Clin. exp. Immunol. fl992) 88, 295-300
Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy N, YANO, M. ENDOH. M. MIYAZAKI. F, YAMAUCHI. Y. NOMOTO & H. SAKAI Department of Internal Medicine. School of Medicine. Tokai University, Isehara, Kanagawa, Japan (Accepted for publication 18 December 1991)
SUMMARY In order to elucidate the factors responsible for altered inimunoglobuHn production in patients with IgA nephropalhy (IgAN). the in vitro effeets of IL-4 and interferon-gamma (IFN-y) on the synthesis of IgH and IgA by peripheral blood mononuclcar cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was signifieantly increased in palienis with IgA nephropathy compared with eontrols. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL-4 were almost the same both in palients with IgAN and in eontrols. The enhaneemenl rale of IL-4-induced IgE and IgA synthesis was signihcanlly lower in IgAN than in the controls, suggesting in vivo preaetivation of PBMC in IgAN patients. IFN-7 suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN, These results suggested ihat altered IL-4 action might be involved in the development oflgA nephropathy. Keywords
IgA nephropathy
lL-4
IFN-y
IgE
INTRODLCTION IgA nephropathy (IgAN) is the mosl common primary glomerulonephritis [1.2]. The characteristic feature of this disease is the presence of IgA-dominant deposits in ihe mesangial areas ofthe glomeruli. However, the precise mechanisms of IgA deposition and the development of nephropathy have not yet been elucidated. Several abnormalities ofthe immunologieal status in IgAN. such as polyelonal aetivation of IgA-producing B cells [3], presence of various autoantibodies [4-6] and association with other immunologieal disorders [7-y]. have been reported. Based on these reports, ihc hypothesis that some alteration of B cells might be involved in the development of IgAN has been advocated, IL-4 has pleiotropic actions on growth and differentiation of B cells |10]. Tcells [11], macrophages [12), mast cells [II]. and other haematopoietic progenitor cells [13] in humans and in mice. This lymphokine induces a cell surface antigen. CD23. a low-affinity Fe receptor for IgE on B cells [14,15] or tnonocytes [l(S],and increases the le\ el of soluble CD23 in mononuelear cell culture supernatant [17]. One ofthe best known effects of IL-4 on B cells is induction of immunoglobulin elass switching. especially to IgE [18]. In recent studies, increased expression of CD23 on peripheral blood lymphocytes has been reported in asthma [19]. atopic dermatitis [20). and in other atopie diseases [21.22] as well as in collagen diseases sueh as systemic lupus erythcmalosus (SLE) and rheumatic arthritis [23], Other reports showed elevated levels of lL-4 in sera from patients with
IgA progressive systemic sclerosis [24], and expression of IL-4 mRNA in allergen-induced late-phase cutaneous reactions in atopie subjects [25], A study using I L-4-transgenie miee revealed thai IL-4 induces allcrgic-like inflammatory disease in systemic organs [26]. These studies strongly suggested that IL-4 plays some role in the development ofdiseases wiih allergie changes. The aim of ihe present study was to elueidate the role of IL-4 in altered production of IgE and IgA by peripheral blood mononuciear cells (PBMC) from patients with IgAN.
