Goodpasture's syndrome has been noted to occur more frequently in smokers (Salama et al., 2001). Lazor et al. (2007) reported that 89% of their patients with Goodpasture's syndrome were active smokers. In another study, alveolar hemorrhage was present in 100% of patients who smoked and in only 20% of nonsmokers with Goodpasture's disease (Donaghy & Rees, 1983). No significant difference in circulating anti-GBM antibody titers was found between smokers and nonsmokers, suggesting that cigarette smoking may increase the permeability of lung capillaries and thus expose alveolar basement membranes to circulating anti-GBM antibodies (Donaghy & Rees, 1983; Klasa et al., 1988). Other inhaled substances may also be associated with anti-GBM disease, including cocaine (García-Rostan y Pérez et al., 1997; Lazor et al., 2007), hard metals such as inert tungsten carbide and cobalt (Lechleitner et al., 1993), smoke inhalation (Klasa et al., 1988) and possibly volatile hydrocarbon solvents (Beirne & Brennan, 1972; Bombassei & Kaplan, 1992). In particular, hydrocarbon exposure may influence the development of alveolar hemorrhage (Churchill et al., 1983; Bonzel et al., 1987). Another environmental factor, alemtuzumab, which is a humanized anti-CD52 monoclonal antibody, recently was identified as a cause of anti-GBM disease (Clatworthy et

Genetic factors appear to play a role in susceptibility to anti-GBM disease. As a genetic factor of anti-GBM disease, the human leukocyte antigen (HLA) complexes are known to influence susceptibility to anti-GBM disease. A strong association with HLA-DR2 specificity has been confirmed (Rees et al., 1978). In HLA genotyping, DRB1\*1501 (the serologically defined HLA-DR2 gene) and DRB1\*1502 (HLA-DR15 gene) allele at the DRB1 locus is associated with anti-GBM disease in Caucasians (Fisher et al., 1997), Chinese (Yang et al., 2009) and Japanese (Kitagawa et al., 2008). The strongest association was with HLA DRB1\*1501 but, when the effect of this gene was excluded, subsequent analysis revealed an increased frequency of DRB1\*04 and DRB1\*03 and a decreased frequency of DRB1\*07 and DRB1\*01 (Phelps & Rees, 1999). Other genetic influences of anti-GBM disease have been identified, including the immunoglobulin heavy chain Gm locus that encodes the constant region of the IgG heavy chain (Rees et al., 1984), polymorphisms of FCGR genes that encode the Fc receptor for IgG (FcγR) (Zhou et al., 2010a, 2010b) and kallikrein genes (Liu et al.,

General malaise (fatigue), weight loss, fever, arthralgia or myalgia may be the initial features of anti-GBM disease in a pattern similar to but much less prominent than that in systemic vasculitis. Symptoms relating to anemia may also occur even in the absence of significant

**3.4 Environmental and genetic factors**

al., 2008).

2009).

hemoptysis.

**4. Clinical symptoms**

anti-GBM antibody production (Blue & Lange, 1975).

The frequencies of patients with each symptom are expressed as percentages. \*All investigated patients had Goodpasture's syndrome. Abbreviations: N.A., not available.

Table 2. Investigations of clinical symptoms in anti-GBM disease at the initial presentation.

The principal clinical features relate to the development of renal failure due to RPGN or alveolar hemorrhage (Table 2). Hemoptysis is the predominant symptom of alveolar hemorrhage. Alveolar hemorrhage may cause severe impairment of oxygenation, so intensive care and artificial ventilation are sometimes needed. The mild lung symptoms are only dry cough and shortness of breath. Although one-third to two-thirds of patients with anti-GBM disease demonstrate alveolar hemorrhage in general, in our survey, 23.4% (11/47) of patients with anti-GBM disease suffered from alveolar hemorrhage (Hirayama et al., 2008). A minority of patients exhibited macrohematuria. Anirua or oliguria was seen in 17- 62% of patients at presentation, and these findings suggested a poorer prognosis (Levy et al., 2001; Hudson et al., 2003).

### **5. Laboratory examinations**

In general, all patients with anti-GBM disease had microscopic hematuria on urinalysis. Proteinuria is modest, but can be heavier when the disease has a more subacute course. In our survey (Hirayama et al., 2008), the mean 24-hour excretion of urinary protein in renallimited anti-GBM disease was 2.1±3.0 g and that of Goodpasture's syndrome was 3.7±3.2 g.

Anti-Glomerular Basement Membrane Disease 259

was 82±45 mm/h. The mean serum C-reactive protein (CRP) level in renal-limited anti-GBM disease was 8.5±7.2 mg/dl, and that in Goodpasture's syndrome was 8.2±8.1 mg/dl. In comparison with other forms of RPGN, such as micropolyangiitis (MPA) and Wegener's granulomatosis (WG), there was no difference in inflammation markers such as leukocyte count, ESR and serum CRP. However, in patients with anti-GBM disease, the mean level of s-Cr at the time of diagnosis was higher than that in patients with MPA (4.54±3.13 mg/dl) or WG (3.84±3.24 mg/dl). Therefore, early diagnosis of anti-GBM disease is very important. Although obvert hemoptysis may not be immediately present in patients with Goodpasture's syndrome and alveolar hemorrhage may not be immediately obvious in radiological examinations, an elevated alveolo-arterial oxygen difference (AaPO2) can be a sensitive indicator of alveolar hemorrhage. An elevated red blood cell count in bronchoalveolar lavages, as detected by bronchoscopy, is useful information for the diagnosis of alveolar hemorrhage,

The diagnosis of anti-GBM disease is dependent on the detection of anti-GBM antibodies in either the circulation or the kidney tissue. These serum antibodies are usually detected using an enzyme-linked immunosorbent assay or radioimmunoassay method. The antibodies have not been reported to occur in the absence of disease, and false negatives are rare when appropriate checks are performed. In our survey (Hirayama et al., 2008), 91.5% (43/47) of patients with anti-GBM disease were diagnosed via the detection of serum anti-GBM

In serological examinations, other autoantibodies were not usually detected. However, in our survey (Hirayama et al., 2008), anti-nuclear antibodies were detected in 11.8% of renallimited anti-GBM disease and in 27.3% of patients with Goodpasture's syndrome. Anti-DNA antibody was not detected in renal-limited anti-GBM disease, but it was detected in 22.2% of patients with Goodpasture's syndrome. Moreover, anti-neutrophil cytoplasmic antibodies (ANCA) were detected in 12.8% (5/39) of patients with anti-GBM disease; a perinuclear pattern was detected in all five anti-GBM disease patients with ANCA, and a cytoplasmic pattern was detected in one. Anti-GBM antibody and ANCA coexisted in 15 - 50% of cases of anti-GBM disease described in the previous literature (Jayne et al., 1990; Bosch et al., 1991; Yang et al., 2005; Rutgers et al., 2005; Levy et al., 2004). Other studies revealed that patients with double-positive antibodies were predominantly MPO-ANCA, older and male (Jayne et al., 1990; Bosch et al., 1991; Yang et al., 2005; Rutgers et al., 2005). In our survey (Hirayama et al., 2008), the age at onset of patients with double-positive antibodies was higher (mean age, 52.6 years), but female-dominant (male : female = 1 : 4). The prognosis of patients with double-positive antibodies varied; the renal and patient survival rates of patients with double-positive antibodies were reported to be either better (Jayne et al., 1990; Bosch et al., 1991), not significantly different (Yang et al., 2005), or worse (Rutgers et al., 2005; Levy et al., 2004) than those of patients with anti-GBM antibody alone. In our survey (Hirayama et al., 2008), the prognosis of patients with double-positive antibodies was poor; two died and the remaining three required maintenance hemodialysis. Alveolar hemorrhage was observed in two of five patients with double-positive antibodies,

Kidneys were usually normal-sized or enlarged due to inflammation. In our survey (Hirayama et al., 2008), ultrasonography showed that 61.0% of patients with anti-GBM

but lung biopsy does not contribute to this diagnosis (Lazor et al., 2007).

and three of them had interstitial pneumonitis.

**6. Imaging examinations**

antibodies.

Amounts of urinary protein and serum creatinine levels are expressed as means ± standard deviation or medians with ranges. Frequency of end-stage renal failure at presentation is expressed as a percentage. To convert serum creatinine in mg/dL to μmol/L, multiply by 88.4. Abbreviations: ESRD, end-stage renal disease; N.A., not available.

Table 3. Investigations of renal findings in anti-GBM disease at the initial presentation.

Unfortunately, most patients with anti-GBM disease had renal failure at the time of diagnosis, and the number of patients needing dialysis was not a few (Table 3). In our survey (Hirayama et al., 2008), the mean serum creatinine (s-Cr) level in renal-limited anti-GBM disease was 7.07±4.21 mg/dl, while that in Goodpasture's syndrome was 7.99±4.31 mg/dl. Hemodialysis therapy had already been initiated in 59.6% (28/47) of the anti-GBM disease patients before the start of immunosuppressive treatments.

Anemia was observed in most patients with anti-GBM disease, and the mean hemoglobin concentration in renal-limited anti-GBM disease was 8.8±1.7 g/dl, while that in Goodpasture's syndrome was 7.5±1.1 g/dl. The mean erythrocyte sedimentation rate (ESR) in renal-limited anti-GBM disease was 105±44 mm/h, and that in Goodpasture's syndrome

Shah et al. 2002 Various N.A. 6.62 35%

Herody et al. 1993 France N.A. N.A. 55%

Andrassy et al. 1991 Germany 6.4 12.8 67%

Merkel et al. 1994 Germany N.A. 11.41 ± 5.64 71%

Daly et al. 1996 Ireland N.A. 5.1 ± 6.8 50%

Segelmark et al. 2003 Sweden N.A. 8.94 46%

Savage et al. 1986 United Kingdom N.A. N.A. 64%

Williams et al. 1988 United Kingdom N.A. 11.80 ± 4.67 70%

Levy et al. 2001 United Kingdom N.A. 3.59 55%

Briggs et al. 1979 United States 2.6 ± 0.5 5.94 ± 7.11 33%

Johnson et al. 1985 United States 4.3 ± 5.2 4.87 ± 6.93 12%

Jennette 2003 United States 1.67 ± 3.35 9.7 ± 7.2 N.A. (0.20- 16.20) (0.8 - 50) Walker et al. 1985 Australia 1.4 6.56 45%

Simpson et al. 1982 New Zealand N.A. 5.37 ± 5.22 10%

Teague et al. 1978 New Zealand N.A. N.A. 14%

Cui et al. 2005 China N.A. N.A. 71%

Hirayama et al 2008 Japan 2.4 ± 3.0 7.29 ± 4.19 60%

Amounts of urinary protein and serum creatinine levels are expressed as means ± standard deviation or medians with ranges. Frequency of end-stage renal failure at presentation is expressed as a percentage. To convert serum creatinine in mg/dL to μmol/L, multiply by 88.4. Abbreviations: ESRD, end-stage

Unfortunately, most patients with anti-GBM disease had renal failure at the time of diagnosis, and the number of patients needing dialysis was not a few (Table 3). In our survey (Hirayama et al., 2008), the mean serum creatinine (s-Cr) level in renal-limited anti-GBM disease was 7.07±4.21 mg/dl, while that in Goodpasture's syndrome was 7.99±4.31 mg/dl. Hemodialysis therapy had already been initiated in 59.6% (28/47) of the anti-GBM

Anemia was observed in most patients with anti-GBM disease, and the mean hemoglobin concentration in renal-limited anti-GBM disease was 8.8±1.7 g/dl, while that in Goodpasture's syndrome was 7.5±1.1 g/dl. The mean erythrocyte sedimentation rate (ESR) in renal-limited anti-GBM disease was 105±44 mm/h, and that in Goodpasture's syndrome

Table 3. Investigations of renal findings in anti-GBM disease at the initial presentation.

disease patients before the start of immunosuppressive treatments.

(Hong Kong) N.A. 6.96 ± 6.41 50%

Serum creatinine (mg/dL)

1.2 1.27 41% (0 – 35.0) (0.61 – 21.47) (11/28)

(0 – 15.3) (6.1 – 16.5) (2/3)

(0.2 – 3.5) (0.19 – 22.96) (20/28)

(1.9 – 3.5) (0.8 – 30.0) (6/18)

(0 – 22.0) (0.9 - 25.0) (2/17)

(0.4 – 5.4) (1.24 – 32.35) (10/22)

(0.1 – 12.2) (1.00 – 16.80) (28/47)

(N.A.) (27/78)

(N.A.) (20/40)

(5.44 – 12.34) (36/79)

(1.60 – 18.37) (7/10)

(0.6 – 10.9) (39/71)

(0.68 – 19.80) (2/20)

(1.19 – 22.09) (5/10)

ESRD (%)

(16/29)

(69/108)

(4/29)

(69/97)

Authors Year Nation Urinary protein (g/day)

Switzerland

Lazor et al. 2007 France &

Li et al. 2004 China

renal disease; N.A., not available.

was 82±45 mm/h. The mean serum C-reactive protein (CRP) level in renal-limited anti-GBM disease was 8.5±7.2 mg/dl, and that in Goodpasture's syndrome was 8.2±8.1 mg/dl. In comparison with other forms of RPGN, such as micropolyangiitis (MPA) and Wegener's granulomatosis (WG), there was no difference in inflammation markers such as leukocyte count, ESR and serum CRP. However, in patients with anti-GBM disease, the mean level of s-Cr at the time of diagnosis was higher than that in patients with MPA (4.54±3.13 mg/dl) or WG (3.84±3.24 mg/dl). Therefore, early diagnosis of anti-GBM disease is very important.

Although obvert hemoptysis may not be immediately present in patients with Goodpasture's syndrome and alveolar hemorrhage may not be immediately obvious in radiological examinations, an elevated alveolo-arterial oxygen difference (AaPO2) can be a sensitive indicator of alveolar hemorrhage. An elevated red blood cell count in bronchoalveolar lavages, as detected by bronchoscopy, is useful information for the diagnosis of alveolar hemorrhage, but lung biopsy does not contribute to this diagnosis (Lazor et al., 2007).

The diagnosis of anti-GBM disease is dependent on the detection of anti-GBM antibodies in either the circulation or the kidney tissue. These serum antibodies are usually detected using an enzyme-linked immunosorbent assay or radioimmunoassay method. The antibodies have not been reported to occur in the absence of disease, and false negatives are rare when appropriate checks are performed. In our survey (Hirayama et al., 2008), 91.5% (43/47) of patients with anti-GBM disease were diagnosed via the detection of serum anti-GBM antibodies.

In serological examinations, other autoantibodies were not usually detected. However, in our survey (Hirayama et al., 2008), anti-nuclear antibodies were detected in 11.8% of renallimited anti-GBM disease and in 27.3% of patients with Goodpasture's syndrome. Anti-DNA antibody was not detected in renal-limited anti-GBM disease, but it was detected in 22.2% of patients with Goodpasture's syndrome. Moreover, anti-neutrophil cytoplasmic antibodies (ANCA) were detected in 12.8% (5/39) of patients with anti-GBM disease; a perinuclear pattern was detected in all five anti-GBM disease patients with ANCA, and a cytoplasmic pattern was detected in one. Anti-GBM antibody and ANCA coexisted in 15 - 50% of cases of anti-GBM disease described in the previous literature (Jayne et al., 1990; Bosch et al., 1991; Yang et al., 2005; Rutgers et al., 2005; Levy et al., 2004). Other studies revealed that patients with double-positive antibodies were predominantly MPO-ANCA, older and male (Jayne et al., 1990; Bosch et al., 1991; Yang et al., 2005; Rutgers et al., 2005). In our survey (Hirayama et al., 2008), the age at onset of patients with double-positive antibodies was higher (mean age, 52.6 years), but female-dominant (male : female = 1 : 4). The prognosis of patients with double-positive antibodies varied; the renal and patient survival rates of patients with double-positive antibodies were reported to be either better (Jayne et al., 1990; Bosch et al., 1991), not significantly different (Yang et al., 2005), or worse (Rutgers et al., 2005; Levy et al., 2004) than those of patients with anti-GBM antibody alone. In our survey (Hirayama et al., 2008), the prognosis of patients with double-positive antibodies was poor; two died and the remaining three required maintenance hemodialysis. Alveolar hemorrhage was observed in two of five patients with double-positive antibodies, and three of them had interstitial pneumonitis.

