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Clinical Immunology (2008) 126, 260–269

Increase in B-cell-activation factor (BAFF) and IFN-γproductions by tonsillar mononuclear cells stimulatedwith deoxycytidyl-deoxyguanosineoligodeoxynucleotides (CpG-ODN) in patients with IgAnephropathyTakashi Goto, Nobuyuki Bandoh ⁎, Tomoki Yoshizaki, Hayabusa Nozawa,Miki Takahara, Seigo Ueda, Tatsuya Hayashi, Yasuaki Harabuchi

Department of Otolaryngology – Head and Neck Surgery, Asahikawa Medical College, 2-1-1-1 Midorigaoka Higashi,Asahikawa, Hokkaido, 078-8510, Japan

Received 21 July 2007; accepted with revision 2 November 2007Available online 14 January 2008

⁎ Corresponding author. Fax: +81 166E-mail address: bando@asahikawa-

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2007.11.003

Abstract IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, isrecognized as a tonsil-related diseases since it often gets worse after and/or during acute tonsillitisand the disease progression is often prevented by tonsillectomy. Although several reports showedan increase in IgA production of tonsillar mononuclear cells (TMCs), its mechanism has not yet beenfully clarified. Recently, B-cell-activation factor (BAFF), which stimulates B-cell proliferation andimmunoglobulin production, was identified. Unmethylated deoxycytidyl-deoxyguanosine oligo-deoxynucleotide (CpG-ODN), which is able to mimic the immunostimulatory activity of microbialDNA, is known to be involved in the production of immunoglobulins and some cytokines. In thisstudy, we focused on roles of BAFF and IFN-γ in IgA production of TMCs stimulated with CpG-ODN inIgAN patients. Two-color flow cytometric analysis revealed that the intercellular expression of IFN-γ on the T-cells freshly isolated from tonsils was significantly higher in IgAN patients than in non-IgAN patients (p=0.032). The spontaneous productions of IgA and IFN-γ of TMCs were significantlyhigher in IgAN patients than in non-IgAN patients (p=0.023 and p=0.02). Under stimulation withCpG-ODN, the productions of IgA, BAFF and IFN-γ of TMCs were significantly higher in IgAN patientsthan in non-IgAN patients (p=0.013, p=0.005 and p=0.039). The IgA production of TMCsstimulated by CpG-ODN was inhibited by the treatment with anti-BAFF antibody and/or anti-IFN-γantibody. Under stimulation with IFN-γ, the BAFF expression on the CD1c cells and the BAFFproduction of TMCs were significantly higher in IgAN patients than in non-IgAN patients (p=0.004and p=0.042). These data suggest that hyper-immune response to microbial DNAmay be present in

KEYWORDSBAFF;CpG-ODN;IFN-γ;IgA nephropathy;Tonsil-related disease;Tonsillitis

68 2559.med.ac.jp (N. Bandoh).

7 Elsevier Inc. All rights reserved.

261BAFF and IFN-γ productions in tonsils with IgA nephropathy

IgAN patients and may lead to hyperproduction of BAFF up-regulated by IFN-γ, resulting inhyperproduction of IgA in IgAN patients.© 2007 Elsevier Inc. All rights reserved.

Introduction

IgA nephropathy (IgAN), which is characterized by mesangialIgA deposits, is the most common form of primary glomer-ulonephritis in the world. The course of IgAN was initiallyconsidered benign, but now it is recognized that progress toend-stage renal failure often occurs in these patients over aperiod of 20 years. The disease often gets worse after and/orduring upper respiratory tract infections, particularly, acutetonsillitis. Tonsillectomy has been reported to be aneffective treatments for IgAN patients [1,2]. Recently,clinical comparison studies between tonsillectomy patientsand non-tonsillectomy patients observed for over 10 yearsshowed that renal survival rates of tonsillectomy patientswere statistically higher than those of non-tonsillectomypatients, concluding that tonsillectomy was an independentfactor for long-term renal survival in patients with IgAN[3,4]. Therefore, IgAN is now recognized as a tonsil-relateddiseases.

Several alterations related to IgA class switching in IgANhave been reported: namely, elevation of serum IgA,increases in circulating IgA immune complexes [5], increasesin IgA-bearing cells in peripheral blood, and enhancement ofin vitro IgA production by peripheral blood mononuclear cells[6]. Thus, it is believed that hyperproduction of IgA plays akey role in the pathogenesis of IgAN.

In tonsils of IgAN patients, IgA-positive plasma cellsincreased compared to those from recurrent tonsillitispatients [2]. The polymeric IgA1, which is a common subclassin the deposition of glomerular mesangium in IgAN, wasproduced dominantly in tonsillar lymphocytes of IgANpatients [7]. The polymeric IgA1 in sera decreased aftertonsillectomy in IgAN patients [1]. In vitro studies showedthat tonsillar lymphocytes of IgAN patients revealed asignificantly higher proliferation and IgA production inresponse to the Haemophilus parainfluenzae, which ismore commonly isolated in tonsils with IgAN and is depositedin the glomerular mesangium of IgAN patients [8–10]. Thus,tonsil is known to be one of the sites of hyperproduction ofIgA in IgAN patients.

Initial occurrence of IgAN pathogenesis is known to occurby aberrant mucosal immune response of upper respiratorytracts, especially of tonsils, to chronic environmentalantigens from bacteria and viruses. In IgAN patients,streptococcal M proteins, which are the most commonpathogens of acute tonsillitis, provoke lymphocyte produc-tion of cytokine TGF-β that enhances the number of IgA-producing plasma cells [11]. H. parainfluenzae antigens alsoprovoke IgA production of tonsillar lymphocytes [8–10]. Avariety of viruses have been related to exacerbation of IgAN.In mice models, coxsackie B4 virus inoculated repeatedlyinto mice induces depositions of IgA and viral gene in themesangium similar to IgA nephropathy coxsackie B4 virus[12]. These facts emphasize that the innate immuneresponse may be involved in the onset of IgAN.

