Clin. exp. Immunol (1991) 86, 471-477
Surface charge distribution is a determinant of antigen deposition in the renal glomerulus: studies employing 'charge-hybrid' molecules S. R. BATSFORD, M. J. MIHATSCH*, M. RAWIEL, T. M. SCHMIEDEKE & A. VOGT Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany, and *Institute of Pathology, Basel, Switzerland
(Acceptedfor publication 18 June 1991)
SUMMARY The deposition of antigens and immune complexes (IC) in the renal glomerulus is charge-dependent. The demonstration that molecules of net anionic charge, but with discrete positively charged regions, exhibit affinity for the glomerular basement membrane (GBM) extends this concept. Charge hybrid (polar) molecules were constructed by covalently coupling small polycations (lysozyme or linear poly-L-lysine chains with a mean of 17 and 20 residues) to larger polyanions (ovalbumin or human serum albumin (HSA)). Although the products were of overall net anionic charge they still bound to glomerular structures. Immunofluorescence studies performed after i.v. injection of the samples into rats revealed that HSA: poly-L-lysine had the highest affinity. Radioisotopic measurements showed uptake of HSA: poly-L-lysine to be a function of the number of lysine residues; binding of HSA: polyL-lysine2O was 2-5 times higher than HSA: poly-L-lysine17 (P 160 kD)
Alone*
Plus antibodyt
+
(+)
(+)
+
+
2+-3+
ND ND ND
2+ 2+ 3+
* t Stained for HSA* or rabbit IgGt (mean of three rats/fraction). Antigen alone = 5 mg/100 g body weight intravenously, kidneys removed 15 min later; with antibody= 1 mg antigen plus 0 25-0-5 ml antiserum (i.v., per 100 g body weight) 15 min later, kidneys removed after 1 h. Staining patterns and controls-see Results section. I 1:1 product. ND, not done.
subsequent staining was with amido black. Isoelectric focusing (IEF) was performed in slab gels of 5 5% polyacrylamide using an ampholyte gradient of pH 4-5-9-8. HSA: poly-L-lysine preparations precipitated spontaneously in the electrofocusing gel, regardless of the point of application. Analysis of these latter samples was done by electrophoresis in uncharged agarose (IEF grade, Pharmacia) in veronal buffer (pH 8-2, 0.05 M) using cytochrome C (pI 10-7), ribonuclease (pl 9-5), whale myoglobin (pI 8 3), horse myoglobin (pI 7-3), HSA (pI 5-2) and ovalbumin (pI 4 2) as markers.
Immunofluorescence Immunofluorescence was performed on 5 gm cryostat sections fixed in acetone. The intensity of glomerular fluorescence was estimated on a 0 to + 3 scale on coded sections. Electronmicroscopy Renal tissue was fixed in 2-5% buffered glutaraldehyde for 2 h. Epon embedded ultrathin sections were stained with uranyl acetate and lead citrate. Statistical analysis This was performed with the aid of two-way analysis ofvariance or the Mann-Whitney U-test as appropriate.
