- Email: [email protected]
Activation of the lectin complement pathway in Henoch-Schönlein purpura nephritis
Activation of the Lectin Complement Pathway in Henoch-Schönlein Purpura Nephritis Satoshi Hisano, MD, Misao Matsushita, PhD, Teizo Fujita, MD, and Hiroshi Iwasaki, MD ● Background: We previously reported the existence of complement activation through the alternative and lectin pathways in patients with immunoglobulin A (IgA) glomerulonephritis (GN). The current study aims to elucidate the correlation between each complement pathway and clinicopathologic findings in patients with Henoch-Schönlein purpura nephritis (HSPN). Methods: Immunohistologic staining was performed on renal specimens obtained from 31 patients with HSPN and 20 controls as non-IgA GN by using antibodies against IgG, IgA, IgA1, IgA2, IgM, C1q, C3c, C4, fibrinogen, factor B, C4-binding protein (C4-bp), C5b-9, CD59, mannose-binding lectin (MBL), and MBL-associated serine protease-1 (MASP-1). Results: No control showed deposition of any antibody. In 16 patients with mesangial IgA1/IgA2 codeposits, mesangial deposits of C3c, C4, factor B, C4-bp, C5b-9, CD59, MBL, and MASP-1 were found. In the remaining 15 patients with mesangial IgA1 deposits alone, no mesangial deposits of C4 or MBL/MASP-1 were found, and mesangial deposits of C3c, factor B, C5b-9, and CD59 were evident in 11 patients. Glomerular deposits of fibrinogen were detected in 15 of 16 patients with IgA1/IgA2 codeposits and only 6 of 15 patients with IgA1 deposits. Severity of glomerular changes and degrees of hematuria and proteinuria at latest follow-up were greater in patients with IgA1/IgA2 codeposits than in those with IgA1 deposits. Conclusion: Complement activation through both the alternative and lectin pathways is found in patients with HSPN. Complement activation is promoted in situ in the glomerulus. MBL/MASP-1 may be associated with glomerular deposition of fibrinogen. Complement activation through the lectin pathway may contribute to the development of advanced glomerular injuries and prolonged urinary abnormalities in patients with HSPN. Am J Kidney Dis 45:295-302. © 2004 by the National Kidney Foundation, Inc. INDEX WORDS: Henoch-Schönlein purpura nephritis (HSPN); immunoglobulin A1 (IgA1); immunoglobulin A2 (IgA2); mannose-binding lectin (MBL); mannose-binding lectin–associated serine protease-1 (MASP-1); lectin pathway.
H
ENOCH-SCHÖNLEIN purpura nephritis (HSPN) is a systemic disease that shows hematuria and proteinuria associated with purpuric rash, abdominal pain, and/or arthralgia.1 HSPN is characterized histologically by predominant immunoglobulin A (IgA) deposition and, simultaneously, C3 deposition in the mesangium on immunofluorescence microscopy, similar to IgA glomerulonephritis (GN).2,3 Recently, a new pathway of complement activation, the lectin pathway, which is initiated by mannose-binding lectin (MBL), ficolins, MBL-associated serine protease-1, -2, -3 (MASP-1, MASP-2, and MASP-3), and a truncated form of MASP-2, was reported.4-9 Our previous study immunohistologically showed that alternative pathway–involved complement activation was associated with mesangial deposits of IgA1 alone in patients with IgA GN, and in those with mesangial IgA1 and IgA2 deposits, both the alternative and lectin pathways were involved in complement activation.10 We first reported that mesangial IgA2 deposits might activate the lectin pathway in patients with IgA GN.10 Complement activation through the lectin pathway was found in 20% to 50% of patients with IgA GN.10-12 Endo et al13 reported complement activation through the lectin pathway in 8 of
10 patients with HSPN. However, the number of their patients is too small and correlation between the lectin pathway and clinicopathologic findings in patients with HSPN is not fully clarified. The aim of our study is to elucidate the correlation between each complement pathway and clinicopathologic findings in patients with HSPN. METHODS Kidney tissue was obtained by means of percutaneous renal biopsy in 31 patients with HSPN, aged 4 to 18 years, between 1998 and 2001. HSPN was diagnosed as hematuria and proteinuria associated with such characteristic manifes-
From the Department of Pathology, School of Medicine, Fukuoka University, Fukuoka; Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka; and Department of Biochemistry, Fukushima Medical University, Fukushima, Japan. Received June 15, 2004; accepted in revised form October 18, 2004. Originally published online as doi:10.1053/j.ajkd.2004.10.020 on December 29, 2004. Address reprint requests to Satoshi Hisano, MD, Department of Pathology, School of Medicine, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-0180, Japan. E-mail: [email protected] © 2004 by the National Kidney Foundation, Inc. 0272-6386/04/4502-0008$30.00/0 doi:10.1053/j.ajkd.2004.10.020
American Journal of Kidney Diseases, Vol 45, No 2 (February), 2005: pp 295-302
295
296
tations as purpuric rash, abdominal pain, and/or joint pain.1 Controls were used as non-IgA GN (10 patients with minimal change nephrotic syndrome, 10 patients with thin basement membrane disease). Informed consent was obtained from patients and/or their parents before renal biopsy. On approval from the Human Ethics Review Committee of Fukuoka University (Fukuoka, Japan), this study protocol was performed. Light microscopy was evaluated on sections stained with periodic acid–Schiff and periodic acid–methenamine silver. The histological severity of glomerular lesions and tubulointerstitial lesions (TILs) was determined according to the previous report,14 modified by us. Degrees of glomerular hypercellularity (GH; endocapillary hypercellularity and/or mesangial hypercellularity), mesangial matrix expansion (MME), segmental lesions (SLs; crescent formation, tuft adhesion to Bowman’s capsule, and/or segmental sclerosis), glomerular sclerosis (GS), and TILs (tubular atrophy and interstitial fibrosis) were evaluated. Endocapillary hypercellularity is defined as 3 or more leukocytic cells in the capillary space, and mesangial hypercellularity is defined as 3 or more nuclei in the mesangial area. GS was determined as a sclerotic lesion occupying 50% or more of the glomerular area. Each index was semiquantitatively evaluated. Degrees of GH and MME in each glomerulus were evaluated as a point as follows: 0, no lesion; 1, involving 10% to 30% of the glomerular area; 2, involving 31% to 50% of the glomerular area; and 3, involving more than 50% of glomerular area. Points of individual glomerulus were added and divided by the number of glomeruli in each biopsy specimen, ie, an average of points in each biopsy. Degrees of SLs, GS, and TILs were evaluated as a point according to the percentage of glomeruli showing SLs and GS per total glomeruli and tubulointerstitium showing tubular atrophy and fibrosis in the cortical area as follows, respectively: 0, no lesion; 1, involving 10% to 30% of total glomeruli and cortical area, respectively; 2, involving 31% to 50% of total glomeruli and cortical area, respectively; and 3, involving more than 50% of total glomeruli and cortical area, respectively. All patients were administered prednisolone and/or warfarin. Prednisolone was administered initially at a dose of 2 mg/kg/d for 4 weeks followed by a dose of 2 mg/kg/d on alternative days for 4 weeks, then tapered off for 12 to 24 months. Warfarin was administered daily at a dose of 1 to 3 mg/d for 12 to 24 months. Clinical records examined at the time of biopsy and latest follow-up, including blood pressure, urinalysis, and serum protein and serum creatinine levels, were obtained from the pediatricians who followed up these 31 children.
