aDepartment of Internal Medicine, National Defense Medical College, Saitama 359-8513, JapanbHemodialysis Center, Chofu Hospital, Tokyo 182-0034, JapancDepartment of Preventive Medicine and Public Health, National Defense Medical College, Saitama 359-8513, JapandDepartment of Pediatrics, National Hospital Organization, Nishisaitama-Chuo National Hospital, Saitama 359-1151, JapaneDepartment of Nephrology, Showa University School of Medicine, Tokyo 142-8666, JapanfDepartment of Pediatrics, Yamanashi Medical University, Yamanashi 409-3898, JapangDepartment of Pediatrics, Juntendo University Nerima Hospital, Tokyo 177-8521, JapanhDepartment of Nephrology, Sendai Shakaihoken Hospital, Miyagi 981-8501, Japan
Received 7 September 2009; revised 16 February 2010; accepted 17 February 2010
Neutrophil;Plasmin;Streptococcal pyrogenicexotoxin B
Summary The nephritis-associated plasmin receptor is a recently identified nephritogenic antigenassociated with acute poststreptococcal glomerulonephritis and proposed to play a pathogenic role,but its precise glomerular localization in acute poststreptococcal glomerulonephritis has not beenelucidated. We therefore analyzed renal biopsy sections from 10 acute poststreptococcalglomerulonephritis patients by using immunofluorescence staining with anti–nephritis-associatedplasmin receptor antibody and various markers of glomerular components. Nephritis-associatedplasmin receptor was detected in the glomeruli of all patients, and double staining for nephritis-associated plasmin receptor and collagen IV showed nephritis-associated plasmin receptor to bepredominantly on the inner side of the glomerular tufts. Nephritis-associated plasmin receptor–positive areas within glomerular tufts were further characterized with markers for neutrophils,mesangial cells, endothelial cells, and macrophages. In 6 of the patients, nephritis-associated plasminreceptor staining was seen mainly in neutrophils and to a lesser degree in mesangial and endothelialcells. In the other 4 patients, nephritis-associated plasmin receptor staining was seen mainly inmesangial cells and to a lesser degree in neutrophils and endothelial cells. In all patients,macrophages showed little staining. Elevated plasmin activity in glomerular neutrophils wasidentified by combining in situ zymography staining for plasmin activity and immunofluorescencestaining for neutrophils. The glomerular localizations of nephritis-associated plasmin receptor andanother nephritogenic antigen, streptococcal pyrogenic exotoxin B, were compared by doubleimmunofluorescence staining and found to be similar. These findings indicate the nephritogenic
⁎ Corresponding author. Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan.E-mail address: [email protected] (T. Oda).
Acute poststreptococcal glomerulonephritis (APSGN), asequel of streptococcus infection and the prototypic acutenephritic syndrome [1-3], is recently rare in the industrial-ized world but is often found in rural and Aboriginalcommunities [4]. Because of the characteristic manifesta-tions of this disease (onset of symptoms 1-3 weeks afterstreptococcal infection, decrement in serum complementtiter, and glomerular deposition of C3 and/or immunoglob-ulin [Ig] G in the early phase), its initiation is widelythought to involve the immunologic response to strepto-coccal antigens. The most popular theory of the pathogenicmechanism of APSGN has been the immune complextheory, which involves the glomerular deposition of so-called nephritogenic streptococcal antigen and subsequentformation of immune complexes in situ and/or thedeposition of circulating antigen-antibody complexes[1-4]. Glomerular immunoglobulin deposition, however,is often not prominent in this disease. Furthermore, thereason for the difference between the site of glomerular cellinfiltration and the site of immune complex deposition isunclear: the major site of inflammation is the inner side ofthe glomerular tufts (glomerular endocapillary proliferationis the histologic hallmark of this disease), whereas in theearly phase of this disease, the immune complexes locatemainly on the outer (subepithelial) side of the glomerulartufts [3,5]. Another type of human glomerulonephritis withsubepithelial immune complex deposition, membranousnephropathy, is rarely accompanied by endocapillary cellinfiltration. Thus, the mechanism of the prominent glomer-ular endocapillary proliferation occurring in APSGN isunknown [2,3]; and the identity of the causative antigen(s)remains a matter of debate [6-11]. We recently isolated andcharacterized a nephritogenic antigen from group Astreptococcus [12-15] that we call the nephritis-associatedplasmin receptor (NAPlr) and is homologous to the group Astreptococcus plasmin receptor also known as streptococcalglyceraldehyde-3-phosphate dehydrogenase (GAPDH),which has been shown in vitro to bind plasmin and maintainits proteolytic activity by protecting it from physiologicinhibitors like α2-antiplasmin [16]. Glomerular deposits ofNAPlr are present in essentially all patients with early-phaseAPSGN [14]. The localization of NAPlr differs from that ofC3 or IgG [15] but is almost identical to that of plasminactivity [17], suggesting that deposited NAPlr inducesglomerular damage by keeping active plasmin trapped inthe glomeruli. The precise intraglomerular localization ofNAPlr in APSGN patients, however, has not been determined.
