Lower level of synovial fluid interferon-gamma in HLA-B27-positive than in HLA-B27-negative patients with Chlamydia trachomatis reactive arthritis.

OBJECTIVES: To compare the synovial fluid (SF) concentrations of various cytokines in rheumatoid arthritis (RA) and in reactive arthritis, and to look for a correlation between cytokine levels and the presence of HLA-B27 antigen in reactive arthritis patients. METHODS: Concentrations of interleukin (IL) 10, IL-12, IL-18, interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) were determined by commercially available enzyme-linked immunosorbent assays (ELISA) in the SF from 48 patients with reactive arthritis, 33 with RA and 13 with osteoarthritis (non-inflammatory controls). RESULTS: The SF concentrations of IL-10 were significantly lower in patients with reactive arthritis (median 2.3 pg/ml) than in RA patients (median 14.6 pg/ml). The SF levels of IFN-gamma were not significantly different but the ratios of IFN-gamma to IL-10 were significantly higher in patients with reactive arthritis (median 9.2) than in RA patients (median 0.83). When the subset of patients with Chlamydia trachomatis reactive arthritis was considered, the SF concentration of IFN-gamma was significantly lower in HLA-B27-positive (median 2.9 pg/ml) than in HLA-B27-negative patients (median 42.4 pg/ml). After 2 yr of follow-up, two HLA-B27-positive patients, who had low SF levels of IFN-gamma, had a chronic course of arthritis, whereas after 1 yr all HLA-B27-negative patients had complete resolution of arthritis. CONCLUSIONS: The lower IFN-gamma concentrations in HLA-B27-positive patients with C. trachomatis reactive arthritis could be related to the tendency of these patients to have more severe or chronic arthritis.

Indirect evidence of intra-articular immunoglobulin G synthesis in patients with Chlamydia trachomatis reactive arthritis.

OBJECTIVES: To investigate whether B-cell stimulation occurs in joints of Chlamydia trachomatis reactive arthritis patients by comparing the immunoglobulin G (IgG) anti-C. trachomatis antibody responses in serum and synovial fluid (SF). METHODS: The number and spectrum of C. trachomatis antigens recognized by paired serum and SF samples from 16 patients with C. trachomatis reactive arthritis and 20 patients with other inflammatory arthropathies independent of this bacteria, were studied by immunoblotting. The responses to five different Chlamydia antigens were also determined in enzyme-linked immunosorbent assays. RESULTS: In C. trachomatis reactive arthritis patients, a higher number of C. trachomatis antigens was recognized by SF (17.6+/-5.1) than by serum (11.1+/-6.3) IgG and a higher intensity of SF IgG binding to the outer membrane protein 2 (OMP2) was observed. CONCLUSIONS: These results suggest an intra-articular IgG production and a possible role of some Chlamydia antigens like OMP2 in the pathogenesis of C. trachomatis reactive arthritis.

Role of galactosylation in the renal pathogenicity of murine immunoglobulin G3 monoclonal cryoglobulins.

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins. (Blood. 2001;97:3537-3543)

Role of neutrophils in murine cryoglobulinemia.