Bacterial DNA is the most common antigen that provokesinnate immune response via toll-like receptor (TLR)-9. Themicrobial genome, compared to vertebrate DNA, containsmore unmethylated deoxycytidyl-deoxyguanosine (CpG)[13]. Oligodeoxynucleotides (ODNs) with CpG (CpG-ODN)are able to perfectly mimic the immunostimulatory activityof microbial DNA [13]. In vitro stimulation with CpG-ODNinduces peripheral blood lymphocytes to produce IgM, IgGand IgA [14] and to secrete IL-6 and IFN-γ [15]. Intranasaladministration of CpG DNA into mice induces strong IgAantibody response in the sera and upper respiratory tracts[16]. Thus, it is suggested that CpG-ODN, which is in themicrobial DNA, is a possible candidate for antigenic factorsin the initial occurrence of IgAN pathogenesis.

B-cell-activation factor (BAFF), also called B-lymphocytestimulator (BLyS), is a member of the TNF family critical forthe maintenance of normal B-cell development and home-ostasis [17]. BAFF is expressed in monocytes, macrophages,dendritic cells, and neutrophils [18,19], and it has amechanism by which macrophages and dendritic cellsdirectly regulate human B-cell activation [18]. Upon stimula-tion with IFN-γ, IFN-α and bacterial antigens, BAFF isinduced from mature dendritic cells to be released[18,20,21], then BAFF costimulates B-cell proliferation andimmunoglobulin production [18,20]. Recently, it has beenreported that the BAFF-transgenic mice showed an increasein serum IgA levels as well as a deposition of IgA immunecomplexes in the renal glomerular mesangium [22], suggest-ing that BAFF may be involved in the development of IgAN.However, there is little information regarding role of BAFF intonsils with IgAN.

In this study, we focused on the role of BAFF in IgAproduction of TMCs stimulated with CpG-ODN in IgANpatients. Using TMCs from IgAN and control patients, weinvestigated: (i) whether CpG-ODN stimulation provokesproduction of IgA and BAFF by TMCs, (ii) whether BAFF isinvolved in IgA production, and (iii) what cytokine isassociated with IgA and BAFF production.

Materials and methods

Patients and samples

All patients were studied at the Asahikawa Medical College.There were 2 study groups undergoing tonsillectomy, theIgAN group and the non-IgAN group. IgAN was diagnosed bynephrologists based on renal biopsy findings including thepresence of predominant deposition of IgA in the mesangiumarea and the proliferation of the mesangial cells and matrix.None of the patients were treated with anti-platelet drugs,anti-inflammatory drugs, corticosteroids, and/or immuno-suppressants before tonsillectomy. The non-IgAN group wascomposed of patients with chronic tonsillitis. Patients withchronic tonsillitis required tonsillectomy because of

262 T. Goto et al.

recurrent episodes (3 or more times per year) of acutetonsillitis and/or a persistent sore throat. Patients withchronic tonsillitis had no renal diseases. All patients signedinformed consent for therapy and tissue studies. TheInstitutional Review Board approved the study.

Cell preparation

Tonsils obtained by tonsillectomy were manually cut intosmall pieces in phosphate-buffered saline (PBS) with 100 U/mlof penicillin and 100 μg/ml of streptomycin (Life Technologies,Gaithersburg, MD, USA), and the cells were passed through acell strainer (BD Biosciences, Bedford, MA, USA). Tonsillarmononuclear cells (TMCs) were isolated by the gradientcentrifugation method using Ficoll Paque Plus® (AmershamPharmacia Biotech, Piscataway, NJ, USA), as describedpreviously [23]. The cells were washed 5 times with sterilePBS and counted. The viability of the cell suspensionswasmorethan 95%.

Cell culture

TMCs were suspended at a concentration of 1×106 per ml in3 ml of RPMI 1640 medium (GIBCO, Grand Island, NY, USA)supplemented with 10% fetal calf serum (FCS; GIBCO),100 U/ml of penicillin, and 100 μg/ml of streptomycin. Thecells were cultured either without any stimulation or withCpG-ODN (5′-TCGTCGTTTTCGGCGCGCGCCG-3′; ODN2395;Hokkaido System Science Co, Ltd., Sapporo, Japan) in 6-well culture plates in an atmosphere of 5% CO2 at 37 °C.After 3 days in culture, the culture supernatant fluids werecollected and subjected to IgA, BAFF, and cytokine produc-tion determinations, respectively. For the inhibition assay,TMCs were cultured with 1 mM of CpG-ODN together withanti-BAFF antibody (R&D Systems, Minneapolis, MN, USA)and/or anti-IFN-γ antibody (R&D Systems) for 3 days, andthen the cultured supernatant fluids were subjected to IgAand BAFF productions. For stimulation with IFN-γ, TMCswere cultured with 100 U/ml recombinant IFN-γ (PBLBiomedical, Piscataway, NJ, USA) for 3 days, and then thecells were collected and subjected to the determination ofBAFF expression.