Immunohistologic Staining Immunohistologic staining was performed according to our previous report.10 Fluorescein isothiocyanate (FITC)labeled anti-IgG, IgA, IgM, C1q, C3c, fibrinogen polyclonal antibodies (Dako, Copenhagen, Denmark), FITC-labeled anti-C4 polyclonal antibody (MBL; Medical and Biological Laboratories, Nagoya, Japan), and anti-factor B polyclonal antibody (Biogenesis, Poole, UK) were used. Monoclonal antibodies against human IgA1 (NIF2; Skybio, Bedfordshire, UK), IgA2 (2E2, Skybio), C4-binding protein (C4-bp; 11-2D3; Fuji Yakuhin , Toyama, Japan), C5b-9 (Dako), and CD59 (Cosmobio, Tokyo, Japan) were used. Monoclonal
HISANO ET AL
antibodies against human MBL (3E7) and MASP-1 (4C2) were provided from T.F., coinvestigator, as previously reported.15,16 Sections cut at a thickness of 4 m from frozen tissues were immersed into Tris-buffered saline (TBS) 3 times for 5 minutes. Diluted FITC-labeled polyclonal antibodies (IgG, IgA, IgM, C3c, C1q, C4, and fibrinogen), IgA1 and IgA2 monoclonal antibodies, MBL monoclonal antibody, MASP-1 monoclonal antibody, C4-bp monoclonal antibody, C5b-9 monoclonal antibody, CD59 monoclonal antibody, and factor B polyclonal antibody were overlaid on the sections. Sections were incubated at 4°C overnight. Sections on which FITC-labeled polyclonal antibodies were overlaid were observed on immunofluorescence microscopy. Immunohistochemical studies for monoclonal antibodies (IgA1, IgA2, MBL, MASP-1, C4-bp, CD5b-9, and CD59) and factor B polyclonal antibody were performed using the avidin-biotin peroxidase method, as previously reported.10 After treatment in 0.5% hydrogen peroxide in methanol solution to quench endogenous peroxidase activity, sections overlaid by monoclonal antibodies were incubated with biotin-labeled antibody against mouse IgG (Dako), and those overlaid by factor B polyclonal antibody were incubated with biotin-labeled antibody against rabbit IgG (Dako) at room temperature for 30 minutes. After washing in TBS, sections were incubated with avidin-biotin-peroxidase complex (Dako) at room temperature for 30 minutes. After 3 washes in TBS, sections were incubated with peroxidaselabeled avidin for 30 minutes. Sections were treated with 3,3-diaminobenzidine (Dohjin, Kyoto, Japan), counterstained with hematoxylin, and observed using a light microscope. The intensity of immunohistologic deposits in glomeruli was based semiquantitatively on a scale of 0 to 3⫹: 0, negativity of the glomerular area; 1⫹, approximately 50% positivity of the glomerular area; 2⫹, 51% to 75% positivity of the glomerular area; and 3⫹, 76% or more positivity of the glomerular area. Immunohistochemical specificity was confirmed by means of an absorption test with antibodies and replacement of primary antibodies to nonimmunized sera according to our previous report.10 As a negative staining control, nonimmune normal serum or TBS was substituted for the primary antibodies according to the previous report.10,17
Statistical Analysis Data are expressed as mean ⫾ SD. Differences in mean values between groups was examined for statistical significance by using the Mann-Whitney U test. Association of categorical variables was examined by means of chi-square test. P less than 5% is regarded as a statistically significant difference.
RESULTS
Immunohistochemical Characterization of Glomerular Deposits Specificity of IgA1 and IgA2 monoclonal antibodies and correct identification of IgA2 without cross-reacting with IgA1 were confirmed by
LECTIN PATHWAY IN HSPN
297
means of the immunoabsorption test, as in our previous report.10 Marked IgA1 and IgA2 deposition in the mesangium was found (Fig 1A and B, respectively). MBL and MASP-1 colocalization in the mesangium was detected by using FITC-labeled monoclonal antibody against MASP-1 and tetramethylrhodamine isothiocyanate isomer R–labeled polyclonal antibody against MBL in the same section (Fig 1C to E). Mesangial deposition of factor B, C4-bp, C5b-9, CD59, and C4 is shown in Fig 1F to J, respectively. Phenotypic Profiles of IgA1 and IgA1/IgA2 Mesangial Deposits Phenotypic profiles of mesangial deposits of IgA1 alone and IgA1/IgA2 codeposits are listed in Table 1. No specimen from patients with minimal change nephrotic syndrome and thin basement membrane disease showed glomerular deposition of any antibody. Mesangial IgA1 deposits were detected in the 31 patients with HSPN. Mesangial IgA2 deposits were detected in 16 patients. Patients with HSPN were divided into 2 groups, as listed in Table 1. In 16 of 31 patients with HSPN, mesangial deposits of both IgA1 and IgA2 were detected, accompanied by mesangial deposits of C3c, MBL, MASP-1, C4-bp, and C5b-9 in 16 patients; mesangial deposits of factor B and CD59 in 15 patients; and mesangial deposits of C4 in 14 patients. The remaining 15 patients with mesangial deposits of IgA1 alone showed no mesangial deposits of C4 or MBL/MASP-1, accompanied by mesangial deposits of C3c, factor B, C5b-9, and CD59 in 11 patients. Mesangial C4-bp deposits were evident in 6 patients. Mesangial fibrinogen deposits were detected in 15 of 16 patients with mesangial deposits of both IgA1and IgA2, whereas fibrinogen deposits were detected in only 6 of 15 patients with mesangial deposits of IgA1 alone (chi-square test, P ⬍ 0.005). Clinical and Histopathologic Findings Associated With IgA1 and IgA1/IgA2 Mesangial Deposits Clinical and histopathologic findings associated with IgA1 and IgA1/IgA2 mesangial deposits are listed in Table 2. Duration of follow-up was 12 to 60 months (mean, 20.5 ⫾ 10.6 months). Degrees of hematuria and proteinuria at latest
Fig 1. Mesangial deposits of (A) IgA1, (B) IgA2, (C) MASP-1, (D) MBL, (E) merge of MSAP-1 and MBL, (F) factor B, (G) C4-bp, (H) C5b-9, (I) CD59, and (J) C4. Immunohistologic staining shown in A and B was performed in serial sections of the same case. Colocalization of (C) MBL and (D) MASP-1 in the mesangium is shown. (Original magnification ⴛ400.)
298
Table 1. Phenotype Profiles of IgA1 and IgA1/IgA2 Mesangial Deposits Urinalysis at Biopsy
Patient No.
Immunofluorescence
Immunoperoxidase
Proteinuria (g/dL)
GH
MME
SLs
GS
TILs
IgG/IgA
IgM/C1q
C3/C4
Fibrinogen
MBL/MASP-1
Factor B/C4-bp
C5b-9/CD59
50 50 30 25 35 40 20 30 30 50 80 20 30 50 15
300 120 50 500 100 450 100 100 200 100 300 50 100 100 150
1.1 0.5 0.4 0.4 0.4 0.8 0.9 0.6 1.5 0.9 1.1 1 0.2 1 0.8
0 0.1 0 0 0.2 0.1 0.1 0 0 0.3 0 0 0.3 0.1 0.2
3 1 1 0 1 1 1 1 1 1 1 0 1 3 1
0 0 0 0 1 0 0 0 0 1 0 0 1 0 0
0 0 0 1 0 0 0 1 0 1 0 0 1 0 0
⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫹/⫹ ⫺/⫹ ⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫺/⫹ ⫺/2⫹ ⫺/3⫹ ⫹/⫹ ⫺/⫹ ⫺/3⫹ ⫺/3⫹
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺
⫹/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺
⫺ ⫺ 2⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ 2⫹ ⫺ ⫺ ⫹ ⫺
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺
⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹
⫺/⫺ ⫺/⫺ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹
100 80 30 10 15 300 80 30 50 50 50 15 60 5 10 50
10 200 80 80 80 500 300 80 240 30 30 70 50 30 30 50
0.6 1 0.5 0.4 1 1 1 0.7 0.7 0.6 0.7 0.4 0.5 0.4 0.3 1.3
0.5 0.9 0.3 0.2 0 0.1 0.1 0.1 0.1 0.4 0.5 0.3 0.3 0.3 0 0.8
0 0 1 0 1 2 2 1 1 0 1 0 0 1 0 1
1 1 1 1 0 0 0 1 0 1 1 0 1 0 0 1
0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0
⫹/3⫹ ⫹/2⫹ ⫺/2⫹ ⫺/2⫹ ⫺/2⫹ ⫹/3⫹ ⫺/2⫹ ⫹/2⫹ ⫺/2⫹ ⫺/3⫹ ⫹/2⫹ ⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫺/2⫹ ⫺/2⫹
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺
⫹/⫺ ⫹/⫹ ⫹/⫺ ⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/2⫹ ⫹/⫹ 2⫹/⫹ 2⫹/⫹ ⫹/2⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹ ⫹ ⫹ ⫹ ⫹ 2⫹ ⫹ 2⫹ ⫹ 2⫹ ⫹ ⫹ 2⫹ 2⫹ ⫹ ⫺
⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹/⫹ ⫹/⫹ ⫺/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹/⫹ ⫹/⫹ ⫹/⫺ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
Abbreviation: RBC/HPF, red blood cells per high-power field.
