Nephrol Dial Transplant (2004) 19: 574–579 DOI: 10.1093/ndt/gfg616 Original Article Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy Hiroshi Ohtani 1 , Hideki Wakui 1 , Atsushi Komatsuda 1 , Shin Okuyama 1 , Rie Masai 1 , Nobuki Maki 1 , Akihiro Kigawa 1 , Ken-ichi Sawada 1 and Hirokazu Imai 1,2 1 The Third Department of Internal Medicine, Akita University School of Medicine, Akita and 2 The Department of Nephrology and Rheumatology, Aichi Medical University, Aichi, Japan Abstract Background. Several studies have shown a pre domi- nant glomerul ar depos ition of IgG4 in patie nts with idio pathic memb ranous nephro pathy (MN), where as signi ﬁcant depositi ons of other IgG subcl asses have been shown in patients with lupus-associated MN and bucillamine-induced MN. Methods. We exami ned the dis tri but ion pat tern s ofglomerular IgG subclass deposits in 10 patients with malignancy-associated MN (M-MN) and in 15 patients wi th idiopathic MN by immunoﬂuorescence (IF) microscopy. Results. The glo mer ula r IF int ens iti es of IgG1 and IgG2 were sig niﬁcan tly stronger in the mal ignanc y group than in the idiopathic group ( P<0. 05) . In contrast, the re were no dif fer enc es in glo mer ula r IF intensities of IgG3 and IgG4 between the two groups. Conclusion. Our ﬁndings suggest that the distribution patt ern s of gl omerular IgG subc lass deposi ts are dif fere nt in idi opa thi c MN and M- MN. The stro ng IF intensity of glomerular IgG1 and IgG2 in M-MN may provide a possible predictor for this condition. Keywords: IgG subclasses; immune complex; immuno- ﬂuorescence; malignancy; membranous nephropathy Introduction Membranous nephropathy (MN), the common cause of the adult nephrotic syndrome, is characterized by glomerular subepithelial IgG deposits. Human IgG is div ide d int o fou r subcla sses (IgG1, IgG2, IgG3 and IgG4) according to antigenic differences in their heavy chains. Each subclass differs in terms of several aspects: encodi ng genes, molecula r weigh ts, isoel ectric poin t and interaction with complements . These IgG sub- cl asses are pro duced pref ere nt iall y in response to different antigens . A number of studies have shown differing distribu- tion patterns of glomerular IgG subclass deposits in patients with glomerular diseases [3–8]. For example, a predo minan t glomerular depositi on of IgG4 is char- acteristic of idiopathic MN [3,4,6,7,9]. In the case oflupus-associated MN, several reports have shown the glomerular deposition of IgG1, IgG2 and IgG3, as well as IgG4 [6,7]. Furthermore, Nagahama et al.  showed glomerular deposition of IgG2 and/or IgG3, in addi- tion to IgG4, in bucillamine-induced MN. Altogther, these ﬁnd ing s sugges t tha t the pat hog enesis of idi o- pathic MN is different from that of some secondary forms of MN. It has been documented that 10% of MN cases are associ ate d wi th mal ign anc y [10 –12]. In fac t, several investigators have reported both resolution and exacer- bation of malignancy-associated MN (M-MN) during tumour treatment in certain patients [10,13,14]. These ﬁndings suggested a causal relationship between MN and malignancy. In the present study, we examined by immunoﬂuo- rescence (IF) microscopy the distribution of glomerular IgG subclass deposits in 10 patients with M-MN and in 15 pat ients wi th idi opa thi c MN. We fou nd tha t the glomerular IF intensity of IgG1 and IgG2 in the malignancy group was signiﬁcantly stronger than in the idiopathic group. These ﬁndings suggest that M-MN and idiopathic MN result from different immunologi- cal mechanisms. Subjects and methods Patients with biopsy-proven MN were selected for review in this study. The biopsies were performed at Akita University Correspondence and offprint requests to : Hiroshi Ohtani, MD, Third Depa rtment of Internal Medicine, Aki ta Univers ity Scho ol ofMedicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. Email: [email protected]Nephrol Dial Transplant Vol. 19 No. 3 ß ERA–EDTA 2004; all rights reserved by g u esto n A p ril3, 2012http ://n dt. o x fo rdjo u rals. o rg /D o w n lo adedfro m
1The Third Department of Internal Medicine, Akita University School of Medicine, Akita and2The Department of Nephrology and Rheumatology, Aichi Medical University, Aichi, Japan
Background. Several studies have shown a predomi-nant glomerular deposition of IgG4 in patients withidiopathic membranous nephropathy (MN), whereassignificant depositions of other IgG subclasses havebeen shown in patients with lupus-associated MN andbucillamine-induced MN.Methods. We examined the distribution patterns of glomerular IgG subclass deposits in 10 patients withmalignancy-associated MN (M-MN) and in 15 patientswith idiopathic MN by immunofluorescence (IF)microscopy.Results. The glomerular IF intensities of IgG1 andIgG2 were significantly stronger in the malignancy
group than in the idiopathic group (P<0.05). Incontrast, there were no differences in glomerular IFintensities of IgG3 and IgG4 between the two groups.Conclusion. Our findings suggest that the distributionpatterns of glomerular IgG subclass deposits aredifferent in idiopathic MN and M-MN. The strongIF intensity of glomerular IgG1 and IgG2 in M-MNmay provide a possible predictor for this condition.