Furthermore, Batsford and coworkers [11] recently ana-lyzed a series of APSGN patients and found thatimmunohistochemical glomerular positivity for NAPlrwas far less frequent than that for streptococcal pyrogenicexotoxin B (SPEB). This is critically inconsistent with ourprevious findings [14,15,18]. To confirm the glomerulardeposition of NAPlr and identify its precise localization,we performed single and double immunofluorescencestaining with various glomerular cell markers and evaluatedstaining specificity by using antigen absorption tests. Wealso compared the localizations of SPEB and NAPlr bydouble immunofluorescence staining. The present resultsconfirm the glomerular deposition of NAPlr in APSGNpatients and show that it is found principally in neutrophils,mesangial cells, and endothelial cells. We show here thatthe distributions of NAPlr and SPEB in the glomeruli ofAPSGN patients are similar, and we discuss possiblereasons for this similarity.
2. Materials and methods
2.1. Patients
Renal biopsy tissues were collected from 10 APSGNpatients whose characteristics are listed in Table 1. Allshowed overt symptoms of APSGN such as facial edema,hypertension, and hematuria. Percutaneous needle biopsieswere performed to rule out progressive renal diseases thatcan cause acute nephritic syndrome (eg, IgA nephropathy,membranoproliferative glomerulonephritis, lupus nephri-tis, rapidly progressive glomerulonephritis). The diagnosisof APSGN was based on serologic and bacteriologicevidence of acute streptococcal infection before the onsetof nephritis and on characteristic histologic features of thebiopsy tissue (representative glomerular feature ofAPSGN in hematoxylin and eosin–stained tissue sectionis shown in Fig. 1). The major light microscopic findingin all of these patients was glomerular endocapillaryproliferation, which was accompanied by mesangialproliferation or crescent formation in some patients asshown in Table 1. By routine immunofluorescencemicroscopy, all patients showed positive staining for C3,staining pattern of which was also shown in Table 1. Fivepatients with rapidly progressive glomerulonephritis(defined as the presence of crescents in N60% ofglomeruli) served as disease control. Informed consentwas obtained from each patient in accordance with theprinciples of the Declaration of Helsinki.
Table1
Clin
ical
andhistolog
icprofilesof
patientswith
APSGN
andthesummaryof
NAPlrlocalization
Patient
no.