HISANO ET AL
IgA1 deposits 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 IgA1 ⫹ IgA2 deposits 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Hematuria (RBC/HPF)
Light Microscopy
LECTIN PATHWAY IN HSPN
299
Table 2. Clinical and Histopathologic Findings Associated With IgA1 and IgA1/IgA2 Mesangial Deposits IgA1 Alone (n ⫽ 15)
Age at onset (y) Time between onset and biopsy (mo) Time between biopsy and latest follow-up (mo) Treatment (no. of cases) Prednisolone alone Predinisolone ⫹ warfarin Hematuria (RBC/HPF) At biopsy Latest follow-up Proteinuria (mg/dL) At biopsy Latest follow-up Serum creatinine (mg/dL) At biopsy Latest follow-up Histological index GH MME SLs GS TILs
IgA1 ⫹ IgA2 (n ⫽ 16)
P
8.1 ⫾ 2.9 11.6 ⫾ 9.8 22.6 ⫾ 12.1
10.5 ⫾ 4.3 17.4 ⫾ 8.4 17.6 ⫾ 7.1
NS NS NS
7 8
8 8
NS* NS*
37.0 ⫾ 16.8 2.5 ⫾ 4.5
58.4 ⫾ 70.3 24.8 ⫾ 29.3
181.3 ⫾ 141.7 6.7 ⫾ 11.1
116.3 ⫾ 131.8 77.5 ⫾ 61.8
0.0169 ⬍0.0001
0.5 ⫾ 0.1 0.5 ⫾ 0.1
0.4 ⫾ 0.1 0.5 ⫾ 0.1
NS NS
0.8 ⫾ 0.4 0.1 ⫾ 0.1 1.1 ⫾ 0.8 0.2 ⫾ 0.4 0.3 ⫾ 0.5
0.7 ⫾ 0.3 0.3 ⫾ 0.3 0.7 ⫾ 0.3 0.6 ⫾ 0.5 0.1 ⫾ 0.3
NS 0.0021
NS 0.0075 NS 0.0416 NS
NOTE. Values expressed as mean ⫾ SD. To convert creatinine in mg/dL to mol/L, multiply by 88.4. Abbreviations: RBC/HPF, red blood cells per high-power field; NS, not significant. *Not significant by chi-square test; the remaining data were analyzed using the Mann-Whitney U test.
follow-up were greater in patients with mesangial deposits of both IgA1 and IgA2 compared with those with mesangial deposits of IgA1 alone. Degrees of GH, MME, SLs, GS, and TILs were compared between patients with mesangial deposits of both IgA1 and IgA2 and those with mesangial deposits of IgA1 alone. Mean scores for MME and GS were higher in those with IgA1 and IgA2 deposits than in those with IgA1 deposits alone. Values for the remaining indices were not different between the 2 groups. DISCUSSION
Our current study shows that alternative pathway–involved complement activation is associated with mesangial deposits of IgA1 alone, and in those with mesangial deposits of IgA1 and IgA2, both the alternative and lectin pathways are involved in complement activation. Complement activation through the lectin pathway is associated with mesangial IgA2 deposits in patients with HSPN. Results of the present study are consistent with those of our previous study of patients with IgA GN.10 The lectin pathway is initiated by lectin and ficolins, which have a high affinity to terminal
mannose and N-acetylglucosamine moieties on surfaces of various pathogens, including bacteria, yeast, fungi, and viruses.4-7 Human MBL forms complexes with 3 types of serine protease: MASP-1, MASP-2, and MASP-3.4-9 Our study shows mesangial colocalization of MBL and MASP-1, indicating the existence of an MBL/ MASP-1 complex in the mesangium. Binding of MBL or ficolins to sugar chains leads to activation of C4 and C2 without C1 component.6-8,18 MBL activates the lectin complement pathway through cleavage of C4 and C2.6-8,18 Some studies indicated that MASP-2 activates C4 and C2, whereas MASP-1 activates C3 directly.5,19 Factor B activates the alternative complement pathway in the presence of factor D and properdin.20 C4-bp is a sensitive indicator of C4 consumption and inactivates C4b as a cofactor for factor I.13,21 C5b-9, membrane attack complex, is an end product of complement activation.22 CD59 is the sole inhibitor of the formation of membrane attack complex.22 Mesangial MBL/MASP-1, C4, C4-bp, factor B, C3, C5b-9, and CD59 deposits were detected in patients with mesangial IgA1 and IgA2 deposits. The MBL/MASP-1 complex can activate the complement cascade in the mes-
300
angium after cleavage of C4/C2 and cleaving C3 directly and lead to the formation of a membrane attack complex (cytolytically active C5b-9).23 The presence of C4, C4-bp, C5b-9, and CD59 indicates the occurrence of ongoing complement activation and controlling complement activation in situ. Endo et al13 previously reported that activation products and regulatory proteins were deposited together in the glomerulus in patients with HSPN. In our study, mesangial C4-bp deposits were found in a significant number of patients with mesangial IgA1 and IgA2 deposits compared with those of patients with mesangial IgA1 deposits. This regulatory protein seems to have an inhibitory effect on complement activation through the lectin pathway. The coagulation system is involved in the pathogenesis of HSPN.24 Glomerular fibrin/ fibrinogen deposits are present much more frequently in patients with HSPN than in patients with IgA GN.24 Ono et al25 reported that intraglomerular deposition of fibrin was found in patients with IgA GN and HSPN, and intraglomerular coagulation might progress in accordance with intraglomerular infiltration of macrophages. In our study, glomerular fibrinogen deposition was detected in a remarkable number of patients with mesangial IgA1 and IgA2 deposits in comparison to the number of patients with mesangial IgA1 deposits. This suggests that MBL/MASP-1 may be associated with glomerular fibrinogen deposition. Recently, Swierzko et al26 reported that Ra-reactive factor, a complex of MBL and MASP, activated fibrinogen in vitro, suggesting the involvement of the coagulation system in the process. There is a novel function of MASP-1 showing that MASP-1 cleaves fibrinogen, leading to fibrinopeptide B release.27 Fibrinopeptide B is a potent chemotactic factor for neutrophils, fibroblasts, and monocytes.27 However, the function of MBL/MASP-1 in the coagulation system of HSPN is not fully clarified. Scores for MME and GS were higher in patients with mesangial IgA1 and IgA2 deposits than in those with mesangial deposits of IgA1 alone. However, in our previous study, glomerular MBL/MASP-1 deposition was not a prognostic indicator in patients with IgA GN.10 The discrepancy in histological prognostic indices between the present and the previous study occurs because histological indices were not evalu-
HISANO ET AL
ated semiquantitatively in the previous study, different from the present study. We therefore semiquantitatively reevaluated the histological findings of IgA GN in the previous study. In patients with IgA GN, MME, SLs, and GS scores were higher in patients with mesangial IgA1 and IgA2 deposits than in those with mesangial IgA1 alone (Hisano S, Matsushita M, Takebayashi S, and Fujita T, unpublished data).28 Mesangial MBL/MASP-1 deposits are associated with advanced glomerular injuries. All our patients were administered prednisolone for 12 to 24 months. Hematuria and proteinuria improved by the treatment in patients with mesangial deposits of IgA1 alone despite the short-term follow-up, whereas these urinary abnormalities did not improve thoroughly in patients with mesangial IgA1 and IgA2 deposits despite treatment. Prolongation of urinary abnormalities in patients with mesangial IgA1 and IgA2 may be associated with the initial advanced histological findings. There was no difference in serum creatinine concentrations between the 2 groups throughout the follow-up period. No evidence of renal impairment is considered to be the short-term follow-up of 20.5 months. How does IgA2 trigger activation of the lectin pathway? HSPN and IgA GN are preceded by upper respiratory infection.24 Ogura et al29 reported that Haemophilus parainfluenzae had a role in the cause of IgA GN and HSPN in a subset of patients. MBL is reported to bind directly to a number of microorganisms through carbohydrates expressed on their surfaces.23 After infection caused by certain bacteria or viruses, MBL/MASP-1 may bind to mannose or N-acetylglucosamine of the bacterium or virus.23 Recently, Roos et al30 showed that the lectin pathway was activated by human IgA binding to MBL in vitro. They suggested that the interaction between MBL and IgA contributed to mesangial complement deposition in patients with IgA GN.30 However, they did not elucidate which of IgA1 or IgA2 bound to MBL and activated the lectin pathway. It is well known that the moieties of mannose and N-acetylglucosamine are rich in both IgA1 and IgA2 subclasses in the CH3 domain of the hinge region.31 Therefore, IgA1 or IgA2 has a potential to bind to MBL in the circulation. IgA likely is produced preferentially by carbohydrate antigens.32 It potentially is pos-