Membranous nephropathy (MN), the common causeof the adult nephrotic syndrome, is characterized byglomerular subepithelial IgG deposits. Human IgG isdivided into four subclasses (IgG1, IgG2, IgG3 andIgG4) according to antigenic differences in their heavy
chains. Each subclass differs in terms of several aspects:
encoding genes, molecular weights, isoelectric pointand interaction with complements . These IgG sub-classes are produced preferentially in response todifferent antigens .
A number of studies have shown differing distribu-tion patterns of glomerular IgG subclass deposits inpatients with glomerular diseases [3–8]. For example, apredominant glomerular deposition of IgG4 is char-acteristic of idiopathic MN [3,4,6,7,9]. In the case of lupus-associated MN, several reports have shown theglomerular deposition of IgG1, IgG2 and IgG3, as wellas IgG4 [6,7]. Furthermore, Nagahama et al .  showedglomerular deposition of IgG2 and/or IgG3, in addi-
tion to IgG4, in bucillamine-induced MN. Altogther,these findings suggest that the pathogenesis of idio-pathic MN is different from that of some secondaryforms of MN.
It has been documented that 10% of MN cases areassociated with malignancy [10–12]. In fact, severalinvestigators have reported both resolution and exacer-bation of malignancy-associated MN (M-MN) duringtumour treatment in certain patients [10,13,14]. Thesefindings suggested a causal relationship between MNand malignancy.
In the present study, we examined by immunofluo-rescence (IF) microscopy the distribution of glomerularIgG subclass deposits in 10 patients with M-MN and
in 15 patients with idiopathic MN. We found thatthe glomerular IF intensity of IgG1 and IgG2 in themalignancy group was significantly stronger than in theidiopathic group. These findings suggest that M-MNand idiopathic MN result from different immunologi-cal mechanisms.
Subjects and methods
Patients with biopsy-proven MN were selected for review inthis study. The biopsies were performed at Akita University
Correspondence and offprint requests to: Hiroshi Ohtani, MD, ThirdDepartment of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. Email:[email protected]
Hospital and its affiliated hospitals from 1985 to 2002. In all,we found 311 cases with MN. Among them, 15 patients(4.8%) were diagnosed as having M-MN by the following
criteria: (i) the existence of malignancy discovered at thetime of or after the biopsy, as well as in treated cases; or
(ii) the reduction or disappearance of proteinuria aftertherapy for malignancy. We selected 10 of these 15 casesthat had adequate frozen tissues available for stainingglomerular IgG subclass deposits. In addition, 15 cases
were selected randomly from patients with idiopathic MN inwhom malignancy was excluded using basic routine cancerscreening procedures.
Renal biopsy specimens were processed using standard
light and electron microscopy techniques. MN lesions wereclassified into four stages based on microscopy findings. Forthe IF studies, frozen renal tissues were cut into 4mm sectionswith a cryostat, and were stained with fluorescein isothio-
cyanate (FITC)-conjugated rabbit monospecific antibodiesagainst human IgG, IgA and IgM heavy chains, and lightchains, C3, C1q and fibrinogen (DAKO A/S, Glostrup,
Denmark) at a 10-fold dilution. The sections were alsostained with FITC-conjugated mouse monoclonal antibodiesagainst human IgG1 (Fc), IgG2 (Fab), IgG3 (F(ab’)2) and
IgG4 (pFc) (The Binding Site Ltd, Birmingham, UK) at a10-fold dilution. The names of theclones of theses monoclonalantibodies were: anti-human IgG1, 8c/6–39; anti-humanIgG2, HP6014; anti-human IgG3, HP6050; and anti-human
IgG4, HP6023. IF intensity was graded as: negative (score 0),weakly positive (score 0.5), 1þ (score 1.0), 2þ (score 2.0) and3þ (score 3.0). This assessment was performed by three
observers that were blind to the background data. The inter-observer comparisons showed good agreement ( values :0.72, 0.73 and 0.87). When different scores were obtainedfrom the observers, only the majority scores were adopted.
We compared the intensity of IgG subclasses in the M-MNand idiopathic MN groups using Mann–Whitney U -tests,
because the data did not show a Gaussian distribution.
Background of patients
Profiles of the 25 patients in this study are shownin Table 1. Six M-MN patients and eight idiopathicMN patients presented with nephrotic range protein-uria (>3.5 g/day). The time between clinical onset of proteinuria and recognition of the tumour ranged from7 days to 9 months. All patients had solid tumours of various types. In terms of treatment, tumours werecompletely resected in five patients. In six M-MN
patients, proteinuria improved after the treatment of the tumour. The remaining four patients were nottreated because they had multiple metastasis at the timeof diagnosis. Proteinuria persisted in these patients, andall of them died due to the malignancy.