Age/sex
Onset
a
(d)
UP
U-RBC
(cells/HPF)
Creatinine
(mg/dL
)CH50
(U/m
L)
ASO
(U/m
L)
Light
microscop
yb
IFforC3c
NAPlrlocalizationd
Mes
Cres
Cap
Mes
Neu
MΦ
Mes
End
137
/F8
+++
50-99
1.7
15.2
691
−+
+++
−+++
±+
±2
21/M
8+++
30-49
0.8
5.1
226
−−
++
+±
±+++
±3
44/F
8+
5-9
0.9
16.9
1373
+−
+++
+++
−+
±4
48/M
8+
5-9
1.0
6.7
254
+−
++
+±
++
+5
16/M
12+++
N10
02.2
23.9
1530
+−
++
+++
++
+6
16/M
14+++
30-49
1.0
9.4
536
+−
++
++
+++
±+
+7
10/M
20+
30-49
1.3
12.5
539
++
+++
+±
++
+8
9/M
20++
N10
00.6
12.0
214
−−
++
++
±±
+++
+9
44M
∼20
e+
10-19
0.9
19.2
601
+−
+++
++
−+
+10
45/M
32+++
20-29
1.7
4.7
166
++
+++
++
±+
+
Abb
reviations:U
P,urinary
protein;U-RBC,urinary
redbloo
dcells;H
PF,high-po
werfield;CH50
,50%
hemolyticun
itof
complem
ent;ASO,anti-streptolysinO;M
es,m
esangial;C
res,crescent;C
ap,capillary;
Neu,neutroph
il;MΦ,macroph
age;
End
,end
othelialcell.
aDurationbetweendiseaseon
setandrenalbiop
sy.
bThe
existenceor
absenceof
glom
erular
mesangial
proliferationandcrescent
form
ationun
derlig
htmicroscop
ywas
identifiedas
+or
−.cThe
levelof
immun
ofluorescence(IF)staining
forC3alon
gcapillary
oron
mesangial
area
was
graded
as−,
+,++,or
+++.
dIntraglomerular
NAPlr-positive
portionwas
identifiedby
doub
lestaining
asneutroph
il,macroph
age,mesangial
cell,
orendo
thelialcell.
eAccuratedate
couldno
tbe
obtained
becausetheon
setdate
was
notrecorded
exactly
.
1278 T. Oda et al.
2.2. Immunofluorescence staining for NAPlr andantigen absorption tests
Unfixed cryostat sections of APSGN and rapidlyprogressive glomerulonephritis patients were stained withfluorescein isothiocyanate (FITC)–conjugated polyclonalrabbit anti-NAPlr antibody by using procedures describedpreviously [14], and selected sections were also stained withFITC-conjugated monoclonal mouse anti-NAPlr antibody(1F10) (Abcam Inc, Cambridge, MA). The specificity of the1F10 has been confirmed by Western blotting [19].
Antibody absorption tests were used to rule out cross-reactivity between the polyclonal anti-NAPlr antibody andhuman GAPDH. Blue A-Sepharose (Amersham Bios-ciences, Piscataway, NJ) was used to isolate humanGAPDH from the red blood cells of a healthy volunteer asdescribed by Thompson et al [20]. Polyclonal anti-NAPlrantibody was incubated with a 5-fold excess concentrationof human GAPDH for 2 hours. Unless otherwise stated, allprocedures were performed at 27°C. On some sections,direct immunofluorescence staining with absorbed anti-NAPlr antibody was performed, which was compared withthat with nonabsorbed anti-NAPlr antibody.
2.3. Double immunofluorescence staining for NAPlrand glomerular markers
Double staining for NAPlr and collagen IV was used todetermine the localization of NAPlr relative to theglomerular basement membrane. After being washed inphosphate-buffered saline (PBS), sections were incubated ina mixture of FITC-conjugated rat anti-human collagen IV α3chain antibody (a gift from Dr Yoshikazu Sado, ShigeiMedical Research Institute, Okayama, Japan) and AlexaFluor 594–conjugated rabbit anti-NAPlr antibody for 30minutes, washed in PBS, and mounted. The rabbit anti-NAPlr antibody was labeled with Alexa Fluor 594 by using aprotein labeling kit (Molecular Probes, Inc, Eugene, OR) asdescribed elsewhere [15].