Two-color flow cytometry

Two-color immunofluorescence was performed as describedpreviously [23]. For BAFF expression, 1 million TMCs werereacted with 20 μl of 0.1 mg/ml phycoerythrin (PE)-labeledanti-CD19 antibody (Beckman Coulter, Marseilles, France) orwith 10 μl of 10 μg/ml PE-labeled anti-CD1c antibody(Miltenyi Biotec) together with fluorescein isothiocyanate(FITC)-labeled 10 μl of 0.1 mg/ml anti-BAFF antibody (AlexisBiochemicals, San Diego, CA) for 20 min at 4 °C. Isotypemouse IgG2a (Biodesign International, Saco, ME, USA) wasused as a control. The cells werewashed three times and thensubjected to flow cytometric analysis using a FACS Caliburanalyzer and CellQuest software (Becton Dickinson, San Jose,CA, USA). All dilutions andwashings were done in ice-cold PBScontaining 0.1% sodium azide and 2% FCS. The data weredisplayed as a contour cytogram and mean fluorescenceintensity was corrected for isotype control antibody.

For the expression of BAFF receptors, 1 million TMCs werereacted with 10 μl of 0.1 mg/ml anti-BCMA (AlexisBiochemicals), 5 μl of 0.1 mg/ml anti-TACI (Alexis Biochem-icals) or 10 μl of 0.1 mg/ml anti-BR3 antibody (AlexisBiochemicals) for 20 min at 4 °C. After washing three times,the cells were reacted with FITC-labeled anti-mouse IgGantibody (Alexis Biochemicals). The cells were then washedthree times and reacted with 20 μl of 0.1 mg/ml PE-labeledanti-CD19 antibody (Beckman Coulter) for 20 min at 4 °C. Tomeasure surface BAFF-binding activity, TMC were reactedwith 80 μl of 0.5 mg/ml human BAFF protein fused to mouseCD8 (Alexis Biochemicals), 10 μl of 0.1 mg/ml FITC-labeledanti-mouse CD8 antibody (BD PharMingen, San Diego, CA,USA), and PE-labeled anti-CD19 antibody (Beckman Coulter),subsequently.

For intracellular staining with IFN-γ, 1 million TMCs wereincubated with 40 ng/ml of phorbol 12-myristate 13-acetate(PMA), 2 μg/ml of ionomycin and 20 μg/ml of Brefeldin Asolution (Sigma Aldrich, St. Louis, MO, USA) for 8 h. The cellswere harvested and stained with 20 μl of 0.1 mg/ml PE-labeled anti-CD3 antibody (Beckman Coulter) together with20 μl of 0.1 mg/ml FITC-labeled anti-IFN-γ antibody (Beck-man Coulter) according to the manufacturer's protocol(Beckman Coulter).

Enzyme-linked immunosorbent assay

Commercialized enzyme-linked immunosorbent assay (ELISA)kits were used for measurements of IgA (Bethyl Laboratories,Montgomery, TX, USA), IL-6, IFN-γ and IFN-α (R&D systems,Minneapolis, MN), and BAFF (Bender Medsystem, Vienna,Austria). The culture supernatants were plated in the wellsand analyzed according to the manufacturer's instructions asdescribed previously [23]. Briefly, the 96-well flat-bottomedplates coated with mouse anti-human IgA, cytokines or BAFFantibodies were incubated with 200 μl of PBS containing 4%bovine serum albumin (BSA) for 2 h at 37 °C to cover theunreacted sites. The wells were washed with PBS containing0.05% polysorbate 20 (Tween-20; PBS-T) and incubatedovernight with 100 μl of supernatant culture fluids at anadequate dilution at 4 °C. Each sample was assayed induplicate. After washing three times with PBS-T, the plateswere then incubated sequentially with 100 μl of biotinylatedanti-human IgA, anti-cytokines or anti-BAFF antibodies atadequate dilution and with 100 μl of peroxidase-conjugatedstreptavidin diluted 1:1000 in PBS-Twith 1% BSA at 37 °C for60 min each. After each reaction, the plates were washedthree times with PBS-T. The wells were then reacted with100 μl of substrate solution containing 0.1 mg/ml tetra-methylbenzidine and 0.003% H2O2. After 10 min of incubationat room temperature, the reaction was stopped by adding100 μl of 5 N sulfuric acid. The optical density of eachwell wasmeasured by an automated spectrophotometer (SLT-Labin-struments, Grödig, Austria) at 450 nm. A serial-dilutedstandard solution was run in each plate and the cytokinelevel in the sample was read from the standard curve.

Statistical analysis

The data were expressed as median (25th–75th percentiles).Two group comparisons were tested using nonparametric test

263BAFF and IFN-γ productions in tonsils with IgA nephropathy

procedures such as Mann–Whitney U test and Wilcoxon'ssigned rank test. Statistical tests were based on a level ofsignificance pb0.05.

Results

Clinical characteristics and outcome of patients

Sixty-six patients undergoing tonsillectomy were the sub-jects of this study. The IgAN group consisted of 37 patients(14 males and 23 females) aged from 16 to 66 years withmedian age of 31 years. The non-IgAN group consisted of 29patients with chronic tonsillitis (11 males and 18 females)aged from 11 to 58 years with median age of 30 years. In anycomparison analyses in this study, age and gender distribu-tions were not different between IgAN and non-IgAN groups.According to the prognostic criteria for IgAN published by ajoint committee of the Special Study Group on ProgressiveGlomerular Diseases [24], renal lesions were classified intofour grades based on light microscopic findings. In our series,groups I, II, III and IV had 3, 8, 22 and 4 patients respectively.After tonsillectomy, patients were followed up over12 months with median periods of 21months (12–61months).Fifteen (41%) patients were in remission. No patient hadrenal failure nor did they required dialysis. At least 12monthsafter tonsillectomy, heamaturia disappeared in 20 (54%)patients and proteinuria disappeared in 25 (68%) patients.

