Clearance and organ localization of small DNA anti-DNA immune complexes in mice

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.     

Rapid blood clearance of injected mouse IgG2a in SCID mice

SCID mice were found to have rapid blood clearance of injected mouse IgG2a antibodies (Ab), while IgG1 Ab were cleared normally. This effect varied depending on the strain of SCID mice, being very rapid in most Taconic ICR mice and slower in C.B-17 mice. A similar effect was previously described in nude mice, and shown to be due to the very low concentrations of endogenous IgG2a and IgG2b in these mice. Therefore, IgG2a and IgG2b concentrations were assayed in sera from 30 SCID mice: the concentrations were very low, with one exception. Rapid blood clearance in all strains could be strongly inhibited by injection of large amounts of irrelevant IgG2a. Therefore, the low endogenous IgG2a and IgG2b concentrations are responsible for the rapid blood clearance rate of injected IgG2a in these mice, as in nude mice. However, the relatively slow blood clearance of injected IgG2a in certain SCID mice (C.B-17 mice and rare Taconic ICR mice), was not generally due to higher levels of IgG2a or IgG2b, but rather to some other factor that has not been identified. F(ab')(2) Ab fragments were not cleared rapidly, which supports other evidence that clearance is via the CD64 Fcgamma receptor. This rapid clearance of IgG2a affects the ability of such Ab to target tumors, and was also shown to affect the maximum tolerated dose (MTD) of radiolabeled IgG2a Ab, labeled with either (125)I or (111)In. Mice with faster blood clearance had a lower MTD, probably due to the fact that Ab uptake was in the bone as well as the spleen. This effect must be taken into consideration in experiments in which SCID mice are injected with IgG2a or IgG2b Ab.     

A novel role of complement factor C1q in augmenting the presentation of antigen captured in immune complexes to CD8+ T lymphocytes

Ag-IgG immune complexes (IC) are efficiently taken up, and Ag-derived peptides are subsequently processed and presented by APC. In vitro experiments indicate that IgG Fc Receptors (FcgammaR) facilitate the efficient uptake of IC by dendritic cells. Previous experiments showed that the cross-presentation of Ag-derived peptides after s.c. administration of IC is FcgammaR-dependent. To study the role of different FcgammaR and complement in MHC class I Ag presentation after i.v. administration, we used mice deficient for FcgammaRs and complement components. These mice were injected with CFSE-labeled OVA-specific CD8+ T cells followed by administration of IC composed of OVA and rabbit anti-OVA IgG i.v. to measure MHC class I presentation of OVA-derived peptides. The Ag presentation was partly reduced in FcRgamma-chain-deficient mice, but not affected in FcgammaRI/II/III-deficient mice, complement factor C3-deficient mice, or FcgammaRI/II/III x C3-deficient mice. Importantly, CD8+ T cell proliferation was significantly reduced in mice deficient for C1q. This proliferation could be restored when IC were incubated with purified human C1q before injection. Likewise, purified C1q could strongly enhance the uptake and presentation of IC by dendritic cells in vitro. Heat inactivation abrogated the C1q-mediated uptake of IC. In addition, in vivo uptake of OVA-IC in the spleen was significantly reduced in C1q-deficient mice compared with wild-type mice. Together, these results indicate a novel function of C1q, which is present in high levels in the bloodstream, by directly enhancing the uptake and MHC class I presentation of Ag captured in IC by APC to CD8+ T cells.     