To identify intraglomerular NAPlr staining, we used 4murine monoclonal antibodies from Dako Cytomation(Glostrup, Denmark) as markers of intraglomerular cellcomponents: anti-human α-smooth muscle actin antibody1A4 to mark mesangial cells, anti-human CD31 antibodyJC70A to mark endothelial cells, anti-human neutrophilelastase antibody NP57 to mark neutrophils, and anti-humanCD68 antibody EBM11 to mark macrophages. After thesectionswere stainedwith FITC-conjugated rabbit anti-NAPlrantibody, they were fixed in 4% paraformaldehyde for 5minutes, blocked with 7% nonfat skim milk in PBS for 5minutes, and incubated with one of the monoclonal antibodiesfor 1 hour. Bound antibodies were visualized by incubatingthe sections in Alexa Fluor 594–conjugated goat anti-mouseIgG antibody (Molecular Probes) for 30 minutes. Digitalimages of the stained sections were obtained with a confocalmicroscope (Zeiss LSM 510; Carl Zeiss, Göttingen,
Fig. 1 A representative photomicrograph of light microscopicimage of hematoxylin and eosin staining in a patient (patient 5) withAPSGN (original magnification ×260).
1279Localization of NAPlr in APSGN
Germany). Double staining was assessed as follows: − for nodouble staining, ± for less than 10% of the NAPlr-positive areabeing double positive, + for 10% to 30% of the NAPlr-positivearea being double positive, ++ for 31% to 50% of the NAPlr-positive area being double positive, and +++ for greater than50% of the NAPlr-positive area being double positive.
2.4. Double staining for neutrophils andplasmin activity
Plasmin activity in glomerular neutrophils was evaluatedby performing in situ zymography for plasmin activity [17]and indirect immunofluorescence staining for neutrophilelastase sequentially in the cryostat sections of APSGN andrapidly progressive glomerulonephritis patients. After sec-tions were fixed in 4% paraformaldehyde for 5 minutes, theywere incubated for 30 minutes with a reaction mixturecontaining 0.1% Fast Violet B and 0.5 mmol/L p-toluenesulfonyl-L-lysine α-naphthyl ester (Torii Pharmaceu-tical Co, Ltd, Tokyo, Japan) in 67 mmol/L phosphate buffer.Indirect immunofluorescence staining for neutrophil elastasewas then performed in the same manner as described aboveexcept that Alexa Fluor 488–conjugated goat anti-mouseIgG antibody (Molecular Probes) was used as the secondaryantibody. Light microscope images of plasmin activity andfluorescence microscope images of neutrophil elastasestaining were acquired, digitized, and merged.
2.5. Double immunofluorescence staining for NAPlrand SPEB
Because of the limited availability of renal biopsy sectionsfor the present series of patients, localization profiles of
NAPlr and SPEB were compared by double immunofluo-rescence staining in renal biopsy sections from only 6 of theAPSGN patients (patients 1, 4, 5, 7, 9, and 10). Cryostatsections were fixed for 5 minutes in 4°C acetone, incubatedfor 60 minutes in a mixture of Alexa Fluor 594–conjugatedrabbit anti-NAPlr antibody and FITC-conjugated rabbit anti-zymogen/SPEB antibody (a gift from Dr Stephen Batsford,Department of Immunology, Institute of Medical Microbi-ology, Freiburg, Germany), washed in PBS, and mounted.Digital images were obtained with a confocal microscope(Zeiss LSM 510, Carl Zeiss). To rule out an influence ofantibody mixing on the staining results, several consecutivesections were also stained for either NAPlr or SPEB.
