Erythrogenic toxin type B and its precursor isolated from nephritogenic streptococci induce leukocyte infiltration in normal rat kidneys
Maritza Romero,
Jesus Mosquera,
Ernesto Novo1,
Lucas Fernandez2 and
Gustavo Parra2
Instituto de Investigaciones Clinicas,
1 Instituto de Investigaciones Odontologicas and
2 Centro de Cirugia Experimental, Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela
Correspondence and offprint requests to:
Jesus A. Mosquera, MD, Instituto de Investigaciones Clinicas, Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela.
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Abstract
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Background. Leukocyte infiltration is a common feature in renal biopsies from patients with acute post-streptococcal glomerulonephritis (APSGN). Cationic streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) have been implicated in the pathogenesis of the disease, and the presence of ETB has been evidenced in renal biopsies from patients with APSGN. The present studies were performed to determine the effect of the ETBP and ETB on renal leukocyte infiltration and the mechanism(s) implicated in the phenomenon.
Methods. Male SpragueDawley rats were injected intrarenally with 100 µg of ETB or ETBP. Animals were sacrificed at 1, 6 and 24 h after injection and renal samples were studied by indirect immunofluorescence for the presence of leukocyte common antigen (LCA+) cells, C3, monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1), and by direct immunofluorescence for the presence of immunoglobulins. ETB and ETBP were tested for chemotactic effect and migration inhibition factor (MIF) activity by chemotaxis under agarose and agarose microdroplet methods, respectively. Streptococcal proteins were also tested for the capacity to induce MIF activity in rat glomerular cultures. To test for the influence of cationic charge on renal LCA+ cell infiltration, rats were injected with cationized ferritin or polyethyleneimine (PEI) and sacrificed 1 h later.
Results. An increased number of LCA+ cells was found in glomeruli and interstitial areas in ETB- or ETBP-injected animals. ETB and ETBP showed chemotactic and MIF activity on neutrophils and macrophages, and ETBP induced MIF activity in supernatants of glomerular cultures. Data obtained from C3, MCP-1, ICAM-1 or immunoglobulin renal staining in experimental animals were not significantly different when compared to control values. Cationized compounds failed to induce LCA+ cell infiltration; however, an increased number of glomerular LCA+ cells was observed after PEI perfusion.
Conclusions. ETB and ETBP induce renal LCA+ cell infiltration during a short period after intrarenal injection, and this finding could be mediated by chemotactic and MIF activities. These observations could be relevant in the early events of pathogenesis of APSGN.
Keywords: kidney; leukocyte infiltration; streptococcal erythrogenic toxin
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Introduction
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Acute post-streptococcal glomerulonephritis (APSGN) is a diffuse proliferative endocapillary nephritis that occurs in the convalescent period of a group A streptococcal infection [1]. One of the relevant histological features of APSGN is the abnormal glomerular and interstitial leukocyte infiltration [2,3]. It has been documented that the streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) react preferentially with APSGN sera, and these proteins have been found in renal biopsies of patients with APSGN [46]. On the basis of these observations, we have studied the effect of intrarenal injection of ETB or ETBP on the induction of renal leukocyte infiltration. Our findings demonstrated that ETB and ETBP are capable of inducing glomerular and interstitial leukocyte infiltration in normal rat kidneys.
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Subjects and methods
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Animals
All experiments were performed on male SpragueDawley rats of body weight varying from 150 to 200 g (Instituto Venezolano de Investigaciones Cientificas, IVIC, Venezuela).
