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From the Departments of Nephrology* andImmunohematology and Blood Transfusion,Leiden University Medical Center, Leiden, The Netherlands; and theDepartment of Pathology and LaboratoryMedicine, University of Calgary, Calgary,Alberta, Canada
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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1 chain of collagen type VI in association with the 5
chain of collagen type IV. In conclusion, LEW recipients of
F344 kidney grafts produce IgG1 antibodies against donor type perlecan
and 1(VI)/5(IV) collagen and develop transplant glomerulopathy.
These data implicate an important role for the humoral immune response
in the development of glomerulopathy during chronic
rejection.
A well-established model to study CR in renal allografts is the F344 to LEW rat model. All LEW recipients of F344 grafts develop acute rejection at approximately day 30 resulting in 50% graft loss. The surviving animals show histopathological and functional characteristics of CR from day 50. The reverse combination, ie, LEW kidneys transplanted into F344 rats all exhibit long-term surviving kidney grafts in the absence of histological abnormalities, despite early acute rejection episodes. In this model, antibody responses specific for lymph node-derived lymphocytes have been described.9 These antibodies disappeared at 8 weeks after Tx and were described to activate neutrophils and resulted in T cell activation. In addition, a humoral immune response against undefined tissue antigens has been reported previously in this model.10 However, the nature, kinetics, and the specificity of these kidney-specific antibodies has remained elusive.
In chronic cardiac allograft rejection with graft vasculopathy, alloantibodies are mainly directed against the endothelium. In a previous report of chronic renal allograft rejection with transplant glomerulopathy in the rat using the same model, hardly any antibodies against donor endothelial cells were detected.11
Because the GBM is frequently duplicated in CR, we hypothesize that it may be a target of the humoral immune response in CR. In the present study, we investigated the kinetics and specificity of the anti-GBM antibody response after Tx.
Previous experiments have shown the development of anti-tubular basement membrane (TBM) antibodies after allogeneic kidney transplantation but such anti-TBM antibodies did not result in tissue damage or tubulointerstitial inflammation.12 In the present study we focus on anti-GBM antibodies generated after Tx, because these antibodies could play a role in the pathogenesis of glomerular lesions.
We observed that anti-GBM antibodies in the LEW recipients of F344
grafts are exclusively of the IgG1 isotype, donor-type GBM specific,
and are reactive with F344 GBM preparations after elution from
transplanted rat kidneys. Furthermore, proteomics revealed that the
antigens recognized by post Tx sera are the heparan sulfate
proteoglycan perlecan and the 1 chain of collagen type VI in
association with the 5 chain of collagen type IV.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Male inbred Fisher (F344, RT1lv1) and Lewis (LEW, RT1l) rats weighing 250 g were purchased from Harlan, Horst, The Netherlands. Animals had free access to water and standard rat chow. Animal care and experimentation were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Kidney Transplantation
Kidney transplantations were performed under halothane anesthesia as previously described.10,11 The left kidney of the recipient was removed and a donor kidney was transplanted in the orthotopic position; a patch of the donor aorta and of the inferior vena cava were anastomosed to the recipient aorta and inferior vena cava, respectively. The donor ureter was anastomosed end-to-end to the ureter of the recipient. The remaining native right kidney was removed 7 days after transplantation. Postoperatively, animals received 1 mg/kg body weight of temgesic subcutaneously (buprenorphine-hydrochloride; Schering-Plough B.V., Amstelveen, The Netherlands) for pain relief.
Blood samples were collected weekly by tail vein puncture and sera were stored at -80°C. All rats were housed in metabolic cages once a week to collect urine for assessment of microalbuminuria that was measured on a Hitachi-911 nephelometer (Hitachi, Tokyo, Japan).
LEW rats that had received a F344 kidney graft were sacrificed on days 7 (n = 3), 14 (n = 3), 30 (n = 6), 60 (n = 6), and 90 (n = 6) after transplantation and sera and kidneys were collected. Similarly, F344 rats received a LEW kidney and were sacrificed on days 60 (n = 6) and 100 (n = 2), respectively.
To investigate the effect of acute rejection on antibody formation and development of transplant glomerulopathy three LEW recipients of F344 grafts received low-dose cyclosporine A (CsA) subcutaneously (Sandimmune; Novartis Pharma, Basel, Switzerland, 1.5 mg/kg body weight) 5 days a week for 4 weeks and remained afterward without further treatment until sacrifice on day 100.
Histology
Tissue samples were fixed in methyl Carnoy’s solution,11 embedded in paraffin, sectioned, and stained with periodic acid-Schiff, hematoxylin and eosin, or trichrome. All kidney sections were scored blindly by a renal pathologist using a semiquantitative scale (0 to 3); mesangiolysis was scored as described previously;13 and glomerulitis, glomerulosclerosis, and transplant glomerulopathy were scored as described in the Banff working classification.13,14 Histological changes were compared using the Kruskal-Wallis one-way analysis of variance on ranks using Duna’s comparison between multiple groups. P values <0.05 were considered significant.