3. Results
3.1. Single staining for NAPlr and evaluation ofstaining specificity
Staining with polyclonal anti-NAPlr antibody (Fig. 2A)showed glomerular positivity in all APSGN patientsanalyzed in the present study, although the staining patternand intensity varied, and diffuse (ie, glomerular as well as intubulointerstitial) nuclear background staining was ob-served in several sections. Similar results were observedwith monoclonal anti-NAPlr antibody (Fig. 2B), but boththe specific staining and background staining wererelatively weak. On the other hand, glomerular NAPlrstaining was not found in any biopsies of rapidlyprogressive glomerulonephritis patients stained with eitherpolyclonal anti-NAPlr antibody or monoclonal anti-NAPlrantibody (data not shown). We previously reported thatpreabsorption of polyclonal anti-NAPlr antibody withrecombinant plasmin receptor or pretreatment of tissuesections with unlabeled anti-NAPlr antibody diminishedglomerular NAPlr immunofluorescence staining [14,15].Because NAPlr is the same molecule as streptococcalGAPDH, the nonspecific positivity due to the cross reactionwith human GAPDH is a matter of concern. In the presentstudy, however, absorption of the polyclonal anti-NAPlrantibody with human GAPDH did not affect the glomerularNAPlr staining (Fig. 2C), indicating that the anti-NAPlrantibody does not react with human GAPDH and stainsNAPlr (ie, streptococcal GAPDH) specifically.
3.2. Double staining for NAPlr andglomerular markers
Double staining for NAPlr and collagen IV showed thatglomerular NAPlr positivity was localized predominantly onthe inner side of glomerular tufts (Fig. 3A-C) and to a lesserdegree on the parietal epithelial cells lining Bowman capsule.NAPlr staining was rarely detected on the outer side ofglomerular tufts (subepithelial sites or podocytes). The
Fig. 3 NAPlr localization relative to the glomerular basement membrane. Confocal microscopy images of double immunofluorescencestaining for collagen IV (A, FITC) and NAPlr (B, Alexa Fluor 594) with a merged image (C) in an APSGN patient (patient 9). NAPlrimmunofluorescence was mostly seen on the inner side of glomerular tufts and rarely seen on the outer side of glomerular tufts (subepithelialsite or podocytes) (scale bar = 10 μm).
Fig. 2 Glomerular deposition of NAPlr in a patient (patient 5) with APSGN. Representative photomicrographs of immunofluorescencestaining for NAPlr (A) with polyclonal rabbit anti-NAPlr antibody, (B) with monoclonal mouse anti-NAPlr antibody (1F10), or (C) withpolyclonal rabbit anti-NAPlr antibody that had been preabsorbed with isolated human GAPDH (original magnification ×260).
1280 T. Oda et al.
intratuft NAPlr positivity was therefore further investigatedby double staining with markers of glomerular intratuft cellcomponents. Representative microphotographs of doublestaining are shown in Fig. 4A-L, and the results aresummarized in the Table 1. In 6 of the 10 patients (1, 3, 5,6, 9, and 10), NAPlr staining was localized mainly inneutrophils (Fig. 4A-C) and to a lesser degree in mesangialand endothelial cells (Fig. 4G-I). In the other 4 patients(patients 2, 4, 7, and 8), NAPlr staining was localized mainlyin mesangial cells (Fig. 4J-l) and to a lesser degree inneutrophils and endothelial cells. In all patients, some
ig. 4 Precise intraglomerular localization of NAPlr by double staining. Representative confocal microscopy images of doublemunofluorescence staining for NAPlr (A, D, G, J; FITC) and various markers of intratuft glomerular cell components (B, neutrophils:
eutrophil elastase; E, macrophages: CD68; H, endothelial cells: CD31; K, mesangium: α-smooth muscle actin) (Alexa Fluor 594). In mostatients, NAPlr immunofluorescence was localized predominantly in neutrophils (A-C, patient 1). Macrophages showed only minor NAPlrmunofluorescence in all patients (D-F, double-positive portion is indicated by arrow, patient 1). NAPlr immunofluorescence was alsocalized in endothelial cells (G-I, double-positive portions are indicated by arrows, patient 5). In some patients, the mesangium showed the
Fimnpimlo
most NAPlr staining (J-L, patient 8) (scale bar = 20 μm).
macrophages were positive for NAPlr staining (Fig. 4D-F);but this staining was minor. As summarized in Table 1, nosignificant correlation was found between glomerular NAPlrdistributions with certain clinical or histologic features ofAPSGN patients.