#### **6. Imaging examinations**

Kidneys were usually normal-sized or enlarged due to inflammation. In our survey (Hirayama et al., 2008), ultrasonography showed that 61.0% of patients with anti-GBM

Anti-Glomerular Basement Membrane Disease 261

Fig. 3. Periodic acid-Schiff methenamine silver (PAM)−stained glomerulus in a patient with

The disruption of the capillary walls, segmental necrosis and cellular crescent formation are observed. Rupture of the basement membrane of Bowman's capsule and periglomerular infiltration of inflammatory cells are also observed. Interstitial edema with infiltration of

Various degrees of crescent formation are observed in more than 90% of patients with anti-GBM disease. In Europe, the United States and Asian-Pacific, including Japan, the mean percentage of glomeruli showing crescent formation ranged from 40% to 100%, and about 70% to 100% of patients with anti-GBM disease had more than 50% crescentic glomeruli (Figure 4). Anti-GBM disease is pathologically the most severe form of glomerulonephritis

Although tubules are usually normal, epithelial flattening is revealed in the severe acute phase. In the chronic phase, tubules in the area of severe injury undergo atrophy and some disappear. Acute tubulitis sometimes occurs if there is a linear staining of tubular basement membranes for IgG. Interstitial edema with infiltration of inflammatory cells is predominant in the acute phase, whereas interstitial fibrosis is revealed in the chronic phase. Interstitial infiltrates are composed of neutrophils, eosinophils, lymphocytes, monocytes and macrophages. If Bowman's capsules are disrupted, inflammatory cells infiltrate around glomeruli and have a granulomatous appearance. Acute inflammation of renal vessels,

(Holdsworth et al., 1985; Jennette, 2003, Hirayama et al., 2008).

anti-GBM disease.

inflammatory cells is revealed.

disease had kidneys of normal size, while atrophic kidneys were observed in 12.2% of patients and enlarged kidneys were observed in 26.8%. There were no specific morphological abnormalities on any type of renal imaging examinations.

In cases with Goodpasture's syndrome, shadows usually involve the central lung fields with peripheral and upper-lobe sparing on chest radiography or computed tomography (Figure 2). Although the shadows are generally symmetrical, they can be markedly asymmetrical.

Fig. 2. Chest computed tomography in a patient with Goodpasture's syndrome.

Symmetrical shadows involved the central lung fields with peripheral sparing. Bilateral pleural effusions due to hypervolemia in acute kidney injury were also observed.

### **7. Pathological findings**

A renal biopsy is essential in suspected anti-GBM disease both to confirm the diagnosis and to assess the renal prognosis. Glomerular fibrinoid necrosis and crescent formation with linear staining of the glomerular capillary walls for IgG are the histological hallmarks of anti-GBM disease.

#### **7.1 Light microscopic findings**

The histological pattern of disease starts with mesangial expansion and hypercellularity. It progresses to focal and segmental glomerulonephritis with infiltration by inflammatory cells, accompanied by segmental necrosis with prominent breaks in the GBM. Later, glomeruli develop an extensive crescent formation composed of parietal epithelial cells and macrophages in association with the destruction of the GBM (Figure 3). The crescents are usually at the same stage of evolution.

disease had kidneys of normal size, while atrophic kidneys were observed in 12.2% of patients and enlarged kidneys were observed in 26.8%. There were no specific

In cases with Goodpasture's syndrome, shadows usually involve the central lung fields with peripheral and upper-lobe sparing on chest radiography or computed tomography (Figure 2). Although the shadows are generally symmetrical, they can be markedly

morphological abnormalities on any type of renal imaging examinations.

Fig. 2. Chest computed tomography in a patient with Goodpasture's syndrome.

pleural effusions due to hypervolemia in acute kidney injury were also observed.

Symmetrical shadows involved the central lung fields with peripheral sparing. Bilateral

A renal biopsy is essential in suspected anti-GBM disease both to confirm the diagnosis and to assess the renal prognosis. Glomerular fibrinoid necrosis and crescent formation with linear staining of the glomerular capillary walls for IgG are the histological hallmarks of

The histological pattern of disease starts with mesangial expansion and hypercellularity. It progresses to focal and segmental glomerulonephritis with infiltration by inflammatory cells, accompanied by segmental necrosis with prominent breaks in the GBM. Later, glomeruli develop an extensive crescent formation composed of parietal epithelial cells and macrophages in association with the destruction of the GBM (Figure 3). The crescents are

asymmetrical.

**7. Pathological findings**

**7.1 Light microscopic findings**

usually at the same stage of evolution.

anti-GBM disease.

Fig. 3. Periodic acid-Schiff methenamine silver (PAM)−stained glomerulus in a patient with anti-GBM disease.

The disruption of the capillary walls, segmental necrosis and cellular crescent formation are observed. Rupture of the basement membrane of Bowman's capsule and periglomerular infiltration of inflammatory cells are also observed. Interstitial edema with infiltration of inflammatory cells is revealed.

Various degrees of crescent formation are observed in more than 90% of patients with anti-GBM disease. In Europe, the United States and Asian-Pacific, including Japan, the mean percentage of glomeruli showing crescent formation ranged from 40% to 100%, and about 70% to 100% of patients with anti-GBM disease had more than 50% crescentic glomeruli (Figure 4). Anti-GBM disease is pathologically the most severe form of glomerulonephritis (Holdsworth et al., 1985; Jennette, 2003, Hirayama et al., 2008).

Although tubules are usually normal, epithelial flattening is revealed in the severe acute phase. In the chronic phase, tubules in the area of severe injury undergo atrophy and some disappear. Acute tubulitis sometimes occurs if there is a linear staining of tubular basement membranes for IgG. Interstitial edema with infiltration of inflammatory cells is predominant in the acute phase, whereas interstitial fibrosis is revealed in the chronic phase. Interstitial infiltrates are composed of neutrophils, eosinophils, lymphocytes, monocytes and macrophages. If Bowman's capsules are disrupted, inflammatory cells infiltrate around glomeruli and have a granulomatous appearance. Acute inflammation of renal vessels,

Anti-Glomerular Basement Membrane Disease 263

Fig. 5. Direct immunofluorescence for IgG in a patient with anti-GBM disease.

the part of the cellular crescent is not.

**7.3 Electroscopic findings**

crescentic glomerulonephritis.

**8.1 Therapeutic plasmapheresis**

antibodies by the use of immunosuppressive agents.

**8. Treatments**

Linear staining for IgG along glomerular capillary walls is observed, but staining for IgG at

The rupture of GBM with variable degrees of endothelial swelling and lucent expansion of the subendothelial zone is common urtrasturactural findings in the acute phase of anti-GBM disease. Rupture of Bowman's capsule, focal effacement of epithelial foot processes and accumulation of epithelial cells and macrophages in Bowman's spaces are also observed. Occasionally, neutrophils are identified in capillaries, especially at sites where GBM is disrupted. Those findings are also observed in pauci-immune crescentic glomerulonephritis, but electron-dense deposits are absent, unlike the case with immune complex-type

As the pathogenesis of anti-GBM disease became clear, treatment regimens were designed to remove the circulating pathogenic anti-GBM antibodies by therapeutic plasma exchange, attenuate the pathogenic antibody-mediated glomerular inflammatory responses by administration of corticosteroids and suppress further production of these pathogenic

To remove the circulating pathogenic anti-GBM antibodies, therapeutic plasma exchange is recommended as the initial treatment. The effectiveness of therapeutic plasmapheresis for improving renal function has been reported. In the most commonly used regimens, plasma exchange of 4 L of plasma for 5% human albumin was performed daily for 14 days or until

except for glomerular capillaries, is not typical for anti-GBM disease, unless the case has concurrent ANCA (Bosch et al., 1991).

Fig. 4. Previous investigations of crescent formation in anti-GBM disease and ANCAassociated vasculitis.

Each bar shows the frequency of patients with 50% or more crescents in anti-GBM disease (purple) and ANCA-associated vasculitis (blue). The numbers show the mean percentage of glomeruli showing crescent formation in anti-GBM disease. Abbreviations: UK, United Kingdom; USA, United States; NZ, New Zealand.

#### **7.2 Immunofluorescence findings**

The immunohistologic feature of anti-GBM disease is linear staining of the glomerular capillary walls for IgG (Figure 5). IgG1 is the predominant IgG subclass in staining of the glomerular capillary walls (Bowman et al., 1987; Segelmark et al., 1990). Linear staining for IgM and IgA is less common, but rare cases with anti-GBM disease have linear staining only for IgA and circulating IgA-class anti-GBM antibodies in the absence of IgG-class anti-GBM antibodies in the serum or staining in glomeruli (Border et al., 1979; Gris et al., 1991; Borza et al., 2005). Granular or discontinuous linear staining for C3 is observed in most cases with anti-GBM disease, but glomerular staining for C3 is negative for some cases (Wilson and Dixon, 1973). Irregular staining for fibrin is observed in portions of glomerular necrosis and cellular crescents.

Linear staining of tubular basement membranes for IgG sometimes occurs (Lehman et al., 1975; Andres et al., 1978).

except for glomerular capillaries, is not typical for anti-GBM disease, unless the case has

Fig. 4. Previous investigations of crescent formation in anti-GBM disease and ANCA-

Each bar shows the frequency of patients with 50% or more crescents in anti-GBM disease (purple) and ANCA-associated vasculitis (blue). The numbers show the mean percentage of glomeruli showing crescent formation in anti-GBM disease. Abbreviations: UK, United

The immunohistologic feature of anti-GBM disease is linear staining of the glomerular capillary walls for IgG (Figure 5). IgG1 is the predominant IgG subclass in staining of the glomerular capillary walls (Bowman et al., 1987; Segelmark et al., 1990). Linear staining for IgM and IgA is less common, but rare cases with anti-GBM disease have linear staining only for IgA and circulating IgA-class anti-GBM antibodies in the absence of IgG-class anti-GBM antibodies in the serum or staining in glomeruli (Border et al., 1979; Gris et al., 1991; Borza et al., 2005). Granular or discontinuous linear staining for C3 is observed in most cases with anti-GBM disease, but glomerular staining for C3 is negative for some cases (Wilson and Dixon, 1973). Irregular staining for fibrin is observed in portions of glomerular necrosis and

Linear staining of tubular basement membranes for IgG sometimes occurs (Lehman et al.,

79

74

77

78

79

56

0 10 20 30 40 50 60 70 80 90 100

41

66

87

100

concurrent ANCA (Bosch et al., 1991).

Hirayama et al. (2008, Japan) Cui et al. (2005, China) Li et al. (2004, China) Simpson et al. (1982, NZ) Teague et al. (1978, NZ) Walker et al. (1985, Australia) Holdsworth et al. (1985, Australia)

**7.2 Immunofluorescence findings**

Kingdom; USA, United States; NZ, New Zealand.

associated vasculitis.

cellular crescents.

1975; Andres et al., 1978).

Fischer et al. (2006, USA) Jennette (2003, USA) Johnson et al. (1985, USA) Briggs et al. (1979, USA) Levy et al. (2001, UK) Williams et al. (1988, UK) Savage et al. (1986, UK) Segelmark et al. (2003 Sweden) Daly et al. (1996, Ireland) Merkel et al. (1994, Germany) Herody et al. (1993, France) Shah et al. (2002, various)

Fig. 5. Direct immunofluorescence for IgG in a patient with anti-GBM disease.

Linear staining for IgG along glomerular capillary walls is observed, but staining for IgG at the part of the cellular crescent is not.

### **7.3 Electroscopic findings**

The rupture of GBM with variable degrees of endothelial swelling and lucent expansion of the subendothelial zone is common urtrasturactural findings in the acute phase of anti-GBM disease. Rupture of Bowman's capsule, focal effacement of epithelial foot processes and accumulation of epithelial cells and macrophages in Bowman's spaces are also observed. Occasionally, neutrophils are identified in capillaries, especially at sites where GBM is disrupted. Those findings are also observed in pauci-immune crescentic glomerulonephritis, but electron-dense deposits are absent, unlike the case with immune complex-type crescentic glomerulonephritis.

### **8. Treatments**

As the pathogenesis of anti-GBM disease became clear, treatment regimens were designed to remove the circulating pathogenic anti-GBM antibodies by therapeutic plasma exchange, attenuate the pathogenic antibody-mediated glomerular inflammatory responses by administration of corticosteroids and suppress further production of these pathogenic antibodies by the use of immunosuppressive agents.

#### **8.1 Therapeutic plasmapheresis**

To remove the circulating pathogenic anti-GBM antibodies, therapeutic plasma exchange is recommended as the initial treatment. The effectiveness of therapeutic plasmapheresis for improving renal function has been reported. In the most commonly used regimens, plasma exchange of 4 L of plasma for 5% human albumin was performed daily for 14 days or until

Anti-Glomerular Basement Membrane Disease 265

to the undetectable range. However, these treatments cannot yet be recommended as a first-

Most patients without treatment died shortly after diagnosis of anti-GBM disease; the survival rate at 12 months was 4%, and the renal survival rate was 2% (Benoit et al., 1963). Although mortality has improved by the introduction of intense immunosuppression, renal survival remains very poor because of the delayed diagnosis of anti-GBM disease or delayed

The prognosis for patients with anti-GBM disease is poor; the survival rate at 6-12 months was 67-94%, but the renal survival rate was 15-58% in Europe, the United States, China and

Herody et al. 1993 France OCS+CYC+AZA 29 50 93 41

Merkel et al. 1994 Germany OCS+CYC+PE 35 57 89 29 Daly et al. 1996 Ireland IS+PE 40 67 98 20 Segelmark et al. 2003 Sweden OCS+CYC+PE 79 24 66 25 Peters et al. 1982 United Kingdom IS+PE 41 56 76 39 Savage et al. 1986 United Kingdom IS+PE 108 52 78 20 Levy et al. 2001 United Kingdom OCS+CYC+PE 71 62 77 53 Proskey et al. 1970 United States IS 56 100 77 23 Wilson et al. 1973 United States IS 53 60 53 13 Beirne et al. 1977 United States IS 29 54 42 17 Briggs et al. 1979 United States IS(+PE) 18 61 84 22 Johnson et al 1985 United States OCS+CYC 9 78 89 22

Walker et al. 1985 Australia IS+PE 22 62 59 45 Teague et al. 1978 New Zealand IS+PE 29 100 64 31

Cui et al. 2005 China IS+PE 97 58 92 22 Hirayama et al 2008 Japan OCS+CYC 21 14 86 24

Abbreviations: N, number of patients; AH, alveolar hemorrhage; IS, immunosuppressants (including methylprednisolone pulse therapy, oral corticosteroids, cyclophosphamide or azathioprine); PE, plasma exchange; OCS, oral corticosteroids; CYC, cyclophosphamide; AZA, azathioprine.

Table 4. Investigations of treatments for anti–GBM antibody disease.

Switzerland OCS+CYC+PE 24 100 100 58

(Hong Kong) IS+PE 10 40 70 15

(%)

OCS+CYC+PE 8 100 100 75

no treatment 8 100 63 25 OCS+AZA 4 100 100 50 OCS+CYC+PE 8 100 100 63

OCS+CYC+PE 22 36 68 14

1-year survival (%) Patient Renal

Authors Year Nation Treatment N AH

line therapy because no randomized controlled trials have been carried out.

**9. Prognosis**

**9.1 Outcomes**

Japan (Table 4).

initiation of induction therapies.

Lazor et al. 2007 France &

Simpson et al. 1982 New Zealand

Li et al. 2004 China

the circulating anti-GBM antibodies were no longer detected (Lockwood et al., 1976). In the presence of alveolar hemorrhage, 300-400 ml of fresh-frozen plasma was given at the end of each treatment.