Reagents
Monoclonal antibody (mAb) against rat leukocyte common antigen (LCA) and tetramethylrhodamine isothiocyanate (TRIC)-conjugated F(ab'2) rat anti-mouse IgG were obtained from Accurate Chemical and Scientific Corp. (Westbury, NY). Polyclonal antibodies goat anti-rat C3, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG, FITC-conjugated goat anti-rabbit IgG, biotin-conjugated rabbit anti-goat IgG and FITC-labelled rabbit anti-rat immunoglobulins were purchased from Cappel Scientific Division (West Chester, PA). MAbs against rat intercellular adhesion molecule-1 (ICAM-1) were from Seikagaku Corp., Tokyo, Japan; rabbit antibody against rat monocyte chemotactic protein-1 (MCP-1) was from Biosource International, USA; TRIC-labelled extravidin, RPMI 1640, fetal bovine serum (FBS), bovine serum albumin and ovalbumin were from Sigma Chemical Corp., St. Louis, MO; polyethyleneimine (PEI: mol wt. 40 000) was from Polysciences, Ltd, Eppelheim, Germany; minimal essential medium (MEM) and thioglycollate medium were from Gibco, Grand Island, NY; Seaplate agarose was from FMC Bioproducts, Rockland, ME; and OCT compound was from Lab Tek Products, Division Miles Laboratories Inc., IL. 125I was purchased from NEN Research Products (DuPont Company, DE) and supplied as a Na[125I] in isotonic solution with a specific activity of 17.4 Ci/mg. Cationized horse ferritin (pI>9.5) was kindly supplied by Dr Arnold Vogt and Dr Stephen Batsford from Institute of Immunology, Zentrum für Hygiene der Universität Freiburg, Germany.
Isolation of ETBP and ETB
ETB and EBTP were kindly donated by Dr Arnold Vogt and Dr Stephen Batsford and isolated as follows: Two ß-haemolytic group A streptococcal strains isolated from patients with APSGN were used. The bacteria were kept on blood agar at 4°C and pre-cultured in chemically defined medium (CDM), pH 6.9 at 37°C for 6 h. Afterwards, 20 ml of bacterial culture were added to 180 ml of fresh CDM and cultured until the culture was cloudy (~6 h). A 200 ml aliquot of the pre-culture was added to the main culture medium (800 ml of CDM and 1000 ml of Todd-Hewitt, 1% glucose, pH 6.9). Bacteria were cultured for several hours and pH was maintained by the addition of a solution of 0.5 M NaOH containing 50% glucose. Then, the pH was reduced to 5.9 and the culture was left for a further 12 h; pH was maintained with a 0.5 M NaOH solution containing 50% glucose. The culture was centrifuged (10 800 g for 15 min) and the supernatant was sterile filtered, concentrated by a factor of 1000 in a Amicon system using an YM-5 membrane and finally run on a Mono-S cation exchange column (Pharmacia HR 5/5) in a FPLC system. The conditions were: pH 6.0, flow rate 1 ml/min, buffer gradient 3.5250 mM MES. The ETBP eluted before ETB and the fractions were tested in SDSPAGE under non-reducing conditions. Then, streptococcal antigens were run in an isoelectric focusing gel to determine pl. Under these conditions, ETBP was 44 kDa, pl 8.2, and ETB was 30 kDa, pl 9.0. Polyclonal antibodies to both proteins were raised in rabbits by repeated immunization.
Experiments to demonstrate glomerular and interstitial leukocyte infiltration after intrarenal injection of ETBP or ETB
To explore the possibility that ETBP or ETB are involved in the leukocyte infiltration of the kidney, a group of rats was injected in the left kidneys via the aorta with 500 µl of a solution of ETBP or ETB in normal saline solution (100 µg/500 µl). That concentration was chosen since previous investigators [711] have reported intrarenal injection of several cationic antigens at doses ranging from 20 to 500 µg to induce in situ immune complex glomerulonephritis in rats. Before injection, the aorta was clamped just below the right renal artery. Animals were sacrificed at 1, 6 and 24 h (each time point, n=46) and, afterwards, left kidneys were perfused and samples of renal cortexes were obtained, embedded in OCT compound (Tissue Tek), frozen in acetone and dry ice and maintained at -70°C until use for immunofluorescent studies. Control animals (n=10) were sacrificed at 1, 6 and 24 h after renal injection of 500 µl of normal saline solution. In order to determine the effect of non-relevant anionic proteins on LCA+ cell infiltration, a group of rats (n=4) was injected with a solution of native ovalbumin (100 µg/500 µl) and sacrificed 1 h later.