LECTIN PATHWAY IN HSPN
sible that IgA2 production is enhanced by carbohydrates of some bacteria or viruses. Increased IgA2 levels after infection may bind to MBL/ MASP-1 and may form an IgA2-MBL/MASP-1 complex in the circulation. The IgA2-MBL/ MASP-1 complex subsequently may be trapped in the mesangium and thus may activate the lectin pathway in situ in the glomerulus. The IgA2-MBL/MASP-1 complex is an important defense factor of the immune system for bacteria or viruses, but it also may have an unfavorable role in disease progression. Mesangial deposits of IgA1 were detected in all 31 patients. Therefore, results suggest that IgA1-containing immune complexes may have a pathogenic role in HSPN, as some investigators previously reported.24 From our current study, we could not elucidate how complement activation through the lectin pathway may contribute to the development of advanced glomerular injury. Additional investigation is needed to elucidate the association of lectin pathway–involved complement activation with the development of advanced glomerular injuries in patients with HSPN. ACKNOWLEDGMENT The authors thank Dr James C.M. Chan, Professor of Pediatrics, University of Vermont, The Barbara Bush Children’s Hospital, Portland, ME, for helpful advice.
REFERENCES 1. White RHR, Yoshikawa N: IgA nephropathy and Henoch-Schönlein nephritis, in Barratt TM, Avner ED, Harmon WF (eds): Pediatric Nephrology (ed 4). Baltimore, MD, Lippincott Williams & Wilkins, 1999, pp 691-706 2. Yoshikawa N, Ito H, Yoshiya K, et al: HenochSchönlein nephritis and IgA nephropathy in children: A comparison of clinical course. Clin Nephrol 27:233-237, 1987 3. Wald BF: Is Henoch-Schönlein purpura the systemic form of IgA nephropathy? Am J Kidney Dis 12:373-377, 1988 4. Matsushita M, Fujita T: Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J Exp Med 176:1497-1502, 1992 5. Thiel S, Vorup-Jensen T, Stover CM, et al: A second serine protease associated with mannose-binding lectin that activates complement. Nature 386:506-510, 1997 6. Takahashi M, Endo Y, Fujita T, Matsushita M: A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. Int Immunol 11:859-863, 1999 7. Stover CM, Thiel S, Thelen M, et al: Two constituents of the initiation complex of the mannan-binding lectin
301
activation pathway of complement are encoded by a single structural gene. J Immunol 162:3481-3490, 1999 8. Matsushita M, Fujita T: Ficolins and the lectin complement pathway. Immunol Rev 180:78-85, 2001 9. Dahl MR, Thiel M, Matsushita T, et al: MASP-3 and its association with distinct complexes of the mannanbinding lectin complement activation pathway. Immunity 15:127-135, 2001 10. Hisano S, Matsushita M, Fujita T, Endo Y, Takebayashi S: Mesangial IgA2 deposits and lectin pathway-mediated complement activation in IgA glomerulonephritis. Am J Kidney Dis 38:1082-1088, 2001 11. Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T: Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy. Nephrol Dial Transplant 13:19841990, 1998 12. Matsuda M, Shikata K, Wada J, et al: Deposition of mannan binding protein and mannan binding proteinmediated complement activation in the glomeruli of patients with IgA nephropathy. Nephron 80:408-413, 1998 13. Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T: Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis. Am J Kidney Dis 35:401-407, 2000 14. Katafuchi R, Kiyoshi Y, Oh Y, Uesugi N, Yanase T, Fujimi S: Glomerular score as a prognosticator in IgA nephropathy: Its usefulness and limitation. Clin Nephrol 49:1-8, 1998 15. Matsushita M, Takahashi A, Hatsuse H, Kawakami M, Fujita T: Human mannose-binding protein is identical to a component of Ra-reactive factor. Biochem Biophys Res Commun 183:645-651, 1992 16. Terai I, Kobayashi K, Matsushita M, Fujita T, Matsuno K: ␣2-Macroglobulin binds to and inhibits mannosebinding protein-associated serine protease. Int Immunol 7:1579-1584, 1995 17. Nagata M, Akioka Y, Tsunoda Y, et al: Macrophage in childhood IgA nephropathy. Kidney Int 48:527-535, 1995 18. Matsushita M, Endo Y, Fujita T: MASP-1 (MBLassociated serine protease 1). Immunobiology 199:340-347, 1998 19. Matsushita M, Kuriya M, Hamasaki N, Tsujimura M, Shiraki H, Fujita T: Activation of the lectin complement pathway by H-ficolin (Hakata antigen). J Immunol 168:35023506, 2002 20. Huang Y, Krein PM, Muruve DA, Winston BW: Complement factor B gene regulation: Synthesis effects of TNF-alpha and IFN-gamma in macrophages. J Immunol 169:2627-2635, 2002 21. Endo M, Ohi H, Satomura A, et al: Regulation of in situ complement activation via the lectin pathway in patients with IgA nephropathy. Clin Nephrol 55:185-191, 2001 22. Harris CL, Hanna SM, Mizuno M, Holt DS, Marchbank KJ, Morgan BP: Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: Tissue distribution and functional comparison. Immunology 109:117-126, 2003 23. Turner MW: Mannose-binding lectin: The pluripotent molecule of the innate immune system. Immunol Today 17:532-540, 1996
302
24. Davin J-C, Ten Berge IJ, Weening JJ: What is the difference between IgA nephropathy and Henoch-Schönlein purpura? Kidney Int 59:823-834, 2001 25. Ono T, Muso E, Suyama K, et al: Intraglomerular deposition of intact cross-linked fibrin in IgA nephropathy and Henoch-Schönlein purpura nephritis. Nephron 74:522528, 1996 26. Swierzko AS, Cedzynski M, Kirikae T, et al: Role of the complement-lectin pathway in anaphylactoid reaction induced with lipopolysaccharide in mice. Eur J Immunol 33:2842-2852, 2003 27. Hajela K, Kojima M, Ambrus G, et al: The biological functions of MBL-associated serine protease (MASPs). Immunobiology 205:467-475, 2002 28. Hisano S, Matsushita M, Takebayashi S, Fujita T: Activation of the lectin complement pathway in IgA glomerulonephritis [in Japanese]. Jpn J Nephrol 45:194A, 2003 (abstr)
HISANO ET AL
29. Ogura Y, Suzuki S, Shirakawa T, et al: Haemophilus parainfluenzae antigen and antibody in children with IgA nephropathy and Henoch-Schönlein nephritis. Am J Kidney Dis 36:47-52, 2000 30. Roos A, Bouwman LH, van Giglwijk-Janssen DJ, Faber-Krol MC, Stahl GL, Daha MR: Human IgA activates the complement system via the mannan-binding lectin pathway. J Immunol 167:2861-2868, 2001 31. Amore A, Cirina P, Conti G, Brusa P, Peruzzi L, Coppo R: Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells. J Am Soc Nephrol 12:1862-1871, 2001 32. Russel MW, Lue C, Van Den Wall Bakke AWL, Moldoveanu Z, Mestecky J: Molecular heterogeneity of human IgA antibodies during immune response. Clin Exp Immunol 87:1-6, 1992
H
ENOCH-SCHÖNLEIN purpura nephritis (HSPN) is a systemic disease that shows hematuria and proteinuria associated with purpuric rash, abdominal pain, and/or arthralgia.1 HSPN is characterized histologically by predominant immunoglobulin A (IgA) deposition and, simultaneously, C3 deposition in the mesangium on immunofluorescence microscopy, similar to IgA glomerulonephritis (GN).2,3 Recently, a new pathway of complement activation, the lectin pathway, which is initiated by mannose-binding lectin (MBL), ficolins, MBL-associated serine protease-1, -2, -3 (MASP-1, MASP-2, and MASP-3), and a truncated form of MASP-2, was reported.4-9 Our previous study immunohistologically showed that alternative pathway–involved complement activation was associated with mesangial deposits of IgA1 alone in patients with IgA GN, and in those with mesangial IgA1 and IgA2 deposits, both the alternative and lectin pathways were involved in complement activation.10 We first reported that mesangial IgA2 deposits might activate the lectin pathway in patients with IgA GN.10 Complement activation through the lectin pathway was found in 20% to 50% of patients with IgA GN.10-12 Endo et al13 reported complement activation through the lectin pathway in 8 of