MATERIALS AND METHODS Patients Twenty-six patients with IgAN and 22 age-matched patients with non-IgA prolifcrative glomerulonephrilis (PGN) were selected randomly from patients who were diagnosed by open renal biopsy at Ihe Division of Nephrology and Metabolism. Department of Internal Medieine, School of Medicine. Tokai University. Isehara, The histopathological changes seen in patients with IgAN and PGN were classified into four grades: grade 1, minimal stage; grade 2, slight stage: grade 3, moderate slage: and grade 4, advanced stage. Healthy members of the Institute's staff whose age and sex distributions were similar to those of the patients served as controls. Patients and control profiles are summarized in Table 1. Cell cultures in vitro immunogtobuiin synthesis studies were performed using a modification ofthe method of Waldmann and Broder [27]. PBMC were isolated from heparinized venoas blood by Fieoll-
Correspondence: Naohiro Yano, MI), Department oT Internal Medicine, Tokai University, Isehara, Kanagawa, 259-11, Japan,
295
296
N. YanoQ\a\. Table L Data of patients with IgA nephropathy fIgAN)and proliferative gtomenilonephritis (PGN) and controls
Age (years) Group
Controls ( H = 19)
Mean
Range
38'I 406 .18 2
18 72 18 72
Sex (F:M)
Serum creatinine (mg/dl)
Urinary protein g/day)
Glomerular filtration rate (ml/min)
tO:l6 12:20 10:9
1-3 + 0-5
0-64 ±0 36
70 3 ±12 0 68-4 + 20-1
Paqiie (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient eentrifugation. Un fractionated PBMC were suspended in Iscove's modified Duibccco's medium (IMDM; Ginro. Grand Island. NY) supplemented with lO"--.. fetal calf serum (FCS). PBMC (I X 10") were cultured for 14days with [nediiim alone or in the presence of IOO U/ml of recombinant human lL-4 (rhIL-4) (HIL-4-C; Genzyme, Boston, MA) with or withotit 1000 U ml of interferon-gamma (IFN-/; Imunomax-y. Shionogi Pharm. Co., Osaka. Japan) in I5-ml sterile plastic tubes (Sumitomo Medical Co.. Tokyo, Japan). An additional set of conlrol cultures was treated with 50 figjml of cycloheximide (CHX: Sigma, St Louis. MO) for evaluation of preformed immunoglobulins. Preformed IgE was subtracted from the IgE concentration present in the incubated cultures to determine the net IgE concentration.
reagents were added and measurements were performed as described above. Quaiitiftcation oJ .scrum levels ot IgA ttntl IgE Serum levels of igA and IgE were determined by la.ser nephelometry and latex photometric immunoassay (LPiA-100; Iatron, Tokyo. Japan), respectively. The results were expressed as mg/dl Ibr IgA and IU/ml for IgE. The limit olsensitivity for IgE was 5 U/ml. Statistical analysis The Mann-Whitney f-test was used to evaluate the significance of the data, P anti-human IgA (Cappel, West Chester. PA), diluted 1:200 in carbonate butler for IX hours at 4 C. After washing, the plates were blocked >Aith I"'.. BSA in PBS. Then, the culture supernatants diluted to 1:5, 1: 10 or 1:20 and serially diluted normal human serum with a known IgA concentration as standard were added. After incubating for 18 h at 4 C. alkaliphosphatase-conjtigated atlinity purilied goat anti-human IgA (Sigma) diluted 1:2()0 wus added lor 5 h at room temperature. The plates were washed five times. Then the colour-developing
The sensitivity ofthe in vitro assays wa.s 10 pg/ml for IgE und 3 ng ml for IgA. C^oncentnitions from 10 to lOU U/ml of rhIL-4 were found to induce dose-dependent IgA or IgE synthesis during 14-day PBMCcultures from four normal donors (Fig. I).
1000
- 1000
IOO
10
10
25
50 IOO 200
SOO 1000
lL-4 concentratKw (U/mll
I'ig. I. Dose response curve of in vitro IgH (•) and IgA (O) synthesis with rhlL-4 .•Jiimulation. One-million PBMC from four normal donors were cullured in I ml of Iscove's modilicd Dulbceco's medium supplemented with U)'\, heat-inactivated FCS and were stimulated with increasing concentrations of 10 1000 tl,ml of rh IL-4 on day 0, Cultures were terminated 14 days aflcr s[imulatit>n and assayed for humunoglobulin content. IgE and IgA concentrations in all lour cultures reached plateaus from 100 U/mi of rhlL-4. Results are mean + s.d.