Experimental design Affinity of charge-hybrid molecules for the renal glomerulus. Groups of rats (n = 3) were given 5 mg/ 100 g body weight ofeach sample intravenously (see Table 1), the kidneys were removed 15 min later and binding was assessed by immunofluorescence using the appropriate FITC-labelled antibody. In the case of HSA: poly-L-lysine, fractions separated on Sephacryl S.300
473
Antigen charge distribution and IC formation were studied as well, three rats were tested per peak. As a control, DMS-treated HSA was also given to two rats. Formation of glomerular immune complexes. In these experiments 1 mg/l00 g body weight of the charge hybrid (see Table 1) was given intravenously, followed by 0-25-0-5 ml of anti-HSA antiserum after 15 min. Renal tissue was removed I h later for immunofluorescence studies; three rats were studied per sample. Preliminary experiments had shown that when a dose of I mg/ l00 g was exceeded and antibody was given subsequently, acute toxicity (especially lung haemorrhage) was seen. Increasing the quantity of antiserum given (from 0 5 ml/100 g body weight) did not alter the intensity of immunofluorescent staining seen-apparently saturating conditions had been reached. The persistence of the immune complex was also studied. Nine rats received 1 mg/100 g body weight of HSA:poly-Llysine2O followed by 0 5 ml/100 g body weight of rabbit antiHSA 15 min later. Groups of three rats were killed after I h, 24 h and 14 days and the kidneys were examined for the presence of HSA, rabbit-IgG, rat IgG and rat C3 by immunofluorescence. Renal tissue was taken for electron microscopy on day 14. As controls, two rats were given DMS-treated HSA followed by anti-HSA as above and examined by immunofluorescence at I h; and four rats were given HSA: poly-L-lysine20 followed by normal rabbit serum; staining for rabbit IgG and HSA was performed I h later in two rats and at 14 days in the two remaining, which were also examined by electron microscopy. Influence of size of the poly-L-lysine chain on glomerular formation and persistence of immune complexes. Two probes were available, HSA: poly-L-lysinel7 and HSA: poly-L-lysine20. Rats were given I mg/I00 g body weight of HSA:poly-L-lysine (17 or 20) intravenously followed by 0 25 ml/100 g body weight of rabbit anti-HSA intravenously 15 min later. In both groups four rats were killed I h, 2 h and 4 h later, both kidneys were removed, the glomeruli were isolated and the residual radioactivity measured. In the case of HSA:poly-L-lysine20 a further two rats were examined at 24 h and 3 days. Kinetic studies on persistence of HSA: poly-L-lysine2o in glomeruli, blood and other organs. Rats received 5 mg/100 g body weight of '25I HSA:poly-L-lysine2O (containing 1 mg/100 g body weight '3'I BSA to allow correction for blood contamination) intravenously and at 1 h, 4 h and 24 h three rats were killed. The radioactivity in each kidney, the isolated glomeruli, liver, spleen, heart, lung and blood were counted. Immunofluorescent staining was also performed on renal tissue. In a further three rats also given 5 mg/100 g body weight, serial serum samples were collected after 15 min, 1, 4, 8, 24 and 48 h. Effect of prior administration of a small polycation (PEI 1200) on glomerular binding of HSA :poly-L-lysine2o. A group of five rats was given 0-5 mg/100 g body weight of PEI 1200 (Polysciences, Warrington, PA; dissolved in PBS) intravenously followed immediately by a mixture of '3'I HSA:poly-Llysine2O (1 mg/100 g body weight) and 1251 BSA (I mg/100 g body weight) (to allow correction for blood contamination). The animals were killed after 15 min and the kidneys, liver and a blood sample were taken and counted, as were the glomeruli after isolation. In the control group (n=5) PBS was substituted for PEI. Effect of glomerular deposition of HSA :poly-L-lysine2o-anti HSA complex on urinary protein excretion. Immune complex formation was induced in five rats as above and the urinary protein excretion was measured, commencing 2 days before and continuing for 14 days after administration.
. . . . . . . . . . . . . . . . .. . . . .
2 4 6 3 5 Fig. 1. Agarose electrophoresis: (1) cytochrome C (pl 10-7); (2) whale myoglobin (pl 8-3); (3) horse myoglobin (pl 7 3); (4) HSA (pl 5-2); (5) HSA:poly-L-lysine2O; (6) HSA:poly-L-lysine17. The point of application is arrowed. Note that both HSA:poly-L-lysine samples are anionic.
RESULTS
Preparation and analysis of charge hybrid probes The small polycation lysozyme could be coupled to the larger polyanions, ovalbumin or HSA with DMS. The crude ovalbumin: lysozyme and HSA: lysozyme preparations obtained from chromatofocusing were separated by gel filtration on a Sephadex G. 100 column to give a highly enriched 1: I fraction. In the case of HSA the relatively small size differences between monomeric HSA and 1: 1 products necessitated repeated purification. The samples were further analysed on SDS-PAGE to confirm both the purity and the values for the Stokes-Einstein radius obtained by gel filtration; the overall net charge was determined by isoelectric focusing, results in Table 1. The reaction products formed between HSA and poly-L-lysine chains proved to be more heterogeneous, and polymerization between HSA molecules was more prominent than with the other polycations used, presumably due to the poly-L-lysine chains. The material could be separated on a Sephacryl S.300 column into three peaks, corresponding to 1, 2 and 3 or more HSA molecules linked to poly-L-lysine. As before, SDS-PAGE analysis was used to assess purity and size distribution of the products. Using radiolabelled poly-L-lysine and HSA it could be shown that the ratio of HSA to poly-L-lysine was almost unity in both the crude reaction product and the three peaks obtained, so it is justified to assume that only one poly-L-lysine chain was attached to each HSA molecule on average, even when HSA polymers were produced. The isoelectric maxima of the HSA: poly-L-lysine20 fraction were determined from its electrophoretic mobility in agarose (Fig. 1). The results are shown in Table 1. Affinity of charge-hybrid molecules for the glomerular capillary wall in vivo The affinity of ovalbumin: lysozyme (1: 1 product, see above) was extremely low, HSA: lysozyme (1: 1 product, see above) showed clear binding and HSA: poly-L-lysine2o had the highest
