IgA, - Hindawi Publishing Corporationdownloads.hindawi.com/journals/mi/1996/265928.pdfduction of...

Research Paper

Mediators of Inflammation, , 191-195 (1996)

DEPOSmONS of IgA in the renal glomerular mesan-gial area are a hallmark of IgA nephropathy, andare thought to be crucial for the onset of inflam-mation processes in IgA nephropathy. In thisreport we show that human mesangial cells (MC)in vitro bind IgA and that binding of IgAenhances the production of IL-6 by MC. Further-more we show that the size of IgA is crucial in itscapability to enhance IL-6 production. Mono-meric IgA does not affect basic IL-6 production,whereas dimeric and polymeric IgA enhance IL-6production up to 3- to 9-fold respectively. Addi-tional studies demonstrate that enhanced IL-6production by MC is not accompanied byincreased proliferation of human mesangial cells,a f’mding which is distinct from that found withrat mesangial cells. Taken together, thesefmdings suggest that deposition of dimeric andpolymeric IgA in the mesangial area of humankidneys in IgA nephropathy may amplify localinflammation.

Key words: IgA, Interleukin 6, Renal mesangial cells

Dimeric and polymeric IgA, but notmonomeric IgA, enhance theproduction of 11_-6 by human renalmesangial cells

T. J. F. Reterink,cA W. E. M. Schroeijers,L. A. van Es and M. R. Daha

Department of Nephrology, Leiden UniversityHospital, C3P, P.O. Box 9600, 2300 RC Leiden,the Netherlands

CACorresponding Author

Introduction

Immunoglobulin A (IgA) is the predominantimmunoglobulin in human secretions and thesecond most important immunoglobulin in thecirculation on a quantitative basis. 1’2 Depositionsof IgA in the glomerular mesangial area of thekidney, as found in IgA nephropathy, are thoughtto play a crucial role in the inflammatory pro-cesses in this disease. The deposited IgA ismainly of the IgA1 subclass and is thought to bederived from the circulation.4 Also co-depositionsof IgG and complement factors, such as comple-ment component 3 (C3) are routinely seen inrenal biopsies of patients with IgA nephropathy.5

Elevated serum levels of IgA,6 increased produc-tion of IgA1,7 and a hyperresponse for IgA1 aftervaccination7 is found in patients with IgAnephropathy. The mechanisms of IgA depositionin the kidney are still unclear. However, adelayed clearance of IgA has been suggested.Recently, a specific receptor for IgA was de-scribed in rat and human renal glomerularmesangial cells (CD89).9 It is not known whetherCD89 plays a role in IgA deposition in the kidneyduring IgA nephropathy. It is interesting thatphagocytic blood cells of patients with IgAnephropathy seem to be hampered in CD89-mediated clearance of IgA.10

Mesangial depositions of IgA have been asso-ciated with increased levels of IL-6 in the urine ofpatients with IgA nephropathy.6 IL-6 is a potent,

(C) 1996 Rapid Science Publishers

multifunctional cytokine, which has multiple bio-logical function activities on a wide variety oftissues and cells, such as B- and T-lymphocytes,myeloma cells, haematopoietic stem cells,hepatocytes, fibroblasts11 and rat glomerularmesangial cells. 12 It was shown that IL-6 is pro-duced by a variety of cells, such as macrophages,lymphocytes, fibroblasts and endothelial cells.13-17 Also, it has been shown that MC are able toproduce IL-6,18-2 which acts as an autocrinegrowth factor for rat mesangial cells in vitro.2’2

In this study we demonstrate that humanMC bind IgA and produce IL-6 upon stimulationwith IgA in vitro. The degree of production ofIL-6 is dependent on the size of the IgA. Onlydimeric and polymeric IgA enhance IL-6 produc-tion by MC. The IgA-induced production of IL-6is inhibitable by cycloheximide, which indicatesde novo synthesis. Furthermore we show thathuman MC do not proliferate upon stimulationwith IgA.

