Dimeric and polymeric IgA, but not monomeric IgA, enhance the production of IL-6 by human renal mesangial cells

Depositions of IgA in the renal glomerular mesangial area are a hallmark of IgA nephropathy, and are thought to be crucial for the onset of inflammation processes in IgA nephropathy. In this report we show that human mesangial cells (MC) in vitro bind IgA and that binding of IgA enhances the production of IL-6 by MC. Furthermore we show that the size of IgA is crucial in its capability to enhance IL-6 production. Monomeric IgA does not affect basic IL-6 production, whereas dimeric and polymeric IgA enhance IL-6 production up to 3- to 9-fold respectively. Additional studies demonstrate that enhanced IL-6 production by MC is not accompanied by increased proliferation of human mesangial cells, a finding which is distinct from that found with rat mesangial cells. Taken together, these fmdings suggest that deposition of dimeric and polymeric IgA in the mesangial area of human kidneys in IgA nephropathy may amplify local inflammation.


Introduction
Immunoglobulin A (IgA) is the predominant immunoglobulin in human secretions and the second most important immunoglobulin in the circulation on a quantitative basis. 1 '2 Depositions of IgA in the glomerular mesangial area of the kidney, as found in IgA nephropathy, are thought to play a crucial role in the inflammatory processes in this disease. The deposited IgA is mainly of the IgA1 subclass and is thought to be derived from the circulation. 4 Also co-depositions of IgG and complement factors, such as complement component 3 (C3) are routinely seen in renal biopsies of patients with IgA nephropathy. 5 Elevated serum levels of IgA, 6 increased production of IgA1, 7 and a hyperresponse for IgA1 after vaccination 7 is found in patients with IgA nephropathy. The mechanisms of IgA deposition in the kidney are still unclear. However, a delayed clearance of IgA has been suggested. Recently, a specific receptor for IgA was described in rat and human renal glomerular mesangial cells (CD89). 9 It is not known whether CD89 plays a role in IgA deposition in the kidney during IgA nephropathy. It is interesting that phagocytic blood cells of patients with IgA nephropathy seem to be hampered in CD89mediated clearance of IgA. 10 Mesangial depositions of IgA have been associated with increased levels of IL-6 in the urine of patients with IgA nephropathy. 6 IL-6 is a potent, (C) 1996 Rapid Science Publishers multifunctional cytokine, which has multiple biological function activities on a wide variety of tissues and cells, such as Band T-lymphocytes, myeloma cells, haematopoietic stem cells, hepatocytes, fibroblasts 11 and rat glomerular mesangial cells. 12 It was shown that IL-6 is produced by a variety of cells, such as macrophages, lymphocytes, fibroblasts and endothelial cells. [13][14][15][16][17] Also, it has been shown that MC are able to produce IL-6,18-2 which acts as an autocrine growth factor for rat mesangial cells in vitro. 2'2 In this study we demonstrate that human MC bind IgA and produce IL-6 upon stimulation with IgA in vitro. The degree of production of IL-6 is dependent on the size of the IgA. Only dimeric and polymeric IgA enhance IL-6 production by MC. The IgA-induced production of IL-6 is inhibitable by cycloheximide, which indicates de novo synthesis. Furthermore we show that human MC do not proliferate upon stimulation with IgA.