10 patients with HSPN. However, the number of their patients is too small and correlation between the lectin pathway and clinicopathologic findings in patients with HSPN is not fully clarified. The aim of our study is to elucidate the correlation between each complement pathway and clinicopathologic findings in patients with HSPN. METHODS Kidney tissue was obtained by means of percutaneous renal biopsy in 31 patients with HSPN, aged 4 to 18 years, between 1998 and 2001. HSPN was diagnosed as hematuria and proteinuria associated with such characteristic manifes-
From the Department of Pathology, School of Medicine, Fukuoka University, Fukuoka; Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, Hiratsuka; and Department of Biochemistry, Fukushima Medical University, Fukushima, Japan. Received June 15, 2004; accepted in revised form October 18, 2004. Originally published online as doi:10.1053/j.ajkd.2004.10.020 on December 29, 2004. Address reprint requests to Satoshi Hisano, MD, Department of Pathology, School of Medicine, Fukuoka University, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-0180, Japan. E-mail: [email protected] © 2004 by the National Kidney Foundation, Inc. 0272-6386/04/4502-0008$30.00/0 doi:10.1053/j.ajkd.2004.10.020
American Journal of Kidney Diseases, Vol 45, No 2 (February), 2005: pp 295-302
295
296
tations as purpuric rash, abdominal pain, and/or joint pain.1 Controls were used as non-IgA GN (10 patients with minimal change nephrotic syndrome, 10 patients with thin basement membrane disease). Informed consent was obtained from patients and/or their parents before renal biopsy. On approval from the Human Ethics Review Committee of Fukuoka University (Fukuoka, Japan), this study protocol was performed. Light microscopy was evaluated on sections stained with periodic acid–Schiff and periodic acid–methenamine silver. The histological severity of glomerular lesions and tubulointerstitial lesions (TILs) was determined according to the previous report,14 modified by us. Degrees of glomerular hypercellularity (GH; endocapillary hypercellularity and/or mesangial hypercellularity), mesangial matrix expansion (MME), segmental lesions (SLs; crescent formation, tuft adhesion to Bowman’s capsule, and/or segmental sclerosis), glomerular sclerosis (GS), and TILs (tubular atrophy and interstitial fibrosis) were evaluated. Endocapillary hypercellularity is defined as 3 or more leukocytic cells in the capillary space, and mesangial hypercellularity is defined as 3 or more nuclei in the mesangial area. GS was determined as a sclerotic lesion occupying 50% or more of the glomerular area. Each index was semiquantitatively evaluated. Degrees of GH and MME in each glomerulus were evaluated as a point as follows: 0, no lesion; 1, involving 10% to 30% of the glomerular area; 2, involving 31% to 50% of the glomerular area; and 3, involving more than 50% of glomerular area. Points of individual glomerulus were added and divided by the number of glomeruli in each biopsy specimen, ie, an average of points in each biopsy. Degrees of SLs, GS, and TILs were evaluated as a point according to the percentage of glomeruli showing SLs and GS per total glomeruli and tubulointerstitium showing tubular atrophy and fibrosis in the cortical area as follows, respectively: 0, no lesion; 1, involving 10% to 30% of total glomeruli and cortical area, respectively; 2, involving 31% to 50% of total glomeruli and cortical area, respectively; and 3, involving more than 50% of total glomeruli and cortical area, respectively. All patients were administered prednisolone and/or warfarin. Prednisolone was administered initially at a dose of 2 mg/kg/d for 4 weeks followed by a dose of 2 mg/kg/d on alternative days for 4 weeks, then tapered off for 12 to 24 months. Warfarin was administered daily at a dose of 1 to 3 mg/d for 12 to 24 months. Clinical records examined at the time of biopsy and latest follow-up, including blood pressure, urinalysis, and serum protein and serum creatinine levels, were obtained from the pediatricians who followed up these 31 children.
Immunohistologic Staining Immunohistologic staining was performed according to our previous report.10 Fluorescein isothiocyanate (FITC)labeled anti-IgG, IgA, IgM, C1q, C3c, fibrinogen polyclonal antibodies (Dako, Copenhagen, Denmark), FITC-labeled anti-C4 polyclonal antibody (MBL; Medical and Biological Laboratories, Nagoya, Japan), and anti-factor B polyclonal antibody (Biogenesis, Poole, UK) were used. Monoclonal antibodies against human IgA1 (NIF2; Skybio, Bedfordshire, UK), IgA2 (2E2, Skybio), C4-binding protein (C4-bp; 11-2D3; Fuji Yakuhin , Toyama, Japan), C5b-9 (Dako), and CD59 (Cosmobio, Tokyo, Japan) were used. Monoclonal
HISANO ET AL
antibodies against human MBL (3E7) and MASP-1 (4C2) were provided from T.F., coinvestigator, as previously reported.15,16 Sections cut at a thickness of 4 m from frozen tissues were immersed into Tris-buffered saline (TBS) 3 times for 5 minutes. Diluted FITC-labeled polyclonal antibodies (IgG, IgA, IgM, C3c, C1q, C4, and fibrinogen), IgA1 and IgA2 monoclonal antibodies, MBL monoclonal antibody, MASP-1 monoclonal antibody, C4-bp monoclonal antibody, C5b-9 monoclonal antibody, CD59 monoclonal antibody, and factor B polyclonal antibody were overlaid on the sections. Sections were incubated at 4°C overnight. Sections on which FITC-labeled polyclonal antibodies were overlaid were observed on immunofluorescence microscopy. Immunohistochemical studies for monoclonal antibodies (IgA1, IgA2, MBL, MASP-1, C4-bp, CD5b-9, and CD59) and factor B polyclonal antibody were performed using the avidin-biotin peroxidase method, as previously reported.10 After treatment in 0.5% hydrogen peroxide in methanol solution to quench endogenous peroxidase activity, sections overlaid by monoclonal antibodies were incubated with biotin-labeled antibody against mouse IgG (Dako), and those overlaid by factor B polyclonal antibody were incubated with biotin-labeled antibody against rabbit IgG (Dako) at room temperature for 30 minutes. After washing in TBS, sections were incubated with avidin-biotin-peroxidase complex (Dako) at room temperature for 30 minutes. After 3 washes in TBS, sections were incubated with peroxidaselabeled avidin for 30 minutes. Sections were treated with 3,3-diaminobenzidine (Dohjin, Kyoto, Japan), counterstained with hematoxylin, and observed using a light microscope. The intensity of immunohistologic deposits in glomeruli was based semiquantitatively on a scale of 0 to 3⫹: 0, negativity of the glomerular area; 1⫹, approximately 50% positivity of the glomerular area; 2⫹, 51% to 75% positivity of the glomerular area; and 3⫹, 76% or more positivity of the glomerular area. Immunohistochemical specificity was confirmed by means of an absorption test with antibodies and replacement of primary antibodies to nonimmunized sera according to our previous report.10 As a negative staining control, nonimmune normal serum or TBS was substituted for the primary antibodies according to the previous report.10,17
Statistical Analysis Data are expressed as mean ⫾ SD. Differences in mean values between groups was examined for statistical significance by using the Mann-Whitney U test. Association of categorical variables was examined by means of chi-square test. P less than 5% is regarded as a statistically significant difference.