297
Altered immunoglobulin ."ivnlhcsis by PBMC in IgAN P
Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy N, YANO, M. ENDOH. M. MIYAZAKI. F, YAMAUCHI. Y. NOMOTO & H. SAKAI Department of Internal Medicine. School of Medicine. Tokai University, Isehara, Kanagawa, Japan (Accepted for publication 18 December 1991)
SUMMARY In order to elucidate the factors responsible for altered inimunoglobuHn production in patients with IgA nephropalhy (IgAN). the in vitro effeets of IL-4 and interferon-gamma (IFN-y) on the synthesis of IgH and IgA by peripheral blood mononuclcar cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was signifieantly increased in palienis with IgA nephropathy compared with eontrols. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL-4 were almost the same both in palients with IgAN and in eontrols. The enhaneemenl rale of IL-4-induced IgE and IgA synthesis was signihcanlly lower in IgAN than in the controls, suggesting in vivo preaetivation of PBMC in IgAN patients. IFN-7 suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN, These results suggested ihat altered IL-4 action might be involved in the development oflgA nephropathy. Keywords
IgA nephropathy
lL-4
IFN-y
IgE
INTRODLCTION IgA nephropathy (IgAN) is the mosl common primary glomerulonephritis [1.2]. The characteristic feature of this disease is the presence of IgA-dominant deposits in ihe mesangial areas ofthe glomeruli. However, the precise mechanisms of IgA deposition and the development of nephropathy have not yet been elucidated. Several abnormalities ofthe immunologieal status in IgAN. such as polyelonal aetivation of IgA-producing B cells [3], presence of various autoantibodies [4-6] and association with other immunologieal disorders [7-y]. have been reported. Based on these reports, ihc hypothesis that some alteration of B cells might be involved in the development of IgAN has been advocated, IL-4 has pleiotropic actions on growth and differentiation of B cells |10]. Tcells [11], macrophages [12), mast cells [II]. and other haematopoietic progenitor cells [13] in humans and in mice. This lymphokine induces a cell surface antigen. CD23. a low-affinity Fe receptor for IgE on B cells [14,15] or tnonocytes [l(S],and increases the le\ el of soluble CD23 in mononuelear cell culture supernatant [17]. One ofthe best known effects of IL-4 on B cells is induction of immunoglobulin elass switching. especially to IgE [18]. In recent studies, increased expression of CD23 on peripheral blood lymphocytes has been reported in asthma [19]. atopic dermatitis [20). and in other atopie diseases [21.22] as well as in collagen diseases sueh as systemic lupus erythcmalosus (SLE) and rheumatic arthritis [23], Other reports showed elevated levels of lL-4 in sera from patients with
IgA progressive systemic sclerosis [24], and expression of IL-4 mRNA in allergen-induced late-phase cutaneous reactions in atopie subjects [25], A study using I L-4-transgenie miee revealed thai IL-4 induces allcrgic-like inflammatory disease in systemic organs [26]. These studies strongly suggested that IL-4 plays some role in the development ofdiseases wiih allergie changes. The aim of ihe present study was to elueidate the role of IL-4 in altered production of IgE and IgA by peripheral blood mononuciear cells (PBMC) from patients with IgAN.