Experiments to determine the effect of cationic compounds on renal leukocyte infiltration
To determine whether electrostatic forces are involved in the phenomena observed, some experiments, aimed at determining the effect of the highly cationic compounds (PEI and cationized ferritin) on kidney leukocyte infiltration, were performed. Five hundred µl of a 0.1% PEI solution (mol. wt 40 000, pH 7.3, 400 mosmol; n=5) or 500 µl of a solution of cationized horse ferritin (100 µg/500 µl; n=5) were injected in the left kidney of rats as described above. After 1 h, animals were sacrificed and perfused kidney samples were processed for immunofluorescent studies as described below. Controls represent animals injected with a 400 mosmol, pH 7.3 saline solution (n=5) or with normal saline solution (n=5).
Isolation of cells
Neutrophils.
Peritoneal neutrophils (PMN) were harvested from SpragueDawley rats 5 h after a peritoneal injection of 10 ml of 3% thioglycollate medium. Cells were washed, resuspended in RPMI 1640 containing 10% heat-inactivated FBS and used for chemotaxis experiments.
Monocytes/macrophages.
Peritoneal macrophages were obtained following the same procedure as for PMN, except that thioglycollate was left in the peritoneal cavity for 4 days.
Chemotactic assay
The chemotactic effect of ETB and ETBP on PMN and macrophages was assessed using a modified version of the under agarose method described by Nelson et al. [12]. Briefly, 1.2% agarose (seaplate agarose) was prepared in Dulbeccos phosphate-buffered saline (PBS; DPBS), containing 10% heat-inactivated normal rat serum. PMN or macrophages were resuspended at 1x106/15 µl/well in RPMI 1640, 10% FBS. To test the chemotactic activity of ETB (ranging from 100 to 0.01 ng/ml) or ETBP (ranging from 50 to 0.01 µg/ml), 15 µl of cationic antigens in RPMI 1640 were added to wells in agarose. Incubation was performed at 37°C, 5% CO2 for 2 (PMN) or 18 h (macrophages). Quantitation of chemotaxis was performed using a stereoscopic microscope provided with a micrometer eyepiece. Chemotactic distance was considered as the linear distance that the cells migrated toward the cationic streptococcal proteins. Spontaneous migration was considered as the distance that the cells migrated toward the control medium. The chemotactic index was calculated by dividing the chemotactic distance by the spontaneous migration distance.
Isolation and culture of rat glomeruli
Kidneys of SpragueDawley rats were perfused by aorta puncture with 30 ml of Hank's solution and 10 ml of RPMI 1640 medium containing 10% FBS; after this, renal cortexes were dissected out from the perfused kidneys and glomeruli were obtained by pressing the minced renal cortexes through sieves of graded sizes. Glomeruli were suspended in Hank's solution containing 25 U/ml of collagenase type II, 0.01 mg/ml of DNase type I (Sigma Chemical Co.) and 1% HEPES, and incubated for 10 min at 37°C. The glomerular suspension was then washed three times and incubated on 24-well tissue culture plates (Costar, Cambridge, MA) at 1x104 glomeruli/ml in RPMI 1640 medium supplemented with L-glutamine, penicillin/streptomycin, HEPES and 10% FBS. Glomeruli were incubated with different concentrations of ETBP (ranging from 70 to 5 µg/ml) for 24 h at 37°C, 5% CO2. Afterwards, supernatants were collected, filtered and tested for migration inhibition factor (MIF) activity as described below. Controls represent supernatants from glomerular cultures treated as described but without ETBP. A solution of ETBP ranging from 70 to 5 µg/ml was tested to evaluate MIF activity from the cationic antigen used to stimulate glomerular cultures.