RESULTS
Immunohistochemical Characterization of Glomerular Deposits Specificity of IgA1 and IgA2 monoclonal antibodies and correct identification of IgA2 without cross-reacting with IgA1 were confirmed by
LECTIN PATHWAY IN HSPN
297
means of the immunoabsorption test, as in our previous report.10 Marked IgA1 and IgA2 deposition in the mesangium was found (Fig 1A and B, respectively). MBL and MASP-1 colocalization in the mesangium was detected by using FITC-labeled monoclonal antibody against MASP-1 and tetramethylrhodamine isothiocyanate isomer R–labeled polyclonal antibody against MBL in the same section (Fig 1C to E). Mesangial deposition of factor B, C4-bp, C5b-9, CD59, and C4 is shown in Fig 1F to J, respectively. Phenotypic Profiles of IgA1 and IgA1/IgA2 Mesangial Deposits Phenotypic profiles of mesangial deposits of IgA1 alone and IgA1/IgA2 codeposits are listed in Table 1. No specimen from patients with minimal change nephrotic syndrome and thin basement membrane disease showed glomerular deposition of any antibody. Mesangial IgA1 deposits were detected in the 31 patients with HSPN. Mesangial IgA2 deposits were detected in 16 patients. Patients with HSPN were divided into 2 groups, as listed in Table 1. In 16 of 31 patients with HSPN, mesangial deposits of both IgA1 and IgA2 were detected, accompanied by mesangial deposits of C3c, MBL, MASP-1, C4-bp, and C5b-9 in 16 patients; mesangial deposits of factor B and CD59 in 15 patients; and mesangial deposits of C4 in 14 patients. The remaining 15 patients with mesangial deposits of IgA1 alone showed no mesangial deposits of C4 or MBL/MASP-1, accompanied by mesangial deposits of C3c, factor B, C5b-9, and CD59 in 11 patients. Mesangial C4-bp deposits were evident in 6 patients. Mesangial fibrinogen deposits were detected in 15 of 16 patients with mesangial deposits of both IgA1and IgA2, whereas fibrinogen deposits were detected in only 6 of 15 patients with mesangial deposits of IgA1 alone (chi-square test, P ⬍ 0.005). Clinical and Histopathologic Findings Associated With IgA1 and IgA1/IgA2 Mesangial Deposits Clinical and histopathologic findings associated with IgA1 and IgA1/IgA2 mesangial deposits are listed in Table 2. Duration of follow-up was 12 to 60 months (mean, 20.5 ⫾ 10.6 months). Degrees of hematuria and proteinuria at latest
Fig 1. Mesangial deposits of (A) IgA1, (B) IgA2, (C) MASP-1, (D) MBL, (E) merge of MSAP-1 and MBL, (F) factor B, (G) C4-bp, (H) C5b-9, (I) CD59, and (J) C4. Immunohistologic staining shown in A and B was performed in serial sections of the same case. Colocalization of (C) MBL and (D) MASP-1 in the mesangium is shown. (Original magnification ⴛ400.)
298
Table 1. Phenotype Profiles of IgA1 and IgA1/IgA2 Mesangial Deposits Urinalysis at Biopsy
Patient No.
Immunofluorescence
Immunoperoxidase
Proteinuria (g/dL)
GH
MME
SLs
GS
TILs
IgG/IgA
IgM/C1q
C3/C4
Fibrinogen
MBL/MASP-1
Factor B/C4-bp
C5b-9/CD59
50 50 30 25 35 40 20 30 30 50 80 20 30 50 15
300 120 50 500 100 450 100 100 200 100 300 50 100 100 150
1.1 0.5 0.4 0.4 0.4 0.8 0.9 0.6 1.5 0.9 1.1 1 0.2 1 0.8
0 0.1 0 0 0.2 0.1 0.1 0 0 0.3 0 0 0.3 0.1 0.2
3 1 1 0 1 1 1 1 1 1 1 0 1 3 1
0 0 0 0 1 0 0 0 0 1 0 0 1 0 0
0 0 0 1 0 0 0 1 0 1 0 0 1 0 0
⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫹/⫹ ⫺/⫹ ⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫺/⫹ ⫺/2⫹ ⫺/3⫹ ⫹/⫹ ⫺/⫹ ⫺/3⫹ ⫺/3⫹
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺
⫹/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫺/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺
⫺ ⫺ 2⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ 2⫹ ⫺ ⫺ ⫹ ⫺
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺
⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫺ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹
⫺/⫺ ⫺/⫺ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹
100 80 30 10 15 300 80 30 50 50 50 15 60 5 10 50
10 200 80 80 80 500 300 80 240 30 30 70 50 30 30 50
0.6 1 0.5 0.4 1 1 1 0.7 0.7 0.6 0.7 0.4 0.5 0.4 0.3 1.3
0.5 0.9 0.3 0.2 0 0.1 0.1 0.1 0.1 0.4 0.5 0.3 0.3 0.3 0 0.8
0 0 1 0 1 2 2 1 1 0 1 0 0 1 0 1
1 1 1 1 0 0 0 1 0 1 1 0 1 0 0 1
0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0
⫹/3⫹ ⫹/2⫹ ⫺/2⫹ ⫺/2⫹ ⫺/2⫹ ⫹/3⫹ ⫺/2⫹ ⫹/2⫹ ⫺/2⫹ ⫺/3⫹ ⫹/2⫹ ⫹/2⫹ ⫺/2⫹ ⫹/2⫹ ⫺/2⫹ ⫺/2⫹
⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫹/⫺ ⫺/⫺ ⫺/⫺
⫹/⫺ ⫹/⫹ ⫹/⫺ ⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/2⫹ ⫹/⫹ 2⫹/⫹ 2⫹/⫹ ⫹/2⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹ ⫹ ⫹ ⫹ ⫹ 2⫹ ⫹ 2⫹ ⫹ 2⫹ ⫹ ⫹ 2⫹ 2⫹ ⫹ ⫺
⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹/⫹ ⫹/⫹ ⫺/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
⫹/⫹ ⫹/⫹ ⫹/⫺ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ 2⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹
Abbreviation: RBC/HPF, red blood cells per high-power field.