Expressions of BAFF and BAFF receptors on freshlyisolated TMC

First, we investigated the expression of BAFF and BAFFreceptors on freshly isolated TMCs. Results are summarizedin Table 1. In all TMCs, BAFF expression was detected in1.34% (0.92–2.45%) cells [median (25th–75th percentiles)].Two-color flow cytometric analysis of BAFF and CD1c showedthat BAFF expression was detected only in CD1c-positivetonsillar cells at 5.10% (2.73–9.58%) and was not detected inany CD1c-negative cells. Since CD1c expresses both dendriticcells and resting B-cells, we performed two-color flowcytometric analysis of BAFF and CD19. The analysis showed

Table 1 Expressions of BAFF and BAFF receptors on tonsillar mo

Population IgAN

CD1c cellsBAFF expression (%) 5.10 (2.73–9.58)

CD19 cellsBAFF expression (%) 0.28 (0.20–0.49)BR3 expression (%) 99.1 (95.9–99.8)TACI expression (%) 48.1 (37.0–51.9)BCMA expression (%) 20.4 (14.8–25.1)BAFF binding activity (%) 80.9 (79.2–82.6)

Values were expressed as medians (25th–75th percentiles) of the percMann–Whitney U test was used to determine the p-value. ns: not signia Freshly isolated tonsillar mononuclear cells (TMC) from IgAN patient

anti-CD1c or anti-CD19 antibody and together with either FITC-labeleBAFF-binding activity on CD19 cells was measured by reacting withmouse CD8 antibody and together with PE-labeled anti-CD19 antibod

that BAFF expression was not detected in any CD19-positivecells. There was no significant difference on the BAFFexpression of CD1c-positive cells between IgAN patients(n=15) and non-IgAN patients (n=8).

In two-color flow cytometric analysis of BAFF-bindingreceptors and CD19, the BR3 was expressed on CD19-positivecells at a higher percentage [99.1% (95.9–99.8%)], whereasthe TACI was expressed at a much lower level [48.1% (37.0–51.9%)] than BR3, and the BCMA was expressed at the lowestlevel [20.4% (14.8–25.1%)]. There were no significantdifferences among the percentages of expressions of BR3,TACI, and BCMA on whole TMCs, CD19-positive and -negativepopulations between IgAN patients (n=15) and non-IgANpatients (n=8; Table 1). Two-color flow cytometric analysisof BAFF-binding activity and CD19 showed that BAFF-bindingactivity was detected in the majority of CD19-positive cells[80.9% (79.2–82.6%)], but was not detected in any CD19-negative population. There was no difference in the surfaceBAFF-binding activity on CD19-positive cells between IgANpatients (n=15) and non-IgAN patients (n=8; Table 1).

Increase in BAFF and IgA productions of TMC byCpG-ODN stimulation in IgAN patients

Preliminary study of 1 to 7 days of culture with 0.25, 0.5, 1.0,2.0 μM of CpG-ODN revealed that the highest levels of BAFFand cytokines in the supernatants were found after 3 days inculture with 1.0 μM of CpG-ODN. Thus, we performed CpG-ODN stimulation study under this condition.

The BAFF production of TMCs without any stimulation wasnot different between IgAN patients (n=6) and non-IgANpatients (n=10). However, the BAFF production of TMCsstimulated with CpG-ODN was significantly higher in IgANpatients than in non-IgAN patients [0.44 ng/ml (0.30–0.53 ng/ml) vs. 0.24 ng/ml (0.20–0.29 ng/ml), p=0.005;Fig. 1a]. The BAFF stimulation index was calculated using thisformula: the BAFF levels with CpG-ODN/the BAFF levelswithout any stimulation. The BAFF stimulation index wassignificantly higher in IgAN patients than in non-IgAN patients[1.22 (1.19–1.25) vs. 1.03 (0.96–1.18), p=0.039; Table 2].

The IgA production of TMCs without any stimulation wassignificantly higher in IgAN patients (n=6) than in non-IgAN

nonuclear cells a

Non-IgAN p-value

5.56 (1.50–8.60) ns

0.47 (0.29–0.94) ns99.0 (96.9–99.6) ns40.9 (37.3–44.9) ns25.1 (14.9–32.0) ns81.8 (73.4–83.3) ns

entages on TMCs.ficant.s (n=15) and non-IgAN patients (n=8) were reacted with PE-labeledd anti-BAFF, anti-BR3, anti-TACI or anti-BCMA antibodies. Surfacehuman BAFF protein fused to mouse CD8 and FITC-labeled anti-y. TMCs were then analyzed by two-color flow cytometry.

Figure 1 Productions of (a) BAFF and (b) IgA by tonsillar mononuclear cells (TMCs) stimulated with CpG-ODN. TMC from IgANpatients (n=6) and in non-IgAN patients (n=10) were cultured for 3 days with or without stimulation of 1.0 μM CpG-ODN. The BAFF andIgA levels in the culture supernatants were measured in enzyme-linked immunosorbent assay (ELISA). BAFF production wassignificantly higher in IgAN patients (n=6) than in non-IgAN patients (p=0.005; a). IgA production was significantly higher in IgANpatients than in non-IgAN patients in the presence and absence of CpG-ODN (p=0.013 and p=0.023; b). The median values aredisplayed as short bars (-). Mann–Whitney U test were used to determine the p-value. ns: not significant. (c) IgA production by TMCstimulated with CpG-ODN in the presence of anti-BAFF antibody. TMC from 5 donors were cultured for 3 days with 1.0 μM of CpG-ODNtogether with 1.25, 2.5, 5.0 and 10 mg/ml of anti-BAFF antibody. The IgA production decreased in a dose dependent manner of anti-BAFF antibody and the level was significantly lower at the concentration of 10 mg/ml of anti-BAFF antibody (p=0.003). Values areexpressed as mean±SD. Wilcoxon's signed rank test was used to determine the p-value.