To reduce the replacement of plasma, anti-GBM antibody removal has been modified. Immunoadsorption to remove circulating IgG immunoglobulins without the need for protein substitution during daily treatments may also be beneficial in Goodpasture's disease. Anecdotal case reports suggest that it may be an alternative to plasmapheresis in patients with severe renal failure (Laczika et al., 2000). There was a case report of Goodpasture's syndrome that we treated with double filtration plasmapheresis combined with immunosuppression therapy (Nagasu et al., 2009). In that therapy, the removal efficiency for the anti-GBM antibody was 24 to 60% for each procedure.

### **8.2 Corticosteroids**

To attenuate the pathogenic antibody-mediated glomerular inflammatory responses, corticosteroid is also a key element of this treatment. According to the most commonly used regimens, oral dosing of prednisolone at 1 mg/kg/day ideal body weight (maximum 80 mg daily) continues for at least 2 weeks, after which the dose is reduced every second week to 30 mg by 8 weeks. After that, the dosages of prednisolone are tapered to 2.5-5.0 mg/week and maintained at 7.5-10 mg/kg/day. Oral corticosteroids have generally been continued for at least 6 months. Intravenous administration of methylprednisolone 10 mg/kg (500- 1000 mg) once daily for 1-3 days has been advocated for patients with severe alveolar hemorrhage or very rapid deterioration of renal function (Johnson et al., 1985).

#### **8.3 Immunosuppressive agents**

To further suppress the production of pathogenic anti-GBM antibodies, a combination of immunosuppressive agents is usually given. Among these immunosuppressive agents, cyclophosphamide is usually administered. According to the most commonly used regimens, the oral dose is 2-3 mg/kg/day (this is rounded down to the nearest 50 mg; reduced to 2 mg/kg/day in patients over 55 years) for 3 months. This administration is stopped if white blood cell counts fall below 4,000/μL. In such cases the agent is restarted at a lower dose once the white blood cell counts return above 4,000/μL. Intravenous cyclophosphamide (IVCY) is not usually administered, but it may be useful for a refractory case of the standard therapy (Baumgartner et al., 1995).

Although azathioprine is sometimes used as maintenance therapy, it alone does not provide adequate immunosuppression to modify the disease.

#### **8.4 Therapeutic options for refractory diseases**

There is very little study on the treatment of refractory anti-GBM disease. Cyclosporine is controversial; at 6 mg/kg/day it was effective for an anti-GBM disease patient treated with corticosteroid, cyclophosphamide and plasma exchange (Querin et al., 1992), whereas it was not useful (Pepys et al., 1982). Small numbers of case reports of successful outcomes with mycophenolic acid or mycophenolate mofetil in patients unresponsive to or intolerant of standard therapy have been published (Garcia-Canton et al., 2000; Kiykim et al., 2010; Malho et al., 2010). Rituximab, a chimeric monoclonal anti-CD20 antibody, was effective for a case of relapsed anti-GBM disease that was resistant to standard treatment (Arzoo et al., 2002). In that case, rituximab (375 mg/m2) was administered once a week for 6 consecutive weeks; the symptoms completely resolved and anti-GBM antibody titers were decreased from 51 U/mL to the undetectable range. However, these treatments cannot yet be recommended as a firstline therapy because no randomized controlled trials have been carried out.

### **9. Prognosis**

264 An Update on Glomerulopathies – Clinical and Treatment Aspects

the circulating anti-GBM antibodies were no longer detected (Lockwood et al., 1976). In the presence of alveolar hemorrhage, 300-400 ml of fresh-frozen plasma was given at the end of

To reduce the replacement of plasma, anti-GBM antibody removal has been modified. Immunoadsorption to remove circulating IgG immunoglobulins without the need for protein substitution during daily treatments may also be beneficial in Goodpasture's disease. Anecdotal case reports suggest that it may be an alternative to plasmapheresis in patients with severe renal failure (Laczika et al., 2000). There was a case report of Goodpasture's syndrome that we treated with double filtration plasmapheresis combined with immunosuppression therapy (Nagasu et al., 2009). In that therapy, the removal

To attenuate the pathogenic antibody-mediated glomerular inflammatory responses, corticosteroid is also a key element of this treatment. According to the most commonly used regimens, oral dosing of prednisolone at 1 mg/kg/day ideal body weight (maximum 80 mg daily) continues for at least 2 weeks, after which the dose is reduced every second week to 30 mg by 8 weeks. After that, the dosages of prednisolone are tapered to 2.5-5.0 mg/week and maintained at 7.5-10 mg/kg/day. Oral corticosteroids have generally been continued for at least 6 months. Intravenous administration of methylprednisolone 10 mg/kg (500- 1000 mg) once daily for 1-3 days has been advocated for patients with severe alveolar

To further suppress the production of pathogenic anti-GBM antibodies, a combination of immunosuppressive agents is usually given. Among these immunosuppressive agents, cyclophosphamide is usually administered. According to the most commonly used regimens, the oral dose is 2-3 mg/kg/day (this is rounded down to the nearest 50 mg; reduced to 2 mg/kg/day in patients over 55 years) for 3 months. This administration is stopped if white blood cell counts fall below 4,000/μL. In such cases the agent is restarted at a lower dose once the white blood cell counts return above 4,000/μL. Intravenous cyclophosphamide (IVCY) is not usually administered, but it may be useful for a refractory

Although azathioprine is sometimes used as maintenance therapy, it alone does not provide

There is very little study on the treatment of refractory anti-GBM disease. Cyclosporine is controversial; at 6 mg/kg/day it was effective for an anti-GBM disease patient treated with corticosteroid, cyclophosphamide and plasma exchange (Querin et al., 1992), whereas it was not useful (Pepys et al., 1982). Small numbers of case reports of successful outcomes with mycophenolic acid or mycophenolate mofetil in patients unresponsive to or intolerant of standard therapy have been published (Garcia-Canton et al., 2000; Kiykim et al., 2010; Malho et al., 2010). Rituximab, a chimeric monoclonal anti-CD20 antibody, was effective for a case of relapsed anti-GBM disease that was resistant to standard treatment (Arzoo et al., 2002). In that case, rituximab (375 mg/m2) was administered once a week for 6 consecutive weeks; the symptoms completely resolved and anti-GBM antibody titers were decreased from 51 U/mL

efficiency for the anti-GBM antibody was 24 to 60% for each procedure.

hemorrhage or very rapid deterioration of renal function (Johnson et al., 1985).

each treatment.

**8.2 Corticosteroids**

**8.3 Immunosuppressive agents**

case of the standard therapy (Baumgartner et al., 1995).

adequate immunosuppression to modify the disease.

**8.4 Therapeutic options for refractory diseases**

Most patients without treatment died shortly after diagnosis of anti-GBM disease; the survival rate at 12 months was 4%, and the renal survival rate was 2% (Benoit et al., 1963). Although mortality has improved by the introduction of intense immunosuppression, renal survival remains very poor because of the delayed diagnosis of anti-GBM disease or delayed initiation of induction therapies.

#### **9.1 Outcomes**

The prognosis for patients with anti-GBM disease is poor; the survival rate at 6-12 months was 67-94%, but the renal survival rate was 15-58% in Europe, the United States, China and Japan (Table 4).

Abbreviations: N, number of patients; AH, alveolar hemorrhage; IS, immunosuppressants (including methylprednisolone pulse therapy, oral corticosteroids, cyclophosphamide or azathioprine); PE, plasma exchange; OCS, oral corticosteroids; CYC, cyclophosphamide; AZA, azathioprine.

Table 4. Investigations of treatments for anti–GBM antibody disease.

Anti-Glomerular Basement Membrane Disease 267

renal function has been reported, the prognosis for patients with this disease is poor. To improve the prognosis, it may be necessary to detect this disease in earlier stages and to

The authors acknowledge the participants in this Japanese nationwide survey and the members of the RPGN Clinical Guidelines Committee of Japan. The authors thank Dr. Miho Nagai, Dr. Yujiro Ogawa, Dr. Shogo Fujita, Dr. Homare Shimohata and Prof. Masaki Kobayashi (Tokyo Medical University Ibaraki Medical Center) and Dr. Joichi Usui (University of Tsukuba) for their assistance. The Japanese nationwide RPGN survey was supported by a grant-in-aid from the Research Fund for the Special Study Group on

Adler, S.; Bruns, F.J., Fraley, D.S. & Segel, D.P. (1981). Rapid progressive glomerulonephritis:

Adler, S.; Chen, X. & Eng, B. (1990). Control of rat glomerular epithelial cell growth in vitro. *Kidney International*, Vol.37, No.4, (April 1990), pp. 1048-1054, ISSN 0085-2538 Andrassy, K.; Küster, S., Waldherr, R. & Ritz, E. (1991). Rapidly progressive

Andres, G.; Brentjens, J., Kohli, R., Anthone, R., Anthone, S., Baliah, T., Montes, M.,

Angangco, R.; Thiru, S., Esnault, V.L., Short, A.K., Lockwood, C.M. & Oliveira, D.B. (1994).

Arzoo, K.; Sadeghi, S. & Liebman, H.A. (2002). Treatment of refractory antibody mediated

Atkins, R.C.; Holdsworth, S.R., Glasgow, E.F. & Matthews, F.E. (1976). The macrophage in

Baricos, W.H.; Cortez, S.L., Le, Q.C., Wu, L.T., Shaw, E., Hanada, K. & Shah, S.V. (1991).

Baumgartner, I.; Gmür, J., Fontana, A., Widmer, U. & Walter, E. (1995). Recovery from life

Vol.43, No.1, (January 1995), pp. 68-70, ISSN 0301-0430

*Transplantation*, Vo.9, No.6, (June 1994), pp.630-636, ISSN 0931-0509

relapse after prolonged remission. *Archives of Internal Medicine*, Vol.141, No.7, (June

glomerulonephritis: analysis of prevalence and clinical course. *Nephron*, Vol.59,

Mookerjee, B.K., Prezyna, A., Sepulveda, M., Venuto, R. & Elwood, C. (1978). Histology of human tubulo-interstitial nephritis associated with antibodies to renal basement membranes. *Kidney International,* Vol.13, No. 6, (June 1978), pp. 480-491,

Does truly 'idiopathic' crescentic glomerulonephritis exist? *Nephrology Dialysis*

autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab). *Annals of the Rheumatic Diseases*, Vol.61, No.10, (October 2002), pp. 922-924, ISSN 0003-4967

human rapidly progressive glomerulonephritis. *The Lancet*, Vol.1, No.7964, (April

Evidence suggesting a role for cathepsin L in an experimental model of glomerulonephritis. *Archives of Biochemistry and Biophysics*, Vol.288, No.2, (August

threatening pulmonary hemorrhage in Goodpasture's syndrome after plasmapheresis and subsequent pulse dose cyclophosphamide. *Clinical Nephrology*,

Progressive Glomerular Disease, the Ministry of Health, Labor and Welfare, Japan.

1981), pp. 852-854, ISSN 0003-9926

No.2, (October 1991), ISSN 0028-2766

1976), pp. 830-832, ISSN 0140-6736

1991), pp. 468-472, ISSN 0003-9861

ISSN 0085-2538

treat it without delay.

**12. References**

**11. Acknowledgment**

Renal function improves in 15-75% of patients with anti-GBM disease through the combination of plasma exchange with corticosteroids and immunosuppressive agents, whereas the renal survival rates of anti-GBM disease patients treated with immunosuppressive agents alone ranged from 2-22%. Improvement of renal function is usually evident within days of the start of plasma exchange. However, it should be emphasized that this regimen has never been properly assessed by a prospective randomized controlled trial because of the rarity and acuteness of the condition. The only reported randomized controlled trial was very small and used lower doses of both plasma exchange and cyclophosphamide than those that are generally used in practice.

Although the effectiveness of treatment using therapeutic plasma exchange combined with immunosuppressive agents to improve renal function has been reported, only half of patients with anti-GBM disease had been treated with plasma exchange in our survey (Hirayama et al., 2008). Therefore, there was no significant difference in the renal survival rates between anti-GBM antibody disease patients treated with and without plasma exchange (P = 0.683 by the Log-rank Mantel-Cox test). Moreover, there was no significant difference in mortality between anti-GBM antibody disease patients treated with and without plasma exchange (P = 0.109).

#### **9.2 Predictors of survival**

The best predictors of renal survival are s-Cr at the initiation of treatment and the mean percentage of crescent formations. Renal function improves coincidentally with the introduction of plasma exchange in about 80-95% of patients with s-Cr levels less than or equal to 5.7-6.8 mg/dL (500-600 μmol/L), but in far fewer of those with higher s-Cr levels or those who require dialysis. Unfortunately, most patients with anti-GBM disease had renal failure at the time of diagnosis, and the mean percentage of crescent formation was high in anti-GBM disease patients. Therefore, in most patients with anti-GBM disease, the diagnosis may have been made too late to improve renal function by combination therapy.

#### **9.3 Relapse/recurrence**

Relapses of anti-GBM disease are rarely observed, in contrast to most other autoimmune kidney diseases. The anti-GBM antibodies seem to disappear spontaneously after 12-18 months (Levy et al., 1996). However, several reports demonstrated recurring cases with anti-GBM disease (Adler et al., 1981; Hind et al., 1984; Klasa et al., 1988; Levy et al., 1996). In our survey (Hirayama et al., 2008), relapse or recurrence was also rare in patients with anti-GBM disease (13.9%) in comparison with patients with ANCA-associated vasculitis, such as WG (29.4%) and MPA (29.3%). Therefore, remission induction therapy is more important in anti-GBM disease. The mean time to recurrence is estimated to be 4.3 years (range, 1-10 years), and that late recurrence may occur with a frequency of 2-14%. During relapses, circulating anti-GBM antibodies often reappear. The combination of plasmapheresis and immunosuppressive agents as re-remission induction therapy is also successful in relapsing cases (Levy et al., 1996).

### **10. Conclusion**

Anti-GBM disease is a rare but well-characterized glomerulonephritis. It occurs across all racial groups but is most common in Caucasians. Although the effectiveness of treatment using therapeutic plasma exchange combined with immunosuppressive agents to improve renal function has been reported, the prognosis for patients with this disease is poor. To improve the prognosis, it may be necessary to detect this disease in earlier stages and to treat it without delay.

### **11. Acknowledgment**

266 An Update on Glomerulopathies – Clinical and Treatment Aspects

Renal function improves in 15-75% of patients with anti-GBM disease through the combination of plasma exchange with corticosteroids and immunosuppressive agents, whereas the renal survival rates of anti-GBM disease patients treated with immunosuppressive agents alone ranged from 2-22%. Improvement of renal function is usually evident within days of the start of plasma exchange. However, it should be emphasized that this regimen has never been properly assessed by a prospective randomized controlled trial because of the rarity and acuteness of the condition. The only reported randomized controlled trial was very small and used lower doses of both plasma

Although the effectiveness of treatment using therapeutic plasma exchange combined with immunosuppressive agents to improve renal function has been reported, only half of patients with anti-GBM disease had been treated with plasma exchange in our survey (Hirayama et al., 2008). Therefore, there was no significant difference in the renal survival rates between anti-GBM antibody disease patients treated with and without plasma exchange (P = 0.683 by the Log-rank Mantel-Cox test). Moreover, there was no significant difference in mortality between anti-GBM antibody disease patients treated with and

The best predictors of renal survival are s-Cr at the initiation of treatment and the mean percentage of crescent formations. Renal function improves coincidentally with the introduction of plasma exchange in about 80-95% of patients with s-Cr levels less than or equal to 5.7-6.8 mg/dL (500-600 μmol/L), but in far fewer of those with higher s-Cr levels or those who require dialysis. Unfortunately, most patients with anti-GBM disease had renal failure at the time of diagnosis, and the mean percentage of crescent formation was high in anti-GBM disease patients. Therefore, in most patients with anti-GBM disease, the diagnosis

Relapses of anti-GBM disease are rarely observed, in contrast to most other autoimmune kidney diseases. The anti-GBM antibodies seem to disappear spontaneously after 12-18 months (Levy et al., 1996). However, several reports demonstrated recurring cases with anti-GBM disease (Adler et al., 1981; Hind et al., 1984; Klasa et al., 1988; Levy et al., 1996). In our survey (Hirayama et al., 2008), relapse or recurrence was also rare in patients with anti-GBM disease (13.9%) in comparison with patients with ANCA-associated vasculitis, such as WG (29.4%) and MPA (29.3%). Therefore, remission induction therapy is more important in anti-GBM disease. The mean time to recurrence is estimated to be 4.3 years (range, 1-10 years), and that late recurrence may occur with a frequency of 2-14%. During relapses, circulating anti-GBM antibodies often reappear. The combination of plasmapheresis and immunosuppressive agents as re-remission induction therapy is also successful in relapsing

Anti-GBM disease is a rare but well-characterized glomerulonephritis. It occurs across all racial groups but is most common in Caucasians. Although the effectiveness of treatment using therapeutic plasma exchange combined with immunosuppressive agents to improve

may have been made too late to improve renal function by combination therapy.

exchange and cyclophosphamide than those that are generally used in practice.

without plasma exchange (P = 0.109).