474
S. R. Batsford et al.
Fig. 3. Electron micrograph of section of glomerular capillary wall from a rat given 1 mg of HSA: poly-L-lysine followed by 0 5 ml of rabbit antiHSA (both i.v., per 100 g body weight, interval 15 min). Tissue taken after 14 days. Note discrete electron dense deposits in both subepithelial (large arrows) and subendothelial locations (small arrows) (x 6800).
Table 3. Quantity of HSA: pOly-L-lysine in glomerular bound immune complex
Time
Fig. 2. (a) Glomerulus from a rat given 5 mg/100 g body weight of HSA:poly-L-lysine20 15 min earlier intravenously, stained with FITClabelled anti-HSA antiserum; note capillary/mesangial pattern ( x 370). (b) Glomerulus from a rat given I mg of HSA: lysozyme followed by 0 5 ml of rabbit anti-HSA (both i.v., per 100 g body weight, interval 15 min). One hour later renal tissue was removed and stained with FITC antiHSA. Note capillary/mesangial pattern ( x 370). (c) Glomerulus from a rat given 1 mg of HSA: poly-L-lysine20 followed by 0-5 ml of rabbit antiHSA (both i.v., per 100 g body weight, interval 15 min). One hour later renal tissue was removed and stained with FITC anti-rabbit IgG. Note intense deposition of antibody along the glomerular capillary wall ( x 370). (d) As (c) but higher magnification; note granular pattern of immune complex deposition ( x 930).
1 h 2h 4h 24h 3 days
HSA: poly-L-lysine2O
HSA: poly-L-lysineI7
0-57+0 15 0-44+012 0 24+0 04
025 +009t 0-14+0-04t
0.08* 0.06*
0-10+0-07t ND ND
* Only two rats studied. P< 0-001 in comparison to HSA: poly-L-lysine20 (two-way analysis of variance). ND, not done. Values are means of four animals (pg ± 2 s.e.m.). Rats given 1 mg of antigen followed by 0-25 ml of antiserum 15 min later (i.v., per 100 g body weight).
affinity (Table 1). The staining pattern of ovalbumin: lysozyme was restricted to some speckles in a mesangial distribution, and in the case of HSA: lysozyme and HSA: poly-L-lysine deposition was in a mesangial/capillary pattern (Fig. 2a). Analysis of Table 2. Glomerular immune histology in rats given HSA:poly-L-lysine20 and anti-HSA
Intensity of fluorescence (mean of three rats) Time
HSA
Rabbit-IgG
Rat-IgG
Rat C3
Ih 24 h 14 days
+ (+)
++ ++ +
+
+ +
+
(+) +
(+)
Rats were given I mg of antigen followed by 0 5 ml of antiserum 15 min later (i.v. and per 100 g body weight).
subfractions of HSA: poly-L-lysine (see Table 1) did not reveal noteworthy differences. For all further experiments the product obtained after chromatofocusing (size heterogeneous) was used. HSA which had been subjected to cross-linking with DMS produced only very faint staining in some mesangial regions. Formation ofglomerular immune complexes All the charge hybrids tested were accessible to circulating antibody when bound in the glomeruli, resulting in IC forma-
tion (Table 1). Complexes induced with HSA: lysozyme-antiHSA were distributed in a mesangial/capillary pattern (Fig. 2b); deposition of HSA: poly-L-lysine20-anti-HSA was mainly along the capillary wall (Fig. 2c) where it was granular in nature (Fig.