Histological findings for each case in both of the groupsare shown in Table 2. Mesangial proliferation wasabsent or mild in all the patients. In addition, the meanstages of MN did not differ between the two groups.
The IF intensity of IgG, IgA, IgM, , , C3, C1q andfibrinogen was similar in the two groups.
IF intensity of glomerular IgG subclass deposits
IF intensities of glomerular IgG subclass deposits in thetwo groups are shown in Table 3. In addition,representative IF findings are shown in Figures 1 and 2.
In Figure 3, the glomerular IF intensities of each IgGsubclass from the two groups are shown. The intensitiesof IgG1 and IgG2 in the M-MN group were signifi-cantly stronger than in the idiopathic MN group(P<0.05). In further studies, we examined whetherthe glomerular IF intensity of IgG1 and IgG2 could beused as a predictor of M-MN. The potential values of IgG1 and IgG2 glomerular staining for distinguishingM-MN from idiopathic MN are illustrated in Table 4.When scoring levels were set at 1.0 for both IgG1 andIgG2 IF intensities, the sensitivity and specificity were70 and 80%, respectively.
In a previous IF study that used a set of anti-IgGsubclass monoclonal antibodies, we found strikingdifferences in the distribution of glomerular IgGsubclass deposits between patients with membranopro-liferative glomerulonephritis, idiopathic MN and lupusnephritis . The present study used the same set of antibodies and showed differing distribution patternsof glomerular IgG subclass deposits in patients withM-MN and idiopathic MN.
Idiopathic MN is now regarded as an IgG4-mediateddisease . In contrast, there are several reports that
lupus-associated MN [6,7] and bucillamine-inducedMN  are characterized by other IgG subclassglomerular deposits. These observations suggest thatimmune complexes containing other than IgG4 may beimportant factors in certain secondary forms of MN.
Approximately 10% of MN cases are associated withmalignancy, and diagnosis of MN is sometimes thefirst clue to the presence of malignancy [10–12].Brueggemeyer et al .  found that the incidence of malignancy in MN patients was almost five timesgreater than in a control population. Also, the findingsthat tumour treatment produced resolution of thenephrotic syndrome and that relapse occurred followingtumour recurrence or metastasis suggest a causal
relationship between MN and malignancy [10,13,14].Furthermore, tumour-related antigens or antibodies tothese antigens have been detected in the glomerulardeposits in some cases of M-MN [16–18], althoughtumour-specific antigen–antibody complexes have notyet been identified.
To our knowledge, there have been no detailedanalyses of glomerular IgG subclass deposits in M-MN.In the present study, we showed a more dominantglomerular deposition of IgG1 and IgG2 in patientswith M-MN than in those with idiopathic MN. Incontrast, there were no differences in glomerular IF
intensity of IgG4 between the two groups. Interestingly,Ullenhag et al .  demonstrated IgG1, IgG2 andIgG4 responses against vaccination with the recombi-nant carcinoembryonic antigen in combination withgranulocyte–macrophage colony-stimulating factor. A
recent review by Holdsworth et al .  assessed therelevance of Th1/Th2 responses to a variety of antigenswith IgG subclass profiles in mice and humans. HumanIgG1 and IgG2 are Th1-related isotypes [interleukin(IL)-12 and interferon- driven], while IgG4 is a Th2-related isotype (IL-4 driven). In malignant diseases,both the tumour itself and several tumour-relatedproducts can act as antigens. Certain ILs activatedby these various antigens may stimulate IgG1- andIgG2-mediated mechanisms (Th1-type), as well as theIgG4-mediated mechanism (Th2-type).
Several investigators have indicated that there is aclear association between MN and malignancy [10–12].This association is of clinical importance since protein-
uria may be the first sign of occult malignancy.Although the present study had a limited methodo-logical approach and small sample size, the datain Table 4 indicate that the glomerular IF intensity of IgG1 and IgG2 could be used as a possible predictor of M-MN. In addition, it would be prudent to analyseglomerular IgG subclass deposits in patients with MN,especially in those over the age of 40 who have clinicalor laboratory evidence of possible occult malignancy.When a strong glomerular deposition of IgG1 andIgG2 is observed, we recommend careful examinationfor malignancy in such cases.
Table 2. Histological findings in 10 patients with M-MN and 15 patients with idiopathic MN
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Received for publication: 31.3.03Accepted in revised form: 18.9.03
IgG1 IgG2 IgG3 IgG4
M I M I M I M I
Fig. 3. IF intensities of glomerular IgG subclasses in patients withM-MN and idiopathic MN. Box and whisker plots of the data arein Table 3. The lower bar indicates the lower extreme; the upperbar, the upper extreme; the lower line of the box, the lower quartile;the upper line of the box, the upper quartile; and the circle showsthe median. The intensities of IgG1 and IgG2 in the malignancygroup (M) are significantly stronger than in the idiopathic group(I). *P<0.05.
Table 4. IF intensity of glomerular IgG1 and IgG2 deposits in 10patients with M-MN and 15 patients with idiopathic MN