Murine IgG3 anti-IgG2a rheumatoid factor (RF) monoclonal antibodies (mAb) with cryoglobulin activity, are able to induce, in normal mice, skin leukocytoclastic vasculitis and lupus-like glomerulonephritis resembling 'wire-loop' lesions (subendothelial immune deposits). The development of glomerular, but not skin, lesions in immunoglobulin-deficient mice (lacking the corresponding IgG2a autoantigen) receiving IgG3 RF cryoglobulins indicates that the RF activity of IgG3 monoclonal cryoglobulins and subsequent formation of IgG3-IgG2a immune complexes play a critical role in the development of skin vasculitis. In contrast, nephritogenic activity is solely contributed by IgG3-associated cryoglobulin activity. Polymorphonuclear leukocyte (PMN) infiltration is one of the major pathologic changes observed in both types of lesions. Treatment with mAbs against the adhesion molecules leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) (both known for their involvement in PMN-endothelial cell interaction) inhibits the development of skin vascular lesions. However, it has no effect on the generation of glomerulonephritis. Apparently, adhesion molecule requirements for PMN interaction with glomerular capillary endothelial cells are different from those for PMN infiltration of the skin. However, the PMN depletion experiment has clearly shown that PMNs play an active role in the development of 'wire-loop' glomerular lesions. In the absence of the glomerular PMN infiltration, IgG3 RF cryoglobulins induce a different type of glomerular lesion, characterized by voluminous intracapillary thrombi and mesangial deposits, yet lacking subendothelial deposits. This is consistent with the fact that the latter lesions can be induced by certain IgG3 mAbs, which are unable to provoke glomerular PMN infiltration. Finally, the activation of the complement system does not appear to play a major role in either skin or glomerular lesions induced by IgG3 RF cryoglobulins.

Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies.

Protease nexin 1 (PN-1), a potent serpin-class antiprotease, is thought to be synthesized in the murine kidney. However, neither the cellular localization of PN-1 synthesis nor its role has yet been defined. To address these questions, we determined by in situ hybridizations RNase protection assay and immunoblotting, the sites of PN-1 mRNA accumulation in normal mouse kidneys and the modulation of PN-1 expression in several pathological conditions. In normal kidneys, PN-1 mRNA was detected primarily in glomeruli, most likely in mesangial cells. The glomerular expression of PN-1 was substantially enhanced not only in lupus-like glomerulonephritis (induced by IgG3 monoclonal rheumatoid factors or occurring spontaneously in lupus-prone mice), but also in mild glomerular lesions associated with intracapillary thrombi induced by IgG3 anti-trinitrophenyl monoclonal antibodies. In contrast, no modulation of PN-1 mRNA levels was observed during the course of lipopolysaccharide-induced acute tubular necrosis. A constitutive PN-1 gene expression and its up-regulation during glomerular injury suggest a possible role for PN-1 in glomerular biology. In view of its high inhibitory activity towards thrombin, mesangial PN-1 may be involved in the control of glomerular coagulation following initial glomerular injuries.

Polymorphonuclear leukocytes play a key role in the generation of "wire-loop" lesions induced by a murine IgG3 rheumatoid factor.

Murine IgG3 anti-IgG2a rheumatoid factor (RF) monoclonal antibodies (mAb) with cryoglobulin activity are able to induce skin leukocytoclastic vasculitis and glomerulonephritis resembling "wire-loop" glomerular lesions in normal mice. Since polymorphonuclear leukocyte (PMN) infiltration is one of the major pathological changes observed in both types of lesions, we determined the role of PMN and complement in the generation of these two different lesions, induced by 6-19 IgG3 RF mAb, by interfering with adhesion molecules known for their involvement of PMN-endothelial cell interaction or by depleting mice of their PMN or C3. Our results have demonstrated that first, the PMN-endothelial cell interaction mediated by leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) was crucial for the generation of 6-19 RF mAb-induced skin leukocytoclastic vasculitis, but not for glomerular lesions; second, PMN played an active role in the development of "wire-loop" glomerular lesions; in the absence of the PMN glomerular infiltration, 6-19 RF mAb induced a different type of glomerular lesions, characterized by voluminous intracapillary thrombi and mesangial deposits, but not subendothelial deposits; and third, the activation of the complement system did not appear to play a major role in both skin and glomerular lesions.

Induction of plasminogen activator inhibitor type 1 in murine lupus-like glomerulonephritis.

