Low-molecular weight C1q-binding immunoglobulin G in patients with systemic lupus erythematosus consists of autoantibodies to the collagen-like region of C1q.

The majority of C1q-binding IgG in sera of some patients with systemic lupus erythematosus (SLE) cosediments with monomeric IgG. This study was undertaken to provide definitive proof that the low-molecular weight C1q-binding IgG consists of autoantibodies to C1q. Monomeric C1q-binding IgG was isolated from five SLE plasmas by C1q affinity chromatography and gel filtration. All C1q-binding IgG preparations and their F(ab')2 fragments bound to both C1q and the collagen-like region of C1q by an ELISA. To rule out the possibility that small DNA-antiDNA immune complexes caused this binding activity, Fab' fragments of the C1q-binding IgG preparations were digested with DNase I to degrade any DNA. The Fab' fragments continued to bind to C1q and its collagen-like region after this treatment. C1q-binding IgG was heterogenous on isoelectric focusing. Interaction of C1q-binding IgG with solid-phase C1q was retained in 1 M NaCl, whereas the binding of DNA or heat-aggregated IgG to solid-phase C1q was abrogated or markedly diminished. The association constant of C1q-binding IgG with solid-phase C1q was 2.7 X 10(7) M-1. We conclude that low-molecular weight C1q-binding IgG in the studied patients with SLE consists of autoantibodies to the collagen-like region of C1q.

Evidence for conductive Cl- pathway in the basolateral membrane of rabbit renal proximal tubule S3 segment.

The mechanism of Cl- exit was examined in the basolateral membrane of rabbit renal proximal tubule S3 segment with double-barreled, ion-selective microelectrodes. After the basolateral Cl-/HCO3- exchanger was blocked by 2'-disulfonic acid, a bath K+ step from 5 to 20 mM induced 26.6 mV depolarization and 7.7 mM increase in intracellular Cl- activities ([Cl(-)]i). K+ channel blockers, Ba2+, and quinine strongly suppressed both the response in cell membrane potentials (Vb) and in (Cl-)i to the bath K+ step, while Cl- channel blockers, A9C (1 mM) and IAA-94 (0.3 mM) inhibited only the latter response by 49 and 74%, respectively. By contrast, an inhibitor of K(+)-Cl- cotransporter, H74, had no effect on the increase in (Cl-)i to the bath K+ step. Furosemide and the removal of bath Na+ were also ineffective, suggesting that (Cl-)i are sensitive to the cell potential changes. Bath Cl- removal in the presence of quinine induced a depolarization of more than 10 mV and a decrease in (Cl-)i, and IAA-94 inhibited these responses similarly in the bath K+ step experiments. These results indicate that a significant Cl- conductance exists in the basolateral membrane of this segment and functions as a Cl- exit mechanism.

Distribution of beta 2-adrenergic receptor mRNA expression along the hamster nephron segments.

Distribution of beta 2-adrenergic receptor mRNA expression along the microdissected hamster nephron segments was examined by the reverse transcription-polymerase chain reaction (RT-PCR) technique. Conventional RT-PCR using a set of primers on separate exons could not be applied for the detection of beta 2-adrenergic receptor mRNA because of its intronless nature. We used the 'rapid amplification of cDNA ends' protocol [(1985) Proc. Natl. Acad. Sci. USA 85, 8998-9002] as a maneuver for RT-PCR of an intronless gene. Using this method, we successfully located hamster beta 2-adrenergic receptor mRNA only in glomeruli and early proximal convoluted tubule along the nephron segments tested.

C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q.

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.

Tubuloglomerular feedback response in rats with antithymocyte serum-induced glomerular lesions.

Glomerular function and tubuloglomerular feedback (TGF) responses were studied in rats treated with antithymocyte rabbit serum (ATS). Microscopic findings revealed extensive mesangial cell loss and injury, enlarged capillary lumen, and decreased tortuosity of glomerular capillaries. Whole kidney GFR and SNGFR were lower in ATS-treated rats than in control rats (1.04 +/- 0.04 vs 0.94 +/- 0.03 ml/min/g kidney weight, 32 +/- 2 vs 27 +/- 1 nl/min, respectively) due to a decrease in Kf (0.017 +/- 0.001 vs 0.021 +/- 0.001 nl/s/mmHg). The magnitude of the TGF responses of SNGFR was less in ATS rats than in control rats (17% +/- 4% vs 34% +/- 5%), but that of SFP was not significantly different (18% +/- 2% vs 23% +/- 1%). These results indicate that the mesangial cells are necessary for full expression of the TGF mechanism and suggest the involvement of changes in Kf in the TGF mechanism.