Materials and Methods

Cell culture: Glomerular mesengial cells (MC)were cultured using glomeruli obtained fromhuman foetal kidneys of 12-19 weeks of gesta-tion, by mechanical dissociation and sequential

22 23sieving as described. Approval for the use offoetal kidneys was obtained by informed consentand from the medical ethics committee of thehospital. After sieving, glomerular epithelial cells

Mediators of Inflammation Vol 5 1996 191

T. J. F. Reterink et al.

were removed by digestion with type 1Acollagenase (Sigma, St Louis, MO, USA) for 20min at 37C. After washing, the resulting glom-erular suspensions were resuspended in DMEM(Seromed, Biochrom, Berlin, Germany) supple-mented with 20% heat-inactivated foetal calfserum (FCS) (Gibco, Breda, The Netherlands),plated onto charged plastic culture Primariaflasks (Falcon, Becton Dickinson, San Jose, CA)and cultured at 37C in 5% CO2. After outgrowthof the MC, the hillocks formed were lifted off theculture flasks and explanted into 24-well cultureplates (Greiner, Alphen aan de Rijn, The Nether-lands). MC growing out of the hillocks were sub-cultured in T25 or T75 culture flasks (Greiner).For the experiments MC were used betweensubculture 2 and 8.

Isolation oflgA: Human IgA was isolated by chro-matography using DEAE Sephadex (Pharmacia,Uppsala, Sweden) anion exchange chromato-graphy, and Sephacryl S-300 (Pharmacia) gel fil-tration as described.24 Purity of the final IgApreparations was checked by SDS-poly-acrylamide gel and ELISA for residual IgG andIgM. The IgA preparations were shown to be freeof detectable IgG and IgM by ELISA.

Rat IgA was isolated as described,25 briefly, IgAcontaining ascites from Lewis rats, inoculatedintraperitoneally with 106 viable LO-DNP-45hybridoma cells, was collected and precipitatedwith a final concentration of 50% (NH4)2SO4,

The pellet was resuspended and dialysed againstphosphate-buffered saline (PBS)-2 mM EDTAand IgA-anti-dinitrophenol (DNP) was affinitypurified using a DNP-lysine-coupled Sepharoseaffinity column. After washing, anti-DNP-specificIgA was eluted from the column with 0.1 M DNP.After removal of free DNP by chromatography onDowex 1 (1 x 2 400), the IgA containing frac-tions were pooled, concentrated and subjectedto gel filtration chromatography on a Sephacryl S-300 column to yield monomeric, dimeric- andpolymeric IgA. The purified IgA preparationswere dialysed against PBS and were shown to bedevoid of detectable amounts of IgA and IgM, asdetected by ELISA.

Radiolabelling of IZ_: Human dimeric IgA wasradiolabelled with ">I using Iodo-Beads (Pierce,Rockford, IL) according to the manufacturer’sinstructions. Non-incorporated iodine was separ-ated from protein by gel filtration using Sepha-dex G25 (Pharmacia). The specific activity was 3l.tCi/l.tg protein.

Binding of IgA to MC: After growing to subcon-fluency in 48-well plates, MC were washed three

times with PBS/0.5% bovine serum albumin(BSA), and three wells were trypsinized to countthe number of cells present at the beginning ofthe assay, using a Coulter Counter (Coulter Elec-tronics, Mijdrecht, The Netherlands). Subse-quently, MC were incubated for 16 h at 4C witha dose response of iodinated human IgA in 300btl DMEM/0.5%BSA in triplicate. After incubation,the wells were washed three times with coldPBS/0.5%BSA to remove non-bound radioactivity.After washing, 300 l.tl 1 M NaOH was added perwell, to detach and solubilize the cells and sub-sequently, the amount of radioactivity was meas-ured and calculated per l.tg/105 cells. All datawere corrected for nonspecific binding.

IL-6production: Subconfluent 48-well plates withMC were washed three times with phosphatebuffered saline (PBS) and cultured for an addi-tional 48 h in DMEM/0.5%FCS to induce a quies-cent state. After washing with DMEM/0.5%FCS,fixed concentrations of IgA were added to thecells in DMEM/0.5%FCS in triplicate. As a positivecontrol, MC were stimulated with 100 ng/ml LPS.After 72 h of stimulation, supernatants were har-vested and assessed for IL-6 production in theB9-bioassay, using the I1-6-dependent murinehybridoma cell line B9,20’26 kindly provided byDr L. A. Aarden (CLB, Amsterdam, The Nether-lands) in combination with the Cell Titer 96assay (Promega, Leiden, The Netherlands) tomeasure proliferation. Serial dilutions of humanrecombinant IL-6 were used as a standard.