MIF activity assay
MIF activity was assayed by the agarose microdroplet method [13,14]. Briefly, guinea pig peritoneal macrophages were obtained by injection of 20 ml of sterile liquid paraffin in the guinea pig peritoneal cavity. After 3 days, peritoneal exudate was obtained by washing the peritoneal cavity, and cells were washed and resuspended in MEM. For determination of MIF activity, 50x106 cells were pelleted by centrifugation and mixed with 100 µl of 0.2% seaplate agarose in medium 199 containing 1% newborn bovine serum, and 1 µl of the mixture was dispensed in wells of the 96 flat-bottom wells of a microtitre culture plate. The microdroplets were allowed to solidify and then 100 µl of the following samples were added: (i) ETB or ETBP ranging from 200 to 6.25 µg/ml in MEM containing 10% FBS; (ii) supernatants from untreated and ETBP-treated glomerular cultures; and (iii) ETBP ranging from 70 to 5 µg/ml (doses used to stimulated glomerular cultures). Afterwards, the horizontal and vertical diameters of the microdroplets were measured by a light microscope (Leitz, Germany) provided with a micrometer eyepiece. A new measurement was done after 24 h of incubation at 37°C in 5% CO2. The area of migration was calculated by substracting the area of the original droplet from the area after 24 h incubation, and MIF activity was expressed as the percentage of the inhibited migration, as follows:
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Renal immunofluorescent studies
To analyse the presence of different antigens in the glomeruli and interstitium in untreated and ETB/ETBP-treated rats, cryostat sections (4 µm) from kidneys were air dried, fixed in cold acetone for 10 min and washed with PBS for 5 min. Thereafter, sections were stained with primary mAbs against LCA or ICAM-1, then a secondary antibody, TRIC-labelled F(ab')2 rat anti-mouse IgG, was used to localize the first antibody. Renal sections were also stained with a polyclonal antibody goat anti-rat C3 and sequentially reacted with biotin-conjugated rabbit anti-goat IgG and TRIC-labelled extravidin, or with a rabbit antibody against rat MCP-1, and FITC-labelled goat anti-rabbit IgG. To localize the ETBP, renal sections were reacted with a polyclonal rabbit antibody against ETBP and then an FITC-labelled goat anti-rabbit IgG was used as a secondary antibody. To determine rat immunoglobulin deposition in the kidney, renal frozen sections were incubated with FITC-labelled rabbit anti-rat immunoglobulins. Sections were incubated with the respective antibodies for 30 min and mounted with p-phenylenediamine to retard fluorescence fading. Renal samples were observed at least by two investigators, and >15 glomeruli were examined per renal sample.
Radiolabelling and renal localization of ETB
ETB and ovalbumin were labelled with 125I by the chloramine-T method [15]. Intrarenal binding of cationic ETB was determined by the method used by Wilson and Dixon [16], with modifications. Since the intrarenal injection of cationic molecules together with anionic molecules could induce binding of anionic molecules to renal structures [17], 1 ml of a solution of 125I-labelled ETB (100 µg/ml) or 125I-labelled ovalbumin (100 µg/ml) was perfused into the left kidney in different animals at the rate of 1 ml/min. Ovalbumin was used as anionic control [17]. Rats were sacrificed at 1 h after perfusion and the left kidneys were obtained, homogenized and washed in cold PBS until no radioactivity was detected in the supernatants. Homogenized tissues were weighted and counted in a gamma counter [16]. The results are expressed as ngs of ETB or ovalbumin/mg of renal tissue.
Statistical analysis
Results were analysed by analysis of variance with Dunnet post-test and MannWhitney test. The results are expressed as means±SEM. Two-tailed P<0.05 between groups was considered significant.
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Results
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Kidney leukocyte infiltration induced by the intrarenal injection of ETBP or ETB was examined by indirect immunofluorescence. An increased number of LCA+ cells in the glomerulus and interstitium was found at 1 and 6 h after injection of ETB. LCA+ cells were increased after ETBP injection at 1 and 24 h; however, the values fell to lower levels at 6 h (Table 1
, Figure 1
). At the fluorescence microscopy level, streptococcal cationic antigens were localized in the glomerulus and interstitium of kidney samples (Figure 2
). Renal binding of 125I-labelled ETB and 125I-labelled ovalbumin after intrarenal injection is shown in Figure 3
. Radioactivity in the left kidney injected with ETB was higher (4.16±1.48 ng/mg) than that from animals injected with 125I-labelled native ovalbumin (0.94±0.26 ng/mg). In addition to low renal binding, ovalbumin failed to increase the number of renal LCA+ cells at 1 h after intrarenal injection (ovalbumin: glomerulus 1.7±0.24; interstitium 9.43±0.49; saline: glomerulus 2.27±0.17; interstitium 8.54±0.64).

