Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy

Hiroshi Ohtani1, Hideki Wakui1, Atsushi Komatsuda1, Shin Okuyama1, Rie Masai1, Nobuki Maki1, Akihiro Kigawa1, Ken-ichi Sawada1 and Hirokazu Imai1,2

1The Third Department of Internal Medicine, Akita University School of Medicine, Akita and 2The Department of Nephrology and Rheumatology, Aichi Medical University, Aichi, Japan

Correspondence and offprint requests to: Hiroshi Ohtani, MD, Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Akita 010-8543, Japan. Email: Ohtanih{at}doc.med.akita-u.ac.jp



   Abstract
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 Abstract
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 Subjects and methods
 Results
 Discussion
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Background. Several studies have shown a predominant glomerular deposition of IgG4 in patients with idiopathic membranous nephropathy (MN), whereas significant depositions of other IgG subclasses have been shown in patients with lupus-associated MN and bucillamine-induced MN.

Methods. We examined the distribution patterns of glomerular IgG subclass deposits in 10 patients with malignancy-associated MN (M-MN) and in 15 patients with idiopathic MN by immunofluorescence (IF) microscopy.

Results. The glomerular IF intensities of IgG1 and IgG2 were significantly stronger in the malignancy group than in the idiopathic group (P<0.05). In contrast, there were no differences in glomerular IF intensities of IgG3 and IgG4 between the two groups.

Conclusion. Our findings suggest that the distribution patterns of glomerular IgG subclass deposits are different in idiopathic MN and M-MN. The strong IF intensity of glomerular IgG1 and IgG2 in M-MN may provide a possible predictor for this condition.

Keywords: IgG subclasses; immune complex; immunofluorescence; malignancy; membranous nephropathy



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Membranous nephropathy (MN), the common cause of the adult nephrotic syndrome, is characterized by glomerular subepithelial IgG deposits. Human IgG is divided into four subclasses (IgG1, IgG2, IgG3 and IgG4) according to antigenic differences in their heavy chains. Each subclass differs in terms of several aspects: encoding genes, molecular weights, isoelectric point and interaction with complements [1]. These IgG subclasses are produced preferentially in response to different antigens [2].

A number of studies have shown differing distribution patterns of glomerular IgG subclass deposits in patients with glomerular diseases [38]. For example, a predominant glomerular deposition of IgG4 is characteristic of idiopathic MN [3,4,6,7,9]. In the case of lupus-associated MN, several reports have shown the glomerular deposition of IgG1, IgG2 and IgG3, as well as IgG4 [6,7]. Furthermore, Nagahama et al. [8] showed glomerular deposition of IgG2 and/or IgG3, in addition to IgG4, in bucillamine-induced MN. Altogther, these findings suggest that the pathogenesis of idiopathic MN is different from that of some secondary forms of MN.

It has been documented that 10% of MN cases are associated with malignancy [1012]. In fact, several investigators have reported both resolution and exacerbation 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 immunofluorescence (IF) microscopy the distribution of glomerular IgG subclass deposits in 10 patients with M-MN and in 15 patients with idiopathic MN. We found that 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 immunological mechanisms.



   Subjects and methods
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Patients with biopsy-proven MN were selected for review in this study. The biopsies were performed at Akita University 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 the time of or after the biopsy, as well as in treated cases; or (ii) the reduction or disappearance of proteinuria after therapy for malignancy. We selected 10 of these 15 cases that had adequate frozen tissues available for staining glomerular IgG subclass deposits. In addition, 15 cases were selected randomly from patients with idiopathic MN in whom malignancy was excluded using basic routine cancer screening procedures.

Renal biopsy specimens were processed using standard light and electron microscopy techniques. MN lesions were classified into four stages based on microscopy findings. For the IF studies, frozen renal tissues were cut into 4 µm sections with a cryostat, and were stained with fluorescein isothiocyanate (FITC)-conjugated rabbit monospecific antibodies against human IgG, IgA and IgM heavy chains, {kappa} and {lambda} light chains, C3, C1q and fibrinogen (DAKO A/S, Glostrup, Denmark) at a 10-fold dilution. The sections were also stained with FITC-conjugated mouse monoclonal antibodies against human IgG1 (Fc), IgG2 (Fab), IgG3 (F(ab')2) and IgG4 (pFc) (The Binding Site Ltd, Birmingham, UK) at a 10-fold dilution. The names of the clones of theses monoclonal antibodies were: anti-human IgG1, 8c/6–39; anti-human IgG2, HP6014; anti-human IgG3, HP6050; and anti-human IgG4, HP6023. IF intensity was graded as: negative (score 0), weakly positive (score 0.5), 1+ (score 1.0), 2+ (score 2.0) and 3+ (score 3.0). This assessment was performed by three observers that were blind to the background data. The inter-observer comparisons showed good agreement ({kappa} values [15]: 0.72, 0.73 and 0.87). When different scores were obtained from the observers, only the majority scores were adopted.

We compared the intensity of IgG subclasses in the M-MN and idiopathic MN groups using Mann–Whitney U-tests, because the data did not show a Gaussian distribution.



   Results
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Background of patients
Profiles of the 25 patients in this study are shown in Table 1. Six M-MN patients and eight idiopathic MN patients presented with nephrotic range proteinuria (>3.5 g/day). The time between clinical onset of proteinuria and recognition of the tumour ranged from 7 days to 9 months. All patients had solid tumours of various types. In terms of treatment, tumours were completely resected in five patients. In six M-MN patients, proteinuria improved after the treatment of the tumour. The remaining four patients were not treated because they had multiple metastasis at the time of diagnosis. Proteinuria persisted in these patients, and all of them died due to the malignancy.