HISANO ET AL
IgA1 deposits 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 IgA1 ⫹ IgA2 deposits 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Hematuria (RBC/HPF)
Light Microscopy
LECTIN PATHWAY IN HSPN
299
Table 2. Clinical and Histopathologic Findings Associated With IgA1 and IgA1/IgA2 Mesangial Deposits IgA1 Alone (n ⫽ 15)
Age at onset (y) Time between onset and biopsy (mo) Time between biopsy and latest follow-up (mo) Treatment (no. of cases) Prednisolone alone Predinisolone ⫹ warfarin Hematuria (RBC/HPF) At biopsy Latest follow-up Proteinuria (mg/dL) At biopsy Latest follow-up Serum creatinine (mg/dL) At biopsy Latest follow-up Histological index GH MME SLs GS TILs
IgA1 ⫹ IgA2 (n ⫽ 16)
P
8.1 ⫾ 2.9 11.6 ⫾ 9.8 22.6 ⫾ 12.1
10.5 ⫾ 4.3 17.4 ⫾ 8.4 17.6 ⫾ 7.1
NS NS NS
7 8
8 8
NS* NS*
37.0 ⫾ 16.8 2.5 ⫾ 4.5
58.4 ⫾ 70.3 24.8 ⫾ 29.3
181.3 ⫾ 141.7 6.7 ⫾ 11.1
116.3 ⫾ 131.8 77.5 ⫾ 61.8
0.0169 ⬍0.0001
0.5 ⫾ 0.1 0.5 ⫾ 0.1
0.4 ⫾ 0.1 0.5 ⫾ 0.1
NS NS
0.8 ⫾ 0.4 0.1 ⫾ 0.1 1.1 ⫾ 0.8 0.2 ⫾ 0.4 0.3 ⫾ 0.5
0.7 ⫾ 0.3 0.3 ⫾ 0.3 0.7 ⫾ 0.3 0.6 ⫾ 0.5 0.1 ⫾ 0.3
NS 0.0021
NS 0.0075 NS 0.0416 NS
NOTE. Values expressed as mean ⫾ SD. To convert creatinine in mg/dL to mol/L, multiply by 88.4. Abbreviations: RBC/HPF, red blood cells per high-power field; NS, not significant. *Not significant by chi-square test; the remaining data were analyzed using the Mann-Whitney U test.
follow-up were greater in patients with mesangial deposits of both IgA1 and IgA2 compared with those with mesangial deposits of IgA1 alone. Degrees of GH, MME, SLs, GS, and TILs were compared between patients with mesangial deposits of both IgA1 and IgA2 and those with mesangial deposits of IgA1 alone. Mean scores for MME and GS were higher in those with IgA1 and IgA2 deposits than in those with IgA1 deposits alone. Values for the remaining indices were not different between the 2 groups. DISCUSSION
Our current study shows that alternative pathway–involved complement activation is associated with mesangial deposits of IgA1 alone, and in those with mesangial deposits of IgA1 and IgA2, both the alternative and lectin pathways are involved in complement activation. Complement activation through the lectin pathway is associated with mesangial IgA2 deposits in patients with HSPN. Results of the present study are consistent with those of our previous study of patients with IgA GN.10 The lectin pathway is initiated by lectin and ficolins, which have a high affinity to terminal
mannose and N-acetylglucosamine moieties on surfaces of various pathogens, including bacteria, yeast, fungi, and viruses.4-7 Human MBL forms complexes with 3 types of serine protease: MASP-1, MASP-2, and MASP-3.4-9 Our study shows mesangial colocalization of MBL and MASP-1, indicating the existence of an MBL/ MASP-1 complex in the mesangium. Binding of MBL or ficolins to sugar chains leads to activation of C4 and C2 without C1 component.6-8,18 MBL activates the lectin complement pathway through cleavage of C4 and C2.6-8,18 Some studies indicated that MASP-2 activates C4 and C2, whereas MASP-1 activates C3 directly.5,19 Factor B activates the alternative complement pathway in the presence of factor D and properdin.20 C4-bp is a sensitive indicator of C4 consumption and inactivates C4b as a cofactor for factor I.13,21 C5b-9, membrane attack complex, is an end product of complement activation.22 CD59 is the sole inhibitor of the formation of membrane attack complex.22 Mesangial MBL/MASP-1, C4, C4-bp, factor B, C3, C5b-9, and CD59 deposits were detected in patients with mesangial IgA1 and IgA2 deposits. The MBL/MASP-1 complex can activate the complement cascade in the mes-
300
angium after cleavage of C4/C2 and cleaving C3 directly and lead to the formation of a membrane attack complex (cytolytically active C5b-9).23 The presence of C4, C4-bp, C5b-9, and CD59 indicates the occurrence of ongoing complement activation and controlling complement activation in situ. Endo et al13 previously reported that activation products and regulatory proteins were deposited together in the glomerulus in patients with HSPN. In our study, mesangial C4-bp deposits were found in a significant number of patients with mesangial IgA1 and IgA2 deposits compared with those of patients with mesangial IgA1 deposits. This regulatory protein seems to have an inhibitory effect on complement activation through the lectin pathway. The coagulation system is involved in the pathogenesis of HSPN.24 Glomerular fibrin/ fibrinogen deposits are present much more frequently in patients with HSPN than in patients with IgA GN.24 Ono et al25 reported that intraglomerular deposition of fibrin was found in patients with IgA GN and HSPN, and intraglomerular coagulation might progress in accordance with intraglomerular infiltration of macrophages. In our study, glomerular fibrinogen deposition was detected in a remarkable number of patients with mesangial IgA1 and IgA2 deposits in comparison to the number of patients with mesangial IgA1 deposits. This suggests that MBL/MASP-1 may be associated with glomerular fibrinogen deposition. Recently, Swierzko et al26 reported that Ra-reactive factor, a complex of MBL and MASP, activated fibrinogen in vitro, suggesting the involvement of the coagulation system in the process. There is a novel function of MASP-1 showing that MASP-1 cleaves fibrinogen, leading to fibrinopeptide B release.27 Fibrinopeptide B is a potent chemotactic factor for neutrophils, fibroblasts, and monocytes.27 However, the function of MBL/MASP-1 in the coagulation system of HSPN is not fully clarified. Scores for MME and GS were higher in patients with mesangial IgA1 and IgA2 deposits than in those with mesangial deposits of IgA1 alone. However, in our previous study, glomerular MBL/MASP-1 deposition was not a prognostic indicator in patients with IgA GN.10 The discrepancy in histological prognostic indices between the present and the previous study occurs because histological indices were not evalu-
HISANO ET AL
ated semiquantitatively in the previous study, different from the present study. We therefore semiquantitatively reevaluated the histological findings of IgA GN in the previous study. In patients with IgA GN, MME, SLs, and GS scores were higher in patients with mesangial IgA1 and IgA2 deposits than in those with mesangial IgA1 alone (Hisano S, Matsushita M, Takebayashi S, and Fujita T, unpublished data).28 Mesangial MBL/MASP-1 deposits are associated with advanced glomerular injuries. All our patients were administered prednisolone for 12 to 24 months. Hematuria and proteinuria improved by the treatment in patients with mesangial deposits of IgA1 alone despite the short-term follow-up, whereas these urinary abnormalities did not improve thoroughly in patients with mesangial IgA1 and IgA2 deposits despite treatment. Prolongation of urinary abnormalities in patients with mesangial IgA1 and IgA2 may be associated with the initial advanced histological findings. There was no difference in serum creatinine concentrations between the 2 groups throughout the follow-up period. No evidence of renal impairment is considered to be the short-term follow-up of 20.5 months. How does IgA2 trigger activation of the lectin pathway? HSPN and IgA GN are preceded by upper respiratory infection.24 Ogura et al29 reported that Haemophilus parainfluenzae had a role in the cause of IgA GN and HSPN in a subset of patients. MBL is reported to bind directly to a number of microorganisms through carbohydrates expressed on their surfaces.23 After infection caused by certain bacteria or viruses, MBL/MASP-1 may bind to mannose or N-acetylglucosamine of the bacterium or virus.23 Recently, Roos et al30 showed that the lectin pathway was activated by human IgA binding to MBL in vitro. They suggested that the interaction between MBL and IgA contributed to mesangial complement deposition in patients with IgA GN.30 However, they did not elucidate which of IgA1 or IgA2 bound to MBL and activated the lectin pathway. It is well known that the moieties of mannose and N-acetylglucosamine are rich in both IgA1 and IgA2 subclasses in the CH3 domain of the hinge region.31 Therefore, IgA1 or IgA2 has a potential to bind to MBL in the circulation. IgA likely is produced preferentially by carbohydrate antigens.32 It potentially is pos-
LECTIN PATHWAY IN HSPN
sible that IgA2 production is enhanced by carbohydrates of some bacteria or viruses. Increased IgA2 levels after infection may bind to MBL/ MASP-1 and may form an IgA2-MBL/MASP-1 complex in the circulation. The IgA2-MBL/ MASP-1 complex subsequently may be trapped in the mesangium and thus may activate the lectin pathway in situ in the glomerulus. The IgA2-MBL/MASP-1 complex is an important defense factor of the immune system for bacteria or viruses, but it also may have an unfavorable role in disease progression. Mesangial deposits of IgA1 were detected in all 31 patients. Therefore, results suggest that IgA1-containing immune complexes may have a pathogenic role in HSPN, as some investigators previously reported.24 From our current study, we could not elucidate how complement activation through the lectin pathway may contribute to the development of advanced glomerular injury. Additional investigation is needed to elucidate the association of lectin pathway–involved complement activation with the development of advanced glomerular injuries in patients with HSPN. ACKNOWLEDGMENT The authors thank Dr James C.M. Chan, Professor of Pediatrics, University of Vermont, The Barbara Bush Children’s Hospital, Portland, ME, for helpful advice.