264 T. Goto et al.

patients (n=10) [370.4 ng/ml (303.2–433.7 ng/ml) vs.249.1 ng/ml (200.7–274.9 ng/ml), p=0.023; Fig. 1b]. TheIgA production of TMCs stimulated with CpG-ODN was alsosignificantly higher in IgAN patients than in non-IgAN patients[436.4 ng/ml (402.2–453.9 ng/ml) vs. 268.5 ng/ml (193.6–339.7 ng/ml), p=0.013]. The IgA stimulation index wassignificantly higher in IgAN patients than in non-IgAN patients[1.46 (1.31–1.55) vs. 1.08 (0.91–1.26), p=0.028; Table 2].

Table 2 Stimulation indices of BAFF, IgA and cytokine productioncells with stimulation of CpG-ODN or IFN-γ a

IgAN

CpG-ODN stimulationBAFF production 1.22 (1.19–1.25IgA production 1.46 (1.31–1.55IFN-γ production 2.69 (1.96–3.76IFN-α production 2.83 (2.43–4.64IL-6 production 2.19 (1.68–4.34

IFN-γ stimulationBAFF expression on CD1c cells 1.49 (1.21–1.78BAFF production 2.43 (1.56–2.89

Stimulation index (SI) was calculated with this formula: the levels of prValues were expressed as median (25th–75th percentiles).Mann–Whitney U test was used to determine the p-value. ns: not signia Tonsillar mononuclear cells from IgAN patients (n=6 for BAFF an

stimulation, n=7 for BAFF expression and production by IFN-γ stimulaand n=6 for cytokine production by CpG-ODN stimulation, n=5 for BAfor 3 days with or without stimulation of 1.0 μM CpG-ODN or 100 Usupernatants were measured in enzyme-linked immunosorbent assaycolor flow cytometry and each value was expressed as a percentage

To confirm whether or not BAFF is involved in IgAproduction of TMCs stimulated with CpG-ODN, TMCs from 5donors were cultured with CpG-ODN together with 1.25,2.5, 5.0 and 10 mg/ml of anti-BAFF antibody. The IgAproduction decreased in a dose-dependent manner of anti-BAFF antibody and the level was significantly lower at aconcentration of 10 mg/ml of anti-BAFF antibody (p=0.003;Fig. 1c).

by tonsillar mononuclear cells and of BAFF expression on CD1c

Non-IgAN p-value

) 1.03 (0.96–1.18) 0.04) 1.08 (0.91–1.26) 0.03) 2.30 (1.27–3.17) 0.02) 3.34 (2.45–4.29) ns) 2.03 (1.65–4.41) ns

) 1.22 (1.05–1.66) 0.05) 2.10 (1.37–2.34) 0.02

oduction or expression with stimulus/the levels without stimulus.

ficant.d IgA productions and n=8 for cytokine production by CpG-ODNtion) and in non-IgAN patients (n=10 for BAFF and IgA productionsFF expression and production by IFN-γ stimulation) were cultured/ml IFN-γ. The levels of BAFF, IgA and cytokines in the culture(ELISA). The BAFF expression on CD1c cells was analyzed by two-of BAFF/CD1c double-positive cells in CD1c-positive cells.

265BAFF and IFN-γ productions in tonsils with IgA nephropathy

Increase in IFN-γ production of TMCs by CpG-ODNstimulation in IgAN patients

Next we investigated how CpG-ODN stimulation influencescytokine productions such as IFN-γ, IFN-α and IL-6 that arerelated to BAFF production. The IFN-γ production of TMCswithout any stimulation was significantly higher in IgANpatients (n=8) than in non-IgAN patients (n=6) [553.1 pg/ml(260.2–782.0 pg/ml) vs. 362.7 pg/ml (46.7–465.9 pg/ml),p=0.02; Fig. 2a]. The IFN-γ production of TMCs stimulatedwith CpG-ODN was also significantly higher in IgAN patientsthan in non-IgAN patients [887.9 pg/ml (774.4–1013 pg/ml)vs. 693.8 pg/ml (220.4–1024.2 pg/ml), p=0.039]. The IFN-γstimulation index was significantly higher in IgAN patientsthan in non-IgAN patients [2.69 (1.96–3.76) vs. 2.30 (1.27–3.17), p=0.02; Table 2].

The CpG-ODN stimulation enhanced the IFN-α and IL-6production of TMCs. However, there was no statisticaldifference on IFN-α and IL-6 productions of TMCs betweenIgAN patients (n=8) and non-IgAN patients (n=6) [IFN-α:232.7 pg/ml (147.2–401.9 pg/ml) vs. 279.3 pg/ml (176.1–276.2 pg/ml) and IL-6: 909.4 pg/ml (668.5–1031 pg/ml) vs.899.8 pg/ml (861.0–1024 pg/ml); Figs. 2b and c]. The IFN-αand IL-6 stimulation indexes also did not differ between IgANand non-IgAN patients [IFN-α: 2.83 (2.43–4.64) vs. 3.34(2.45–4.29) and IL-6: 2.19 (1.68–4.34) vs. 2.03 (1.65–4.41);Table 2].