**9.2 Predictors of survival**

**9.3 Relapse/recurrence**

cases (Levy et al., 1996).

**10. Conclusion**

The authors acknowledge the participants in this Japanese nationwide survey and the members of the RPGN Clinical Guidelines Committee of Japan. The authors thank Dr. Miho Nagai, Dr. Yujiro Ogawa, Dr. Shogo Fujita, Dr. Homare Shimohata and Prof. Masaki Kobayashi (Tokyo Medical University Ibaraki Medical Center) and Dr. Joichi Usui (University of Tsukuba) for their assistance. The Japanese nationwide RPGN survey was supported by a grant-in-aid from the Research Fund for the Special Study Group on Progressive Glomerular Disease, the Ministry of Health, Labor and Welfare, Japan.

### **12. References**

Anti-Glomerular Basement Membrane Disease 269

Briganti, E.M.; Dowling, J., Finlay, M., Hill, P.A., Jones, C.L., Kincaid-Smith, P.S., Sinclair, R.,

Briggs, W.A.; Johnson, J.P., Teichman, S., Yeager, H.C. & Wilson, C.B. (1979).

Churchill, D.N.; Fine, A. & Gault, M.H. (1983). Association between hydrocarbon exposure

Clatworthy, M.R.; Wallin, E.F. & Jayne, D.R. (2008). Anti-glomerular basement membrane

Cui, Z.; Zhao, M.H., Xin, G. & Wang, H.Y. (2005). Characteristics and prognosis of Chinese

Daly, C.; Conlon, P.J., Medwar, W. & Walshe, J.J. (1996). Characteristics and outcome of anti-

Date, A.; Raghavan, R., John, T.J., Richard, J., Kirubakaran, M.G. & Shastry, J.C.N. (1987).

Donaghy, M. & Rees, A.J. (1983). Cigarette smoking and lung haemorrhage in

*The Lancet*, Vol. 2, No.8364, (December 1983), pp. 1390–1393, ISSN 0140-6736 Fisher, M.; Pusey, C.D., Vaughan, R.W. & Rees, A.J. (1997). Susceptibility to anti-glomerular

García-Cantón, C.; Toledo, A., Palomar, R., Fernandez, F., Lopez, J., Moreno, A., Esparza, N.,

García-Rostan y Pérez, G.M.; García Bragado, F. & Puras Gil, A.M. (1997). Pulmonary

Goodpasture E.W. (1919). The significance of certain pulmonary lesions in relation to the

Vol.338, No.2, (August 2009), pp. 148-151), ISSN 0002-9629

*International*, Vol.51, No.1, (January 1997), pp. 222–229, ISSN 0085-2538 Fischer, E.G. & Lager, D.J. (2006). Anti-glomerular basement membrane glomerulonephritis:

*Practice*, Vol.99, No.2, (February 2005), pp. c49-c55, ISSN 1660-2110

Vol.18, No.1, (January 1996), pp. 105-112, ISSN 0886-022X

No.3, (March 2006), pp. 445-450, ISSN 0002-9173

No.6, (June 2000), pp. 920-922, ISSN 0931-0509

692-697, ISSN 1320-5463

(July 2001), pp. 1364–1367, ISSN 0931-0509

348–361, ISSN 0025-7974

0033-5622

1983), pp. 169-172, ISSN 0028-2766

(August 2008), pp. 768-769, ISSN 0028-4793

McNeil, J.J. & Atkins, R.C. (2001). The incidence of biopsy-proven glomerulonephritis in Australia. *Nephrology Dialysis Transplantation,* Vol.16, No.7,

Antiglomerular basement membrane antibody-mediated glomerulonephritis and Goodpasture's syndrome. *Medicine (Baltimore),* Vol.58, No.5, (September 1979), pp.

and glomerulonephritis. An appraisal of the evidence. *Nephron*, Vol.33, No.3, (June

disease after alemtuzumab. *The New England Journal of Medicine*, Vol.359, No.7,

patients with anti-glomerular basement membrane disease. *Nephron Clinical*

glomerular basement membrane disease: a single-center experience. *Renal Failure,*

Renal disease in adult Indians: a clinicopathological study of 2827 patients. *The Quarterly Journal of Medicine*, Vol.64, No.245, (September 1987), pp. 729-737, ISSN

glomerulonephritis caused by autoantibodies to glomerular basement membrane.

basement membrane disease is strongly associated with HLA-DRB1 genes. *Kidney*

a morphologic study of 80 cases. *American Journal of Clinical Patholology*, Vol.123,

Suria, S., Rossique, P., Diaz, J.M. & Checa, D. (2000). Goodpasture's syndrome treated with mycophenolate mofetil. *Nephrology Dialysis Transplantation*, Vol.15,

hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature. *Pathology International*, Vo.47, No.10, (October 1997), pp.

etiology of influenza. *The American Journal of the Medical Sciences*, Vol.158, No.6, (1919), pp. 863-870, (republished in *The American Journal of the Medical Sciences*,

Beirne, G.J.; Wagnild, J.P., Zimmerman, S.W., Macken, P.D. & Burkholder, P.M. (1977).

Beirne, G.J. & Brennan J.T. (1972). Glomerulonephritis associated with hydrocarbon

Blue, W.T. & Lange, C.F (1975). Increased immunologic reactivity between human

Bombassei, G.J. & Kaplan, A.A. (1992). The association between hydrocarbon exposure and

Bonzel, K.E.; Müller-Wiefel, D.E., Ruder, H., Wingen, A.M., Waldherr, R. & Weber, M.

Border, W.A.; Baehler, R.W., Bhathena, D. & Glassock, R.J. (1979). IgA antibasement

Borza, D.B.; Netzer, K.O., Leinonen, A., Todd, P., Cervera, J., Saus, J. & Hudson, B.G. (2000).

Borza, D.B.; Chedid, M.F., Colon, S., Lager, D.J., Leung, N. & Fervenza, F.C. (2005).

*Kidney Diseases*, Vol.45, No.2, (February 2005), pp. 397-406, ISSN 0272-6386 Bosch, X.; Mirapeix, E., Font, J., Borrellas, X., Rodríguez, R., López-Soto, A., Ingelmo, M. &

Boucher, A.; Droz, D., Adafer, E. & Noel, L.H. (1987). Relationship between the integrity of

Bowman, C.; Ambrus, K. & Lockwood, C.M. (1987). Restriction of human IgG subclass

(September 1977), pp.349–381, ISSN 0025-7974

(September 1963), pp. 424–444, ISSN 0002-9343

pp. 306–309, ISSN 0022-1767

pp. 141–153, ISSN 0271-3586

ISSN 0301-0430

ISSN 0023-6837

0009-9104

(May 1987), pp. 296-300, ISSN 0340-6199

Vol.91, No.1, (July 1979), pp. 21-25, ISSN 0003-4819

Vol.275, No.8, (February 2000), pp. 6030–6037, ISSN 0021-9258

Idiopathic crescentic glomerulonephritis. *Medicine (Baltimore),* Vol.56, No.5,

solvents: mediated by antiglomerular basement membrane antibody. *Archives of Environmental Health*, Vol.25, No.5, (November 1972), pp. 365–369, ISSN 0003-9896 Benoit, F.L.; Rulon, D.B., Theil, G.B., Doolan, P.D. & Watten, R.H. (1963). Goodpasture's

syndrome: A clinicopathologic entity. *The American Journal of Medicine*, Vol.58,

glomerular basement membrane and group A type 12 streptococcal cell membrane after carbohydrase treatment. *Journal of Immunology*, Vol.114, No.1, (January 1975),

anti-glomerular basement membrane antibody-mediated disease (Goodpasture's syndrome). *American Journal of Industrial Medicine*, Vol.21, No.2, (February 1992),

(1987). Anti-glomerular basement membrane antibody-mediated glomerulonephritis due to glue sniffing. *European Journal of Pediatrics*, Vol.146, No.3,

membrane nephritis with pulmonary hemorrhage. *Annals of Internal Medicine*,

The Goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain. *The Journal of Biological Chemistry*,

Recurrent Goodpasture's disease secondary to a monoclonal IgA1-kappa antibody autoreactive with the alpha1/alpha2 chains of type IV collagen. *American Journal of*

Revert, L. (1991). Prognostic implication of anti-neutrophil cytoplasmic autoantibodies with myeloperoxidase specificity in anti-glomerular basement membrane disease. *Clinical Nephrology*, Vol.36, No.3, (September 1991), pp. 107-113,

Bowman's capsule and the composition of cellular crescents in human crescentic glomerulonephritis. *Laboratory Investigation*, Vol.56, No.5, (May 1987), pp. 526–533,

expression in the population of auto-antibodies to glomerular basement membrane. *Clinical and Experimental Immunology*, Vol.69, No.2, (August 1987), pp. 341-349, ISSN

Anti-Glomerular Basement Membrane Disease 271

glomerular basement membrane antibody-mediated disease. *Nephrology Dialysis Transplantation*, Vol. 23, No.10, (October 2008), pp. 3126–3129, ISSN 0931-0509 Kitching, A.R., Tipping, P.G. & Holdsworth, S.R. (1999). IL-12 directs severe renal injury,

crescent formation and Th1 responses in murine glomerulonephritis. *European Journal of Immunology*, Vol.29, No.1, (January 1999), pp. 1–10, ISSN 0014-2980 Kiykim, A.A.; Horoz, M. & Gok, E. (2010). Successful treatment of resistant antiglomerular

basement membrane antibody positivity with mycophenolic acid. *Internal Medicine*,

recurrence after a five-year remission. Case report and review of the literature. *The American Journal of Medicine*, Vol.84, No.4, (April 1988), pp. 751–755, ISSN 0002-9343

Immunoadsorption in Goodpasture's syndrome. *American Journal of Kidney Diseases*,

Suppression of experimental crescentic glomerulonephritis by the interleukin-1 receptor antagonist. *Kidney International*, Vol.43, No.2, (February 1993), pp. 479–485,

R. & Atkins, R.C. (1997). TNF-alpha up-regulates renal MIF expression in rat crescentic glomerulonephritis. *Molecular Medicine*, Vol.3, No.2, (February 1997), pp.

Groupe d'Etudes et de Recherche sur les Maladies Orphelines Pulmonaires (GERMOP); Swiss Group for Interstitial and Orphan Lung Diseases (SIOLD). (2007). Alveolar hemorrhage in anti-basement membrane antibody disease: a series of 28 cases. *Medicine (Baltimore)*, Vol.86, No.3, (March 2007), pp. 181-193, ISSN 0025-

glomerulonephritis induced by anti-glomerular membrane antibody in tumor necrosis factor-deficient mice. *Laboratory Investigation*, Vol.78, No.12, (December

syndrome. Unusual presentation after exposure to hard metal dust. *Chest*, Vol.103,

in human nephritis. *American Journal of Medicine*, Vol.58, No.6, (June 1975), pp. 765-

membrane antibody in the pathogenesis of human glomerulonephritis. *The Journal of Experimental Medicine*, Vol.126, No.6, (December 1967), pp. 989-1004. 0022-1007 Levy, J.B.; Lachmann, R.H. & Pusey, C.D. (1996). Recurrent Goodpasture's disease. *American Journal of Kidney Diseases*, Vol.27, No.4, (April 1996), pp. 573-578, ISSN 0272-6386 Levy, J.B.; Turner, A.N., Rees, A.J. & Pusey, C.D. (2001). Long-term outcome of anti-

glomerular basement membrane antibody disease treated with plasma exchange

Klasa, R.J.; Abboud, R.T., Ballon, H.S. & Grossman, L. (1988). Goodpasture's syndrome:

Laczika, K.; Knapp, S., Derfler, K., Soleiman, A., Horl, W.H. & Druml, W. (2000).

Lan, H.Y.; Nikolic-Paterson, D.J., Zarama, M., Vannice, J.L. & Atkins, R.C. (1993).

Lan, H.Y.; Yang, N., Metz, C., Mu, W., Song, Q., Nikolic-Paterson, D.J., Bacher, M., Bucala,

Lazor, R.; Bigay-Gamé, L., Cottin, V., Cadranel, J., Decaux, O., Fellrath, J.M. & Cordier, J.F.;

Le Hir, M.; Haas, C., Marino, M. & Ryffel, B. (1998). Prevention of crescentic

Lechleitner, P.; Defregger, M., Lhotta, K., Tötsch, M. & Fend, F. (1993). Goodpasture's

Lehman, D.H.; Wilson, C.B. & Dixon, F.J. (1975). Extraglomerular immunoglobulin deposits

Lerner, R.A.; Glassock, R.J. & Dixon, F.J. (1967). The role of anti-glomerular basement

Vol.49, No.6, (June 2010), pp. 577-580, ISSN 0918-2918

Vol.36, No.2, (August 2000), pp. 392–395, ISSN 0272-6386

ISSN 0085-2538

7974

136–144, ISSN 1076-1551

796., ISSN 0002-9343

1998), pp. 1625–1631, ISSN 0023-6837

No.3, (March 1993), pp. 956–957, ISSN 0012-3692

Grcevska, L. & Polenakovic, M. (1995). Crescentic glomerulonephritis as renal cause of acute

Gris, P.; Pirson, Y., Hamels, J., Vaerman, J.P., Quoidbach, A. & Demol, H. (1991).

He, C.J.; Rondeau, E., Medcalf, R.L., Lacave, R., Schleuning, W.D. & Sraer, J.D. (1991).

Heaf, J.; Løkkegaard, H. & Larsen, S. (1999). The epidemiology and prognosis of

Herody, M.; Bobrie, G., Gouarin, C., Grünfeld, J.P. & Noel, L.H. (1993). Anti-GBM disease:

Hind, C.R.K.; Bowman, C., Winearles, C.G. & Lockwood, C.M. (1984). Recurrence of

Holdsworth, S.; Boyce, N., Thomson, N.M. & Atkins, R.C. (1985). The clinical spectrum of

Hudson, B.G.; Tryggvason, K., Sundaramoorthy, M. & Neilson, E.G (2003). Alport's

Johnson, J.P.; Moore, J. Jr., Austin, H.A. 3rd., Balow, J.E., Antonovych, T.T. & Wilson, C.B.

Kalluri, R.; Sun, M.J., Hudson, B.G. & Neilson, E.G. (1996). The Goodpasture autoantigen.

Kaneko, Y.; Sakatsume, M., Xie, Y., Kuroda, T., Igashima, M., Narita, I. & Gejyo, F. (2003).

Kitagawa, W.; Imai, H., Komatsuda, A., Maki, N., Wakui, H., Hiki, Y. & Sugiyama, S. (2008).