Three major components of the plasminogen activators (PA)/plasmin system are synthesized physiologically in glomeruli, and can be involved in glomerular proteolysis and extracellular matrix metabolism: tissue-type PA (tPA), urokinase (uPA) and PA inhibitor type 1 (PAI-1). To explore the possible role of a dysregulation of the plasmin protease system in the development and progression of lupus-like glomerulonephritis, we studied the expression of the renal plasmin protease components during the course of the disease, either acute, induced by IgG3 monoclonal cryoglobulins, or chronic, occurring spontaneously in three different lupus-prone mice: (NZBxNZW)F1, BXSB and MRL-lpr/lpr. RNase protection assays and in situ hybridizations revealed a marked glomerular induction of PAI-1 mRNA abundance without any significant changes in renal tPA and uPA mRNA levels in the two different types of lupus-like glomerulonephritis. The overexpression of PAI-1 mRNA occurred in parallel with a significant decrease in glomerular tPA-catalyzed enzymatic activity as determined by zymographic analysis. In addition, a concomitant increase in glomerular expression of transforming growth factor beta 1 (TGF-beta 1) mRNA was observed. The demonstration of a close correlation between the PAI-1 and TGF-beta 1 mRNA levels and the severity of lupus-like glomerular lesions suggests that a pertubation of the glomerular PA/PAI balance, resulting from a marked TGF-beta 1-mediated induction of PAI-1 gene expression, plays an important role in the progression of lupus-like glomerular lesions, leading to glomerulosclerosis.

Molecular and cellular basis for pathogenicity of autoantibodies.

Using two different kinds of monoclonal autoantibodies, anti-mouse RBC (MRBC) autoantibodies and IgG3 rheumatoid factor (RF) cryoglobulins, we have attempted to better define the molecular and cellular basis of the pathogenicity of autoantibodies. Among eight anti-MRBC monoclonal antibodies (mAbs) obtained from NZB mice, only five of them are able to cause anemia. The distinct differences in specificity between pathogenic and non-pathogenic anti-MRBC mAbs emphasize the importance of autoantibody specificity for the pathogenesis of autoimmune hemolytic anemia. Histological examination has revealed that Fc gamma receptor-mediated erythrophagocytosis and sequestration of agglutinated RBC in spleens and livers are the major pathogenic mechanisms of hemolytic anemia. This indicates that the affinity of autoantibodies for the Fc gamma receptors of phagocytes and/or the ability to cause hemagglutination, both of which vary among immunoglobulin isotypes, are additional factors determining the pathogenic activity of anti-MRBC autoantibodies. Studies on a panel of anti-IgG2a RF mAbs derived from MRL-lpr/lpr mice have demonstrated that only the IgG3 isotypes of RF mAb are able to generate cryoglobulins and to induce skin leukocytoclastic vasculitis and glomerulonephritis in normal mice. Although the cryoglobulin activity of RF mAb associated with the IgG3 isotype has been shown to be solely responsible for the generation of glomerular lesions (both RF and cryoglobulin activities are necessary for cutaneous vascular lesions), the absence of nephritogenic activity by some IgG3 monoclonal cryoglobulins supports the idea that qualitative features of cryoglobulins are critical to determine their pathogenic activities. Of interest, IgG3 autoantibodies lacking the cryoglobulin activity may not be harmful, but even protective against the development of IgG3 cryoglobulin-mediated tissue lesions, because they inhibit the cryoglobulin formation of pathogenic IgG3 autoantibodies as a result of their nonspecific IgG3 Fc-Fc interaction. Our results on monoclonal autoantibodies clearly indicate the importance of certain subpopulations of autoantibodies in the pathogenesis of autoantibody-mediated cellular and tissue injuries.

Glomerulopathy induced by IgG3 anti-trinitrophenyl monoclonal cryoglobulins derived from non-autoimmune mice.