Antigen charge distribution and IC formation
475
Table 4. Kinetic studies of HSA: poly-L-lysine2o persistence in blood and glomeruli after i.v. application
Isolatedt glomeruli
Time
Serum* (pg/l00 p1)
Bloodt (pg/100 p1)
(pg/2 kidneys)
15 min 1h 4h 8h 24h 48 h
30-3+8-7 22 0+±57 13-4+3-7 10-6+2-5 4-4+1-5 1-4+0-9
ND 13-8+5-7 7-8+3-6 ND 1-9+1-3 ND
ND 6-6+4-9 2 0+0 5 ND 05+03 ND
Glomerulart immunofluorescence (stained for HSA) ND ++ + ND
(+) ND
Rats were given 5 mg of antigen per 100 g body weight intravenously. * Values from three rats bled serially. t Three rats studied at each timepoint. ND, not done. Values are means + 2 s.e.m. Table 5. Organ distribution of 1251 HSA: poly-L-lysine20
Uptake at timepoint (pg 2 se.m.)
Left kidney Right kidney Liver Spleen Heart Lung
1h
4h
24h
22-4+9-8 21-0+9 73 1118+348 60 0+29 2 7-8+3 9 47-0+28-1
21 5+1-7 16-2+3-0 821-0+270-0 37-6+ 19-7 9-7+2-2 51-4 +22-3
6-9+3-7 6-8+4-1 61-6+ 19-2 2-9+0-7 3-4+ 1-9 13-5 + 1*0
Rats were given 5 mg of antigen per 100 g body weight intravenously. Values corrected for blood contamination.
Table 6. Effect of prior administration of PEI 1200 on tissue uptake of 1251 HSA:poly-L-lysine20
Quantity of 1251 HSA:poly-L-lysine20 bound (pg±2 s.e.m.) pretreated with Organ Isolated glomeruli (both kidneys) Whole kidneys (left + right) Liver
Blood (I ml)
PBS
PEI*
2-1 +0-8
0-52+0 34t
5 5 +2 1
-0 9+0-6$
362-8+91-7
32 5+11-3
43-8+17 9
65-2+19 7
Rats given 0 5 mg PEI followed immediately by 1 mg of antigen (per 100 g body weight intravenously). t P
Surface charge distribution is a determinant of antigen deposition in the renal glomerulus: studies employing 'charge-hybrid' molecules S. R. BATSFORD, M. J. MIHATSCH*, M. RAWIEL, T. M. SCHMIEDEKE & A. VOGT Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany, and *Institute of Pathology, Basel, Switzerland
(Acceptedfor publication 18 June 1991)
SUMMARY The deposition of antigens and immune complexes (IC) in the renal glomerulus is charge-dependent. The demonstration that molecules of net anionic charge, but with discrete positively charged regions, exhibit affinity for the glomerular basement membrane (GBM) extends this concept. Charge hybrid (polar) molecules were constructed by covalently coupling small polycations (lysozyme or linear poly-L-lysine chains with a mean of 17 and 20 residues) to larger polyanions (ovalbumin or human serum albumin (HSA)). Although the products were of overall net anionic charge they still bound to glomerular structures. Immunofluorescence studies performed after i.v. injection of the samples into rats revealed that HSA: poly-L-lysine had the highest affinity. Radioisotopic measurements showed uptake of HSA: poly-L-lysine to be a function of the number of lysine residues; binding of HSA: polyL-lysine2O was 2-5 times higher than HSA: poly-L-lysine17 (P 160 kD)
Alone*
Plus antibodyt
+
(+)
(+)
+
+
2+-3+
ND ND ND
2+ 2+ 3+
* t Stained for HSA* or rabbit IgGt (mean of three rats/fraction). Antigen alone = 5 mg/100 g body weight intravenously, kidneys removed 15 min later; with antibody= 1 mg antigen plus 0 25-0-5 ml antiserum (i.v., per 100 g body weight) 15 min later, kidneys removed after 1 h. Staining patterns and controls-see Results section. I 1:1 product. ND, not done.
subsequent staining was with amido black. Isoelectric focusing (IEF) was performed in slab gels of 5 5% polyacrylamide using an ampholyte gradient of pH 4-5-9-8. HSA: poly-L-lysine preparations precipitated spontaneously in the electrofocusing gel, regardless of the point of application. Analysis of these latter samples was done by electrophoresis in uncharged agarose (IEF grade, Pharmacia) in veronal buffer (pH 8-2, 0.05 M) using cytochrome C (pI 10-7), ribonuclease (pl 9-5), whale myoglobin (pI 8 3), horse myoglobin (pI 7-3), HSA (pI 5-2) and ovalbumin (pI 4 2) as markers.