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Fig. 1. Indirect immunofluorescent staining of kidney frozen sections showing leukocyte infiltration at 1 h after intrarenal injection of ETB. An increased number of glomerular (A) and interstitial (B) LCA+ cells was found. Clusters of LCA+ cells were found in some interstitial areas (C).
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Fig. 2. Indirect immunofluorescence for streptococcal ETBP 1 h after intrarenal injection of 100 µg of ETBP. Cationic streptococcal protein was localized in the glomerulus (A) and in interstitial areas (B). Glomeruli (C) and interstitium (D) from renal sections of untreated animals were negative.
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Fig. 3. Binding of 125I-labelled ETB and 125I-labelled ovalbumin to renal tissues of rats. Rats were injected intrarenally with 100 µg of radioactive ETB or ovalbumin and sacrificed 1 h later.
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To determine the possible role of cationic charge on renal leukocyte infiltration, we injected either cationized ferritin or PEI into rat kidneys. As shown in Figure 4
, no significant differences in the number of LCA+ cells were observed in animals injected with cationized ferritin; however, a slightly increased number was observed in the glomeruli of animals injected with PEI.

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Fig. 4. Renal LCA+ cells after intrarenal injection of cationized ferritin or PEI. (A) Glomerular LCA+ cells: an increased number was observed in rats treated with PEI (*P<0.02). (B) Interstitial LCA+ cells: no significant differences were observed between controls and experimental animals. Controls, open bars; experimental animals, hatched bars.
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To determine if ETBP or ETB induce deposition of C3 or up-regulation of ICAM-1 or MCP-1 at the renal level, kidney samples from experimental and control rats were studied by indirect immunofluorescence with relevant monoclonal and polyclonal antibodies. No significant differences in C3 and MCP-1 staining in ETBP/ETB-perfused animals and controls were observed (Tables 2 and 3
). Similar results were obtained when renal biopsies were stained for ICAM-1 (data not shown). To rule out the possibility that immune complexes could be involved in the leukocyte infiltration, the presence of glomerular immunoglobulin deposits was studied by direct immunofluorescent staining. Immunofluorescence findings are summarized in Table 4
. Deposition of immunoglobulins at 1 and 24 h was similar in control and experimental animals, except for one animal injected with ETB (+) and sacrificed 1 h later.
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Table 4. Immunofluorescent staining for glomerular immunoglobulins in renal tissues from controls and rats injected with ETBP or ETB
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Experiments were performed with ETBP or ETB to test their abilities to attract leukocytes and to inhibit leukocyte migration under in vitro conditions. The chemotactic effect was observed in PMN as well as in macrophages. Figures 5 and 6
show the chemotactic index values obtained from chemotactic concentrations of ETBP and ETB. ETBP and ETB also showed MIF activity on guinea pig macrophages (Figure 7
) at concentrations ranging from 200 to 6.25 µg/ml. In addition, ETBP at doses ranging from 70 to 5 µg/ml was capable of inducing MIF activity in glomerular cultures (Figure 8
); however, non-reproducible results were obtained when ETB was used to induce MIF activity in glomerular cultures.

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Fig. 5. In vitro chemotaxis of rat macrophages to cationic streptococcal antigens. Chemotactic response to (A) ETBP and (B) ETB. Data represent mean±SEM from six animals.
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Fig. 6. In vitro chemotaxis of rat neutrophils to cationic streptococcal antigens. Data represent mean±SEM from five animals.
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Fig. 8. Migration inhibition factor activity in supernatants from ETBP-treated isolated glomerular cultures. Data represent the percentage of glomerular MIF activity minus the percentage of ETBP MIF activity.