Materials and Methods
Cell culture: Glomerular mesengial cells (MC) were cultured using glomeruli obtained from human foetal kidneys of 12-19 weeks of gestation, by mechanical dissociation and sequential 22 23 sieving as described.
Approval for the use of foetal kidneys was obtained by informed consent and from the medical ethics committee of the hospital. After sieving, glomerular epithelial cells and cultured at 37C in 5% CO2. After outgrowth of the MC, the hillocks formed were lifted off the culture flasks and explanted into 24-well culture plates (Greiner, Alphen aan de Rijn, The Netherlands). MC growing out of the hillocks were subcultured in T25 or T75 culture flasks (Greiner).
For the experiments MC were used between subculture 2 and 8. Isolation of lgA: Human IgA was isolated by chromatography using DEAE Sephadex (Pharmacia, Uppsala, Sweden) anion exchange chromatography, and Sephacryl S-300 (Pharmacia) gel filtration as described. 24 Purity of the final IgA preparations was checked by SDS-polyacrylamide gel and ELISA for residual IgG and IgM. The IgA preparations were shown to be free of detectable IgG and IgM by ELISA.
Rat IgA was isolated as described, 25 briefly, IgA containing ascites from Lewis rats, inoculated intraperitoneally with 106 viable LO-DNP-45 hybridoma cells, was collected and precipitated with a final concentration of 50% (NH4)2SO4, The pellet was resuspended and dialysed against phosphate-buffered saline (PBS)-2 mM EDTA and IgA-anti-dinitrophenol (DNP) was affinity purified using a DNP-lysine-coupled Sepharose affinity column. After washing, anti-DNP-specific IgA was eluted from the column with 0.1 M DNP. After removal of free DNP by chromatography on Dowex 1 (1 x 2 400), the IgA containing fractions were pooled, concentrated and subjected to gel filtration chromatography on a Sephacryl S-300 column to yield monomeric, dimeric-and polymeric IgA. The purified IgA preparations were dialysed against PBS and were shown to be devoid of detectable amounts of IgA and IgM, as detected by ELISA.
Radiolabelling of IZ_: Human dimeric IgA was radiolabelled with ">I using Iodo-Beads (Pierce, Rockford, IL) according to the manufacturer's instructions. Non-incorporated iodine was separated from protein by gel filtration using Sephadex G25 (Pharmacia). The specific activity was 3 l.tCi/l.tg protein.
Binding of IgA to MC: After growing to subconfluency in 48-well plates, MC were washed three times with PBS/0.5% bovine serum albumin (BSA), and three wells were trypsinized to count the number of cells present at the beginning of the assay, using a Coulter Counter (Coulter Electronics, Mijdrecht, The Netherlands). Subsequently, MC were incubated for 16 h at 4C with a dose response of iodinated human IgA in 300 btl DMEM/0.5%BSA in triplicate. After incubation, the wells were washed three times with cold PBS/0.5%BSA to remove non-bound radioactivity.
After washing, 300 l.tl 1 M NaOH was added per well, to detach and solubilize the cells and subsequently, the amount of radioactivity was measured and calculated per l.tg/105 cells. All data were corrected for nonspecific binding. IL-6 production: Subconfluent 48-well plates with MC were washed three times with phosphate buffered saline (PBS) and cultured for an additional 48 h in DMEM/0.5%FCS to induce a quiescent state. After washing with DMEM/0.5%FCS, fixed concentrations of IgA were added to the cells in DMEM/0.5%FCS in triplicate. As a positive control, MC were stimulated with 100 ng/ml LPS.
After 72 h of stimulation, supernatants were harvested and assessed for IL-6 production in the B9-bioassay, using the I1-6-dependent murine hybridoma cell line B9, 20 Effect of cycloheximide on the production of IL-6 by MC: After growing MC to subconfluency in 48well plates, the cells were washed and incubated further, either in medium alone, or in medium containing 10 I.tg/ml human dimeric IgA, with or without 1 lg/ml cyclohexamide in triplicate. After 72 h, the supernatants were harvested and used in the B9 assay in serial dilutions of 1/20-1/640. Cycloheximide at concentrations of less than 5 ng/ml does not interfere with the B9 assay. To determine whether the cells remained viable during incubation with cycloheximide, the cells were washed with medium and incubated in medium alone for another 72 h. After this second incubation, the supernatants were harvested again, and assessed for IL-6 using the B9 assay. MC proliferation assay: MC, subconfluently plated in 96or 48-well plates, were washed three times with phosphate buffered saline (PBS) and cultured for an additional 48h in DMEM/ 0.5% to induce a quiescent state. After washing with DMEM/0.5%FCS, fixed amounts of the various IgA preparations in DMEM/0.5%FCS were IgA and production of ll-6 added in triplicate. As a positive control, 100 ng/ ml LPS was used. After 7 days of stimulation, the cells were washed, trypsinized and counted by using a Coulter Counter.