Immunofluorescence Immunofluorescence was performed on 5 gm cryostat sections fixed in acetone. The intensity of glomerular fluorescence was estimated on a 0 to + 3 scale on coded sections. Electronmicroscopy Renal tissue was fixed in 2-5% buffered glutaraldehyde for 2 h. Epon embedded ultrathin sections were stained with uranyl acetate and lead citrate. Statistical analysis This was performed with the aid of two-way analysis ofvariance or the Mann-Whitney U-test as appropriate.
Experimental design Affinity of charge-hybrid molecules for the renal glomerulus. Groups of rats (n = 3) were given 5 mg/ 100 g body weight ofeach sample intravenously (see Table 1), the kidneys were removed 15 min later and binding was assessed by immunofluorescence using the appropriate FITC-labelled antibody. In the case of HSA: poly-L-lysine, fractions separated on Sephacryl S.300
473
Antigen charge distribution and IC formation were studied as well, three rats were tested per peak. As a control, DMS-treated HSA was also given to two rats. Formation of glomerular immune complexes. In these experiments 1 mg/l00 g body weight of the charge hybrid (see Table 1) was given intravenously, followed by 0-25-0-5 ml of anti-HSA antiserum after 15 min. Renal tissue was removed I h later for immunofluorescence studies; three rats were studied per sample. Preliminary experiments had shown that when a dose of I mg/ l00 g was exceeded and antibody was given subsequently, acute toxicity (especially lung haemorrhage) was seen. Increasing the quantity of antiserum given (from 0 5 ml/100 g body weight) did not alter the intensity of immunofluorescent staining seen-apparently saturating conditions had been reached. The persistence of the immune complex was also studied. Nine rats received 1 mg/100 g body weight of HSA:poly-Llysine2O followed by 0 5 ml/100 g body weight of rabbit antiHSA 15 min later. Groups of three rats were killed after I h, 24 h and 14 days and the kidneys were examined for the presence of HSA, rabbit-IgG, rat IgG and rat C3 by immunofluorescence. Renal tissue was taken for electron microscopy on day 14. As controls, two rats were given DMS-treated HSA followed by anti-HSA as above and examined by immunofluorescence at I h; and four rats were given HSA: poly-L-lysine20 followed by normal rabbit serum; staining for rabbit IgG and HSA was performed I h later in two rats and at 14 days in the two remaining, which were also examined by electron microscopy. Influence of size of the poly-L-lysine chain on glomerular formation and persistence of immune complexes. Two probes were available, HSA: poly-L-lysinel7 and HSA: poly-L-lysine20. Rats were given I mg/I00 g body weight of HSA:poly-L-lysine (17 or 20) intravenously followed by 0 25 ml/100 g body weight of rabbit anti-HSA intravenously 15 min later. In both groups four rats were killed I h, 2 h and 4 h later, both kidneys were removed, the glomeruli were isolated and the residual radioactivity measured. In the case of HSA:poly-L-lysine20 a further two rats were examined at 24 h and 3 days. Kinetic studies on persistence of HSA: poly-L-lysine2o in glomeruli, blood and other organs. Rats received 5 mg/100 g body weight of '25I HSA:poly-L-lysine2O (containing 1 mg/100 g body weight '3'I BSA to allow correction for blood contamination) intravenously and at 1 h, 4 h and 24 h three rats were killed. The radioactivity in each kidney, the isolated glomeruli, liver, spleen, heart, lung and blood were counted. Immunofluorescent staining was also performed on renal tissue. In a further three rats also given 5 mg/100 g body weight, serial serum samples were collected after 15 min, 1, 4, 8, 24 and 48 h. Effect of prior administration of a small polycation (PEI 1200) on glomerular binding of HSA :poly-L-lysine2o. A group of five rats was given 0-5 mg/100 g body weight of PEI 1200 (Polysciences, Warrington, PA; dissolved in PBS) intravenously followed immediately by a mixture of '3'I HSA:poly-Llysine2O (1 mg/100 g body weight) and 1251 BSA (I mg/100 g body weight) (to allow correction for blood contamination). The animals were killed after 15 min and the kidneys, liver and a blood sample were taken and counted, as were the glomeruli after isolation. In the control group (n=5) PBS was substituted for PEI. Effect of glomerular deposition of HSA :poly-L-lysine2o-anti HSA complex on urinary protein excretion. Immune complex formation was induced in five rats as above and the urinary protein excretion was measured, commencing 2 days before and continuing for 14 days after administration.