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Discussion
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Previous studies have demonstrated that renal biopsies from patients with APSGN frequently show infiltration by peripheral blood leukocytes [2,3]. The present studies provide information on the effect of streptococcal ETB and its precursor on the infiltration of leukocytes (LCA+ cells) in the kidney. Following intrarenal injection of ETB or ETBP in rats, LCA+ cells were increased in glomeruli and interstitial areas but, after 24 h, they decreased or returned to basal levels (Table 1
). ETB and ETBP with higher pl have a charge affinity with renal structures such as the glomerular basement membrane [18] and interstitial vessels (Figure 2
), this makes a local effect of these streptococcal antigens in the kidney conceivable. The presence of ETB in early renal biopsies and circulating antibodies against ETBP has been documented in patients with APSGN [46]. Since ETBP/ETB showed a direct chemotactic effect on PMN and monocytes/macrophages (Figures 5 and 6
), this mechanism could be involved in the increased number of LCA+ cells found in the glomeruli and interstitium. The time course of leukocyte migration in the kidney of ETBP/ETB-perfused rats (higher values at 1 h with decreased values later) was similar to the in vivo PMN migration effect induced in rabbit skin by streptococcal erythrogenic toxin type A [19]. In addition, ETBP/ETB showed MIF-like activity by theirselves (Figure 7
) and ETBP was capable of inducing MIF-like activity in supernatants of normal rat glomerular cultures (Figure 8
), mechanisms to account for increased LCA+ cells in the kidney of ETBP/ETB-perfused rats. Intrinsic and passenger renal cells are capable of producing molecules with MIF activity [2022], therefore, they are susceptible stimulation by cationic streptococcal antigens. In this respect, the capacity of streptococcal erythrogenic toxins to induce cytokine production by blood mononuclear cells has been documented [23,24]. We have no explanation for the decreased values of renal LCA+ cells found at 6 h after intrarenal injection of ETBP. We speculate that ETBP under in vivo conditions can be reduced to ETB with the production of inactive intermediate products [25] and decreased leukocyte infiltration at 6 h. Further ETB activation could be involved in the increased number of LCA+ cells found at 24 h (Table 1
).
Previous reports have suggested that complement activation, intrarenal production of chemotactic citokines and up-regulation of adhesion molecules are important mechanisms of intrarenal leukocyte infiltration during the course of human and experimental proliferative glomerulonephritis [2630]. Our results failed to demonstrate significant increases of renal C3 deposition, MCP-1 or ICAM-1 expression in ETBP/ETB-perfused rats when compared with controls, suggesting that those mechanisms are not involved in the leukocyte infiltration induced by ETBP/ETB in our experimental conditions. However, those mechanisms could be involved during the course of nephritis. To investigate the possibility that circulating anti-streptococcal antibodies could react with renal planted ETB or ETBP, we investigated the presence of renal immunoglobulin deposits in experimental and control animals. We failed to demonstrate immunoglobulin deposits in renal tissues, suggesting that renal immune complexes were not involved in the renal leukocyte infiltration found in our experiments.
Previous studies have shown that the glomerular capillary wall contains fixed negatively charged sites with high affinity for cationic substances [31,32]. ETBP/ETB are highly cationic streptococcal proteins with affinity for the renal glomerulus [18]. Binding of cationic proteins to anionic sites is capable of reducing the net anionic charge in renal structures [33], and this may be an important phenomenon that could influence the attraction of negatively charged leukocytes [34]. To rule out the possibility that the cationic charge of ETBP/ETB is responsible for LCA+ cell infiltration, we perfused rats with cationic ferritin and the strongly cationic PEI. Cationized ferritin did not modify the number of glomerular or interstitial LCA+ cells at 1 h after injection. In agreement with this, Batsford et al. [8] failed to find glomerular alterations after injection of cationized ferritin into rats. However, an unexpected number of glomerular LCA+ cells (control 2.08±0.16; PEI 3.11±0.32) was observed 1 h after PEI intrarenal injection. A possible explanation could be related to the binding of PEI to the endothelial cell sialoprotein coating [35] with further binding of negatively charged leukocytes.