Increase in BAFF expression and production of TMCsby IFN-γ stimulation in IgAN patients

Next we investigated how IFN-γ stimulation acts in BAFFexpression and productions of TMCs. The TMCs were culturedwith 100 U/ml of IFN-γ for 3 days, the cells and supernatants

Figure 2 Productions of (a) IFN-γ, (b) IFN-α and (c) IL-6 by tonsillaIgAN patients (n=8) and in non-IgAN patients (n=6) were culturedcytokine levels in the culture supernatants were measured in enzymTMC was significantly higher in IgAN patients than in non-IgAN patientp=0.02, each; a). However, there were no statistical differencesstimulation of CpG-ODN between IgAN and non-IgAN patients (b, c). TU test was used to determine the p-value. ns: not significant.

were then subjected to expression of BAFF and CD1c, andBAFF production, respectively.

The IFN-γ stimulation enhanced BAFF expression oftonsillar CD1c-cells in both IgAN patients (n=7) and non-IgAN patients (n=5). The BAFF expression of tonsillar CD1c-cells without any stimulation did not differ between IgAN andnon-IgAN patients. Under stimulation with IFN-γ, thepercentage of BAFF-expressing CD1c cells was significantlyhigher in IgAN patients than in non-IgAN patients [11.0%(8.8–12.8%) vs. 7.8% (5.6–9.4%), p=0.044; Fig. 3a]. Thestimulation index of BAFF expression was significantly higherin IgAN patients than in non-IgAN patients [1.49 (1.21–1.78)vs. 1.22 (1.05–1.66), p=0.045; Table 2].

The IFN-γ stimulation enhanced BAFF production of TMCsin both IgAN patients (n=7) and non-IgAN patients (n=5). TheBAFF production of TMCs without any stimulation did notdiffer between IgAN and non-IgAN patients. Under stimula-tion with IFN-γ, BAFF production of TMCs was significantlyhigher in IgAN patients than in non-IgAN patients [1.3 ng/ml(1.0–1.6 ng/ml) vs. 0.8 ng/ml (0.6–0.9 ng/ml), p=0.042;Fig. 3b]. The stimulation index of BAFF production wassignificantly higher in IgAN patients than in non-IgAN patients[2.43 (1.56–2.89) vs. 2.10 (1.37–2.34), p=0.018; Table 2].

To confirm whether IFN-γ is involved in IgA production ofTMCs stimulated with CpG-ODN, TMCs from 6 IgAN patientswere cultured with CpG-ODN together with 10 mg/ml ofanti-IFN-γ antibody and/or 10 mg/ml of anti-BAFF antibody.The IgA production of TMCs stimulated with CpG-ODN[199.3 ng/ml (105.1–221.6 ng/ml)] was significantly inhib-ited by treatment with the anti-IFN-γ antibody [180.1 ng/ml (98.2–209.6 ng/ml), p=0.028], treatment with the anti-BAFF antibody [130.1 ng/ml (89.8–184.1 ng/ml), p=0.028],and treatment mixed with both antibodies [124.9 ng/ml(88.9–149.1 ng/ml), p=0.028]. The inhibition of IgAproduction by the mixture of both antibodies was

r mononuclear cells (TMCs) stimulated with CpG-ODN. TMC fromfor 3 days with or without stimulation of 1.0 μM CpG-ODN. Thee-linked immunosorbent assay (ELISA). The IFN-γ production bys for days with and without stimulation of CpG-ODN (p=0.039 andbetween IFN-α and IL-6 productions by TMC with or withouthe median values are displayed as short bars (-). Mann–Whitney

Figure 3 BAFF expression on CD1c-positive tonsillar cells (a)and BAFF production by tonsillar mononuclear cells (TMCs)stimulated with IFN-γ. TMCs from IgAN patients (n=7) and innon-IgAN patients (n=5) were cultured for 3 days with or withoutstimulation of 100 U/ml IFN-γ. The BAFF expression on CD1ccells was analyzed by two-color flow cytometry and each value isexpressed as a percentage of BAFF/CD1c double-positive cells inCD1c-positive cells. The BAFF levels in the culture supernatantswere measured using enzyme-linked immunosorbent assay(ELISA). (a) The IFN-γ stimulation enhanced BAFF expression oftonsillar CD1c-cells in both IgAN and non-IgAN patients. TheBAFF expression of tonsillar CD1c-cells without stimulation wasnot different between IgAN and non-IgAN patients. Understimulation with IFN-γ, the percentage of BAFF-expressingCD1c-cells was significantly higher in IgAN patients than innon-IgAN patients (p= 0.044). (b) The IFN-γ stimulationenhanced BAFF production of TMCs in both IgAN and non-IgANpatients. The BAFF production of TMCs without stimulation didnot differ between IgAN and non-IgAN patients. Under stimula-tion with IFN-γ, BAFF production of TMCs was significantly higherin IgAN patients than in non-IgAN patients (p=0.042). Themedian values are displayed as short bars (-). Mann–Whitney Utest was used to determine the p-value.

Figure 4 IgA production by tonsillar mononuclear cells (TMCs)stimulated with CpG-ODN in the presence of anti-IFN-ã antibodyand/or anti-BAFF antibody. TMCs from 6 IgAN patients werecultured for 3 days with 1.0 μM of CpG-ODN together with 10 mg/ml of anti-IFN-γ antibody and/or 10 mg/ml of anti-BAFFantibody. The IgA levels in the culture supernatants weremeasured in enzyme-linked immunosorbent assay (ELISA). IgAproduction of TMCs stimulated with CpG-ODN was significantlyinhibited by treatment with the anti-IFN-ã antibody (p=0.028),treatment with the anti-BAFF antibody (p=0.028), and treat-ment mixed with both antibodies (p=0.028). The inhibition ofIgA production by mixture of both antibodies was significantlyhigher than that by the anti-IFN-γ alone (p=0.046), but did notdiffer from that of anti-BAFF antibody alone. Values areexpressed as mean±SD. Wilcoxon's signed rank test was usedto determine the p-value.

