IgA1 immune complexes from pediatric patients with IgA nephropathy activate cultured human mesangial cells

Abstract

Background: Circulating immune complexes (CIC) containing galactose (Gal)-deficient IgA1 from adults with IgA nephropathy (IgAN) induce proliferation of cultured mesangial cells, but activities of CIC from pediatric patients with the disease have not been studied.

Methods: CIC of different sizes were isolated from sera of pediatric and adult IgAN patients and their effects on cultured human mesangial cells (MC) were assessed by measuring cellular proliferation, expression of IL-6 and IL-8 and laminin and phosphotyrosine signaling.

Results: Large CIC from pediatric IgAN patients (>800 kDa) containing Gal-deficient IgA1 stimulated cellular proliferation, whereas in some patients, smaller CIC were inhibitory. Addition of stimulatory and inhibitory CIC to MC differentially altered phosphorylation patterns of three major tyrosine-phosphorylated proteins of molecular mass 37, 60 and 115 kDa. The stimulatory CIC transiently increased tyrosine-phosphorylation of the 37-kDa protein and decreased phosphorylation of the other two proteins, whereas the inhibitory CIC increased phosphorylation of all three proteins. Furthermore, we investigated the influence of IgA1-containing CIC from sera of children with IgAN with clinically active disease (i.e., abnormal urinalysis and/or serum creatinine concentration) or inactive disease (i.e., normal urinalysis and serum creatinine concentration) on the expression of IL-6 and IL-8 genes by mesangial cells. Real-time reverse transcription-polymerase chain reaction results showed that the CIC from a patient with active disease stimulated MC to express the two cytokine genes at higher levels than did the CIC from a patient with inactive disease. Moreover, stimulatory CIC increased production of the extracellular matrix protein laminin.

Conclusion: These data indicate that sera of pediatric IgAN patients contain biologically active CIC with Gal-deficient IgA1.

Fig. 1.

Proliferation of human MC measured by ³H-thymidine incorporation after stimulation with IgA1-containing CIC from sera of (a) four pediatric IgAN patients (P1, full square; P2, full triangle; P3, empty square; P4, full circle) and (b) four adult IgAN patients (A1, full circle; A2, full square; A3, full triangle; A4, empty circle), all collected at the time of macroscopic hematuria (Table 1). All samples exhibited stimulatory CIC of ≥800–900 kDa and some samples also had smaller, inhibitory CIC of ∼700–800 kDa. Staining for PCNA confirmed the results: stimulatory CIC increased expression of PCNA in MC. Inserts in (b) show staining of PCNA-positive and PCNA-negative MC in culture. Dashed lines mark background level of MC proliferation.

Fig. 2.

Western blot of MC lysates with anti-phosphotyrosine antibody. Human MC were incubated with stimulatory (Lanes 2 and 3) or inhibitory (Lanes 4 and 5) immune complexes for 15 min (Lanes 2 and 4) or 1 h (Lanes 3 and 5). Lysate sample from mock-treated control (untreated) MC is in Lane 1. Stimulatory CIC transiently increased phosphorylation of the 37-kDa protein and decreased phosphorylation of the other two proteins, whereas the inhibitory CIC increased phosphorylation of all three proteins. The arrow marks 37-kDa phosphoprotein; arrowheads, the other two phosphoproteins.

Fig. 3.

Transcription of IL-8 and IL-6 genes was determined by real-time RT–PCR using total RNA from human MC incubated for 20 h with stimulatory CIC from sera of two patients with IgAN (Pt columns) or corresponding fractions from a healthy control (Con columns). Pt1 had active disease, whereas Pt2 had an inactive disease (i.e., normal urinalysis). Medium without CIC served as the negative control (n.c. columns). PDGF (PDGF columns) and free uncomplexed Gal-deficient IgA1 myeloma protein (Ale) served as additional controls. IgA1 (Ale) was used in concentrations 25, 50 and 75 μg per well (Ale25, Ale50, Ale75 columns). Expression of specific genes was measured in triplicate and expressed relative to that of β-actin. Data comparisons were performed by the E-method [36] and calculated as mean expression in control MC to the expression in stimulated MC. Means and ranges are shown.

Fig. 4.

Expression of laminin in human MC incubated for 48 h with stimulatory CIC from an IgAN patient or with serum fractions of similar size from a normal control. Lysed MC (same amount of protein was loaded) or culture supernatants (same volume of supernatant was loaded) were separated using 4–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and the blots were probed with polyclonal anti-laminin antibody specific for beta chains.