*of Medicine*, Vol.348, No.25, (June 2003), pp. 2543–2556, ISSN 0028-4793 Jayne, D.R.; Marshall, P.D., Jones, S.J. & Lockwood, C.M. (1990). Autoantibodies to GBM

*International*, Vol.37, No.3, (March 1990), pp. 965-970, ISSN 0085-2538 Jennette, J.C. (2003). Rapidly progressive crescentic glomerulonephritis. *Kidney International*,

*(Baltimore)*, Vol.64, No.4, (July 1985), pp. 219-227, ISSN 0025-7974

Vol.63, No.3, (March 2003), pp. 1164-1177, ISSN 0085-2538

1996), pp. 9062–9068, ISSN 0021-9258

(March 2003), pp. 3377–3385, ISSN 0022-1767

*Nephron*, Vol.58, No.4, (August 1991), pp. 418-424, ISSN 0028-2766

Vol.14, No.8, (August 1999), pp. 1889-1897, ISSN 0931-0509

Vol.40, No.5, (November 1993), pp. 249–255, ISSN 0301-0430

No.4, (April 1984), pp. 244-246, ISSN 0301-0430

1991), pp. 131-140, ISSN 0021-9541

022X

renal failure. *Renal Failure*, Vol.17, No.5, (September 1995), pp. 595-604, ISSN 0886-

Antiglomerular basement membrane nephritis induced by IgA1 antibodies.

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells. *Journal of Cellular Physiology*, Vol.146, No.1, (January

glomerulonephritis in Denmark 1985-1997. *Nephrology Dialysis Transplantation*,

Predictive value of clinical, histological and serological data. *Clinical Nephrology*,

circulating anti-glomerular basement membrane antibody three years after immunosuppressive treatment and plasma exchange. *Clinical Nephrology*, Vol.21,

acute glomerulonephritis and lung haemorrhage (Goodpasture's syndrome). *The Quarterly Journal of Medicine*, Vol.55, No.216, (April 1985), pp. 75-86, ISSN 0033-5622

syndrome, Goodpasture's syndrome, and type IV collagen. *The New England Journal*

and neutrophil cytoplasm in rapidly progressive glomerulonephritis. *Kidney*

(1985). Therapy of anti-glomerular basement membrane antibody disease: analysis of prognostic significance of clinical, pathologic and treatment factors. *Medicine*

Structural delineation of two immunologically privileged epitopes on alpha3(IV) chain of type IV collagen. *The Journal of Biological Chemistry*, Vol.271, No.15, (April

Macrophage metalloelastase as a major factor for glomerular injury in antiglomerular basement membrane nephritis. *Journal of Immunology*, Vol.170, No.6,

The HLA-DRB1\*1501 allele is prevalent among Japanese patients with anti-

glomerular basement membrane antibody-mediated disease. *Nephrology Dialysis Transplantation*, Vol. 23, No.10, (October 2008), pp. 3126–3129, ISSN 0931-0509

Anti-Glomerular Basement Membrane Disease 273

Naumovic, R.; Pavlovic, S., Stojkovic, D., Basta-Jovanovic, G. & Nesic, V. (2009). Renal

The New Zealand Glomerulonephritis Study Group. (1989). The New Zealand

Ooi, J.D.; Holdsworth, S.R. & Kitching, A.R. (2008). Advances in the pathogenesis of

*Autoimmunity*, Vol.31, No.3, (November 2008), pp. 295–300, ISSN 0896-8411 Ophascharoensuk, V.; Pippin, J.W., Gordon, K.L., Shankland, S.J., Couser, W.G. & Johnson,

Ortega, L.G. & Mellors, R.C. (1956). Analytical pathology. IV. The role of localized

Pedchenko, V.; Bondar, O., Fogo, A.B., Vanacore, R., Voziyan, P., Kitching, A.R., Wieslander,

Pepys, E.O.; Rees, A.J. & Pepys, M.B. (1982). Enumeration of lymphocyte populations in

Perez, G.O.; Bjornsson, S., Ross, A.H., Aamato, J. & Rothfield, N. (1974). A mini-epidemic of

Persson, U.; Hertz, J.M., Carlsson, M., Hellmark, T., Juncker, I., Wieslander, J. & Segelmark,

*Transplantation*, Vol.19, No.8, (August 2004), pp. 2030-2035, ISSN 0931-0509 Peters, D.K.; Rees, A.J., Lockwood, C.M. & Pusey, C.D. (1982). Treatment and prognosis in

No.2, (August 1974), pp. 161–173, ISSN 0028-2766

Vol.14, No.3, (September 1982), pp. 513-521, ISSN 0041-1345

2009), pp. 373–377, ISSN 1744-9979

11267-11274, ISSN 0021-9258

ISSN 0085-2538

4793

387-396, ISSN 0272-6386

214, ISSN 0165-2478

(May 1989), pp. 239-246, ISSN 0301-0430

Goodpasture's syndrome. *Therapeutic Apheresis and Dialysis*, Vol.13, No.4, (August

biopsy registry from a single centre in Serbia: 20 years of experience. *Nephrology Dialysis Transplantation,* Vol.24, No.3, (March 2009), pp. 877–885, ISSN 0931-0509 Netzer, K.O.; Leinonen, A., Boutaud, A., Borza, D.B., Todd, P., Gunwar, S., Langeveld, J.P. &

Hudson, B.G. (1999). The Goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain. *The Journal of Biological Chemistry*, Vol.274, No.16, (April 1999), pp.

Glomerulonephritis Study: introductory report. *Clinical Nephrology*, Vol.31, No.5,

Goodpasture's disease: from epitopes to autoantibodies to effector T cells. *Journal of*

R.J. (1998). Role of intrinsic renal cells versus infiltrating cells in glomerular crescent formation. *Kidney International*, Vol.54, No.2, (August 1998), pp. 416–425,

antibodies in the pathogenesis of nephrotoxic nephritis in the rat. *The Journal of Experimental Medicine*, Vol.104, No.1, (July 1956), pp. 151-157, ISSN 0022-1007 Parfrey, P.S.; Hutchinson, T.A., Jothy, S., Cramer, B.C., Martin, J., Hanley, J.A. & Seely, J.F.

(1985). The spectrum of disease associated with necrotizing glomerulonephritis and its prognosis. *American Journal of Kidney Disease*s, Vol.6, No.6, (December 1985), pp.

J., Kashtan, C., Borza, D.B., Neilson, E.G., Wilson, C.B. & Hudson, B.G. (2010). Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis. *The New England Journal of Medicine*, Vol.363, No.4, (July 2010), pp. 343-354, ISSN 0028-

whole peripheral blood of patients with antibody-mediated nephritis during treatment with cyclosporin A. *Immunology Letters*, Vol.4, No.4, (April 1982), pp. 211-

Goodpasture's syndrome clinical and immunological studies. *Nephron*, Vol.13,

M. (2004). Patients with Goodpasture's disease have two normal COL4A3 alleles encoding the NC1 domain of the type IV collagen alpha 3 chain. *Nephrology Dialysis*

antibasement membrane antibody-mediated nephritis. *Transplantation Proceedings*,

and immunosuppression. *Annals of Internal Medicine*, Vol.134, No.11, (June 2001), pp. 1033-1042, ISSN 0003-4819

Levy, J.B.; Hammad, T., Coulthart, A., Dougan, T. & Pusey, C.D. (2004). Clinical features and

Li, L.S. & Liu, Z.H. (2004). Epidemiologic data of renal diseases from a single unit in China:

Liu, K.; Li, Q.Z., Delgado-Vega, A.M., Abelson, A.K., Sánchez, E., Kelly, J.A., Li, L., Liu, Y.,

Lockwood, C.M.; Rees, A.J., Pearson, T.A., Evans, D.J., Peters, D.K. & Wilson, C.B. (1976).

Masugi, M. (1939).  The morphogenesis of

Morita, T.; Suzuki, Y. & Churg, J. (1973). Structure and development of the glomerular

Nagasu, H., Abe, M., Kuwabara, A., Kawai, T., Nishi, Y., Okuda, N. & Sakaguchi, K. (2009).

*International*, Vol.5, No.1, (January 1974), pp. 47–56, ISSN 0085-2538

Immunosuppression and plasma-exchange in the treatment of Goodpasture's syndrome. *The Lancet*, Vol.1, No.7962, (April 1976), pp. 711-715, ISSN 0140-6736 Malho, A.; Santos, V., Cabrita, A., Silva, A.P., Pinto, I., Bernardo, I. & Neves, P.L. (2010).

Severe relapsing Goodpasture's disease successfully treated with mycophenolate mofetil. *International Journal of Nephrology*, Vol.16, (August 2010), pp. 383548. ISSN

antinieren serum. Ein beiträg zur pathogenese der diffusen glomerulonephritis. *Beitrage zur pathologischen anatomie und allgemeinen pathologie*, Vol.92, pp. 429-466 Merkel, F.; Pullig, O., Marx, M., Netzer, K.O. & Weber, M. (1994). Course and prognosis of

anti-basement membrane antibody (anti-BM-Ab)-mediated disease: Report of 35 cases. *Nephrology Dialysis Transplantation*, Vol.9, No.4, (April 1994), pp. 372–376,

glomerular crescents in rapidly progressive glomerulonephritis. *Kidney*

crescent. *The American Journal of Pathology*, Vol.72, No.3, (September 1973), pp. 349–

A case report of efficiency of double filtration plasmapheresis in treatment of

*International*, Vol.66, No.4, (October 2004), pp. 1535-1540, ISSN 0085-2538 Li, F.K.; Tse, K.C., Lam, M.F., Yip, T.P., Lui, S.L., Chan, G.S., Chan, K.W., Chan, E.Y., Choy,

pp. 1033-1042, ISSN 0003-4819

ISSN 0021-9738

2090-2158

ISSN 0931-0509

368, ISSN 0002-9440

No.2, (April 2004), pp. 100-104, ISSN 1320-5358

(September 2004), pp. 920–923, ISSN 0085-2538

and immunosuppression. *Annals of Internal Medicine*, Vol.134, No.11, (June 2001),

outcome of patients with both ANCA and anti-GBM antibodies. *Kidney*

B.Y., Lo, W.K., Chan, T.M. & Lai, K.N. (2004). Incidence and outcome of antiglomerular basement membrane disease in Chinese. *Nephrology (Carlton)*, Vol.9,

analysis based on 13,519 renal biopsies. *Kidney International,* Vol.66, No.3,

Zhou, J., Yan, M., Ye, Q., Liu, S., Xie, C., Zhou, X.J., Chung, S.A., Pons-Estel, B., Witte, T., de Ramón, E., Bae, S.C., Barizzone, N., Sebastiani, G.D., Merrill, J.T., Gregersen, P.K., Gilkeson, G.G., Kimberly, R.P., Vyse, T.J., Kim, I., D'Alfonso, S., Martin, J., Harley, J.B., Criswell, L.A.; Profile Study Group; Italian Collaborative Group; German Collaborative Group; Spanish Collaborative Group; Argentinian Collaborative Group; SLEGEN Consortium, Wakeland, E.K., Alarcón-Riquelme, M.E. & Mohan, C. (2009). Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans. *The Journal of Clinical Investigation*, Vol.119, No.4, (April 2009), pp. 911-923, Goodpasture's syndrome. *Therapeutic Apheresis and Dialysis*, Vol.13, No.4, (August 2009), pp. 373–377, ISSN 1744-9979

Anti-Glomerular Basement Membrane Disease 275

Scheer, R.L. & Grossman, M.A. (1964). Immune aspects of the glomerulonephritis associated

Schena, F.P. & Survey of the Italian Registry of Renal Biopsies. (1997). Frequency of the renal

Segelmark, M.; Butkowski, R. & Wieslander, J. (1990) Antigen restriction and IgG subclasses

Segelmark, M.; Hellmark, T. & Wieslander, J. (2003) The prognostic significance in

Shah, M.K. & Hugghins, S.Y. (2002). Characteristics and outcomes of patients with

Sheerin, N.S.; Springall, T., Carroll, M.C., Hartley, B. & Sacks, S.H. (1997). Protection against

Simpson, I.J.; Doak, P.B., Williams, L.C., Blacklock, H.A., Hill, R.S., Teague, C.A., Herdson,

Sumethkul, V.; Changsirikulchai, S., Radinahamed, P. & Chalermasnyakorn, P. (1999).

*Immunology*, Vol.17, No.4, (December 1999), pp. 281-287, ISSN 0125-877X Tang, Z.; Wu, Y., Wang, Q., Zeng, C., Yao, X., Hu, W., Chen, H., Liu, Z. & Li, L. (2003).

Thomson, N.M.; Holdsworth, S.R., Glasgow, E.F. & Atkins, R.C. (1979). The macrophage in

Timoshanko, J.R.; Holdsworth, S.R., Kitching, A.R. & Tipping, P.G. (2002). IFN-gamma

pp. 1009-1021, ISSN 0003-4819

pp. 1411–1418, ISSN 0038-4348

1997), pp. 403–409, ISSN 0009-9104

(May 1958), pp. 132-144, ISSN 0571-9283

No.2, (February 1979), pp. 223-240, ISSN 0002-9440

Vol.168, No.8, (April 2002), pp. 4135–4141, ISSN 0022-1767

pp. 492-504, ISSN 0085-2538

(December 1990), pp. 991–996, ISSN 0931-0509

0931-0509

ISSN 1660-2110

with pulmonary hemorrhage. *Annals of Internal Medicine*, Vol. 60, No.6, (June 1964),

diseases for 7 consecutive years. The Italian Group of Renal Immunopathology. *Nephrology Dialysis Transplantation*, Vol.12, No.3, (March 1997), pp. 418-426, ISSN

among anti-GBM autoantibodies. *Nephrology Dialysis Transplantation*, Vol.5, No.12,

Goodpasture's disease of specificity, titre and affinity of anti-glomerular-basementmembrane antibodies. *Nephron Clinical Practice*, Vol.94, No., (2003), pp. c59-c68,

Goodpasture's syndrome. *South Medical Journal*, Vol.95, No.12, (December 2002),

anti-glomerular basement membrane (GBM)-mediated nephritis in C3- and C4 deficient mice. *Clinical and Experimental Immunology*, Vol.110, No.3, (December

P.B. & Wilson, C.B. (1982). Plasma exchange in Goodpasture's syndrome. *American Journal of Nephrology*, Vol.2, No.6, (December 1982), pp.301-311, ISSN 0250-8095 Stanton, M.C. & Tange, J.D. (1958). Goodpasture's syndrome (pulmonary haemorrhage

associated with glomerulonephritis). *Australasian Annals of Medicine*, Vol.7, No.2,

Antineutrophil cytoplasmic antibody (ANCA) and rapidly progressive crescentic glomerulonephritis in Thai population. *Asian Pacific Journal of Allergy and*

Clinical spectrum of diffuse crescentic glomerulonephritis in Chinese patients. *Chinese Medical Journal*, Vol.116, No.11, (2003), pp. 1737-1740, ISSN 0366-6999 Teague, C.A.; Doak, P.B., Simpson, I.J., Rainer, S.P. & Herdson, P.B. (1978). Goodpasture's

syndrome: an analysis of 29 cases. *Kidney International*, Vol.13, No.6, (June 1978),

the development of experimental crescentic glomerulonephritis. Studies using tissue culture and electron microscopy. *The American Journal of Pathology*, Vol.94,

production by intrinsic renal cells and bone marrow-derived cells is required for full expression of crescentic glomerulonephritis in mice. *Journal of Immunology*,

Phelps, R.G. & Rees, A.J. (1999). The HLA complex in Goodpasture's disease: a model for

Proskey, A.J.; Weatherbee, L., Easterling, R.E., Greene, J.A. Jr. & Weller, J.M. (1970).

Pusey, C.D. (2003). Anti-glomerular basement membrane disease. *Kidney International*,

Quérin, S.; Schürch, W. & Beaulieu, R. (1992). Ciclosporin in Goodpasture's syndrome.

Rees, A.J.; Peters, D.K., Compston, D.A. & Batchelor, J.R. (1978). Strong association between

Rees, A.J.; Demaine, A.G. &, Welsh, K.I. (1984). Association of immunoglobulin Gm

*Kidney Diseases*, Vol.46, No.2, (August 2005), pp. 253-262, ISSN 0272-6386 Rychlík, I.; Jancová, E., Tesar, V., Kolsky, A., Lácha, J., Stejskal, J., Stejskalová, A., Dusek, J. &

Sado, Y.; Kagawa, M., Naito, I., Ueki, Y., Seki, T., Momota, R., Oohashi, T. & Ninomiya, Y.

