidly recycling to the cell surface. In this paper, we have characterized the intracellular pathway involved in the endocytosis and transport of Fc receptors in the J774 macrophage cell line. The results show that the uptake of multivalent ligands follows the normal pathway of receptor-mediated endocytosis: internalization in clathrin-coated pits and coated vesicles, delivery to endosomes, and finally to acid hydrolase-rich lysosomes. Immunoprecipitation of radiolabeled receptor from Percoll density gradients showed that endocytosis of the IgG complexes also results in the concomitant transport of the receptor to lysosomes. Although uptake of the monovalent Fab fragment had no detectable effect on intracellular receptor distribution, preparations of 2.4G2 Fab rendered multivalent by adsorption to colloidal gold were as effective as the IgG complexes at causing lysosomal accumulation of internalized receptors. Thus, it is likely that the down-regulation and degradation of Fc receptors which occurs during the endocytosis of antibody-antigen complexes is due to the transport of internalized receptors to lysosomes. Moreover, the ability of certain Fc receptor-bound ligands to interfere with receptor recycling and trigger lysosomal transport seems to depend on ligand valency rather than on the presence or absence of Fc domains on intact IgG molecules.

During the reaction of an immune precipitate with fresh human serum, C3 undergoes a number of molecular alterations with the formation of conversion products differing from those obtained when purified components react. Those products which remain in the fluid phase, the subject of the present paper, have been identified by their reaction with monospecific antisera to the three antigenic determinants of C3, A, B, and D, after electrophoresis in agar or polyacrylamide gel. When purified C3 reacts with EAC1,4,2, C3i is found in the fluid phase. C3i, a loose complex of C3a and C3b, is in a conformational state whereby only the A and D antigens, present on its C3b portion, will consume antibody. The B antigen, present on the C3a portion of C3i, is unavailable for combination with antibody until C3i dissociates. In the fluid phase of the reaction of an immune precipitate with whole serum, C3i, C3a, and C3b, formed when purified components react, cannot be found. Instead the end products of the reaction appear to be C3c, which contains the A antigen, and C3d, which contains the D antigen. C3c and C3d are similar to the beta1A and alpha2D produced by the aging of serum but differ in their mobilities in acrylamide gel and in agar. The C3c and C3d generated by an immune precipitate also differ slightly from the C3c and C3d produced by the reaction of trypsin with C3 in whole human serum. As human serum reacts with an immune complex, native C3 appears to undergo a primary alteration before conversion. This alteration results in a molecular species of C3 which is labile at 56 degrees C for 30 min, fails to expose additional A and D antigenic sites upon aging, and which forms beta1A and C3d rather than beta1A and alpha2D during aging. In addition to this altered form of native C3, a new conversion product, C3x, is formed as whole serum reacts with an immune complex. C3x is not found in systems utilizing pure complement components. C3x is like C3 in that it bears all three antigenic determinants but differs in that it has a slightly faster mobility in polyacrylamide gel than does native C3. C3x is not only found in the fluid phase but is also bound to the immune precipitate. Finally, the fluid-phase kinetics of each of the antigens of C3 have been determined as normal human serum reacts with an immune precipitate. These illustrate that nearly the entire population of native C3 molecules undergoes conversion rapidly as manifested by the disappearance of the B antigen from the fluid phase. Moreover, the kinetics of the fluid-phase A and D antigens reflect that the conversion of C3 in serum is quantitatively not the same as when purified C3 reacts with C4,2.

Solid phase immunoadsorbents were prepared by coupling antigens to agarose. With this technique specific antibodies were easily isolated in large amounts. The gammaG-globulin class of antibodies isolated in this manner were not denatured as judged by their normal biological half-life in rabbits. Soluble immune complexes at fivefold antigen excess were prepared from isolated specific antibodies and HSA, human lambda-chains, human lambdaG-globulins, and a Waldenström's macroglobulin as antigens. In all these preparations a characteristic immune complex was encountered that represented the smallest stable antigen-antibody union. In the HSA-anti-HSA system they were found to be AgAb(2) complexes, and Ag(2)Ab complexes in the gammaG-anti-gammaG system. These stable complexes fixed complement ineffectively. Also, a spectrum of larger complexes was present in each system, and these complexes fixed complement effectively. With intact antibodies the disappearance curves of immune complexes from the circulation were composed of three exponential components. The immune complexes larger than AgAb(2) were quickly removed from the circulation with half-lives of 0.09-0.37 hr. Their clearance was not dependent on complement components, in that depletion of complement by cobra venom factor and aggregated gammaG-globulin did not alter the pattern of their removal from the circulation. However, when the interchain disulfide bonds of antibodies were reduced and alkylated, the removal of the lambda-anti-lambda, HSA-anti-HSA, and gammaG-anti-gammaG complexes was altered. In these experiments the disappearance curves were composed of two exponential components and the rapid removal of the greater than AgAb(2) complexes did not occur. The immune complexes prepared from reduced and alkylated antibodies fixed complement ineffectively. The presented data indicate that the rapid removal of circulating immune complexes, containing gammaG-globulin molecules as antibodies, depends primarily on the number of antibodies involved. Furthermore, complement fixation is not involved in the rapid removal of such complexes. Nevertheless, the rapid removal of immune complexes and their ability to fix complement have similarities for optimal function in that both processes require intact interchain disulfide bonds of antibodies and complexes that exceed the AgAb(2) combination.