3.3. Plasmin activity of glomerular neutrophils
Because NAPlr staining was found to be localized mainlyin neutrophils and NAPlr is known to bind to plasmin andmaintain its proteolytic activity in vitro [16], we examined
1281Localization of NAPlr in APSGN
Fig. 5 Glomerular infiltrating neutrophils and plasmin activity in APSGN and rapidly progressive glomerulonephritis. Representativephotomicrographs of double staining for neutrophil elastase (A, D, indirect immunofluorescence staining) and plasmin activity (B, E, in situzymography) from a patient with APSGN (A-C, patient 5) and with rapidly progressive glomerulonephritis (D-F). The same fields wereobserved under fluorescence microscopy (A, D) and light microscopy (B, E) and were merged (C, F). The merged image (C) shows up-regulated plasmin activity in a large portion of glomerular neutrophils in APSGN patients but not in rapidly progressive glomerulonephritispatients (F) (original magnification ×260).
1282 T. Oda et al.
the plasmin activity of glomerular neutrophils and found thatmany were positive for plasmin activity in renal tissues fromAPSGN patients (Fig. 5A-C). On the other hand, glomerularneutrophils were not positive for plasmin activity in renaltissues from rapidly progressive glomerulonephritis patients(Fig. 5D-F), which suggests disease specificity of therelationship between plasmin activity and neutrophils.Adding 0.1 U/μL aprotinin (plasmin inhibitor) to the reactionmixture eliminated this activity, providing further evidencefor the specificity of the in situ zymographic staining (datanot shown).
3.4. Relative distributions of NAPlr and SPEB
Like NAPlr staining, SPEB staining was detected in theglomeruli of all 6 patients whose renal biopsy sectionswere double stained for NAPlr and SPEB. The glomerulardistributions of NAPlr and SPEB were similar but notidentical. The NAPlr staining generally predominated, andmost of the SPEB staining was within NAPlr-positiveareas (Fig. 6A-C). The nonspecific background staining,
however, was also more strongly evident in NAPlrstaining than in SPEB staining. This staining pattern wasconfirmed by staining serial sections for either SPEB orNAPlr (data not shown).
4. Discussion
Identifying the streptococcal antigen(s) causing APSGNis an essential part of elucidating the mechanism of APSGNpathogenesis [6-11,21], and the most likely are NAPlr andSPEB. NAPlr is a 43-kd antigen recently isolated from groupA streptococcus by our laboratory and has been shown tohave plasmin(ogen)-binding capacity [14]. Our proposal thatNAPlr-bound plasmin plays a pathogenic role in thedevelopment of APSGN was based on the identicalglomerular distributions of NAPlr and α2-antiplasmin–resistant plasmin activity [17]. We had also found APSGNpatients to have elevated urinary plasmin activity that isresistant to α2-antiplasmin [22]. Plasmin might damagerenal tissue directly by degrading extracellular matrix
Fig. 6 Similar localization of NAPlr and SPEB in APSGN. Representative confocal microscopy images of double immunofluorescencestaining for SPEB (A, FITC) and NAPlr (B, Alexa Fluor 594) in an APSGN patient (patient 5). The merged image (C) shows that thedistributions of NAPlr and SPEB staining are similar but not identical. Arrows indicate a representative intraglomerular region positive forSPEB but negative for NAPlr (scale bar = 20 μm).
1283Localization of NAPlr in APSGN
proteins such as fibronectin or laminin and might also affectalmost all extracellular matrix proteins indirectly byactivating pro–matrix metalloproteases [23,24]. Plasmincan also mediate inflammation by accumulating andactivating monocytes and neutrophils in situ [25,26]. Thus,we think that glomerular damage may initially be induced byimmunodetectable free NAPlr, which can bind plasmin andmaintain its proteolytic activity, rather than by subepithelialimmune complexes. In this respect, the present finding thatimmunodetectable NAPlr is localized on the inner side ofglomerular tufts (endocapillary) is consistent with thepredominantly endocapillary glomerular inflammation inAPSGN. In other words, endocapillary localization ofimmunodetectable NAPlr might account for the differentsites of glomerular inflammation and immunocomplexdeposition in APSGN.