266 T. Goto et al.

significantly higher than that by the anti-IFN-γ alone(p=0.046), but was not different to that by anti-BAFFantibody alone (Fig. 4).

Higher IFN-γ expression on freshly isolated TMCs inIgAN patients

Finally, we confirmed whether or not the IFN-γ expression onthe tonsillar T-cells increases in vivo. TMCs freshly isolatedfrom tonsils were incubated with PMA and ionomycin, andthen intracellularly with PE-labeled anti-CD3 antibody andFITC-labeled anti-IFN-γ antibody. Two-color flow cytometricanalysis showed that the percentage of CD3/IFN-γ double-positive cells was significantly higher in IgAN patients (n=15)than in non-IgAN patients (n=9) [2.16% (1.51–3.53%) vs.1.36% (0.76–1.96%), p=0.032; Fig. 5].

Discussion

Initial occurrence of IgAN pathogenesis is known to occur bynovel immune response to chronic environmental antigens

from bacteria and viruses such as beta-hemolytic strepto-cocci [11], H. parainfluenzae [8–10], and coxsackie B4 virus[12]. Among such microorganisms, H. parainfluenzae is likelyto be the most suitable candidate for antigenic factors; thatis, H. parainfluenzae is more commonly isolated in tonsils ofIgAN patients than in tonsils of non-IgAN patients [8]. In vitrostudies showed that TMCs of IgAN patients revealed asignificantly higher proliferation and IgA production inresponse to H. parainfluenzae antigen [9,10]. However,bacterial colonization in tonsils of IgAN patients is not alwayscomposed of only H. parainfluenzae. More than 700 bacterialspecies or phenotypes, of which over 50% are cultivated, aredetected in the oropharynx [25]. The DNA genome of suchmicroorganisms contain a high amount of CpG that are ableto mimic the immunostimulatory activity [13]. Thus, themicrobial DNA with hypomethylated CpG motifs, which isrepresented by CpG-ODN, is likely to be the most commonantigen among chronic environmental candidates that haveimmunostimulatory activity. Sunaga et al. [26] previouslydemonstrated that two types of synthetic oligodeoxynucleo-tides enhanced H. parainfluenzae-specific IgA antibodyproduction of TMCs, but they did not attempt to comparebetween IgAN and control. In this study, TMCs of IgAN

Figure 5 Intracellular IFN-γ expression on freshly isolatedtonsillar T-cells. Tonsillar mononuclear cells (TMCs) freshlyisolated from IgAN patients (n=15) and non-IgAN patients(n=9) were treated with PMA and ionomycin, and then stainedfor CD3 and IFN-ã. The IFN-γ expression on CD3 cells wasanalyzed by two-color flow cytometry and each value isexpressed as a percentage of IFN-γ/CD3 double-positive cellsin CD3-positive cells. The percentage of CD3/IFN-ã double-positive cells was significantly higher in IgAN patients than innon-IgAN patients (p=0.032). The median values are displayedas short bar (-). Mann–Whitney U test was used to determine thep-value.

267BAFF and IFN-γ productions in tonsils with IgA nephropathy

patients showed significantly higher productions of IgA andIFN-γ in response to CpG-ODN than TMCs from non-IgANpatients. This suggests that hyper-immune response to CpG-ODN, i.e., microbial DNA, may be involved in tonsils of IgANpatients. In mice models, intranasal immunization with CpG-DNA induces strong systemic and local IgA responses,suggesting that CpG-DNA may act as a good mucosal adjuvantfor IgA response [16]. Also, in IgAN patients, microbial DNA inthe oropharynx is likely to induce strong IgA antibodyresponse. The increase in spontaneous IFN-γ and IgAproductions of TMCs, which was found here and elsewhere[27], as well as the increase in IFN-γ expression of freshlyisolated tonsillar T-cells found here, may represent persis-tent stimulation with microbial DNA in vivo prior to TMCisolation from the body of IgAN patients.

The initial targets for CpG-ODN are plasmacytoid den-dritic cells that express high levels of TLR-9. Plasmacytoiddendritic cells activate myeloid dendritic cells via IFN-α,IFN-ã and IL-6 to produce BAFF [20,21]. BAFF directly bindsB-cells and acts as a critical regulator for B-cell differentia-tion into plasma cells for producing immunoglobulins in theT-cell-independent class-switching pathway [18,20]. In thisstudy, in vitro administration of CpG-ODN enhanced BAFFproduction of TMCs and enhanced production of IgA. Theneutral antibodies to BAFF inhibited IgA production in a dose-dependent manner. These data confirm that BAFF acts as anup-regulator for IgA production by TMCs in response to CpG-ODN. Furthermore, TMCs of IgAN patients showed signifi-

cantly higher BAFF production by CpG-ODN stimulation thanTMCs of non-IgAN patients. Kodama et al. [28] demonstratedthat the number of B-1 cells, which play a role for T-cell-independent IgA production in mucosal tissues [29],increased in the germinal center of tonsils of IgAN patients.It is reported that BAFF has a costimulatory effect with TLRstimulation on activation of B-1 cells [30]. It is suggested,based on these reports and our results, that hyper-immuneresponse to microbial DNA may lead to hyperproduction ofBAFF, resulting in hyperproduction of IgA via activation of B-1cells in tonsils of IgAN patients.