Effect of cycloheximide on the production of IL-6by MC: After growing MC to subconfluency in 48-well plates, the cells were washed and incubatedfurther, either in medium alone, or in mediumcontaining 10 I.tg/ml human dimeric IgA, with orwithout 1 lg/ml cyclohexamide in triplicate. After72 h, the supernatants were harvested and usedin the B9 assay in serial dilutions of 1/20-1/640.Cycloheximide at concentrations of less than 5ng/ml does not interfere with the B9 assay. Todetermine whether the cells remained viableduring incubation with cycloheximide, the cellswere washed with medium and incubated inmedium alone for another 72 h. After this secondincubation, the supernatants were harvestedagain, and assessed for IL-6 using the B9 assay.

MC proliferation assay: MC, subconfluentlyplated in 96- or 48-well plates, were washedthree times with phosphate buffered saline (PBS)and cultured for an additional 48h in DMEM/0.5% to induce a quiescent state. After washingwith DMEM/0.5%FCS, fixed amounts of thevarious IgA preparations in DMEM/0.5%FCS were

192 Mediators of Inflammation Vol 5 1996

IgA andproduction ofll-6

added in triplicate. As a positive control, 100 ng/ml LPS was used. After 7 days of stimulation, thecells were washed, trypsinized and counted byusing a Coulter Counter.

Results

To analyse the binding of IgA to MC, the MCwere incubated at 4C with a dose response of25I-labelled human dimeric IgA for 16 h. Humandimeric IgA was able to bind to MC in a dose-dependent manner. Saturation was reached atapproximately 10 btg/ml (Fig. 1).To investigate whether MC are activated

upon binding of IgA, MC were incubated at37C in triplicate wells for 72 h in medium(DMEM/10%FCS) alone, in medium supple-mented with increasing concentrations ofhuman dimeric IgA, or in medium containing100 ng/ml LPS, as a positive control. After stim-ulation, the amount of IL-6 produced by the MCwas measured in the B9 assay. Basal productionof IL-6 by MC was 1 286 _+ 414 units IL-6 per105 cells. Culture of MC with human dimericIgA resulted in up-regulation of IL-6 productionin a dose-dependent fashion. At the highestconcentration of 100 tg/ml, human dimeric IgAwas able to induce a 2.5-fold up-regulation of3171 __+ 1400 units IL-6 per 105 cells (p <0.05). LPS, as a positive control, induced a pro-duction of 6043 +_ 2386 units IL-6 per 105cells (Fig. 2).To investigate whether the production of IL-6

is dependent on the size of IgA, MC were stimu-lated in triplicate wells for 72 h with mediumalone, 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 cellsat a concentration of 10 l.tg/ml and a nine-foldincrease to 11800 __+ 300 units IL-6 per 105 cellsat a concentration of 100 l.tg/ml. Dimeric IgAwas much less potent in inducing an increaseof IL-6 production. At a concentration of100 g/ml, dimeric IgA induced a production of3 586

___1 471 units IL-6 per 105 cells, whereas

monomeric IgA did not induce a significantincrease in IL-6 production (Fig. 3).To investigate whether the IL-6 produced by

the MC upon IgA stimulation is due to de novosynthesis, MC were incubated in triplicate for72 h in medium, or in medium supplementedwith 10 I.tg/ml human dimeric IgA, both withor without cycloheximide (1 btg/ml), andassessed for IL-6. Cycloheximide was able tocause 97.3% inhibition of IgA-induced IL-6release (p < 0.002) (Fig. 4).

8000

7000

6000

5000

4000

3000

2000

lOO0

p 0.05

medium 0.1 10 100

Concentration

LPS

FIG. 2. Effect of increasing concentrations of human dimeric IgAon the production of IL-6 by MC in vitro. MC were incubated intriplicate with IgA for 72 h. After incubation the supernatantswere assessed for IL-6. The effect of LPS, as a positive control, isalso shown on the right.

1200

" 1000

800

g 600

" 400

.O 200

0 1’0 1 2’0IgA (g/ml)

FIG. 1. Binding of 1251-1gA to MC following incubation of MCwith increasing concentrations of 1251-labelled human dimericIgA for 16 h at 4C. One of four representative experiments isshown. Results are expressed as the mean -t- S.E.M. of triplicatewells.