Release of arachidonic acid by stimulation of opsonic receptors in human monocytes: the FcgammaR and the complement receptor 3 pathways

The role of the opsonic receptors FcgammaR and CR3 on the release of arachidonic acid (AA) by human monocytes was studied using IgG-ovalbumin (OVA) equivalence immune complexes (IC), anti-OVA IgG bound to OVA-coupled latex beads, and C3bi-bound IC. Release of AA was produced by IC and latex-OVA beads bound to IgG, whereas binding of C3bi to IC inhibited the ability of IC to release AA. In contrast, coating of zymosan particles with C3bi enhanced AA release as compared with that produced by non-coated particles. Masking of C3bi on C3bi-bound IC by incubation with anti-C3 IgG resulted in the recovery of their ability to release AA, thereby suggesting that binding of C3b by IC reduces their flogogenic effects, whereas opsonization of microbial walls by complement may enhance their proinflammatory potential. The binding/uptake of opsonized zymosan particles was inhibited by anti-CR3 Ab and C3bi-bound IC, but not by beta-glucan, mannan, and anti-Toll-like receptor 2 Ab. These findings show that cooperative engagement of CR3 on both the lectin-like site involved in beta-glucan binding and the I-domain involved in C3bi binding, as it can be observed in the innate immune response, produces AA release, whereas the unique interaction of C3bi-bound IC with the I-domain of CR3, as it may occur in the adaptive immune response, diverts the IC lattice from a productive interaction with FcgammaR linked to AA release.     

Accelerated nephrotoxic nephritis is exacerbated in C1q-deficient mice

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.     

Transgenic expression of a soluble complement inhibitor protects against renal disease and promotes survival in MRL/lpr mice

To investigate the role of complement in lupus nephritis, we used MRL/lpr mice and a transgene overexpressing a soluble complement regulator, soluble CR1-related gene/protein y (sCrry), both systemically and in kidney. Production of sCrry in sera led to significant complement inhibition in Crry-transgenic mice relative to littermate transgene negative controls. This complement inhibition with sCrry conferred a survival advantage to MRL/lpr mice. In a total of 154 animals, 42.5% transgene-negative animals had impaired renal function (blood urea nitrogen > 50 mg/dl) compared with 16.4% mice with the sCrry-producing transgene (p < 0.001). In those animals that died spontaneously, MRL/lpr mice with the sCrry-producing transgene did not die of renal failure, while those without the transgene did (blood urea nitrogen values of 46.6 +/- 9 and 122 +/- 29 mg/dl in transgene-positive and transgene-negative animals, respectively; p < 0.001). Albuminuria was reduced in those transgenic animals in which sCrry expression was maximally stimulated (urinary albumin/creatinine = 12.4 +/- 4.3 and 36.9 +/- 7.7 in transgene-positive and transgene-negative animals, respectively; p < 0.001). As expected in the setting of chronic complement inhibition, there was less C3 deposition in glomeruli of sCrry-producing transgenic mice compared with transgene-negative animals. In contrast, there was no effect on glomerular IgG deposition, levels of anti-dsDNA Ab and rheumatoid factor, or spleen weights between the two groups. Thus, long-term complement inhibition reduces renal disease in MRL/lpr mice, which translates into improved survival. MRL/lpr mice in which complement is inhibited still have spontaneous mortality, yet this is not from renal disease.     

Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

The role of complement in cryoglobulin-induced immune complex glomerulonephritis

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.     

Genetic dissection of the effects of stimulatory and inhibitory IgG Fc receptors on murine lupus

Immune complex (IC)-mediated tissue inflammation is controlled by stimulatory and inhibitory IgG Fc receptors (FcgammaRs). Systemic lupus erythematosus is a prototype of IC-mediated autoimmune disease; thus, imbalance of these two types of FcgammaRs is probably involved in pathogenesis. However, how and to what extent each FcgammaR contributes to the disease remains unclear. In lupus-prone BXSB mice, while stimulatory FcgammaRs are intact, inhibitory FcgammaRIIB expression is impaired because of promoter region polymorphism. To dissect roles of stimulatory and inhibitory FcgammaRs, we established two gene-manipulated BXSB strains: one deficient in stimulatory FcgammaRs (BXSB.gamma(-/-)) and the other carrying wild-type Fcgr2b (BXSB.IIB(B6/B6)). The disease features were markedly suppressed in both mutant strains. Despite intact renal function, however, BXSB.gamma(-/-) had IC deposition in glomeruli associated with high-serum IgG anti-DNA Ab levels, in contrast to BXSB.IIB(B6/B6), which showed intact renal pathology and anti-DNA levels. Lymphocytes in BXSB.gamma(-/-) were activated, as in wild-type BXSB, but not in BXSB.IIB(B6/B6). Our results strongly suggest that both types of FcgammaRs in BXSB mice are differently involved in the process of disease progression, in which, while stimulatory FcgammaRs play roles in effecter phase of IC-mediated tissue inflammation, the BXSB-type impaired FcgammaRIIB promotes spontaneous activation of self-reactive lymphocytes and associated production of large amounts of autoantibodies and ICs.     