This study provides the evidence that ETBP/ETB from nephritogenic streptococci are involved in renal leukocyte infiltration, probably by mechanisms such as chemotactic and MIF activities. Along with other mechanisms of leukocyte infiltration, LCA+ cell infiltration induced by ETBP/ETB, underline their potential pathogenic role in APSGN.
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Acknowledgments
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The authors thank Dr Arnold Vogt and Stephen Batsford for their generous gift of ETBP, ETB and antibodies against cationic streptococcal proteins. We gratefully acknowledge the assistance of Dr Humberto Martinez in the evaluation of this manuscript. We acknowledge financial support from CONDES (Grant: 146096).
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References
|
---|
-
Rammelkamp CH. Acute poststreptococcal glomerulonephritis. In: Read SE, Zabriskie JB, eds. Streptococcal Diseases and the Immune Response. Academic Press, New York: 1980; 43
-
Parra G, Plat JL, Falk RJ, Rodriguez-Iturbe B, Michael AF. Cell populations and membrane attack complex in glomeruli of patients with post-streptococcal glomerulonephritis: identification using monoclonal antibodies by indirect immunofluorescence. Clin Immunol Immunopathol 1984; 33: 324332[ISI][Medline]
-
Hooke DH, Gee DC, Atkins RC. Leukocyte analysis using monoclonal antibodies in human glomerulonephritis. Kidney Int 1987; 31: 964972[ISI][Medline]
-
Vogt A, Batsford S, Rodriguez-Iturbe B, Garcia R. Cationic antigens in poststreptococcal glomerulonephritis. Clin Nephrol 1983; 20: 271279[ISI][Medline]
-
Vogt A, Schmiedeke T, Stockl F, Sugisaki Y, Mertz A, Batsford S. The role of cationic proteins in the pathogenesis of immune complex glomerulonephritis. Nephrol Dial Transplant 1990; [Suppl. 1]: 69
-
Poon-King R, Bannan J, Viteri A, Cu G, Zabriskie JB. Identification of a extracellular plasmin binding protein from nephritogenic streptococci. J Exp Med 1993; 178: 759763[Abstract]
-
Oite T, Batsford SR, Mihatsch MJ, Takamiya H, Vogt A. Quantitative studies of in situ immune complex glomerulonephritis in the rat induced by planted cationized antigen. J Exp Med 1982; 155: 460474[Abstract/Free Full Text]
-
Batsford SR, Takamiya H, Vogt A. A model of in situ immune complex glomerulonephritis in the rat employing cationized ferritin. Clin Nephrol 1980; 14: 211216[ISI][Medline]
-
Oite T, Shimizu F, Suzuki Y, Vogt A. Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis. Virch Arch (Cell Pathol ) 1985; 48: 107118
-
Sasaki M, Batsford S, Thaiss F, Oite T, Vogt A. Ultrastructural studies in passive in situ immune complex glomerulonephritis. Virch Arch (Cell Pathol ) 1989; 58: 173180
-
Yousif Y, Okada K, Batsford S, Vogt A. Induction of glomerulonephritis in rats with staphylococcal phosphatase: new aspects in post-infectious ICGN. Kidney Int 1996; 50: 290297[ISI][Medline]
-
Nelson RD, Quie PG, Simmons RL. Chemotaxis under agarose; a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 1975; 114: 16501656
-
Borish L, Liu DY, Remold H, Rocklin R. Production and assay of macrophage migration inhibition factor, leukocyte migration inhibitory factor and leukocyte adherent inhibitory factor. In: Rose NR, Friedman H, Fahey J, eds. Clinical Laboratory Immunology. America Society for Microbiology, Washington DC: 1986; 282
-
Harrington JT, Stasny P. Macrophage migration from an agarose droplet: development of micromethod for assay of delay type of hypersensitivity. J Immunol 1973; 110: 752759[ISI][Medline]
-
Hunter WM, Greenwood FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 1962; 194: 495496[ISI]