Increase of cytokine production by pulmonary artery endothelial cells induced by supernatants from monocytes stimulated with autoantibodies against U1-ribonucleoprotein.

OBJECTIVE: To clarify the pathogenetic role of autoantibodies against U1-RNP (ribonucleoprotein) (anti-U1-RNP) in mixed connective tissue disease (MCTD), we examined whether supernatants of monocytes which were stimulated with anti-U1-RNP could induce the production of proinflammatory cytokines by human pulmonary artery endothelial cells (HPAECs). METHODS: Monocytes from MCTD patients (n = 11) and normal volunteers (n = 11) were stimulated with purified antibodies against U1-RNP or double-stranded DNA and their supernatants were added to cultures of HPAECs. Cell-associated cytokines were assayed by an enzyme-linked immunosorbent assay. RESULTS: The supernatants of anti-U1-RNP-stimulated MCTD monocytes significantly up-regulated the cell-associated production of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01) by HPAECs compared with their production by normal IgG-stimulated MCTD monocytes, whereas the cell-associated production of IL-1 beta and TNF-alpha by HPAECs was not up-regulated. The supernatants of anti-U1-RNP-stimulated monocytes from normal volunteers similarly up-regulated the cell-associated production by HPAECs of IL-1 alpha (p < 0.01) and IL-6 (p < 0.01), but not of IL-1 beta and TNF-alpha. Supernatants of monocytes stimulated with the F(ab')2 preparation of anti-U1-RNP antibodies enhanced the amounts of both Il-1 alpha and IL-6 associated with HPAECs almost as effectively as those stimulated with intact autoantibody molecules. Inhibition experiments employing specific anti-cytokine antibodies of anti-U1-RNP-stimulated monocyte supernatants suggested that soluble factors, including cytokines, in monocyte supernatants could enhance the cytokine association with HPAECs. CONCLUSION: Up-regulation by anti-U1-RNP autoantibodies of proinflammatory cytokines associated with vascular endothelial cells may play a role in the immunopathological processes leading to proliferative vasculopathy, a characteristic of MCTD.

Activation of the basolateral Cl- conductance by cyclic AMP in rabbit renal proximal tubule S3 segment.

The regulation of basolateral Cl- conductance in renal proximal tubule was investigated by microelectrodes. Our results suggest that this conductance can be activated by cAMP, while increase in cell Ca2+ or activation of PKC has no effect. These properties of this conductance also suggest that CFTR may exist in the basolateral membrane of proximal tubule.

Shunt nephritis: efficacy of an antibiotic trial for clinical diagnosis.

Coagulase negative staphylococcus, a normal skin flora, is especially nosopoietic under shunt management, because coagulase negative staphylococcus sometimes forms a biofilm around itself at catheter tips in vivo, which shields the organism from the effects of antibiotics. But it is difficult to distinguish this pathogen from a possible confounding contamination of a blood culture. In this article, we report a case, and discuss how a patient with suspected shunt nephritis should be examined and treated. In addition, to further histological and prognostic interpretation, we review the previously reported cases of shunt nephritis in Japanese adults.

The location of binding sites on C1q for DNA.

Previous studies have suggested that C1q reacts with DNA via both the globular region of C1q (GR) and the collagen-like region of C1q (CLR). In this study, the binding of dsDNA and ssDNA to GR and CLR was quantitated by a solid-phase assay. Both dsDNA and ssDNA bound to the GR and CLR of C1q in an ionic strength-dependent manner. Under physiologic salt concentrations, however, dsDNA and ssDNA bound preferentially to CLR and not to GR. The binding of dsDNA to C1q was not affected by heat inactivation of C1q or its exposure to pH 4.45, which abolished the binding of heat-aggregated human IgG (AHG) with C1q. The preincubation of the solid-phase C1q with AHG did not decrease the binding of dsDNA or ssDNA to the solid-phase C1q. These results indicate that the major sites for binding DNA to C1q are located in the CLR of C1q and are not overlapping with those for AHG or immune complexes.

IgG subclasses of antibodies to the collagen-like region of C1q in patients with systemic lupus erythematosus.

Low molecular weight C1q-binding IgG in the sera of patients with systemic lupus erythematosus consists of autoantibodies to the collagen-like region of C1q. In this study, the IgG subclass distribution of these autoantibodies was examined by radial immunodiffusion with polyclonal antibodies specific for each subclass. The purified antibodies to the collagen-like region of C1q possessed the IgG subclass distribution present in normal serum.