MATERIALS AND METHODS Patients Twenty-six patients with IgAN and 22 age-matched patients with non-IgA prolifcrative glomerulonephrilis (PGN) were selected randomly from patients who were diagnosed by open renal biopsy at Ihe Division of Nephrology and Metabolism. Department of Internal Medieine, School of Medicine. Tokai University. Isehara, The histopathological changes seen in patients with IgAN and PGN were classified into four grades: grade 1, minimal stage; grade 2, slight stage: grade 3, moderate slage: and grade 4, advanced stage. Healthy members of the Institute's staff whose age and sex distributions were similar to those of the patients served as controls. Patients and control profiles are summarized in Table 1. Cell cultures in vitro immunogtobuiin synthesis studies were performed using a modification ofthe method of Waldmann and Broder [27]. PBMC were isolated from heparinized venoas blood by Fieoll-
Correspondence: Naohiro Yano, MI), Department oT Internal Medicine, Tokai University, Isehara, Kanagawa, 259-11, Japan,
295
296
N. YanoQ\a\. Table L Data of patients with IgA nephropathy fIgAN)and proliferative gtomenilonephritis (PGN) and controls
Age (years) Group
Controls ( H = 19)
Mean
Range
38'I 406 .18 2
18 72 18 72
Sex (F:M)
Serum creatinine (mg/dl)
Urinary protein g/day)
Glomerular filtration rate (ml/min)
tO:l6 12:20 10:9
1-3 + 0-5
0-64 ±0 36
70 3 ±12 0 68-4 + 20-1
Paqiie (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient eentrifugation. Un fractionated PBMC were suspended in Iscove's modified Duibccco's medium (IMDM; Ginro. Grand Island. NY) supplemented with lO"--.. fetal calf serum (FCS). PBMC (I X 10") were cultured for 14days with [nediiim alone or in the presence of IOO U/ml of recombinant human lL-4 (rhIL-4) (HIL-4-C; Genzyme, Boston, MA) with or withotit 1000 U ml of interferon-gamma (IFN-/; Imunomax-y. Shionogi Pharm. Co., Osaka. Japan) in I5-ml sterile plastic tubes (Sumitomo Medical Co.. Tokyo, Japan). An additional set of conlrol cultures was treated with 50 figjml of cycloheximide (CHX: Sigma, St Louis. MO) for evaluation of preformed immunoglobulins. Preformed IgE was subtracted from the IgE concentration present in the incubated cultures to determine the net IgE concentration.
reagents were added and measurements were performed as described above. Quaiitiftcation oJ .scrum levels ot IgA ttntl IgE Serum levels of igA and IgE were determined by la.ser nephelometry and latex photometric immunoassay (LPiA-100; Iatron, Tokyo. Japan), respectively. The results were expressed as mg/dl Ibr IgA and IU/ml for IgE. The limit olsensitivity for IgE was 5 U/ml. Statistical analysis The Mann-Whitney f-test was used to evaluate the significance of the data, P anti-human IgA (Cappel, West Chester. PA), diluted 1:200 in carbonate butler for IX hours at 4 C. After washing, the plates were blocked >Aith I"'.. BSA in PBS. Then, the culture supernatants diluted to 1:5, 1: 10 or 1:20 and serially diluted normal human serum with a known IgA concentration as standard were added. After incubating for 18 h at 4 C. alkaliphosphatase-conjtigated atlinity purilied goat anti-human IgA (Sigma) diluted 1:2()0 wus added lor 5 h at room temperature. The plates were washed five times. Then the colour-developing
The sensitivity ofthe in vitro assays wa.s 10 pg/ml for IgE und 3 ng ml for IgA. C^oncentnitions from 10 to lOU U/ml of rhIL-4 were found to induce dose-dependent IgA or IgE synthesis during 14-day PBMCcultures from four normal donors (Fig. I).
1000
- 1000
IOO
10
10
25
50 IOO 200
SOO 1000
lL-4 concentratKw (U/mll
I'ig. I. Dose response curve of in vitro IgH (•) and IgA (O) synthesis with rhlL-4 .•Jiimulation. One-million PBMC from four normal donors were cullured in I ml of Iscove's modilicd Dulbceco's medium supplemented with U)'\, heat-inactivated FCS and were stimulated with increasing concentrations of 10 1000 tl,ml of rh IL-4 on day 0, Cultures were terminated 14 days aflcr s[imulatit>n and assayed for humunoglobulin content. IgE and IgA concentrations in all lour cultures reached plateaus from 100 U/mi of rhlL-4. Results are mean + s.d.
297
Altered immunoglobulin ."ivnlhcsis by PBMC in IgAN P