Salama, A.D.; Levy, J.B., Lightstone, L. & Pusey, C.D. (2001). Goodpasture's disease. *The Lancet*, Vol.358, No.9285, (September 2001), pp. 917–920, ISSN 0140-6736 Salant, D.J. (2010). Goodpasture's disease—new secrets revealed. *The New England Journal of*

Saus, J.; Wieslander, J., Langeveld, J.P., Quinones, S. & Hudson, B.G. (1988). Identification of

Saxena, R.; Bygren, P., Rasmussen, N. & Wieslander, J. (1991). Circulating autoantibodies in

*Medicine*, Vol.363, No.4, (July 2010), pp. 388-391, ISSN 0028-4793

Vol.6, No.6, (June 1991), pp. 389-397, ISSN 0931-0509

Vol.64, No.4, (October 2003), pp. 1535–1550, ISSN 0085-2538

*Nephron*, Vol.60, No.3, (March 1992), pp. 355-359, ISSN 0028-2766

(November 1999), pp. 1638–1653, ISSN 0085-2538

No.8071, (May, 1978), pp. 966–968, ISSN 0140-6736

(December 2004), pp. 3040-3049, ISSN 0931-0509

pp. 767-776, ISSN 0021-924X

0267-0623

9343

analyzing susceptibility to autoimmunity. *Kidney International*, Vol.56, No.5,

Goodpasture's syndrome. A report of five cases and review of the literature. *The American Journal of Medicine*, Vol.48, No.2, (February 1970), pp. 162-173, ISSN 0002-

HLA-DRW2 and antibody-mediated Goodpasture's syndrome. *The Lancet*, Vol.1,

allotypes with antiglomerular basement membrane antibodies and their titer. *Human Immunology*, Vol.10, No.4, (August 1984), pp. 213-220, ISSN 0198-8859 Rivera, F.; López-Gómez, J.M., Pérez-García, R. & Spanish Registry of Glomerulonephritis.

(2002). Frequency of renal pathology in Spain 1994–1999. *Nephrology Dialysis Transplantation,* Vol.17, No.9, (September 2002), pp. 1594-1562, ISSN 0931-0509 Rutgers, A.; Slot, M., van Paassen, P., van Breda Vriesman, P., Heeringa, P. & Tervaert, J.W.

(2005). Coexistence of anti-glomerular basement membrane antibodies and myeloperoxidase-ANCAs in crescentic glomerulonephritis. *American Journal of*

Herout V. (2004). The Czech registry of renal biopsies. Occurrence of renal diseases in the years 1994-2000. *Nephrology Dialysis Transplantation*, Vol.19, No.12,

(1998). Organization and expression of basement membrane collagen IV genes and their roles in human disorders. *Journal of Biochemistry*, Vol.123, No.5, (May 1998),

the Goodpasture antigen as the α3(IV) chain of collagen IV. *The Journal of Biological Chemistry*, Vol.263, No.26, (September 1988), pp. 13374-13380, ISSN 0021-9258 Savage, C.O.S.; Pusey, C.D., Bowman, C., Rees, A.J. & Lockwood, C.M. (1986)

Antiglomerular basement membrane antibody mediated disease in the British Isles 1980–4. *British Medical Journal*, Vol.292, No.6516, (February 1986), pp. 301–304, ISSN

patients with extracapillary glomerulonephritis. *Nephrology Dialysis Transplantation*,

**16**

*Japan*

Emiko Takeuchi

**Mixed Hematopoietic**

*Kitasato University School of Medicine*

 **Chimerism Allows Cure of Autoimmune**

**Glomerulonephritis: Its Potential and Risks**

Patients with severe autoimmune lupus glomerulonephritis that is resistant to immunosuppressive therapy need alternative treatment. Recently, bone marrow transplantation (BMT) has been proposed as a potential therapy for refractory autoimmune disease. BMT involves the administration of hematopoietic stem cells, which are self- renewing and capable of giving rise to all mature hematopoietic cell types and possibly some non-hematopoietic cell types. The etiologic and pathogenic bases of many autoimmune diseases ultimately reside in the self-renewing hematopoietic stem cell population. Therefore, the effects of BMT as a treatment for and/or preventive measure against these autoimmune diseases have been investigated extensively (Sykes&Nicolic, 2005). Studies in animal models have shown that the transfer of hematopoietic stem cells can reverse the autoimmune state. The induction of fully allogeneic bone marrow (BM) chimerism, however, is fraught with difficulties. Each of the various methods of inducing fully allogeneic BM chimerism through hematopoietic cell transplantation (HCT) requires a different set of conditions, such as host T cell depletion, donor myeloablation, major histocompatibility complex (MHC) fully matched donor BM, or lethal dose of total body irradiation (TBI). Meeting these conditions is usually a burden on the recipient. Moreover, fully allogeneic BM chimerism is always associated with risks of graft versus host disease (GVHD) and immunodeficiency, which make it less practical for clinical

Accordingly, the induction of mixed allogeneic BM chimerism has been proposed as a treatment for autoimmune disease. Mixed chimerism refers to a state in which allogeneic

In this paper, the advantages of inducing mixed BM chimerism are summarized and a process for inducing peripheral/central tolerance is introduced. Several mechanistic pathways which are thought to be involved in reversing the autoimmune state are then described. Based on our original data, we propose one possible mechanism in which newly developed donor T cells, which have been positively selected in the host thymus and restricted host MHC, are able to regulate auto-reactive B cells through T cell receptor (TCR)/MHC interaction. Finally, we discuss the potential risks associated with fully MHC-

donor hematopoietic cells coexist with recipient cells in host bone marrow.

**1. Introduction**

application.

## **Mixed Hematopoietic Chimerism Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks**

Emiko Takeuchi *Kitasato University School of Medicine Japan*

### **1. Introduction**

276 An Update on Glomerulopathies – Clinical and Treatment Aspects

Tipping, P.G.; Lowe, M.G. & Holdsworth, S.R. (1988). Glomerular macrophages express

Vanacore, R.M.; Ham, A.J., Cartailler, J.P., Sundaramoorthy, M., Todd, P., Pedchenko, V.,

Vanacore, R.M.; Ham, A.J., Voehler, M., Sanders, C.R., Conrads, T.P., Veenstra, T.D.,

Walker, R.G.; Scheinkestel, C., Becker, G.J., Owen, J.E., Dowling, J.P. & Kincaid-Smith, P.

Williams, P.S.; Davenport, A., McDicken, I., Ashby, D., Goldsmith, H.J. & Bone, J.M. (1988).

*of Medicine*, Vol.68, No.257, (September 1988), pp. 727–733, ISSN 0033-5622 Wilson, C.B. & Dixon, F.J. (1973). Anti-glomerular basement membrane antibody-induced

Yang, G.; Tang, Z., Chen, Y., Zeng, C., Chen, H., Liu, Z. & Li, L. (2005). Antineutrophil

Yang, R.; Cui, Z., Zhao, J. & Zhao, M.H. (2009). The role of HLA-DRB1 alleles on

Zhou, X.J.; Lv, J.C., Yu, L., Cui, Z., Zhao, J., Yang, R., Han, J., Hou, P., Zhao, M.H. & Zhang,

*Transplantation*, Vol.25, No.1, (January 2010), pp. 97–101, ISSN 0931-0509 Zhou, X.J.; Lv, J.C., Bu, D.F., Yu, L., Yang, Y.R., Zhao, J., Cui, Z., Yang, R., Zhao, M.H. &

*Immunology*, Vol.22, No.1, (January 2010), pp. 45–51, ISSN 1460-2377 Hirayama, K.; Yamagata, K., Kobayashi, M., Koyama, A. (2008). Anti-glomerular basement

No.5, (October 2008), pp. 339-347, ISSN 1342-1751

Vol.133, No.2, (November 2009), pp. 245–250, ISSN 1521-6616

(August 2008), pp. 22737–22748, ISSN 0021-9258

1259, ISSN 0021-9738

0036-8075

89, ISSN 0033-5622

ISSN 0085-2538

0301-0430

augmented procoagulant activity in experimental fibrin-related glomerulonephritis in rabbits. *The Journal of Clinical Investigation*, Vol.82, No.4, (October 1988), pp. 1253–

Sado, Y., Borza, D.B. & Hudson, B.G. (2008). A role for collagen IV cross-links in conferring immune privilege to the Goodpasture autoantigen: structural basis for the crypticity of B cell epitopes. *The Journal of Biological Chemistry*, Vol.283, No.33,

Sharpless, K.B., Dawson, P.E. & Hudson, B.G. (2009). A sulfilimine bond identified in collagen IV. *Science*, Vol.325, No.5945, (September 2009), pp. 1230–1234, ISSN

(1985). Clinical and morphological aspects of the management of crescentic antiglomerular basement membrane antibody (anti-GBM) nephritis/Goodpasture's syndrome. *The Quarterly Journal of Medicine*, Vol.54, No.213, (January 1985), pp. 75–

Increased incidence of anti-glomerular basement membrane antibody (anti-GBM) nephritis in the Mersey Region, September 1984-October 1985. *The Quarterly Journal*

glomerulonephritis. *Kidney International*, Vol.3, No.2, (February 1973), pp. 74–89,

cytoplasmic antibodies (ANCA) in Chinese patients with anti-GBM crescentic glomerulonephritis. *Clinical Nephrology*, Vol.63, No.6, (June 2005), pp. 423-428, ISSN

susceptibility of Chinese patients with anti-GBM disease. *Clinical Immunology*,

H. (2010a). FCGR2b gene polymorphism rather than FCGR2A, FCGR3A and FCGR3B is associated with anti-GBM disease in Chinese. *Nephrology Dialysis*

Zhang, H. (2010b). Copy number variation of FCGR3A rather than FCGR3B and FCGR2B is associated with susceptibility to anti-GBM disease. *International*

membrane antibody disease in Japan: part of the nationwide rapidly progressive glomerulonephritis survey in Japan. *Clinical and Experimental Nephrology*, Vol.12, Patients with severe autoimmune lupus glomerulonephritis that is resistant to immunosuppressive therapy need alternative treatment. Recently, bone marrow transplantation (BMT) has been proposed as a potential therapy for refractory autoimmune disease. BMT involves the administration of hematopoietic stem cells, which are self- renewing and capable of giving rise to all mature hematopoietic cell types and possibly some non-hematopoietic cell types. The etiologic and pathogenic bases of many autoimmune diseases ultimately reside in the self-renewing hematopoietic stem cell population. Therefore, the effects of BMT as a treatment for and/or preventive measure against these autoimmune diseases have been investigated extensively (Sykes&Nicolic, 2005). Studies in animal models have shown that the transfer of hematopoietic stem cells can reverse the autoimmune state. The induction of fully allogeneic bone marrow (BM) chimerism, however, is fraught with difficulties. Each of the various methods of inducing fully allogeneic BM chimerism through hematopoietic cell transplantation (HCT) requires a different set of conditions, such as host T cell depletion, donor myeloablation, major histocompatibility complex (MHC) fully matched donor BM, or lethal dose of total body irradiation (TBI). Meeting these conditions is usually a burden on the recipient. Moreover, fully allogeneic BM chimerism is always associated with risks of graft versus host disease (GVHD) and immunodeficiency, which make it less practical for clinical application.

Accordingly, the induction of mixed allogeneic BM chimerism has been proposed as a treatment for autoimmune disease. Mixed chimerism refers to a state in which allogeneic donor hematopoietic cells coexist with recipient cells in host bone marrow.

In this paper, the advantages of inducing mixed BM chimerism are summarized and a process for inducing peripheral/central tolerance is introduced. Several mechanistic pathways which are thought to be involved in reversing the autoimmune state are then described. Based on our original data, we propose one possible mechanism in which newly developed donor T cells, which have been positively selected in the host thymus and restricted host MHC, are able to regulate auto-reactive B cells through T cell receptor (TCR)/MHC interaction. Finally, we discuss the potential risks associated with fully MHC-

Mixed Hematopoietic Chimerism

**2.2 How is mixed chimerism induced?**

antigen-specific non-responsiveness (Fig.1).

chimeras.

tolerance (right).

Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks 279

3. Mixed chimerism can be achieved through non-myeloablative regimens, which are generally less toxic than the myeloablative regimens necessary to induce full BM

As explained above, once specific tolerance is established, the state of mixed chimerism is thought to be stable. The difficulty in establishing stable mixed chimerism lies in blocking the first attack of host peripheral T cells on donor bone marrow stem cells until "tolerized" T cells are renewed in the host thymus. Because host T cells play a dominant role in the rejection of allografts, several methods of deleting host T cells through the injection of various lymphocyte-deleting antibodies along with either total body irradiation or immunosuppressive drugs have been attempted (Tomita et al, 1996. Nikolic et al, 2000). These regimens enabled BM engraftment but were a burden on recipients and frequently made them susceptible to infection. The toxicity of the necessary conditioning regimens has

Another method of inducing allogeneic tolerance involves the temporary inhibition of costimulatory interaction between APCs and T cells by injecting blocking antibodies. This method works because T cell activation without proper co-stimulation can induce a state of

Fig. 1. Activation of naïve T cells requires co-stimulation. Binding of the peptide/MHC complex by the TCR and the CD4 or CD8 co-receptor transmits the first signal to the T cell. Activation of naïve T cells requires a second signal, namely, the ligation of B7 molecules (B7.1/B7.2) and CD28, which stimulates the clonal expansion of naïve T cells. Binding of CD40L by CD40 plays a central role effector function in the full differentiation of T cells. This ligation also activates APCs to express B7 molecules (left). Stimulation of CTLA4 with anti-CTLA4 Ab and/or Blocking CD40/CD40L by MR1 induces antigen-specific T cell

chimerism (this point will be discussed in detail in the next section).

precluded the use of this approach in clinical transplantation.

peripheral T cell repertoire that is tolerant toward both donor and host cells. Therefore, GVHD, one of the most important complications of allogeneic BMT, is not seen in mixed

mismatched allogeneic mixed chimerism. This information will help to determine the role that HCT can play in the treatment of autoimmune glomerulonephritis.

#### **2. Bone marrow mixed chimerism**

#### **2.1 What is the advantage of mixed chimerism?**

Mixed chimerism refers to a state in which allogeneic donor hematopoietic cells coexist with recipient cells in host bone marrow, whereas fully allogeneic chimerism refers to a state in which donor hematopoietic cells completely replace recipient cells.

It is known that fully allogeneic chimeras transplanted from a donor with fully mismatched MHC usually reject donor BM, or experience severe GVHD. Even if donor BM cells were safely engrafted in host BM, the resulting fully MHC-mismatched chimeras would develop immunodeficiency. In fully allogeneic chimeras, all mature T cells are supposed to be restricted to the host MHC type, irrespective of their own genetic background. This occurs because thymocytes, the precursors of mature T cells, are positively selected for weak reactivity to the self-peptide/MHC complex in the host thymus; this positive selection is mediated only by thymic cortical epithelial cells and not by bone marrow-derived cells. Therefore, in the periphery, all TCRs have certain affinity to host MHC molecules but not to donor MHC molecules. Thus, if the donor MHC is fully mismatched with the host MHC, there are no peripheral T cells which can interact with peripheral B cells differentiated from donor hematopoietic stem cells which generate donor-type MHC. This is the cause of deficiency in humoral immunity in fully MHC-mismatched allogeneic chimerism (Janeway et al., 2001).

In mixed chimerism, on the other hand, TCR/MHC interactions are at least partially maintained, because B cells differentiated from recipient hematopoietic stem cells are still being generated. Moreover, during intrathymic development, thymocytes that have high affinity to self MHC molecules are deleted from the repertoire in a process known as negative selection. Thymocytes from both the recipient and the donor mature on the thymic epithelium expressing MHC molecules with the recipient haplotype. Nevertheless, the repertoire of T cells, which react with high affinity to MHC molecules with the donor haplotype, eliminated in mixed chimera. This implies that bone marrow-derived cells must be able to induce negative selection. Actually, negative selection in the thymus can be mediated by several different cells. The most important of these are the BMderived dendritic cells and macrophages. In mixed chimera, the dendritic cells and macrophages differentiated from both donor and host hematopoietic stem cells are located in the thymus, where they eliminate T cells with strong reactivity to self-peptides on both donor and host MHC; thus donor- and host-specific tolerance to each other is established.