A complement C3 inhibitor specifically targeted to sites of complement activation effectively ameliorates collagen-induced arthritis in DBA/1J mice

Collagen-induced arthritis (CIA) represents an animal model of autoimmune polyarthritis with similarities to human rheumatoid arthritis, and therapy with various systemic complement-inhibitory proteins has been investigated in this model with varying results. We investigated the use of complement receptor 2 (CR2)-Crry, a complement inhibitor with the ability to target C3 breakdown products deposited in a rheumatic joint. Following induction of CIA in DBA/1J mice, animals were treated with either PBS or CR2-Crry (every other day, every 4 days, or with a single injection). The severity of clinical disease was significantly reduced in all CR2-Crry-treated groups compared with controls. Joints from mice receiving multiple doses of CR2-Crry showed significantly decreased inflammatory cell infiltrate, cartilage damage, pannus formation, and bone damage. CR2-Crry treatment also significantly decreased production of anti-collagen IgG and the inflammatory cytokines TNF-alpha and IL-1beta. IL-10 and IL-1Ra levels were increased in CR2-Crry-treated mice. CR2-Crry localized preferentially in the joints of mice with CIA. Analysis of IgG and C3 deposition in the joints of treated animals indicated that both complement regulation and the modulation of anti-collagen Ab production contributed to the protective effects of CR2-Crry. Of interest, a previous study reported that Crry-Ig, an untargeted counterpart of CR2-Crry, had minimal effect on disease, even when administered at a sufficiently high dose to maintain chronic complement inhibition.     

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.     

Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcgammaRI- and III-deficient mice, as well as the combined FcgammaRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B(-/-) mice displayed higher bacteremic load than C1q(-/-) mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q(-/-) mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B(-/-) mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.     

Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice

Group B Streptococcus (GBS) is the foremost bacterial cause of serious neonatal infections. Protective immunity to GBS is mediated by specific Abs to the organism's capsular polysaccharide Ags. To examine the role of complement in the humoral immune response to type III GBS capsular polysaccharide (III-PS), mice deficient in C3 or in CD21/CD35 (i.e., complement receptors 1 and 2; CR1/CR2) were immunized with III-PS. Mice deficient in C3 or Cr2 had an impaired primary immune response to III-PS. The defective response was characterized by low IgM levels and the lack of an isotype switch from IgM to IgG Ab production. Compared with wild-type mice, C3- and Cr2-deficient mice exhibited decreased uptake of III-PS by follicular dendritic cells within the germinal centers and impaired localization of III-PS to the marginal zone B cells. Complement-dependent uptake of capsular polysaccharide by marginal zone B cells appears necessary for an effective immune response to III-PS. The normal immune response in wild-type mice may require localization of polysaccharide to marginal zone B cells with subsequent transfer of the Ag to follicular dendritic cells.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

IgG2a-mediated enhancement of antibody and T cell responses and its relation to inhibitory and activating Fc gamma receptors