REFERENCES 1. White RHR, Yoshikawa N: IgA nephropathy and Henoch-Schönlein nephritis, in Barratt TM, Avner ED, Harmon WF (eds): Pediatric Nephrology (ed 4). Baltimore, MD, Lippincott Williams & Wilkins, 1999, pp 691-706 2. Yoshikawa N, Ito H, Yoshiya K, et al: HenochSchönlein nephritis and IgA nephropathy in children: A comparison of clinical course. Clin Nephrol 27:233-237, 1987 3. Wald BF: Is Henoch-Schönlein purpura the systemic form of IgA nephropathy? Am J Kidney Dis 12:373-377, 1988 4. Matsushita M, Fujita T: Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J Exp Med 176:1497-1502, 1992 5. Thiel S, Vorup-Jensen T, Stover CM, et al: A second serine protease associated with mannose-binding lectin that activates complement. Nature 386:506-510, 1997 6. Takahashi M, Endo Y, Fujita T, Matsushita M: A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. Int Immunol 11:859-863, 1999 7. Stover CM, Thiel S, Thelen M, et al: Two constituents of the initiation complex of the mannan-binding lectin
301
activation pathway of complement are encoded by a single structural gene. J Immunol 162:3481-3490, 1999 8. Matsushita M, Fujita T: Ficolins and the lectin complement pathway. Immunol Rev 180:78-85, 2001 9. Dahl MR, Thiel M, Matsushita T, et al: MASP-3 and its association with distinct complexes of the mannanbinding lectin complement activation pathway. Immunity 15:127-135, 2001 10. Hisano S, Matsushita M, Fujita T, Endo Y, Takebayashi S: Mesangial IgA2 deposits and lectin pathway-mediated complement activation in IgA glomerulonephritis. Am J Kidney Dis 38:1082-1088, 2001 11. Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T: Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy. Nephrol Dial Transplant 13:19841990, 1998 12. Matsuda M, Shikata K, Wada J, et al: Deposition of mannan binding protein and mannan binding proteinmediated complement activation in the glomeruli of patients with IgA nephropathy. Nephron 80:408-413, 1998 13. Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T: Complement activation through the lectin pathway in patients with Henoch-Schönlein purpura nephritis. Am J Kidney Dis 35:401-407, 2000 14. Katafuchi R, Kiyoshi Y, Oh Y, Uesugi N, Yanase T, Fujimi S: Glomerular score as a prognosticator in IgA nephropathy: Its usefulness and limitation. Clin Nephrol 49:1-8, 1998 15. Matsushita M, Takahashi A, Hatsuse H, Kawakami M, Fujita T: Human mannose-binding protein is identical to a component of Ra-reactive factor. Biochem Biophys Res Commun 183:645-651, 1992 16. Terai I, Kobayashi K, Matsushita M, Fujita T, Matsuno K: ␣2-Macroglobulin binds to and inhibits mannosebinding protein-associated serine protease. Int Immunol 7:1579-1584, 1995 17. Nagata M, Akioka Y, Tsunoda Y, et al: Macrophage in childhood IgA nephropathy. Kidney Int 48:527-535, 1995 18. Matsushita M, Endo Y, Fujita T: MASP-1 (MBLassociated serine protease 1). Immunobiology 199:340-347, 1998 19. Matsushita M, Kuriya M, Hamasaki N, Tsujimura M, Shiraki H, Fujita T: Activation of the lectin complement pathway by H-ficolin (Hakata antigen). J Immunol 168:35023506, 2002 20. Huang Y, Krein PM, Muruve DA, Winston BW: Complement factor B gene regulation: Synthesis effects of TNF-alpha and IFN-gamma in macrophages. J Immunol 169:2627-2635, 2002 21. Endo M, Ohi H, Satomura A, et al: Regulation of in situ complement activation via the lectin pathway in patients with IgA nephropathy. Clin Nephrol 55:185-191, 2001 22. Harris CL, Hanna SM, Mizuno M, Holt DS, Marchbank KJ, Morgan BP: Characterization of the mouse analogues of CD59 using novel monoclonal antibodies: Tissue distribution and functional comparison. Immunology 109:117-126, 2003 23. Turner MW: Mannose-binding lectin: The pluripotent molecule of the innate immune system. Immunol Today 17:532-540, 1996
302
24. Davin J-C, Ten Berge IJ, Weening JJ: What is the difference between IgA nephropathy and Henoch-Schönlein purpura? Kidney Int 59:823-834, 2001 25. Ono T, Muso E, Suyama K, et al: Intraglomerular deposition of intact cross-linked fibrin in IgA nephropathy and Henoch-Schönlein purpura nephritis. Nephron 74:522528, 1996 26. Swierzko AS, Cedzynski M, Kirikae T, et al: Role of the complement-lectin pathway in anaphylactoid reaction induced with lipopolysaccharide in mice. Eur J Immunol 33:2842-2852, 2003 27. Hajela K, Kojima M, Ambrus G, et al: The biological functions of MBL-associated serine protease (MASPs). Immunobiology 205:467-475, 2002 28. Hisano S, Matsushita M, Takebayashi S, Fujita T: Activation of the lectin complement pathway in IgA glomerulonephritis [in Japanese]. Jpn J Nephrol 45:194A, 2003 (abstr)
HISANO ET AL
29. Ogura Y, Suzuki S, Shirakawa T, et al: Haemophilus parainfluenzae antigen and antibody in children with IgA nephropathy and Henoch-Schönlein nephritis. Am J Kidney Dis 36:47-52, 2000 30. Roos A, Bouwman LH, van Giglwijk-Janssen DJ, Faber-Krol MC, Stahl GL, Daha MR: Human IgA activates the complement system via the mannan-binding lectin pathway. J Immunol 167:2861-2868, 2001 31. Amore A, Cirina P, Conti G, Brusa P, Peruzzi L, Coppo R: Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells. J Am Soc Nephrol 12:1862-1871, 2001 32. Russel MW, Lue C, Van Den Wall Bakke AWL, Moldoveanu Z, Mestecky J: Molecular heterogeneity of human IgA antibodies during immune response. Clin Exp Immunol 87:1-6, 1992