-
Wilson CB, Dixon FJ. Antigen quantitation in experimental immune complex glomerulonephritis. I. Acute serum sickness. J Immunol 1970; 105: 279290[ISI][Medline]
-
Vogt A, Schmiedeke T, Stöckl F, Sugisaki Y, Mertz A, Batsford S. The role of cationic proteins in the pathogenesis of immune complex glomerulonephritis. Nephrol Dial Transplant 1990; [Suppl. 1]: 69
-
Vogt A, Mertz A, Bastford S, Rodriguez-Iturbe B. Cationic extracellular streptococcal antigen; affinity to the renal glomerulus. In: Recent Advances in Streptococci and Streptococcal Diseases. Reedbooks Ltd, Berkshire, UK: 1984; 170
-
Kamezawa Y, Nakahara T, Abe Y, Kato I. Increased vascular permeability, erythema, and leukocyte emigration induced in rabbit skin by streptococcal erythrogenic toxin type A. FEMS Microbiol Lett 1990; 56: 159162[Medline]
-
Tesch GH, Nikolic-Paterson DJ, Metz CN et al. Rat mesangial cells express macrophage migration inhibitory factor in vitro and in vivo. J Am Soc Nephrol 1998; 9: 417424[Abstract]
-
Lan HY, Bacher M, Yang N et al. The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat. J Exp Med 1997; 185: 14551465[Abstract/Free Full Text]
-
Parra G, Mosquera J, Rodriguez-Iturbe B. Migration inhibition factor in acute serum sickness nephritis. Kidney Int 1990; 38: 11181124[ISI][Medline]
-
Muller-Aluof U, Alouf JE, Gerlach D, Ozegowski JH, Fitting C, Cavaillon JM. Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide. Infect Immun 1994; 62: 49154921[Abstract]
-
Norrby-Teglund A, Norgren M, Holm SE, Andersson U, Andersson J. Similar cytokine induction profiles of a novel streptococcal exotoxin, MF, and pyrogenic exotoxins A and B. Infect Immun 1994; 62: 37313738[Abstract]
-
Liu TY, Elliot SD. Streptococcal proteinase. In: Boyer P, ed. The Enzymes. Academic Press, New York: 1971; 609
-
Wilson CB, Dixon FJ. The renal response to immunological injury. In: Brenner BM, Rector, eds. The Kidney. W.B. Saunders, Philadelphia: 1986; 800
-
Tang WW, Qi M, Warren JS. Monocyte chemoattractant protein 1 mediates glomerular macrophage infiltration in anti-GBM Ab GN. Kidney Int 1996; 50: 665671[ISI][Medline]
-
Prodjosudjadi W, Gerritsma JSJ, van Es LA, Daha MR, Bruijn JA. Monocyte chemoattractant protein 1 in normal and diseased human kidneys: an immunohistochemical analysis. Clin Nephrol 1995; 44: 148155[ISI][Medline]
-
Roy-Chaudhury P, Wu B, King G, Cambell M, Macleod AM, Haites NE, Simpson JG, Power DA. Adhesion molecule interactions in human glomerulonephritis: importance of the tubulointerstitium. Kidney Int 1996; 49: 127134[ISI][Medline]
-
Brady HR. Leukocyte adhesion molecules and kidney diseases. Kidney Int 1994; 45: 12851300[ISI][Medline]
-
Kanwar YS, Farquhar MG. Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes. J Cell Biol 1979; 81: 137153[Abstract]
-
Rennke HG, Venkatachalam MA. Glomerular permeability: in vivo tracer studies with polyanionic and polycationic ferritins. Kidney Int 1977; 11: 4453[ISI][Medline]
-
Camussi G, Tetta C, Coda R, Segoloni GP, Vercellone A. Platelet-activating factor-induced loss of glomerular anionic charges. Kidney Int 1984; 25: 7381[ISI][Medline]
-
Leabu M, Ghinea N, Muresan V, Colceag J, Hasu M, Simionescu N. Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit. J Submicrosc Cytol 1987; 19: 193208[ISI][Medline]
-
Pilia PA, Swain RP, Williaams AV, Loadholt CB, Ainsworth SK. Glomerular anionic site distribution in nonproteinuric rats. A computer-assisted morphometric analysis. Am J Pathol 1985; 121: 474485[Abstract]
Received for publication: 14. 7.98
Accepted in revised form: 22. 3.99