12000

10000

8000

6000

4000

2000

rl rt mlgArt dlgA

B rt plgA

medium 0.1 10 100

Concentration IgA (lg/ml)

FIG. 3. Effect of increasing concentrations of rat monomeric,dimeric or polymeric IgA on the production of IL-6 by MC invitro. MC were incubated with various IgA preparations in tripli-cate for 72 h. After incubation, the supernatants were harvestedand assessed for IL-6. One of three representative experiments isshown. Results are expressed as the mean

___S.E.M.


T. J. F. Reterink et al.

p 0.006 p 0.002

3o0o

"" 2500

u 15oo

1000

500

Medium Medium+CH IgA IgA+CH

FIG. 4. Effect of cycloheximide on the production of IL-6 by MC/n v/tro. MC were incubated in triplicate with medium alone ormedium supplemented with human dimeric IgA (10 g/ml), withor without cycloheximide (1 Ag/ml). Aer 72 h, the supernatantswere hawested and assessed for IL-6. Results are expressed asthe mean S.E.M.

It has been suggested that IgA is able toinduce proliferation of rat MC in vitro. To inves-tigate whether human MC also proliferate uponstimulation with IgA, MC were incubated inmedium alone (DMEM/0.5%FCS) or with variouspreparations of IgA at a concentration of 10 l-tg/ml. As a positive control 100 ng/ml LPS wasused. After 7 days of culture, the triplicate wellswere trypsinized and the total cell number perwell was determined by using the CoulterCounter. None of the IgA preparations was ableto induce MC proliferation. In contrast, 100 ng/ml LPS was able to induce a relative proliferationof 2.24 4- 0.41 (p < 0.05) (Fig. 5).

Discussion

Glomerular mesangial depositions of IgA in thekidney, together with IgG and complement com-

2.5

1.5O

>

0.05

medium rt mlgA dlgA plgA hu dlgA hu plgA LPS

StimulationFIG. 5. Effect of various preparations of IgA on the proliferationof MC in vitro. MC were incubated with 10 lg/ml of various IgApreparations and LPS, as a positive control. The relative prolifera-tion is defined as the mean number of cells per well incubatedfor 7 days with IgA or LPS, divided by the mean number of cellsper well incubated for 7 days with medium alone. Results areexpressed as the mean -i- SoE.M. of triplicate wells.

ponents such as C3, are thought to be crucial forthe onset of the inflammation process in IgAnephropathy. It has been shown that IgG bindstO MC27’28 and stimulates IL-6 production.18’2 Inthis study, we show that human MC are capableof secreting de novo synthesized IL-6 after stimu-lation with IgA. The IL-6 production of the MC isstrongly dependent on the size of the IgA. Poly-meric rat IgA is able to increase the IL-6 produc-tion by MC up to nine-fold. Dimeric IgA is lesspotent in increasing IL-6 production by MC andonly increased the basal production three-fold. Incontrast with polymeric and dimeric IgA, mono-meric IgA is not able to increase the IL-6 produc-tion.

It has been shown that IL-6 is an autocrinegrowth factor for rat MC.12’21 Even thoughdimeric and polymeric IgA enhance the produc-tion of IL-6 by human MC, we did not observeproliferation of the cells. These findings are inagreement with the observation that human MCdo not proliferate upon stimulation with IL-629’3in vitro. In IL-6 transgenic mice, it was shownthat high plasma concentrations of IL-6 are asso-ciated with mesangial proliferation. In humans,however, it was found that urinary IL-6 levels donot correlate with mesangial proliferative glomer-ulonephritis.2 Our finding, that MC fail to pro-liferate upon stimulation with IgA, is also inaccord with these findings. Therefore, our invitro data suggest that mesangial cell proliferationin humans, as found in IgA nephropathy, is pre-sumably not due to the autocrine effect of IL-6produced by MC upon stimulation with IgA. Theprecise mechanisms by which mesangial pro-liferation is induced remain unclear, suggestingthat other mitogens, or combinations of differentmitogens acting synergistically, induce the mesan-gial proliferation, as seen in IgA nephropathy.

References1. Mestecky J, McGhee JR. Immunoglobulin A (IgA): molecular and cellular

interactions involved in IgA biosynthesis and immune response. AdvImmuno11987; 40: 153-245.