BAFF receptor expressions or BAFF-binding activity on B-cells is proposed as one of the factors for IgA hyperproductionvia BAFF in tonsils of IgAN patients, as shown previously inlymphoma cells [31]. BAFF binds B-cells through BAFFreceptors [32] such as BCMA, BR3 and TACI. Among thesethree receptors, BR3 appears to be a primary BAFF-bindingreceptor responsible for B-cell development and survival [33].We confirmed that BR3 was the principal stimulatory receptorfor tonsillar B-cells, because its high expressionwas associatedwith high surface BAFF binding activity on tonsillar B-cells.However, BAFF receptors were not a cause of hyperproductionof BAFF by TMCs in IgAN patients, because the level ofexpression did not differ between IgAN and non-IgAN patients.

IFN-γ is most likely to be involved in IgA hyperproductionvia BAFF in TMCs of IgAN patients. In this study, theadministration of CpG-ODN enhanced IFN-γ production aswell as IgA production of TMCs. The neutralizing antibody toIFN-γ decreased IgA production on a dose dependent manner.The administration of IFN-γ enhanced expression andproduction of BAFF by TMCs. The inhibition of IgA productionby the mixture of neutralizing antibodies to IFN-ã and BAFFwas significantly higher than the inhibition by anti-IFN-γantibody alone, but did not differ from the inhibition by anti-BAFF antibody alone. These findings confirm that IFN-γinduced by CpG-ODN stimulation may contribute to IgAproduction via up-regulation of BAFF production [18,20,21].The TMCs of IgAN patients showed significantly higher IFN-γproduction by CpG-ODN stimulation than in TMCs of non-IgANpatients. Although the production of IL-6 and IFN-αwere alsoenhanced by CpG-ODN stimulation, the levels did not differbetween IgAN and non-IgAN patients. Thus, hyperproductionof IgA via BAFF in IgAN patients may be caused byhyperproduction of IFN-γ in response to CpG-ODN. Fujiedaet al. [27] also demonstrated that TMCs of IgAN patientsshowed hyperproduction of IFN-γ together with hyperpro-duction of IgA as compared with TMC of patients with chronictonsillitis, supporting that IFN-γ may act for IgA production.However, they did not investigate the pathway via BAFF.

In tonsils, we found that spontaneous IFN-γ and IgAproductions by TMCs were higher in IgAN patients, aspreviously shown in elsewhere [27]. The IFN-γ expressionof freshly isolated tonsillar T-cells was significantly higher inIgAN patients than in non-IgAN patients. With regard toperipheral blood mononuclear cells, the mRNA expressionand in vitro production of IFN-γ were reported to be higherin IgAN patients than in both healthy controls [34] and otherglomerulonephritis patients [35]. The serum level of IFN-γwas also higher in IgAN patients than in healthy controls[36,37], and the level decreased after tonsillectomy [37],suggesting that serum IFN-γmay originate from tonsils. In anexperimental IgAN mice model, the administration of IgA

268 T. Goto et al.

immune complex with IFN-ã led to diffuse proliferativegomerulonephritis with proteinuria and heamaturia [38,39].Treatment with an anti-IFN-γ receptor antibody controlledthe disease [39]. These suggest that IFN-γmay play a role forthe induction of glomerulonephritis in IgAN. All these resultstogether with our results indicate that IFN-γ may act as atrigger in all of the pathogenic processes in IgAN. We shouldnote here that the administration of IFN-γ enhanced theexpression and production of BAFF in CD1c-cells at asignificantly higher level in IgAN patients than in non-IgANpatients. This preliminary finding suggests that hyper-immune response to IFN-γ as well as microbial DNA may beinvolved in IgAN patients. Further immunologic analyses willbe necessary to prove this hypothesis.

We cannot accurately explain here what is responsible forhyper-immune response to microbial DNA leading to hyper-productions of IFN-γ, BAFF and IgA. Nevertheless, it may beassociated with genetic factors such as gene polymorphismsof TLR-9, BAFF, IFN-γ, and IgA regulatory region. Genepolymorphism in the Iα regulatory region of the Ig heavychain is reported to cause enhanced IgA production in someIgAN patients [40]. Recently, gene polymorphisms of the IFN-γ are reported to be associated with the development andprogression of IgAN patients [41,42]. Although IgAN has notbeen analyzed yet, gene polymorphisms of BAFF or TLR-9 arereported to be associated with systemic lupus erythematousand rheumatoid arthritis [43]. Genetic polymorphisms ofTLR-9 are reported to influence the immune response to CpGand contribute to hyper-IgM in primary biliary cirrhosis [44].Further studies and progress in molecular biology will help tofurther elucidate the genetic factors that contribute to thedevelopment and progression of IgAN.

In summary, we demonstrated the following in this studyof tonsils from IgAN patients: (i) the spontaneous IFN-γ andIgA productions by TMC increased; (ii) IFN-γ expression offreshly isolated tonsillar T-cell increased; (iii) production ofIFN-γ, BAFF, and IgA by TMCs was enhanced by stimulationwith CpG-ODN; (iv) IgA production was inhibited by theantibodies to IFN-γ and BAFF; (v) BAFF expression of CD1ccells and the BAFF production of TMCs increased by IFN-γstimulation; and (vi) expression of BAFF-binding receptors onB-cells did not differ. These results suggest that hyper-immuneresponse to microbial DNA may lead to hyperproduction ofBAFF via up-regulation of IFN-γ, resulting in the hyperproduc-tion of IgA in the tonsils of IgAN patients. Such an immuneresponse in tonsils may be a trigger of pathogenesis in IgAN.

Acknowledgments

The authors gratefully thank Dr. Takayuki Fujino and Dr.Tomoya Hirayama at department of nephrology for providingclinical data of IgAN patients. This work was supported by agrant-in-aid (No. 18591855) from the Ministry of Education,Science, Sports, Culture and Technology of Japan.

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