. . . . . . . . . . . . . . . . .. . . . .
2 4 6 3 5 Fig. 1. Agarose electrophoresis: (1) cytochrome C (pl 10-7); (2) whale myoglobin (pl 8-3); (3) horse myoglobin (pl 7 3); (4) HSA (pl 5-2); (5) HSA:poly-L-lysine2O; (6) HSA:poly-L-lysine17. The point of application is arrowed. Note that both HSA:poly-L-lysine samples are anionic.
RESULTS
Preparation and analysis of charge hybrid probes The small polycation lysozyme could be coupled to the larger polyanions, ovalbumin or HSA with DMS. The crude ovalbumin: lysozyme and HSA: lysozyme preparations obtained from chromatofocusing were separated by gel filtration on a Sephadex G. 100 column to give a highly enriched 1: I fraction. In the case of HSA the relatively small size differences between monomeric HSA and 1: 1 products necessitated repeated purification. The samples were further analysed on SDS-PAGE to confirm both the purity and the values for the Stokes-Einstein radius obtained by gel filtration; the overall net charge was determined by isoelectric focusing, results in Table 1. The reaction products formed between HSA and poly-L-lysine chains proved to be more heterogeneous, and polymerization between HSA molecules was more prominent than with the other polycations used, presumably due to the poly-L-lysine chains. The material could be separated on a Sephacryl S.300 column into three peaks, corresponding to 1, 2 and 3 or more HSA molecules linked to poly-L-lysine. As before, SDS-PAGE analysis was used to assess purity and size distribution of the products. Using radiolabelled poly-L-lysine and HSA it could be shown that the ratio of HSA to poly-L-lysine was almost unity in both the crude reaction product and the three peaks obtained, so it is justified to assume that only one poly-L-lysine chain was attached to each HSA molecule on average, even when HSA polymers were produced. The isoelectric maxima of the HSA: poly-L-lysine20 fraction were determined from its electrophoretic mobility in agarose (Fig. 1). The results are shown in Table 1. Affinity of charge-hybrid molecules for the glomerular capillary wall in vivo The affinity of ovalbumin: lysozyme (1: 1 product, see above) was extremely low, HSA: lysozyme (1: 1 product, see above) showed clear binding and HSA: poly-L-lysine2o had the highest
474
S. R. Batsford et al.
Fig. 3. Electron micrograph of section of glomerular capillary wall from a rat given 1 mg of HSA: poly-L-lysine followed by 0 5 ml of rabbit antiHSA (both i.v., per 100 g body weight, interval 15 min). Tissue taken after 14 days. Note discrete electron dense deposits in both subepithelial (large arrows) and subendothelial locations (small arrows) (x 6800).
Table 3. Quantity of HSA: pOly-L-lysine in glomerular bound immune complex
Time
Fig. 2. (a) Glomerulus from a rat given 5 mg/100 g body weight of HSA:poly-L-lysine20 15 min earlier intravenously, stained with FITClabelled anti-HSA antiserum; note capillary/mesangial pattern ( x 370). (b) Glomerulus from a rat given I mg of HSA: lysozyme followed by 0 5 ml of rabbit anti-HSA (both i.v., per 100 g body weight, interval 15 min). One hour later renal tissue was removed and stained with FITC antiHSA. Note capillary/mesangial pattern ( x 370). (c) Glomerulus from a rat given 1 mg of HSA: poly-L-lysine20 followed by 0-5 ml of rabbit antiHSA (both i.v., per 100 g body weight, interval 15 min). One hour later renal tissue was removed and stained with FITC anti-rabbit IgG. Note intense deposition of antibody along the glomerular capillary wall ( x 370). (d) As (c) but higher magnification; note granular pattern of immune complex deposition ( x 930).