Results
To analyse the binding of IgA to MC, the MC were incubated at 4C with a dose response of 25I-labelled human dimeric IgA for 16 h. Human dimeric IgA was able to bind to MC in a dosedependent manner. Saturation was reached at approximately 10 btg/ml (Fig. 1).
To investigate whether MC are activated upon binding of IgA, MC were incubated at 37C in triplicate wells for 72 h in medium (DMEM/10%FCS) alone, in medium supplemented with increasing concentrations of human dimeric IgA, or in medium containing 100 ng/ml LPS, as a positive control. After stimulation, the amount of IL-6 produced by the MC was measured in the B9 assay. Basal production of IL-6 by MC was 1 (Fig. 2).
To investigate whether the production of IL-6 is dependent on the size of IgA, MC were stimulated in triplicate wells for 72 h with medium alone, 0.1, 10 and 100 l.tg/ml rat monomeric, dimeric, and polymeric IgA, or 100 ng/ml LPS. Polymeric IgA was able to induce a three-fold increase to 4 086 1443 units IL-6 per 105 cells at a concentration of 10 l.tg/ml and a nine-fold increase to 11800 __+ 300 units IL-6 per 105 cells at a concentration of 100 l.tg/ml. Dimeric IgA was much less potent in inducing an increase of IL-6 production. At a concentration of 100 g/ml, dimeric IgA induced a production of 3 586 _ _ _ 1 471 units IL-6 per 105 cells, whereas monomeric IgA did not induce a significant increase in IL-6 production (Fig. 3).
To investigate whether the IL-6 produced by the MC upon IgA stimulation is due to de novo synthesis, MC were incubated in triplicate for 72 h in medium, or in medium supplemented with 10 I.tg/ml human dimeric IgA, both with or without cycloheximide (1 btg/ml), and assessed for IL-6. Cycloheximide was able to cause 97.3% inhibition of IgA-induced IL-6 release (p < 0.002) (Fig. 4). It has been suggested that IgA is able to induce proliferation of rat MC in vitro. To investigate whether human MC also proliferate upon stimulation with IgA, MC were incubated in medium alone (DMEM/0.5%FCS) or with various preparations of IgA at a concentration of 10 l-tg/ ml. As a positive control 100 ng/ml LPS was used. After 7 days of culture, the triplicate wells were trypsinized and the total cell number per well was determined by using the Coulter Counter. None of the IgA preparations was able to induce MC proliferation. In contrast, 100 ng/ ml LPS was able to induce a relative proliferation of 2.24 4-0.41 (p < 0.05) (Fig. 5).

Discussion
Glomerular mesangial depositions of IgA in the kidney, together with IgG and complement com- ponents such as C3, are thought to be crucial for the onset of the inflammation process in IgA nephropathy. It has been shown that IgG binds tO MC 27'28 and stimulates IL-6 production. 18'2 In this study, we show that human MC are capable of secreting de novo synthesized IL-6 after stimulation with IgA. The IL-6 production of the MC is strongly dependent on the size of the IgA. Polymeric rat IgA is able to increase the IL-6 production by MC up to nine-fold. Dimeric IgA is less potent in increasing IL-6 production by MC and only increased the basal production three-fold. In contrast with polymeric and dimeric IgA, monomeric IgA is not able to increase the IL-6 production.
It has been shown that IL-6 is an autocrine growth factor for rat MC. 12'21 Even though dimeric and polymeric IgA enhance the production of IL-6 by human MC, we did not observe proliferation of the cells. These findings are in agreement with the observation that human MC do not proliferate upon stimulation with IL-629'3 in vitro. In IL-6 transgenic mice, it was shown that high plasma concentrations of IL-6 are associated with mesangial proliferation. In humans, however, it was found that urinary IL-6 levels do not correlate with mesangial proliferative glomerulonephritis. 2 Our finding, that MC fail to proliferate upon stimulation with IgA, is also in accord with these findings. Therefore, our in vitro data suggest that mesangial cell proliferation in humans, as found in IgA nephropathy, is presumably not due to the autocrine effect of IL-6 produced by MC upon stimulation with IgA. The precise mechanisms by which mesangial proliferation is induced remain unclear, suggesting that other mitogens, or combinations of different mitogens acting synergistically, induce the mesangial proliferation, as seen in IgA nephropathy.