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Table 1. Profiles of 10 patients with M-MN and 15 patients with idiopathic MN

 
Histological findings
Histological findings for each case in both of the groups are shown in Table 2. Mesangial proliferation was absent or mild in all the patients. In addition, the mean stages of MN did not differ between the two groups. The IF intensity of IgG, IgA, IgM, {kappa}, {lambda}, C3, C1q and fibrinogen was similar in the two groups.


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Table 2. Histological findings in 10 patients with M-MN and 15 patients with idiopathic MN

 
IF intensity of glomerular IgG subclass deposits
IF intensities of glomerular IgG subclass deposits in the two groups are shown in Table 3. In addition, representative IF findings are shown in Figures 1 and 2.


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Table 3. IF intensity of glomerular IgG subclass deposits in 10 patients with M-MN and 15 patients with idiopathic MN

 


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Fig. 1. IF study of a patient with M-MN (case 3). IgG1, IgG2 and IgG4 were graded as 2+ (score: 2.0), and IgG3 was 1+ (score: 1.0).

 


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Fig. 2. IF study of a patient with idiopathic MN (case 19). IgG4 was graded as 3+ (score: 3.0), and IgG1, IgG2 and IgG3 were all weakly positive (score: 0.5).

 
In Figure 3, the glomerular IF intensities of each IgG subclass from the two groups are shown. The intensities of IgG1 and IgG2 in the M-MN group were significantly stronger than in the idiopathic MN group (P<0.05). In further studies, we examined whether the glomerular IF intensity of IgG1 and IgG2 could be used as a predictor of M-MN. The potential values of IgG1 and IgG2 glomerular staining for distinguishing M-MN from idiopathic MN are illustrated in Table 4. When scoring levels were set at >=1.0 for both IgG1 and IgG2 IF intensities, the sensitivity and specificity were 70 and 80%, respectively.



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Fig. 3. IF intensities of glomerular IgG subclasses in patients with M-MN and idiopathic MN. Box and whisker plots of the data are in Table 3. The lower bar indicates the lower extreme; the upper bar, the upper extreme; the lower line of the box, the lower quartile; the upper line of the box, the upper quartile; and the circle shows the median. The intensities of IgG1 and IgG2 in the malignancy group (M) are significantly stronger than in the idiopathic group (I). *P<0.05.

 

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Table 4. IF intensity of glomerular IgG1 and IgG2 deposits in 10 patients with M-MN and 15 patients with idiopathic MN

 


   Discussion
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In a previous IF study that used a set of anti-IgG subclass monoclonal antibodies, we found striking differences in the distribution of glomerular IgG subclass deposits between patients with membranoproliferative glomerulonephritis, idiopathic MN and lupus nephritis [7]. The present study used the same set of antibodies and showed differing distribution patterns of glomerular IgG subclass deposits in patients with M-MN and idiopathic MN.

Idiopathic MN is now regarded as an IgG4-mediated disease [9]. In contrast, there are several reports that lupus-associated MN [6,7] and bucillamine-induced MN [8] are characterized by other IgG subclass glomerular deposits. These observations suggest that immune complexes containing other than IgG4 may be important factors in certain secondary forms of MN.

Approximately 10% of MN cases are associated with malignancy, and diagnosis of MN is sometimes the first clue to the presence of malignancy [1012]. Brueggemeyer et al. [11] found that the incidence of malignancy in MN patients was almost five times greater than in a control population. Also, the findings that tumour treatment produced resolution of the nephrotic syndrome and that relapse occurred following tumour recurrence or metastasis suggest a causal relationship between MN and malignancy [10,13,14]. Furthermore, tumour-related antigens or antibodies to these antigens have been detected in the glomerular deposits in some cases of M-MN [1618], although tumour-specific antigen–antibody complexes have not yet been identified.

To our knowledge, there have been no detailed analyses of glomerular IgG subclass deposits in M-MN. In the present study, we showed a more dominant glomerular deposition of IgG1 and IgG2 in patients with M-MN than in those with idiopathic MN. In contrast, there were no differences in glomerular IF intensity of IgG4 between the two groups. Interestingly, Ullenhag et al. [19] demonstrated IgG1, IgG2 and IgG4 responses against vaccination with the recombinant carcinoembryonic antigen in combination with granulocyte–macrophage colony-stimulating factor. A recent review by Holdsworth et al. [20] assessed the relevance of Th1/Th2 responses to a variety of antigens with IgG subclass profiles in mice and humans. Human IgG1 and IgG2 are Th1-related isotypes [interleukin (IL)-12 and interferon-{gamma} driven], while IgG4 is a Th2-related isotype (IL-4 driven). In malignant diseases, both the tumour itself and several tumour-related products can act as antigens. Certain ILs activated by these various antigens may stimulate IgG1- and IgG2-mediated mechanisms (Th1-type), as well as the IgG4-mediated mechanism (Th2-type).

Several investigators have indicated that there is a clear association between MN and malignancy [1012]. This association is of clinical importance since proteinuria may be the first sign of occult malignancy. Although the present study had a limited methodological approach and small sample size, the data in 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 analyse glomerular IgG subclass deposits in patients with MN, especially in those over the age of 40 who have clinical or laboratory evidence of possible occult malignancy. When a strong glomerular deposition of IgG1 and IgG2 is observed, we recommend careful examination for malignancy in such cases.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 31. 3.03
Accepted in revised form: 18. 9.03





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