A number of studies in experimental animal models point to an important role of Fc gamma Rs in autoimmunity and allergy. In this study, we investigate how the production of IgG, an early step in the chain of events leading to inflammation, is regulated by activating and inhibitory Fc gamma Rs. IgG Abs are known to feedback-enhance Ab responses to soluble Ags, and this effect requires activating Fc gamma Rs. To test proliferation of Th cells, mice were adoptively transferred with CD4(+) T cells expressing a transgenic OVA-specific TCR before immunization with IgG2a anti-2,4,6-trinitrophenyl (TNP) plus OVA-TNP or with OVA-TNP alone. IgG2a induced a significant increase in OVA-specific T cell numbers, which preceded the OVA-specific Ab response and was dependent on the Fc gamma chain. The role of the inhibitory Fc gamma RIIB in Ab responses was studied in mice lacking this receptor. Although IgG2a enhanced primary Ab responses, development of germinal centers, and immunological memory in wild-type mice, enhancement was markedly stronger in Fc gamma RIIB(-/-) mice. The presented data are compatible with the hypothesis that the mechanism behind IgG2a-mediated up-regulation of Ab responses involves increased Ag presentation to CD4(+) T cells by Fc gamma R(+) APCs. Our observations also illustrate the intricate immunoregulatory role of IgG Abs. On the one hand, they enhance Ab responses via activating Fc gamma Rs, and on the other hand, they set an upper limit for the same Ab response via Fc gamma RIIB.     

Complement activation contributes to both glomerular and tubulointerstitial damage in adriamycin nephropathy in mice

Adriamycin nephropathy is a model of focal segmental glomerulosclerosis, characterized by proteinuria and progressive glomerulosclerosis and tubulointerstitial damage. In this study, we examined the role of complement in the etiology of adriamycin nephropathy in mice. We used mice deficient in C1q, factor D, C3, and CD59, and compared them with strain-matched controls. C3 deposition occurred in the glomeruli of wild-type mice as early as 48 h following a single i.v. injection of adriamycin. C3-deficient mice developed significantly less proteinuria and less podocyte injury at day 3 postadriamycin than controls, suggesting that complement is important in mediating the early podocyte injury. At later time points, C3-deficient mice were protected from glomerulosclerosis, tubulointerstitial injury, and renal dysfunction. Factor D-deficient mice were also protected from renal disease, confirming the importance of alternative pathway activation in this model. In contrast, C1q-deficient mice developed similar disease to controls, indicating that the complement cascade was not activated via the classical pathway. CD59-deficient mice, which lack adequate control of C5b-9 formation, developed significantly worse histological and functional markers of renal disease than controls. Interestingly, although more C9 deposited in glomeruli of CD59-deficient mice than controls, in neither group was tubulointerstitial C9 staining apparent. We have demonstrated for the first time that alternative pathway activation of complement plays an important role in mediating the initial glomerular damage in this in vivo model of focal segmental glomerulosclerosis. Lack of CD59, which regulates the membrane attack complex, led to greater glomerular and tubulointerstitial injury.     

Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.     

Clearance kinetics and immunochemistry in rabbits of soluble antibody/DNA immune complexes. Effects of antibody class and DNA conformation

We examined the clearance kinetics in rabbits of soluble antibody/DNA immune complexes (IC) containing either IgG or IgM anti-DNA antibodies. Differences in the complement-mediated binding of these IC to rabbit blood cells (platelets) were also studied. Complexation of either double-stranded (ds) or single-stranded (ss) DNA with IgG anti-DNA tends to preclude in vivo DNA recognition mechanisms; the DNA is cleared as part of an IC at a rate slower than that of free DNA. Binding of ds- or ssDNA by IgM anti-DNA antibodies leads to formation of IC which are cleared more like free DNA, and this effect is most evident for ssDNA. However, although both IgG- and IgM-containing IC bound rapidly to blood cells in vivo, significant differences in their immunochemistry were apparent. For example, the DNA in IgM-containing IC was more susceptible to both in vivo and in vitro degradation. In addition, the binding of IgM-containing IC to rabbit platelets and human red blood cells was considerably more labile. Based on this systematic investigation of the soluble antibody/DNA IC that can potentially form in the circulation of a patient with systemic lupus erythematosus, it should be possible to formulate predictions regarding the relative pathogenic potential of these IC.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.