SPEB is a cationic cysteine protease secreted as a 42-kdzymogen and subsequently cleaved to a 28-kd activeproteinase [10,27]. It is an exotoxin produced by pyro-genic streptococci and has attracted considerable atten-tion as an important toxin in severe invasive streptococcalinfections [27], and Poon-King et al [28] have suggestedthat it is also a nephritogenic antigen that plays a role inthe pathogenesis of APSGN. They reported a previouslyidentified nephritogenic antigen for APSGN, termednephritis strain-associated protein, as the same moleculeas SPEB and possessing plasmin-binding capacity. Vogtand his colleagues [7] previously reported that theglomeruli of APSGN patients frequently contained a“cationic extracellular streptococcal antigen” later shownto be the same molecule as SPEB. More recently, Cu et al[10] analyzed the renal biopsy tissues of APSGN patientsby using polyclonal anti-SPEB antibody and foundSPEB in the glomeruli of 67% of the APSGN patientsthey examined.
An evaluation of SPEB and streptococcal GAPDH(NAPlr) as nephritogenic antigens for APSGN was
recently reported by Batsford et al [11], who foundanti-SPEB antibodies in the sera of all 53 of the APSGNpatients in their sample and found SPEB deposition inthe glomeruli of 14 of the 17 renal biopsies of theAPSGN patients they examined. They also found highcolocalization of SPEB and C3 by double immunofluo-rescence staining and found SPEB within electron-densesubepithelial deposits by immunoelectron microscopy.They found that serum antibody responses and glomerulardepositions were far less common for streptococcalGAPDH (NAPlr) than for SPEB. However, we previouslyreported that anti-NAPlr antibody was detected at hightiters in 92% (46/50) of sera from APSGN patients andthat glomerular deposition of NAPlr was observed in100% (25/25) of APSGN patients within 2 weeks afterdisease onset or in 83.7% (36/43) of APSGN patientswithin 30 days after onset [14,15]. Thus, the resultreported by Batsford et al [11] is critically different fromwhat we had found. This discrepancy may be in part dueto the different antibodies used in each of these studies;in their study, they used the anti-streptococcal GAPDHantibody that they generated, whereas in ours, we usedthe anti-NAPlr antibody that we generated. The immu-nostaining results for an antigen not infrequently differwhen different antibodies are used [18].
In the present study, the glomerular localization profilesof SPEB and NAPlr were compared using the sameantibodies and the same methodology in a well-definedgroup of APSGN patients. The similar localizations ofNAPlr and SPEB in glomeruli were unexpected because ourprevious study [15] showed different distributions of C3and NAPlr staining and because Batsford et al [11] reporteda high degree of colocalization of C3 and SPEB staining. Incontrast to NAPlr, SPEB has cationic characteristics; so it isplausible that it would deposit in the subepithelial space andcolocalize with C3. NAPlr and SPEB are surely differentmolecules, although the reported molecular weights are