To summarize, mixed chimerism offers several advantages over full chimerism as a means of treating autoimmune disease:

peripheral T cell repertoire that is tolerant toward both donor and host cells. Therefore, GVHD, one of the most important complications of allogeneic BMT, is not seen in mixed chimeras.

3. Mixed chimerism can be achieved through non-myeloablative regimens, which are generally less toxic than the myeloablative regimens necessary to induce full BM chimerism (this point will be discussed in detail in the next section).

### **2.2 How is mixed chimerism induced?**

278 An Update on Glomerulopathies – Clinical and Treatment Aspects

mismatched allogeneic mixed chimerism. This information will help to determine the role

Mixed chimerism refers to a state in which allogeneic donor hematopoietic cells coexist with recipient cells in host bone marrow, whereas fully allogeneic chimerism refers to a state in

It is known that fully allogeneic chimeras transplanted from a donor with fully mismatched MHC usually reject donor BM, or experience severe GVHD. Even if donor BM cells were safely engrafted in host BM, the resulting fully MHC-mismatched chimeras would develop immunodeficiency. In fully allogeneic chimeras, all mature T cells are supposed to be restricted to the host MHC type, irrespective of their own genetic background. This occurs because thymocytes, the precursors of mature T cells, are positively selected for weak reactivity to the self-peptide/MHC complex in the host thymus; this positive selection is mediated only by thymic cortical epithelial cells and not by bone marrow-derived cells. Therefore, in the periphery, all TCRs have certain affinity to host MHC molecules but not to donor MHC molecules. Thus, if the donor MHC is fully mismatched with the host MHC, there are no peripheral T cells which can interact with peripheral B cells differentiated from donor hematopoietic stem cells which generate donor-type MHC. This is the cause of deficiency in humoral immunity in fully MHC-mismatched allogeneic chimerism (Janeway

In mixed chimerism, on the other hand, TCR/MHC interactions are at least partially maintained, because B cells differentiated from recipient hematopoietic stem cells are still being generated. Moreover, during intrathymic development, thymocytes that have high affinity to self MHC molecules are deleted from the repertoire in a process known as negative selection. Thymocytes from both the recipient and the donor mature on the thymic epithelium expressing MHC molecules with the recipient haplotype. Nevertheless, the repertoire of T cells, which react with high affinity to MHC molecules with the donor haplotype, eliminated in mixed chimera. This implies that bone marrow-derived cells must be able to induce negative selection. Actually, negative selection in the thymus can be mediated by several different cells. The most important of these are the BMderived dendritic cells and macrophages. In mixed chimera, the dendritic cells and macrophages differentiated from both donor and host hematopoietic stem cells are located in the thymus, where they eliminate T cells with strong reactivity to self-peptides on both donor and host MHC; thus donor- and host-specific tolerance to each other is

To summarize, mixed chimerism offers several advantages over full chimerism as a means

1. Mixed chimeras exhibit superior immune-competence across complete MHC barriers. Mixed chimeras possess certain populations of antigen presenting cells (APCs) and B cells which express host-type MHC molecules in the periphery, whereas mixed

2. In mixed chimeras, dendritic cells and macrophages differentiated from both the recipient and the donor hematopoietic stem cells locate to the thymus where they delete both host-reactive and donor-reactive T cells through negative selection, resulting in a

chimeras exhibit normal humoral and cellular immune responses.

that HCT can play in the treatment of autoimmune glomerulonephritis.

which donor hematopoietic cells completely replace recipient cells.

**2. Bone marrow mixed chimerism**

et al., 2001).

established.

of treating autoimmune disease:

**2.1 What is the advantage of mixed chimerism?**

As explained above, once specific tolerance is established, the state of mixed chimerism is thought to be stable. The difficulty in establishing stable mixed chimerism lies in blocking the first attack of host peripheral T cells on donor bone marrow stem cells until "tolerized" T cells are renewed in the host thymus. Because host T cells play a dominant role in the rejection of allografts, several methods of deleting host T cells through the injection of various lymphocyte-deleting antibodies along with either total body irradiation or immunosuppressive drugs have been attempted (Tomita et al, 1996. Nikolic et al, 2000). These regimens enabled BM engraftment but were a burden on recipients and frequently made them susceptible to infection. The toxicity of the necessary conditioning regimens has precluded the use of this approach in clinical transplantation.

Another method of inducing allogeneic tolerance involves the temporary inhibition of costimulatory interaction between APCs and T cells by injecting blocking antibodies. This method works because T cell activation without proper co-stimulation can induce a state of antigen-specific non-responsiveness (Fig.1).

Fig. 1. Activation of naïve T cells requires co-stimulation. Binding of the peptide/MHC complex by the TCR and the CD4 or CD8 co-receptor transmits the first signal to the T cell. Activation of naïve T cells requires a second signal, namely, the ligation of B7 molecules (B7.1/B7.2) and CD28, which stimulates the clonal expansion of naïve T cells. Binding of CD40L by CD40 plays a central role effector function in the full differentiation of T cells. This ligation also activates APCs to express B7 molecules (left). Stimulation of CTLA4 with anti-CTLA4 Ab and/or Blocking CD40/CD40L by MR1 induces antigen-specific T cell tolerance (right).

Mixed Hematopoietic Chimerism

and third-party skin.

Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks 281

Fig. 2. An example of chimerism in peripheral blood lymphocytes (PBL).

The percentage of BALB/c (H-2Dd) donor cells present among PBL of various lineages was

Fig. 3. Donor-specific skin graft tolerance. Donor and third-party skin was grafted 1 day post-BMT. Chimeric mice receiving MHC-mismatched BALB/c BM (◆BMT: n=9) accepted BALB/c skin grafts permanently, while third-party skin was rejected. Mice treated with TBI and anti-CD40L Ab (△: n=5) and mice receiving no treatment (●:n=5) rejected both donor

These results indicate that, with regard to reciprocal tolerant between donor and host, T cells in stable mixed chimeric mice do not reject additional tissue grafts transplanted from the same donor. In lupus patients who suffer from renal disorders, and who are treated by means of kidney transplantation, the induction of specific immunologic tolerance to donor

analyzed through two-color FACS. These data were obtained 24 weeks after BMT.

Recently, Takeuchi et al. have shown that administration of MHC-mismatched donor bone marrow to mice receiving 3Gy TBI one day before BMT and a single injection of Hamster- anti-mouse CD40L monoclonal antibody (MR1, hybridoma) intraperitoneally (i.p.) with BMT permitted the induction of permanent mixed chimerism and tolerance without T cell depletion (Y. Takeuchi et al., 2004). This regimen is quite simple and less toxic than the alternatives, because 3Gy TBI is nonlethal and does not require MHC matching. Therefore we have adopted this regimen for treatment of autoimmune disease in systemic lupus erythematosus (SLE) model mice ("BXSB"mice) and investigated the effect of induction of fully MHC-mismatched bone marrow mixed chimerism (E. Takeuchi & Y. Takeuchi, 2007).

### **3. Treatment of autoimmune glomerulonephritis in BXSB lupus mice**

### **3.1 BM mixed chimerism can be induced in BXSB mice**

BXSB mice spontaneously develop autoimmune disease with features similar to human SLE. The disease is associated with auto-antibodies to self-antigens (Ags) including double strand (ds) DNA, single strand (ss) DNA, anti-platelet antibodies (Abs) and anti-erythrocyte Abs, with accompanying splenomegaly and lymphadenopathy. Immune complex-mediated nephropathy is the hallmark disease associated with the BXSB genotype. Histopathological changes are evident by 10 weeks of age, leading to end-stage renal disease and 70% mortality by 40 weeks of age. We sought to determine whether the simple regimen described above was also effective for the induction of long-term mixed chimerism in BXSB mice. Twenty million normal bone marrow cells from MHCmatched (B6/GFP: H-2b) or -mismatched (BALB/c: H-2d) donors were injected with 2.0mg MR1 (i.p.) to seven-week old BXSB mice (H-2b) that had received a nonlethal dose of 4Gy TBI one day prior to BMT. We increased the TBI dose for BXSB mice from 3 to 4Gy because BXSB mice are more resistant to engraftment than normal recipients are. This regimen allowed the induction of multi-lineage mixed chimerism in 70-90% of host BXSB mice.

As shown in Table 1 and Fig.2, long-term stable chimerism was observed in MHCmismatched chimeric mice. No clinical signs of GVHD were seen during the observation period.

Table 1. The percentage of donor cells among PBL of chimeric mice. (n=9)

To confirm the establishment of donor-specific tolerance, chimeric BXSB mice also received skin grafts from a BM donor and a third party (C3H/HeN, H-2k) one day after BMT. All chimeric BXSB mice accepted the donor skin, but rejected the third-party skin grafts within 20 days (Fig.3), indicating that chimeric BXSB mice acquired donor-specific tolerance without immune-deficiencies (E. Takeuchi, 2011).

Recently, Takeuchi et al. have shown that administration of MHC-mismatched donor bone marrow to mice receiving 3Gy TBI one day before BMT and a single injection of Hamster- anti-mouse CD40L monoclonal antibody (MR1, hybridoma) intraperitoneally (i.p.) with BMT permitted the induction of permanent mixed chimerism and tolerance without T cell depletion (Y. Takeuchi et al., 2004). This regimen is quite simple and less toxic than the alternatives, because 3Gy TBI is nonlethal and does not require MHC matching. Therefore we have adopted this regimen for treatment of autoimmune disease in systemic lupus erythematosus (SLE) model mice ("BXSB"mice) and investigated the effect of induction of fully MHC-mismatched bone marrow mixed chimerism (E. Takeuchi

BXSB mice spontaneously develop autoimmune disease with features similar to human SLE. The disease is associated with auto-antibodies to self-antigens (Ags) including double strand (ds) DNA, single strand (ss) DNA, anti-platelet antibodies (Abs) and anti-erythrocyte Abs, with accompanying splenomegaly and lymphadenopathy. Immune complex-mediated nephropathy is the hallmark disease associated with the BXSB genotype. Histopathological changes are evident by 10 weeks of age, leading to end-stage renal disease and 70% mortality by 40 weeks of age. We sought to determine whether the simple regimen described above was also effective for the induction of long-term mixed chimerism in BXSB mice. Twenty million normal bone marrow cells from MHCmatched (B6/GFP: H-2b) or -mismatched (BALB/c: H-2d) donors were injected with 2.0mg MR1 (i.p.) to seven-week old BXSB mice (H-2b) that had received a nonlethal dose of 4Gy TBI one day prior to BMT. We increased the TBI dose for BXSB mice from 3 to 4Gy because BXSB mice are more resistant to engraftment than normal recipients are. This regimen allowed the induction of multi-lineage mixed chimerism in 70-90% of host BXSB

As shown in Table 1 and Fig.2, long-term stable chimerism was observed in MHCmismatched chimeric mice. No clinical signs of GVHD were seen during the observation

To confirm the establishment of donor-specific tolerance, chimeric BXSB mice also received skin grafts from a BM donor and a third party (C3H/HeN, H-2k) one day after BMT. All chimeric BXSB mice accepted the donor skin, but rejected the third-party skin grafts within 20 days (Fig.3), indicating that chimeric BXSB mice acquired donor-specific tolerance

Table 1. The percentage of donor cells among PBL of chimeric mice. (n=9)

without immune-deficiencies (E. Takeuchi, 2011).

**3. Treatment of autoimmune glomerulonephritis in BXSB lupus mice**

**3.1 BM mixed chimerism can be induced in BXSB mice**

& Y. Takeuchi, 2007).

mice.

period.

Fig. 2. An example of chimerism in peripheral blood lymphocytes (PBL). The percentage of BALB/c (H-2Dd) donor cells present among PBL of various lineages was analyzed through two-color FACS. These data were obtained 24 weeks after BMT.

Fig. 3. Donor-specific skin graft tolerance. Donor and third-party skin was grafted 1 day post-BMT. Chimeric mice receiving MHC-mismatched BALB/c BM (◆BMT: n=9) accepted BALB/c skin grafts permanently, while third-party skin was rejected. Mice treated with TBI and anti-CD40L Ab (△: n=5) and mice receiving no treatment (●:n=5) rejected both donor and third-party skin.

These results indicate that, with regard to reciprocal tolerant between donor and host, T cells in stable mixed chimeric mice do not reject additional tissue grafts transplanted from the same donor. In lupus patients who suffer from renal disorders, and who are treated by means of kidney transplantation, the induction of specific immunologic tolerance to donor

Mixed Hematopoietic Chimerism

inhibited the development of lupus-like disease.

**4. How does BM chimerism reverse the autoimmune state?**

next section.

**4.1 Hypothesis**

(E. Takeuchi et al., 2011)

Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks 283

exhibited histopathology similar to BXSB mice that had received only TBI (data not shown). For semiquantitative histologic analyses, more than 30 glomeruli from each kidney section were examined. Glomerulonephritis was scored on a scale of 0-4, based on the intensity and extent of histopathological changes [0; no glomerular lesions. 1; minimal thickening of the mesangium. 2; noticeable increase in both mesangeal and glomerular capillary cellularity. 3; same as 2 with the addition of superimposed inflammatory exudates and capsular adhesions. 4; obliteration of the glomerular architecture (>70% of glomeruli)]. Mean renal scores of both MHC-matched and –mismatched chimeric mice were significantly better than those of untreated or irradiated control BXSB mice (BALB/c BMT: 0.970.74, GFP/B6 BMT: 0.070.26 vs. untreated BXSB: 2.960.72, TBI: 1.850.77, TBI+MR1: 2.473.4, >30 glomeruli from each kidney section in three mice of each group were evaluated. p<0.05). We also evaluated immune-complex mediated glomerulonephritis through immunofluorescence (IF) staining with anti-mouse complement C3 in 30 glomeruli per renal section (Fig.4. right panels). Untreated mice and irradiated mice exhibited the peripheral loop pattern (Figs. 4H and I), while almost all glomeruli in the sections from chimeric mice showed negative staining (Figs. 4J and K). Because the cause of death in BXSB mice is most often renal failure, the improvement of their glomerulonephritis may have contributed to the prolongation of their life-spans. These results indicate that the induction of mixed chimerism significantly

It should be noted, however, that the induction of mixed chimerism in BXSB mice could not completely eliminate auto-reactive host lymphocytes because our regimen retains certain stem cells and lymphocytes belonging to the host. This naturally leads to the question of how donor cells reverse the host autoimmune state, which is discussed in the

We and several other groups have shown that the induction of bone marrow mixed chimerism is an effective treatment for and/or means of prevention against the development of autoimmune disease. Previous studies have debated the mechanisms that may be responsible for the reversal of the autoimmune state in BM chimerism, but the mechanism of the exclusion of self-reactive lymphocytes has not yet been conclusively identified. Preceding studies have argued about the mechanisms underlying the reversal of the autoimmune state in BM chimerism. In several studies which reversed destructive autoimmune type І diabetes (NOD mice with induced mixed chimerism), the suppression of autoimmune disease was attributed to reciprocal clonal deletion or to anergy induction of T lymphocytes of recipient and donor origin (Mathieu et al., 1997. Nikolic et al., 2004). Other mechanisms have also been proposed, including induction of peripheral anergy, a change in the Th1/Th2 profile, correction of abnormal secretion of cytokines and positive selection of regulatory T cells in the thymus. Among proposed hypotheses, we have focused on the role of cognate TCR/MHC interactions in the pathogenesis of autoimmune disease in BXSB mice

**4.2 The induction of MHC-mismatched chimerism does not suppress anti-DNA Abs**  During the development of their lupus-like autoimmune disease, BXSB mice are known to produce auto-antibodies to self-antigens including dsDNA. We measured serum anti-

antigens would prevent both chronic graft rejection and the side effects associated with chronic, nonspecific immunosuppressive therapy.