2. Heremans JF. Immunoglobulin A. In: Sela M, ed. The Antigens, Volume 2New York: Academic Press, 1974; 365-522.

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4. Valentijn RM, Radl J, Haaijman JJ, et al. Circulating and mesangial secre-tory component-binding IgA1 in primary IgA nephropathy. Kidney Int1984; 26: 760-766.

5. Rodico JL. Idiopathic IgA nephropathy. Kidney Int 1984; 25: 717-729.6.-van den Wall Bake AWL, Daha MR, van der Ark A, Hiemstra P, Radl J.

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9. G6mez-Guerrero C, Gonzglez E, Egido J. Evidence for a specific IgAreceptor in rat and human mesangial cells. J Immuno11993; 151: 7172-7181.

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11. Kishimoto T. The biology of interleukin 6. Blood 1989; "74: 1-10.12. Horii Y, Muraguchi A, Iwano M, et al. Involvement of IL-6 in mesangial

proliferative glomerulonephritis. J Immuno11989; 143: 3949-3955.13. Sironi M, Breviario F, Proserpio P, et al. IL-1 stimulates IL-6 production in

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factor 2, or hybridoma growth factor (IL-6) is expressed and released byhuman epidermal cells and epidemoid carcinoma cell lines. J Immunol1989; 142: 1922-1928.

15. &arden LA, de Groot ER, Schaap OL, Lansdorp PM. Production of hybrid-oma growth factor by human monocytes. Eur J Immunol 1987; 1"7:1411-1416.

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17. van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubira MR,Simpson RJ. Purification and NHi-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas.Proc NatlAcad Sci USA 1986; 83: 9679-9683.

18. G6mez-Guerrero C, L6pez-Armada MJ, Gonzez E, Egido J. Soluble IgAand IgG aggregates are catabolized by cultured rat mesangial cells andinduce production of TNF-0t and IL-6, and proliferation. J Immuno11994;153: 5247-5255.

19. van den Dobbelsteen MEA, van der Woude FJ, Schroeijers WEM, van denWall Bake AWL, van Es LA, Daha MR. Binding of dimeric- and polymericIgA to rat renal mesangial cells enhances the release of intefleukin 6.Kidney Int 1994; 46: 512-519.

20. van den Dobbelsteen MEA, van der Woude FJ, Schroeijers WEM, van EsLA, Daha MR. Soluble aggregates of IgG and immune complexes enhancethe production of IL-6 by renal mesangial cells. Kidney Int 1993; 43:544-553.

21. Reuf C, Budde K, Lacy J, Northemann W, Baumann M, Sterzel RB,

Coleman DL. Interleuldn 6 is an autocrine growth factor for mesangialcells. Kidney Int 1990; 38: 249-257.

22. Striker GE, Striker LJ. Biology of disease. Glomerular cell culture. LabInvest 1985; 53: 122-131.

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24. Hiemstra PS, Gorter A, Stuurman ME, van Es LA, Daha MR. Activation ofthe altemative pathway of complement by human serum IgA. Eur JImmuno11987; 17: 321-326.

25. Rits M, Cormont F, Bazin H, Meykens R, Vaerman JP. Production of IgAsecreting hybridomas with specificity for the 2,4-dinitrophenyl (DNP)hapten. J Immuno11986; 89: 81-87.

26. Helle M, Boeije L, Aarden LA. Functional discrimination between inter-leukin 6 and interleukin 1. EurJ Immuno11988; 18: 1535-1540.

27. van den Dobbelsteen MEA, van der Woude FJ, Schroeijers WEM, KlarMohamad N, van Es LA, Daha MR. Clq, a subunit of the first componentof complement, enhances the binding of aggregated IgG to rat renalmesangial cells. J Immuno11993; 151: 4315-4324.

28. Santiago A, Satriano J, De Candidio S, Holthoffer H, Schreiber R, UnkelessJ, Schlondorff D. A specific Fc, receptor on cultured rat mesangial cells. JImmuno11989; 143: 2575-2582.

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30. Floege J, Topley N, Hoppe J, Barrett TB, Resch K. Mitogenic effect ofplatelet-derived growth factor in human glomerular mesangial cells: mod-ulation and/or suppression of inflammatory cytokines. Clin Exp Immunol1991; 86: 334-341.

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Received 3 January 1996;accepted 22 February 1996


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