1284 T. Oda et al.
close: 43 and 42 kd, respectively. NAPlr was isolated fromcytoplasmic fraction of group A streptococcus, whereasSPEB was isolated from extracellular fraction; amino acidsequences of both molecules were quite different; andstructural and functional properties of both molecules weregenerally different except for several functional propertiessuch as the capacity for binding plasmin. The presentresults are unlikely to be artifactual results or due to crossreactivity of the 2 antibodies because (1) we confirmedsimilar staining patterns of both antigens in serial sectionsby single staining, (2) preabsorption of anti-SPEB antibodywith NAPlr did not affect the staining results (data notshown), (3) preincubation of sections with unlabeled anti-NAPlr antibody did not affect SPEB staining (data notshown), and (4) preincubation of sections with anti-SPEBantibody did not affect NAPlr staining (data not shown).Thus, we could hardly expect the reason for thisdiscrepancy. As suggested by Rodriguez-Iturbe and Bats-ford [1] and by Batsford et al [11], the discrepancy might bedue to the difference in the genetic and demographicbackgrounds of the patients analyzed in the differentstudies. Cu et al [10], however, analyzed the renal tissuesof APSGN patients in the United States and Chile anddescribed the localization of SPEB in mesangial andendocapillary areas as diffuse and granular, similar to thepatterns of NAPlr and SPEB staining observed in thepresent study. Our double-staining results (similar localiza-tions of 2 distinct antigens in the same glomerulus) weresurprising because many researchers, including us, havebeen assuming that APSGN is the result of a singlenephritogenic streptococcal antigen. The present resultsindicate that this assumption may be incorrect. We shouldconsider the possibility that 2 or more antigens interact inthe induction of this disease. Rodriguez-Iturbe and Batsford[1] indicated in their recent review that multiple mechan-isms should be responsible for each case of APSGN andelegantly summarized the potential cooperative roles ofNAPlr and SPEB. It is also interesting that NAPlr andSPEB share a common function. Both bind plasmin,thereby protecting it from physiologic inhibitors, and thusmight cause chemotaxis of inflammatory cells and degra-dation of glomerular basement membranes due to theactivity of plasmin [4,17,28,29]. The binding site onplasmin for these molecules has not been clarified yet.
There have been many studies using single staining toinvestigate the glomerular localization of nephritogenicantigens potentially responsible for APSGN [6,7,10,21].The study reported here is one of the few using doublestaining to identify the precise intraglomerular localization ofnephritogenic antigens. NAPlr may deposit on glomerularmesangial cells and endothelial cells by adhering to matrixproteins [14] and may cause histologic and functional changeon those cells by promoting plasmin-catalyzed proteolysis.With respect to the pathogenic role of NAPlr on neutrophils,the hyperproteolytic state of NAPlr-positive neutrophilsin the present study indicates a possible role of these
neutrophils in the induction of proteolytic glomerulardamage in situ. Regarding the mechanism of localizationof NAPlr on neutrophils, we suggest 2 possibilities. NAPlrmay bind the urokinase-type plasminogen activator receptorexpressed on neutrophils [30,31], which has recently beenshown to be the receptor for streptococcal GAPDH (NAPlr)[32]. Alternatively, NAPlr may be phagocytosed byneutrophils as a foreign bacterial antigen. These mechan-isms, however, would not explain why NAPlr staining wasrarely found in macrophages. Macrophages, like neutrophils,express urokinase-type plasminogen activator receptor[31,33] and engage in phagocytosis; and similar levels ofmacrophage and neutrophil infiltration were observed in theglomeruli of the present series of APSGN patients. Therelative infrequency of NAPlr staining in macrophages mightbe due to different levels of urokinase-type plasminogenactivator receptor expression on the neutrophils and macro-phages of patients with APSGN. Alternatively, it might bedue to the relatively quicker infiltration of neutrophils thanthat of macrophages in this disease [34,35]. Most of theNAPlr deposited in glomeruli might be phagocytosed byneutrophils accumulated in the early phase of the disease,leaving little NAPlr free when macrophages eventuallymigrate into the glomeruli.
In summary, we have confirmed that NAPlr is foundin the glomeruli of APSGN patients and have found itsprincipal locations to be in glomerular neutrophils,mesangial cells, and endothelial cells. We had previouslyfound the location of glomerular NAPlr staining tocorrespond to that of glomerular plasmin activity [17]and, in the present study, have found the patterns of NAPlrand SPEB staining to be similar. Finding out how andwhen these 3 variables colocalize and interact will requirefurther studies.
Acknowledgment
We are grateful to our colleague Ms Yasuko Sugimoto forexpert secretarial assistance.
References
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