We have previously shown that murine IgG3 monoclonal autoantibodies with cryoglobulin activity, derived from lupus-prone mice, are able to induce glomerular lesions resembling the "wire-loop" lesion typically described for human lupus nephritis. In the present study, we have further assessed the nephritogenic potential of four IgG3 anti-hapten, trinitrophenyl (TNP), monoclonal antibodies (mAb) obtained from non-autoimmune mice immunized with TNP-conjugated foreign antigens. Our results showed that two of four IgG3 anti-TNP monoclonal cryoglobulins were capable of inducing glomerular lesions, characterized by voluminous intracapillary thrombi and mesangial deposition of PAS-positive materials, which differed from "wire-loop" lesions generated by IgG3 monoclonal cryoglobulins with autoantibody activities. These anti-TNP monoclonal cryoglobulins, however, failed to induce glomerular lesions when mice were kept at 37 degrees C after the mAb administration. This finding formally proves that the cryoglobulin activity is critically involved in the development of glomerular lesions induced by IgG3 anti-TNP mAb. In addition, we have demonstrated a remarkable difference in the nephritogenic activities of two IgG3 anti-TNP mAb, which exhibit a marked sequence homology in the variable regions of their heavy and light chains (91.5% and 99.1% at the amino acid level, respectively) and an identical isoelectric point. Our results indicate first, that IgG3 monoclonal cryoglobulins are able to generate two different kinds of glomerular lesions, and second, that a subtle difference in variable region sequences may determine not only the nephritogenic activities, but also the type of glomerular lesions mediated by IgG3 cryoglobulins.

Immunoglobulin heavy chain constant region determines the pathogenicity and the antigen-binding activity of rheumatoid factor.

An IgG3 monoclonal antibody, 6-19, derived from unmanipulated MRL/MpJ-lpr/lpr mice, exhibiting cryoglobulin and anti-IgG2a rheumatoid factor activities, induces skin leukocytoclastic vasculitis and glomerulonephritis when injected into normal mice. To determine the role of the gamma 3 heavy chain constant region in the generation of cryoglobulins and associated tissue lesions, we have established an IgG1 class switch variant, clone SS2F8, from the 6-19 hybridoma by sequential sublining. Here we report that the SS2F8 monoclonal antibody, which loses the cryoglobulin activity but retains the rheumatoid factor activity, fails to generate skin and glomerular lesions. The lack of pathogenicity of the IgG1 SS2F8 switch variant is not due to mutations in variable regions, since nucleotide sequence analysis shows no differences between both clones. In addition, we have observed that the IgG1 SS2F8 switch variant exhibits < 10% of the rheumatoid factor activity, as compared with the IgG3 6-19 monoclonal antibody, suggesting that the self-associating property of the gamma 3 isotype promotes antibody-binding activity. The present study indicates that the cryoglobulin activity associated with the gamma 3 isotype is critically involved in the pathogenicity of 6-19 anti-IgG2a rheumatoid factor monoclonal antibody and highlights the pathogenic relevance of autoantibodies of the IgG3 subclass in murine systemic lupus erythematosus.

IgG3 cryoglobulins in autoimmune MRL-lpr/lpr mice: immunopathogenesis, therapeutic approaches and relevance to similar human diseases.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease resembling systemic lupus erythematosus and rheumatoid arthritis. One of the unique serological abnormalities in this strain is remarkably high concentrations of cryoglobulins. Analysis of immunoglobulin components in their cryoglobulins has shown selective enrichment of a particular IgG subclass, IgG3. As IgG3 enrichment is also found in two other cryoglobulins, which are induced after injection with bacterial lipopolysaccharides or infection with malaria, IgG3 apparently represents a major source of murine cryoglobulins. Studies on murine IgG3 monoclonal antibodies (mAbs) have clearly shown that murine IgG3 have the unique physiochemical property to self associate through non-specific IgG3 Fc-Fc interaction, and that most of them can generate monoclonal cryoglobulins. Most strikingly, IgG3 monoclonal cryoglobulins with rheumatoid factor (RF) activity induce extensive pathological manifestations: skin vascular purpura and glomerulonephritis with 'wire loop' lesions. Although the cryoglobulin activity of IgG3 RF mAb is solely responsible for the generation of glomerular lesions (both RF and cryoglobulin activities are necessary for skin vascular lesions), the absence of nephritogenic activity by some IgG3 cryoglobulins supports the idea that qualitative features of cryoglobulins are critical to determine their pathogenic activity. The demonstration of a positive correlation between the production of IgG3 cryoglobulins and the development of lupus nephritis in MRL-lpr/lpr mice further substantiates the pathological importance of cryogenic autoantibodies. On the other hand, it should be emphasised that non-cryogenerating IgG3 autoantibodies may not be harmful, but even protective, as a result of their interaction with pathogenic IgG3 cryoglobulins. Finally, the development of an experimental model of cryoglobulinaemia associated with vascular and glomerular disease certainly represents an invaluable opportunity to study the molecular mechanisms responsible for the generation of cryoglobulins and their associated tissue lesions, and also to assess various therapeutic approaches. Our demonstration that anti-idiotypic mAb can prevent the pathogenic effects of the cryoprecipitable IgG3 RF mAb suggests strongly that such a therapeutic approach might be successful in similar diseases in man.

Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass.

To analyze the involvement of rheumatoid factors (RF) in the generation of cryoglobulins and the development of related tissue injuries, we have established a panel of anti-IgG2a RF mAbs derived from MRL/MpJ-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr, and 129/Sv mice. After injection of hybridoma cells to normal mice, all four IgG3 RF mAbs induced cryoglobulinemia, and various degrees of glomerulonephritis and skin leukocytoclastic vasculitis. In contrast, none of the RF mAbs of the other isotypes generated cryoglobulins or tissue lesions. Since the same observation was obtained with another panel of five clonally related anti-IgG2a RF mAbs of MRL-lpr origin with almost identical heavy and light chain variable (V) regions but five different isotypes, it seems unlikely that the absence of pathogenicity of non-IgG3 RF mAbs was due to differences in fine specificity or V framework regions. In addition, the analysis of serum RF in MRL-lpr mice has demonstrated that a majority of 4 month old MRL-lpr mice produced substantial amounts of IgG3 RF with cryoglobulin activity. Because the cryoglobulin activity is associated with the murine IgG3 heavy chain constant region, RF of this subclass may play a significant role in the development of autoimmune-related tissue injuries, especially in MRL-lpr mice.

Induction of "wire-loop" lesions by murine monoclonal IgG3 cryoglobulins.

We have recently demonstrated that an IgG3 rheumatoid factor (RF) monoclonal antibody (mAb), clone 6-19, derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice, is able to generate cryoglobulins via a non-immunological IgG3 Fc interaction, and to induce an acute glomerulonephritis associated with cryoglobulinemia. Using this experimental model, we have characterized the glomerular lesions induced by the 6-19 RF monoclonal cryoglobulin, in particular the ultrastructural localization of the cryoglobulin deposits. Although their initial localization was confined to the mesangium, the 6-19 cryoglobulins were progressively accumulated in the subendothelial spaces of glomerular capillary walls, leading to the formation of glomerular lesions resembling the "wire-loop" lesion characteristically described for lupus nephritis. In addition, we have found that identical glomerular "wire-loop" lesions were induced by the 6-19-J558 hybrid antibody, composed of the 6-19 gamma 3 heavy chain and J558 lambda 1 light chain, which loses the RF activity, but retains the cryoglobulin activity. These results strongly suggest that the direct deposition of IgG3 cryoglobulins by itself, without involvement of immune complex formation, results in the generation of the classical "wire-loop" lesion characteristic of lupus nephritis. In addition, we have found that similar "wire-loop" lesions were generated by one anti-DNA mAb derived from (NZB x NZW)F1 hybrid mice, and two of four IgG3 mAb of unknown specificities, derived from MRL/MpJ-lpr/lpr mice. The absence of significant glomerular lesions, in spite of large amounts of cryoglobulins, in mice receiving two IgG3 mAb suggests the importance of physicochemical property of cryoglobulins to provoke glomerular lesions.