Anti-vascular endothelial cell antibodies in patients with IgA nephropathy: frequency and clinical significance.

This study examined the frequency of anti-vascular endothelial cell (VEC) antibodies (Ab) in 72 patients with IgA nephropathy (IgAN), and their possible relationship to clinical and histological parameters of the disease. An enzyme immunoassay was developed to measure the binding of sera to endothelial cells grown to a confluent monolayer. Thirty-two percent of IgAN patients had serum anti-VEC activity as compared to 4% of controls (P = 0.004) and 9% of patients with other primary glomerulonephritis (P = 0.017). This was shown to be due to anti-HLA class I Abs in 6 of the 23 IgAN patients, and in the 1 control positive for anti-VEC activity. Hence 17 IgAN patients had anti-VEC Abs, predominantly of the IgA subclass. Stimulation of the endothelial cells with interferon-gamma and interleukin 1 did not increase the binding of these Abs. There was no correlation with circulating immune complex (IC) levels, and removal of ICs in positive sera by ultracentrifugation did not decrease anti-VEC binding. Significant correlations were found between anti-VEC Abs and proteinuria greater than 1 g/day (P = 0.044), as well as IgA anti-VEC Abs and C3 or IgA deposition in renal arterioles (P = 0.048). These IgA Abs may be an important marker of pathogenetic activity in IgAN.

Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport.

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.

Quantitation of circulating immune complexes in human serum by the Raji cell and F(ab')2 anti-C3 micro enzyme immunoassays.

Two micro enzyme immunoassays (microEIA) for circulating immune complexes (CICs) are described. The Raji cell microEIA was similar in sensitivity, reproducibility and specificity to the Raji radioimmunoassay. The F(ab')2 anti-C3 microEIA was comparable to the 2 Raji cell assays. The 2 microEIAs for CICs were used to analyze sera from patients with systemic lupus erythematosus (SLE), the acquired immune deficiency syndrome (AIDS) and from hemophiliacs with AIDS-like symptoms. The microEIAs were very sensitive in detecting CICs in pathologic sera. In contrast, only 3% of normals (n = 30) were positive in the Raji microEIA while none were positive in the F(ab')2 anti-C3 microEIA. The initial high positivity of some normals (9/30) in the F(ab')2 microEIA was due to bovine serum albumin (BSA)-anti-BSA immune complex formation in vitro and was corrected when human albumin was used in buffer preparation instead of BSA. The reagents required for the microEIAs are more stable and less expensive than those required for the RIAs and the need for facilities to deal with 125-labeling and disposal is avoided.