#### **3.2 Induction of mixed chimerism suppressed lupus nephritis in BXSB mice**

Even when transplanted kidneys are engrafted stably, when pre-existing lupus goes untreated, renal disorder will eventually recur. It is also known, however, that the induction of mixed chimerism reverses the autoimmune state. To evaluate the effect of inducing MHCmatched or MHC-mismatched mixed chimerism, individual kidneys were harvested from experimental mice and tissue sections were stained with periodic acid-Schiff (PAS) for histopathologic examination. None of the donor mouse strains were prone to autoimmune disease (Fig.4A). In both the fully MHC-matched and the fully MHC-mismatched chimeric mice groups, lupus glomerulonephritis was significantly ameliorated compared with that in untreated BXSB mice, as revealed by pathological analysis conducted more than 40 weeks after BMT (Figs.4D and E).

Fig. 4. Kidney sections stained with PAS (left panel) and IF with anti-mouse complement C3 (right panel). Kidneys were harvested from normal control mice or 47-50-week-old (40-43 weeks after BMT) BXSB mice with or without the indicated treatments. C57BL/6 control mice (A and G), untreated BXSB mice (B and H), irradiated BXSB mice (C and I), BALB/c BMT chimeric mice (D and J), GFP/B6 BMT chimeric mice (E and K), BALB/cGFP/B6 F1 BMT chimeric mice (F and L).

Both untreated (Fig.4B) and irradiated BXSB mice (Fig.4C) exhibited severe glomerulonephritis with PAS-positive deposits. BXSB mice that had received TBI+MR1 exhibited histopathology similar to BXSB mice that had received only TBI (data not shown). For semiquantitative histologic analyses, more than 30 glomeruli from each kidney section were examined. Glomerulonephritis was scored on a scale of 0-4, based on the intensity and extent of histopathological changes [0; no glomerular lesions. 1; minimal thickening of the mesangium. 2; noticeable increase in both mesangeal and glomerular capillary cellularity. 3; same as 2 with the addition of superimposed inflammatory exudates and capsular adhesions. 4; obliteration of the glomerular architecture (>70% of glomeruli)]. Mean renal scores of both MHC-matched and –mismatched chimeric mice were significantly better than those of untreated or irradiated control BXSB mice (BALB/c BMT: 0.970.74, GFP/B6 BMT: 0.070.26 vs. untreated BXSB: 2.960.72, TBI: 1.850.77, TBI+MR1: 2.473.4, >30 glomeruli from each kidney section in three mice of each group were evaluated. p<0.05). We also evaluated immune-complex mediated glomerulonephritis through immunofluorescence (IF) staining with anti-mouse complement C3 in 30 glomeruli per renal section (Fig.4. right panels). Untreated mice and irradiated mice exhibited the peripheral loop pattern (Figs. 4H and I), while almost all glomeruli in the sections from chimeric mice showed negative staining (Figs. 4J and K). Because the cause of death in BXSB mice is most often renal failure, the improvement of their glomerulonephritis may have contributed to the prolongation of their life-spans. These results indicate that the induction of mixed chimerism significantly inhibited the development of lupus-like disease.

It should be noted, however, that the induction of mixed chimerism in BXSB mice could not completely eliminate auto-reactive host lymphocytes because our regimen retains certain stem cells and lymphocytes belonging to the host. This naturally leads to the question of how donor cells reverse the host autoimmune state, which is discussed in the next section.

### **4. How does BM chimerism reverse the autoimmune state?**

### **4.1 Hypothesis**

282 An Update on Glomerulopathies – Clinical and Treatment Aspects

antigens would prevent both chronic graft rejection and the side effects associated with

Even when transplanted kidneys are engrafted stably, when pre-existing lupus goes untreated, renal disorder will eventually recur. It is also known, however, that the induction of mixed chimerism reverses the autoimmune state. To evaluate the effect of inducing MHCmatched or MHC-mismatched mixed chimerism, individual kidneys were harvested from experimental mice and tissue sections were stained with periodic acid-Schiff (PAS) for histopathologic examination. None of the donor mouse strains were prone to autoimmune disease (Fig.4A). In both the fully MHC-matched and the fully MHC-mismatched chimeric mice groups, lupus glomerulonephritis was significantly ameliorated compared with that in untreated BXSB mice, as revealed by pathological analysis conducted more than 40 weeks

Fig. 4. Kidney sections stained with PAS (left panel) and IF with anti-mouse complement C3 (right panel). Kidneys were harvested from normal control mice or 47-50-week-old (40-43 weeks after BMT) BXSB mice with or without the indicated treatments. C57BL/6 control mice (A and G), untreated BXSB mice (B and H), irradiated BXSB mice (C and I), BALB/c BMT chimeric mice (D and J), GFP/B6 BMT chimeric mice (E and K), BALB/cGFP/B6 F1

Both untreated (Fig.4B) and irradiated BXSB mice (Fig.4C) exhibited severe glomerulonephritis with PAS-positive deposits. BXSB mice that had received TBI+MR1

**3.2 Induction of mixed chimerism suppressed lupus nephritis in BXSB mice**

chronic, nonspecific immunosuppressive therapy.

after BMT (Figs.4D and E).

BMT chimeric mice (F and L).

We and several other groups have shown that the induction of bone marrow mixed chimerism is an effective treatment for and/or means of prevention against the development of autoimmune disease. Previous studies have debated the mechanisms that may be responsible for the reversal of the autoimmune state in BM chimerism, but the mechanism of the exclusion of self-reactive lymphocytes has not yet been conclusively identified. Preceding studies have argued about the mechanisms underlying the reversal of the autoimmune state in BM chimerism. In several studies which reversed destructive autoimmune type І diabetes (NOD mice with induced mixed chimerism), the suppression of autoimmune disease was attributed to reciprocal clonal deletion or to anergy induction of T lymphocytes of recipient and donor origin (Mathieu et al., 1997. Nikolic et al., 2004). Other mechanisms have also been proposed, including induction of peripheral anergy, a change in the Th1/Th2 profile, correction of abnormal secretion of cytokines and positive selection of regulatory T cells in the thymus. Among proposed hypotheses, we have focused on the role of cognate TCR/MHC interactions in the pathogenesis of autoimmune disease in BXSB mice (E. Takeuchi et al., 2011)

### **4.2 The induction of MHC-mismatched chimerism does not suppress anti-DNA Abs**

During the development of their lupus-like autoimmune disease, BXSB mice are known to produce auto-antibodies to self-antigens including dsDNA. We measured serum anti-

Mixed Hematopoietic Chimerism

regulated through TCR/MHC cognate interactions.

Fig. 6. The proposed of T cell surveillance model.

depicted in Fig.6.

**4.4 Do T cells survey auto-reactive antibody production?**

Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks 285

different from those in other groups (data not shown). These results indicated that normal B cells derived from donor BALB/c mice, rather than genetically lupus-prone host-type B cells, were responsible for anti-DNA antibody production in these chimeric BXSB mice. Thus the regulation of auto-antibody production appears to be under MHC-restriction of the host type. Additionally, to confirm which set of B cells (donor-type or host-type) could react with foreign antigens, sheep red blood cells (SRBC) were administered intraperitoneally to five BALB/c chimeric BXSB mice. Three days after immunization, serum anti-SRBC Ab was detected through flow cytometry. All chimeric mice produced antibodies that were reactive with SRBC. As expected, almost all of the detected anti-SRBC Ab was IgMb (host-type), not IgMa (allogeneic donor-type) (data not shown). In mixed chimeras, all peripheral T cells are supposed to be restricted to host MHC, because of positive selection in the host thymus. T cells in fully MHC-mismatched chimeric mice should therefore be capable of cognate interaction with host-type B cells but not with donor-type B cells. Accordingly, only hosttype B cells were activated by antigens through cognate interaction with helper T cells. Donor-type B cells remained "silent" because they could not interact properly with T cells. Interestingly, however, our results indicated the possibility that not only "proper" activation against foreign antigens, but also suppression of auto-reactive antibody production were

Based on these data, we drew the following conclusions: 1. Allogeneic BM chimerism ameliorates autoimmune disease, but fully MHC-mismatched chimerism fails to suppress the production of anti-DNA antibodies. 2. In MHC-mismatched mixed chimeras, anti-DNA antibodies are produced by donor-type B cells rather than host-type B cells. 3. In MHC-mismatched chimera, TCRs are restricted to host-type MHC. Accordingly, T cells can recognize only host B cells but not donor B cells. To tie theses conclusions together, we propose a possible T cell surveillance system of mixed chimerism, as

dsDNA antibody (anti-DNA Ab) levels by means of ELISA to evaluate whether autoreactive Abs were eliminated by the induction of mixed chimerism. Actually, the anti-DNA Ab levels in both MHC-matched and MHC-mismatched chimeric mice were lower than those in untreated or irradiated BXSB mice. Meanwhile, anti-DNA Ab levels in fully MHCmatched mixed chimeric mice (GFP/B6 BMT) were not statistically different from those in normal control B6 mice, but those in fully MHC-mismatched mixed chimeric mice (BALB/c BMT) were significantly higher than those in normal controls. This tendency was even more pronounced when anti-DNA IgM levels in the above groups were compared. There were no significant differences in anti-DNA IgM levels between MHC-mismatched chimeric mice and untreated or TBI+MR1 mice, even though total anti-DNA levels were much lower in chimeric mice than in other groups.

These data indicated that anti-DNA Ab producing cells were still present in the BXSB chimeric mice, though they stopped switching iso-types from IgM to IgG in MHCmismatched chimera. Only in MHC-matched chimeric mice could the expansion of anti-DNA Ab production be suppressed down to a normal level; the induction of fully MHCmismatched chimerism did not completely suppress or eliminate anti-DNA-producing B cells.

#### **4.3 Selective suppression of auto-reactive antibodies in chimeric mice**

In order to distinguish the contributions of donor-type and host-type B cells to anti-dsDNA antibody production, we determined IgM allotypes [IgMa: BALB/c (donor), IgMb: BXSB (host)] in the serum of fully MHC-mismatched (BALB/c→BXSB) chimeric mice 20 weeks after BMT. At this point, the percentage of allogeneic donor B cells in the chimeric mice was 51.011.8%, indicating that allogeneic donor and host B cells had contributed equally to the immune response. Surprisingly, however, we found that the majority of the anti-DNA IgM was IgMa (allogeneic donor-type), whereas IgMb (host-type) anti-DNA antibody production was suppressed (Fig. 5A).

Fig. 5. The ratio of host-type IgM to donor-type IgM. (A) Serum anti-DNA IgM in BALB/c BMT BXSB was measured in each sample by means of ELISA. The percentages of anti-DNA IgMa (donor-type IgM: dark bar) and IgMb (host-type IgM: light bar) add up to the total anti-DNA IgM. (B) Total IgM in each BALB/c BM- transplanted BXSB mouse was measured individually. The percentages of serum IgMa (donor-type IgM: dark bar) and IgMb (hosttype IgM: light bar) add up to the total IgM.

The majority of the total serum IgM, on the other hand, was host-type IgMb (Fig. 5B), suggesting that the production of "normal" serum Ig is dependent on host MHC-restricted T cells. Total serum IgM levels in fully MHC-mismatched chimeric mice were not significantly

dsDNA antibody (anti-DNA Ab) levels by means of ELISA to evaluate whether autoreactive Abs were eliminated by the induction of mixed chimerism. Actually, the anti-DNA Ab levels in both MHC-matched and MHC-mismatched chimeric mice were lower than those in untreated or irradiated BXSB mice. Meanwhile, anti-DNA Ab levels in fully MHCmatched mixed chimeric mice (GFP/B6 BMT) were not statistically different from those in normal control B6 mice, but those in fully MHC-mismatched mixed chimeric mice (BALB/c BMT) were significantly higher than those in normal controls. This tendency was even more pronounced when anti-DNA IgM levels in the above groups were compared. There were no significant differences in anti-DNA IgM levels between MHC-mismatched chimeric mice and untreated or TBI+MR1 mice, even though total anti-DNA levels were much lower in

These data indicated that anti-DNA Ab producing cells were still present in the BXSB chimeric mice, though they stopped switching iso-types from IgM to IgG in MHCmismatched chimera. Only in MHC-matched chimeric mice could the expansion of anti-DNA Ab production be suppressed down to a normal level; the induction of fully MHCmismatched chimerism did not completely suppress or eliminate anti-DNA-producing B

In order to distinguish the contributions of donor-type and host-type B cells to anti-dsDNA antibody production, we determined IgM allotypes [IgMa: BALB/c (donor), IgMb: BXSB (host)] in the serum of fully MHC-mismatched (BALB/c→BXSB) chimeric mice 20 weeks after BMT. At this point, the percentage of allogeneic donor B cells in the chimeric mice was 51.011.8%, indicating that allogeneic donor and host B cells had contributed equally to the immune response. Surprisingly, however, we found that the majority of the anti-DNA IgM was IgMa (allogeneic donor-type), whereas IgMb (host-type) anti-DNA antibody production

Fig. 5. The ratio of host-type IgM to donor-type IgM. (A) Serum anti-DNA IgM in BALB/c BMT BXSB was measured in each sample by means of ELISA. The percentages of anti-DNA IgMa (donor-type IgM: dark bar) and IgMb (host-type IgM: light bar) add up to the total anti-DNA IgM. (B) Total IgM in each BALB/c BM- transplanted BXSB mouse was measured individually. The percentages of serum IgMa (donor-type IgM: dark bar) and IgMb (host-

The majority of the total serum IgM, on the other hand, was host-type IgMb (Fig. 5B), suggesting that the production of "normal" serum Ig is dependent on host MHC-restricted T cells. Total serum IgM levels in fully MHC-mismatched chimeric mice were not significantly

**4.3 Selective suppression of auto-reactive antibodies in chimeric mice**

chimeric mice than in other groups.

was suppressed (Fig. 5A).

type IgM: light bar) add up to the total IgM.

cells.

different from those in other groups (data not shown). These results indicated that normal B cells derived from donor BALB/c mice, rather than genetically lupus-prone host-type B cells, were responsible for anti-DNA antibody production in these chimeric BXSB mice. Thus the regulation of auto-antibody production appears to be under MHC-restriction of the host type. Additionally, to confirm which set of B cells (donor-type or host-type) could react with foreign antigens, sheep red blood cells (SRBC) were administered intraperitoneally to five BALB/c chimeric BXSB mice. Three days after immunization, serum anti-SRBC Ab was detected through flow cytometry. All chimeric mice produced antibodies that were reactive with SRBC. As expected, almost all of the detected anti-SRBC Ab was IgMb (host-type), not IgMa (allogeneic donor-type) (data not shown). In mixed chimeras, all peripheral T cells are supposed to be restricted to host MHC, because of positive selection in the host thymus. T cells in fully MHC-mismatched chimeric mice should therefore be capable of cognate interaction with host-type B cells but not with donor-type B cells. Accordingly, only hosttype B cells were activated by antigens through cognate interaction with helper T cells. Donor-type B cells remained "silent" because they could not interact properly with T cells. Interestingly, however, our results indicated the possibility that not only "proper" activation against foreign antigens, but also suppression of auto-reactive antibody production were regulated through TCR/MHC cognate interactions.

#### **4.4 Do T cells survey auto-reactive antibody production?**

Based on these data, we drew the following conclusions: 1. Allogeneic BM chimerism ameliorates autoimmune disease, but fully MHC-mismatched chimerism fails to suppress the production of anti-DNA antibodies. 2. In MHC-mismatched mixed chimeras, anti-DNA antibodies are produced by donor-type B cells rather than host-type B cells. 3. In MHC-mismatched chimera, TCRs are restricted to host-type MHC. Accordingly, T cells can recognize only host B cells but not donor B cells. To tie theses conclusions together, we propose a possible T cell surveillance system of mixed chimerism, as depicted in Fig.6.

Fig. 6. The proposed of T cell surveillance model.

Mixed Hematopoietic Chimerism

for several isotypes of immunoglobulin.

Allows Cure of Autoimmune Glomerulonephritis: Its Potential and Risks 287