Autoantibodies to the collagen-like region of C1Q deposit in glomeruli via C1Q in immune deposits.

The autoantibodies to the collagen-like region of C1q (CLR), purified from two patients with systemic lupus erythematosus, deposited in mouse glomeruli when human C1q was present in antigen-antibody complexes in glomeruli. The immune deposits with C1q in mouse glomeruli were achieved by the administration of cationized immune complexes containing human C1q. The presented data suggest that the autoantibodies to CLR could enhance the pathogenic role of immune complexes deposited in glomeruli by binding to C1q in immune deposits. These findings may explain the association of autoantibodies to CLR with proliferative lupus nephritis.

Antibodies to the collagen-like region of C1q in sera of patients with autoimmune rheumatic diseases.

Antibodies to the collagen-like region of C1q have recently been observed in sera of patients with systemic lupus erythematosus (SLE). In this study, we documented that these antibodies were present in 47.3% of SLE patient sera, whereas they were uncommon in sera from patients with rheumatoid arthritis (2.8%) and Sjögren's syndrome (12.8%), as well as in normal sera (6.4%). Markedly elevated antibody levels (greater than 4 SD above the normal mean) were observed almost exclusively in sera of patients with SLE. Levels of antibodies to the collagen-like region correlated highly with levels of solid-phase C1q-binding IgG when analyzed by the C1q solid-phase assay for immune complexes (r = 0.87). We previously found that, after sucrose density gradient ultracentrifugation, a predominance of the solid-phase C1q-binding IgG in SLE sera sediments as monomeric IgG. These findings, together with the present data, indicate that reactivity of SLE patients' sera in the C1q solid-phase assay reflects primarily the presence of antibodies to the collagen-like region, and not the presence of immune complexes.

Monocyte-mediated suppression of anti-DNA antibody production in healthy subjects.

Monocyte-depleted mononuclear cells (MNC) from healthy subjects synthesized IgM-class antibody to single-stranded DNA (anti-ssDNA) at significantly higher levels than unfractionated MNC. The increased production of IgM anti-ssDNA by monocyte-depleted normal MNC was inhibited to a greater degree than total IgM by addition of supernatants from autologous monocytes. Moreover, supernatants obtained from normal monocytes cultured with indomethacin retained this suppressive effect on IgM anti-ssDNA antibody production, suggesting that prostaglandin E2 may not be involved in the suppression. These results indicate that B cells programmed to produce IgM anti-ssDNA are present in the normal B cell repertoire, but may be suppressed by monocytes or by a soluble factor (or factors) from the monocytes.

Monocyte-mediated suppression of rheumatoid factor production in normal subjects.

The regulatory role of normal monocytes in the production of rheumatoid factor (RF) was investigated. Monocyte depletion from normal mononuclear cells (MNC) resulted in an elaboration of IgM RF in 18 of 20 subjects. The increased RF production was inhibited by the addition to the cultures of adherent cells or their culture supernatants. These observations demonstrate the suppressive role of normal monocytes in the production of RF. Supernatants obtained from normal monocytes cultured with indomethacin could suppress the RF production, suggesting that prostaglandin E2 may not be involved in the regulation of IgM RF production.

Characterization of C1q-binding IgG complexes in systemic lupus erythematosus.

The molecular size of C1q-binding immunoglobulin (Ig) G complexes in systemic lupus erythematosus (SLE) sera was studied by gel filtration using C1q solid-phase radioimmunoassay (C1q SPRIA). All 15 SLE sera tested contained predominantly small-sized IgG complexes, cofractionating with monomeric IgG. In contrast to heat-aggregated IgG, these small-sized IgG complexes retained C1q-binding activity even after pepsin digestion, exposure to low pH, or reduction and alkylation, suggesting that the F(ab')2 region is involved in C1q-binding activity of these complexes. To see whether anti-C1q antibodies or small antigen-IgG complexes, which bind to C1q via their antigens, are responsible for C1q-binding activity via the F(ab')2 region, the pepsin-digested Ig fractions of SLE sera were fractionated at high salt. C1q-binding activity in the fractions corresponding to the F(ab')2 region increased 2.5- to 3.9-fold at high salt. These results suggest that the C1q-binding, small-sized IgG complexes may be comprised mostly of anti-C1q antibodies and that some of the antibodies, which are dissociated with their antigens at high salt, might be cross-reactive with C1q.