1 h 2h 4h 24h 3 days
HSA: poly-L-lysine2O
HSA: poly-L-lysineI7
0-57+0 15 0-44+012 0 24+0 04
025 +009t 0-14+0-04t
0.08* 0.06*
0-10+0-07t ND ND
* Only two rats studied. P< 0-001 in comparison to HSA: poly-L-lysine20 (two-way analysis of variance). ND, not done. Values are means of four animals (pg ± 2 s.e.m.). Rats given 1 mg of antigen followed by 0-25 ml of antiserum 15 min later (i.v., per 100 g body weight).
affinity (Table 1). The staining pattern of ovalbumin: lysozyme was restricted to some speckles in a mesangial distribution, and in the case of HSA: lysozyme and HSA: poly-L-lysine deposition was in a mesangial/capillary pattern (Fig. 2a). Analysis of Table 2. Glomerular immune histology in rats given HSA:poly-L-lysine20 and anti-HSA
Intensity of fluorescence (mean of three rats) Time
HSA
Rabbit-IgG
Rat-IgG
Rat C3
Ih 24 h 14 days
+ (+)
++ ++ +
+
+ +
+
(+) +
(+)
Rats were given I mg of antigen followed by 0 5 ml of antiserum 15 min later (i.v. and per 100 g body weight).
subfractions of HSA: poly-L-lysine (see Table 1) did not reveal noteworthy differences. For all further experiments the product obtained after chromatofocusing (size heterogeneous) was used. HSA which had been subjected to cross-linking with DMS produced only very faint staining in some mesangial regions. Formation ofglomerular immune complexes All the charge hybrids tested were accessible to circulating antibody when bound in the glomeruli, resulting in IC forma-
tion (Table 1). Complexes induced with HSA: lysozyme-antiHSA were distributed in a mesangial/capillary pattern (Fig. 2b); deposition of HSA: poly-L-lysine20-anti-HSA was mainly along the capillary wall (Fig. 2c) where it was granular in nature (Fig.
Antigen charge distribution and IC formation
475
Table 4. Kinetic studies of HSA: poly-L-lysine2o persistence in blood and glomeruli after i.v. application
Isolatedt glomeruli
Time
Serum* (pg/l00 p1)
Bloodt (pg/100 p1)
(pg/2 kidneys)
15 min 1h 4h 8h 24h 48 h
30-3+8-7 22 0+±57 13-4+3-7 10-6+2-5 4-4+1-5 1-4+0-9
ND 13-8+5-7 7-8+3-6 ND 1-9+1-3 ND
ND 6-6+4-9 2 0+0 5 ND 05+03 ND
Glomerulart immunofluorescence (stained for HSA) ND ++ + ND
(+) ND
Rats were given 5 mg of antigen per 100 g body weight intravenously. * Values from three rats bled serially. t Three rats studied at each timepoint. ND, not done. Values are means + 2 s.e.m. Table 5. Organ distribution of 1251 HSA: poly-L-lysine20
Uptake at timepoint (pg 2 se.m.)
Left kidney Right kidney Liver Spleen Heart Lung
1h
4h
24h
22-4+9-8 21-0+9 73 1118+348 60 0+29 2 7-8+3 9 47-0+28-1
21 5+1-7 16-2+3-0 821-0+270-0 37-6+ 19-7 9-7+2-2 51-4 +22-3
6-9+3-7 6-8+4-1 61-6+ 19-2 2-9+0-7 3-4+ 1-9 13-5 + 1*0
Rats were given 5 mg of antigen per 100 g body weight intravenously. Values corrected for blood contamination.
Table 6. Effect of prior administration of PEI 1200 on tissue uptake of 1251 HSA:poly-L-lysine20
Quantity of 1251 HSA:poly-L-lysine20 bound (pg±2 s.e.m.) pretreated with Organ Isolated glomeruli (both kidneys) Whole kidneys (left + right) Liver
Blood (I ml)
PBS
PEI*
2-1 +0-8
0-52+0 34t
5 5 +2 1
-0 9+0-6$
362-8+91-7
32 5+11-3
43-8+17 9
65-2+19 7
Rats given 0 5 mg PEI followed immediately by 1 mg of antigen (per 100 g body weight intravenously). t P
