Murine cryoglobulinemia: pathogenic and protective IgG3 self-associating antibodies

A murine IgG3 mAb, 6-19, derived from autoimmune MRL-lpr/lpr mice, is a rheumatoid factor (RF) specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intra-peritoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces skin leukocytoclastic vasculitis and acute glomerulonephritis associated with cryoglobulinemia. Because IgG3 interact with each other, we have determined whether noncryoprecipitating IgG3 mAb were able to inhibit the cryoprecipitation of 6-19 mAb and the development of related tissue lesions. In vitro, the cryoprecipitation of 6-19 mAb was almost completely inhibited by a fourfold excess of a noncryoprecipitating non-RF IgG3 (9-106) mAb derived from MRL-lpr/lpr mice. Cryoprecipitation of five other IgG3 mAb was similarly inhibited by the 9-106 mAb, and two other noncryoprecipitating IgG3 mAb, including the 2-6D antinuclear autoantibody, inhibited the cryoprecipitation of 6-19 mAb. In vivo, pretreatment of BALB/c mice with 9-106 or 2-6D mAb prevented the development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was greatly diminished in 9-106 or 2-6D mAb-treated mice, although their serum levels of 6-19 mAb and RF activity were comparable to those of control mice. This indicated that pretreatment with non-cryoglobulin IgG3 inhibited the cryoglobulin generation and cryoglobulin-associated tissue lesions induced by an IgG3 RF cryoglobulin-generating mAb. These results suggest that the balance of formation of IgG3 autoantibodies with or without the cryoglobulin activity may be critical for the development of IgG3 cryoglobulin-mediated tissue lesions in murine lupus, particularly in MRL-lpr/lpr mice.     

Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factors derived from autoimmune MRL/MpJ-lpr/lpr mice

A MRL strain bearing the autosomal recessive mutant gene, lpr (lymphoproliferation), spontaneously develops, in addition to a lupus-like syndrome, unique serological and pathological manifestations. Production of high titers of IgG rheumatoid factors (RF) may be related to the formation of extremely large amounts of cryoglobulins and the development of tissue lesions such as necrotizing polyarteritis, arthritis, and glomerulonephritis. To analyze more directly the relationship of IgG RF to the development of cryoglobulins and tissue injuries, we have established four monoclonal IgG RF secreting hybridomas from unimmunized MRL-lpr/lpr mice and determined their pathogenic effects in normal strains of mice. All the monoclonal IgG RF obtained in this study were of the IgG3 subclass and generated cryoglobulins. However, the fact that not only IgG3 Rf monoclonals but also four of five non-RF IgG3 monoclonals were able to form cryoglobulins, which were composed exclusively of each IgG3 monoclonal, indicates that the IgG3 molecule has a unique physicochemical property to self-associate via nonimmunological interaction and the ability to form cryoglobulins. When the in vivo pathogenic activities of these IgG3 RF and non-RF monoclonals were examined, three of IgG3 RF monoclonals with the specificity to IgG2a were able to induce extensive pathologic manifestations including peripheral vasculitis and glomerulonephritis characteristic of patients with cryoglobulinemia. Our results indicate that the IgG3 itself, independently of its specificity, could be a potential source of cryoglobulins and IgG3 RF, combined with its activity of cryoglobulin formation, may play a significant role in the development of glomerulonephritis and cutaneous vascular lesions of ears and foot pads observed frequently in aged MRL-lpr/lpr mice.     

Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

IgG3 is the major source of cryoglobulins in mice

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.     

Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice.

It is postulated that IFN-gamma confers susceptibility to immunologically mediated tissue injury. To test this hypothesis, we compared the intensity of accelerated anti-glomerular basement membrane glomerulonephritis between wild-type (IFN-gamma+/+) and IFN-gamma gene knockout (IFN-gamma-/-) mice. This disease model is initiated by binding of heterologous (sheep) anti-glomerular basement membrane Abs to the glomeruli of mice preimmunized with sheep IgG. The secondary cellular and humoral immune responses to the planted Ag then lead to albuminuria and glomerular pathology. We found that IFN-gamma-/- mice or IFN-gamma+/+ mice injected with IFN-gamma-neutralizing Ab develop worse albuminuria and glomerular pathology than IFN-gamma+/+ mice. The humoral response to sheep IgG (serum mouse anti-sheep IgG titers and intraglomerular mouse IgG deposits) was comparable in the IFN-gamma+/+ and IFN-gamma-/- groups. In contrast, IFN-gamma-/- mice mounted a stronger cellular immune response (cutaneous delayed-type hypersensitivity reaction) to sheep IgG than IFN-gamma+/+ mice. These findings provide evidence that endogenous IFN-gamma has a protective role in immunologically mediated glomerulonephritis initiated by foreign Ags.     

Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system

Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener''s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ^−/− mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.     

Structural studies of monoclonal human cryoprecipitable immunoglobulins

The light chain type, immunoglobulin class and when possible, heavy chain subclass of eleven monoclonal human cryoglobulins were correlated with the variable region subgroup of their light chains. The variable region subgroups were assigned by determining the primary amino acid sequence for the first 15 amino-terminal residues of these light chains. $\text{5}\text{5}$ IgM cryoglobulins which react with human IgG had light chains of the variable region-III kappa chain subgroup (vK-III). $\text{4}\text{4}$ IgG and $\text{2}\text{2}$ IgM cryoglobulins with undefined antibody specificity had both lambda and kappa light chains none of which were vK-III. The data support the concept that there is marked restriction of the IgM anti-IgG antibody response to the IgG auto-antigen.     

Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand white F1 mice targeted with an anti-DNA heavy chain

Lupus-prone, anti-DNA, heavy (H) chain "knock-in" mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2-3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6-7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vkappa utilization, different from that of nonautoimmune (C57BL/6 x BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vkappa-Jkappa rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.     

BAFF/APRIL inhibition decreases selection of naive but not antigen-induced autoreactive B cells in murine systemic lupus erythematosus

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.     

A critical role for Fc gamma RIIB in the induction of rheumatoid factors

Rheumatoid factors (RF) are autoantibodies with specificity for the Fc portion of IgG, and IgG-containing immune complexes are likely to be the major source of RF autoantigens. Therefore, the activation of RF-producing B cells could be controlled specifically through recognition of IgG immune complexes by the low-affinity IgG FcR, FcgammaRIIB, a potent negative regulator of the BCR. To test this possibility, we determined the development of RF in C57BL/6 (B6) mice lacking FcgammaRIIB, in relation to the H2 haplotype, complement C3, and the Y-linked autoimmune acceleration (Yaa) mutation. FcgammaRIIB-null B6 mice displayed substantial anti-IgG2a RF activities in their sera, in addition to anti-DNA autoantibodies. Their RF and anti-DNA responses were linked to the H2(b) haplotype, but were suppressed almost completely by the H2(d) haplotype. Strikingly, the absence of C3 failed to modulate RF production, but strongly inhibited anti-DNA production. Furthermore, we observed that partial FcgammaRIIB deficiency (i.e., heterozygous level of FcgammaRIIB expression) was sufficient to induce the production of RF and anti-DNA autoantibodies in the presence of the Yaa mutation. In contrast to FcgammaRIIB, the deficiency in another BCR negative regulator, CD22, was unable to promote RF and anti-DNA autoimmune responses in B6 mice. Our results indicate that RF autoimmune responses are critically controlled by FcgammaRIIB, together with the H2(b) and Yaa gene, while C3 regulates positively and specifically anti-DNA, but not RF autoimmune responses.     

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.     

Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation

C1q-deficient (C1qa-/-) mice develop antinuclear Abs and glomerulonephritis (GN) characterized by multiple apoptotic bodies. To explore the contribution of C3 activation to the induction of spontaneous GN, C1qa-/- mice were crossed with factor B- and C2-deficient (H2-Bf/C2-/-) mice. GN was present in 64% of the 45 C1qa/H2-Bf/C2-/- mice compared with 8% of the 65 H2-Bf/C2-/- mice and none of the 24 wild-type controls. IgG was detected in the glomeruli of diseased C1qa/H2-Bf/C2-/- kidneys. However, glomerular staining for C3 was absent. Increased numbers of glomerular apoptotic bodies were detected in undiseased C1qa/H2-Bf/C2-/- kidneys. These findings support the hypothesis that C1q may play a role in the clearance of apoptotic cells without the necessity for C3 activation and demonstrate that the activation of C3 is not essential for the development of GN in this spontaneous model of lupus-like disease.     

A lupus-suppressor BALB/c locus restricts IgG2 autoantibodies without altering intrinsic B cell-tolerance mechanisms

FcgammaR2B-deficient mice develop autoantibodies and glomerulonephritis with a pathology closely resembling human lupus when on the C57BL/6 (B6) background. The same mutation on the BALB/c background does not lead to spontaneous disease, suggesting differences in lupus susceptibility between the BALB/c and B6 strains. An F2 genetic analysis from a B6/BALB cross identified regions from the B6 chromosomes 12 and 17 with positive linkage for IgG autoantibodies. We have generated a congenic strain that contains the suppressor allele from the BALB/c chromosome 12 centromeric region (sbb2(a)) in an otherwise B6.FcgammaR2B(-/-) background. None of the B6.FcgammaR2B(-/-)sbb2(a/a) mice tested have developed IgG autoantibodies in the serum or autoimmune pathology. Mixed bone marrow reconstitution experiments indicate that sbb2(a) is expressed in non-B bone marrow-derived cells and acts in trans. sbb2(a) does not alter L chain editing frequencies of DNA Abs in the 3H9H/56R H chain transgenic mice, but the level of IgG2a anti-DNA Abs in the serum is reduced. Thus, sbb2(a) provides an example of a non-MHC lupus-suppressor locus that protects from disease by restricting the production of pathogenic IgG isotypes even in backgrounds with inefficient Ab editing checkpoints.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

Cutting edge: inducible costimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis.

The CD28 family member inducible costimulator protein (ICOS) has an important role in T cell differentiation and Ig class switching. To investigate the role of ICOS in vivo, ICOS-/- mice were infected s.c. with Leishmania mexicana. While wild-type mice developed large, cutaneous lesions, the growth of lesions and tissue histopathology was significantly delayed in ICOS-/- mice. ICOS-/- mice exhibited marked decreases in both Th1 and Th2 cytokine production and profound defects in L. mexicana-specific Ig isotype class switching to IgG1 and IgG2a and reduced total IgE levels. Our findings indicate that ICOS is a key regulator of both Th1 and Th2 responses and has a role in controlling cutaneous L. mexicana infection.     

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.     

IL-18 has IL-12-independent effects in delayed-type hypersensitivity: studies in cell-mediated crescentic glomerulonephritis

IL-18 (formerly known as IFN-gamma-inducing factor) enhances Th1 responses via effects that are thought to be dependent on and synergistic with IL-12. The potential for IL-18 to exert IL-12-independent effects in delayed-type hypersensitivity (DTH) responses was studied in a model of Th1-directed, DTH-mediated crescentic glomerulonephritis induced by planting an Ag in glomeruli of sensitized mice as well as in cutaneous DTH. Sensitized genetically normal (IL-12(+/+)) mice developed proteinuria and crescentic glomerulonephritis with a glomerular influx of DTH effectors (CD4(+) T cells, macrophages, and fibrin deposition) in response to the planted glomerular Ag. IL-12p40-deficient (IL-12(-/-)) mice showed significant reductions in crescent formation, proteinuria, and glomerular DTH effectors. Administration of IL-18 to IL-12(-/-) mice restored the development of histological (including effectors of DTH) and functional glomerular injury in IL-12(-/-) mice to levels equivalent to those in IL-12(+/+) mice. IL-18 administration to IL-12(-/-) mice increased glomerular ICAM-1 protein expression, but did not restore Ag-stimulated splenocyte IFN-gamma, GM-CSF, IL-2, or TNF-alpha production. Sensitized IL-12(+/+) mice also developed cutaneous DTH following intradermal challenge with the nephritogenic Ag. Cutaneous DTH was inhibited in IL-12(-/-) mice, but was restored by administration of IL-18. IL-12(+/+) mice given IL-18 developed augmented injury, with enhanced glomerular and cutaneous DTH, demonstrating the synergistic effects of IL-18 and IL-12 in DTH responses. These studies demonstrate that even in the absence of IL-12, IL-18 can induce in vivo DTH responses and up-regulate ICAM-1 without inducing IFN-gamma, GM-CSF, or TNF-alpha production.     

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) x C57BL/6)F(1) mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW x C57BL/6)F(1)-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW x C57BL/6)F(1)-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.