Electron Microscopy
Tissue samples were diced into 0.5-mm3 cubes, fixed in 2% glutaraldehyde, and postfixed by immersion in 2% osmium tetroxide solution. After fixation, tissues were washed in 0.1 mol/L (pH 7.4) sodium cacodylate buffer, dehydrated in graded acetone, and embedded in epoxy resin (epon 812), according to the usual procedure, with polymerization being performed at 60°C. One-µm-thick sections were cut by glass knives on a Reichert-Jung Ultracut-E ultramicrotome and stained with 0.5% toluidine blue solution. Ninety- to 100-nm-thin sections were cut on a Reichert-Jung Ultracut-E ultramicrotome with a Diatome diamond knife, stained with uranylacetate (ultrostain 1 solution; Leica Co., Canada) and lead solution (ultrostain 2, Leica Co.). The sections were viewed under a Hitachi 600 electron microscope at 50 kW.
Direct Immunofluorescence
Kidneys removed at different time points were snap-frozen in precooled isobutanol and stored at -150°C. Cryostat sections of 3 µm were acetone-fixed for 10 minutes at room temperature and stored at -20°C. To detect specific rat immunoglobulin subclasses, monoclonal antibodies specific for rat IgA, IgG1, IgG2a, IgG2b, IgG2c, and IgM (Prof. H. Bazin, Leuven University, Leuven, Belgium) were used. These monoclonals were either directly fluorescein isothiocyanate-conjugated or used in combination with a tyramide-fluorescein isothiocyanate amplification15 and kidney sections were subsequently embedded in DABCO-glycerol (1,4-diazabicyclo(2,2,2) octane; Sigma Chemical Co,, St. Louis, MO). Double labeling was performed using a rabbit anti-rat GBM antiserum (prepared in our own laboratory10 ) and bound rabbit antibodies were detected using tetramethyl-rhodamine isothiocyanate-conjugated goat anti-rabbit IgG antibodies (Nordic, Tilburg, The Netherlands).
GBM Isolation
Collagenase-digested GBM preparations of F344 or LEW origin were isolated as described previously.10 Briefly, glomeruli were isolated by dissecting the renal cortex, followed by homogenization and pressing through a series of sieves with decreasing pore size (150 and 106 nm; glomeruli were harvested on a 75-nm sieve). After sonication, membrane fragments were digested with collagenase (Collagenase Type Ia, Sigma) overnight at 37°C in 100 mmol/L of Tris/HCl (pH 7.4) and10 mmol/L of CaCl2. The proteins in the supernatant after centrifugation were used for enzyme-linked immunosorbent assay (ELISA) and Western blot analysis.
GBM ELISA
Ninety-six well ELISA plates (Greiner, Alphen aan de Rÿn,
The Netherlands) were coated overnight with collagenase-digested F344
or LEW GBM preparations (0.3 µg total protein/well) in carbonate
buffer (pH 9.6) at room temperature. After blocking with
phosphate-buffered saline (PBS)/1% bovine serum albumin (w/v), plates
were incubated with serial dilutions of post Tx sera or normal rat sera
in PBS/0.05% (v/v) Tween-20/1% bovine serum albumin (w/v) for 1 hour
at 37°C. Because more than 95% of rat immunoglobulins have 
light
chains,16
antibody binding was detected using a
digoxigenin (DIG)-conjugated (Boehringer-Mannheim, Mannheim, Germany)
mouse monoclonal antibody specific for rat light chains (His8;
Prof. Dr. P. Nieuwenhuis, University of Groningen, Groningen, The
Netherlands) for 1 hour at 37°C. After washing, the wells were
incubated with horseradish peroxidase-conjugated sheep F(ab') fragments
anti-DIG (Boehringer Mannheim) for another hour at 37°C. Finally,
wells were stained with the peroxidase substrate ABTS
(2,2'-amino-bis-3-ethylbenzthiazoline-6-sulfonic acid;
Sigma) in the presence of
H2O2 for 1 hour before the
optical density was measured at 415 nm using a Titertek multiscan plate
reader. To detect specific rat immunoglobulin subclasses, various
monoclonal antibodies were used. As a positive control, purified rat Ig
was coated to the plates and stained with these monoclonal antibodies.
To test for GBM-binding to ELISA plates, coated wells were incubated
with a rabbit anti-rat GBM antiserum.
Elution of Kidney Bound Antibodies
To investigate the specificity of in vivo kidney-bound antibodies, F344 (n = 4) or LEW (n = 3) kidney allografts removed on day 60 were subjected to acid elution. Normal F344 (n = 2) and LEW (n = 2) kidneys were used as controls. Kidneys were perfused with 100 ml of PBS in vivo, removed, and homogenized by pressing pieces of cortex through an 80-mesh sieve generating a sample containing predominantly glomeruli. After sonication, homogenates were incubated for 1 hour at room temperature in citrate buffer (pH 2.5) followed by neutralization with 1 mol/L of NaOH. Samples were tested after overnight dialysis against PBS in the GBM ELISA.
Western Blot Analysis
Collagenase-digested, or nondigested LEW and F344 GBM preparations (6.5 µg total protein) were subjected to 4 to 15% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Criterion Precastgel, Tris-HCL; Bio-Rad Laboratories, Richmond, CA) under reducing or nonreducing conditions, followed by semidry blotting to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Blots were blocked for 2 hours in PBS/1% bovine serum albumin (w/v) at room temperature and washed in PBS/0.05% Tween-20. Blots were subsequently incubated with a 1 in 5 dilution of pooled post Tx sera (day 60) in PBS/Tween-20/0.5% bovine serum albumin overnight at 4°C. After washing, blots were incubated with a DIG-conjugated mouse monoclonal antibody against rat IgG1 for 1 hour at room temperature. Subsequently, blots were incubated with horseradish peroxidase-conjugated sheep F(ab') fragments anti-DIG for another hour at room temperature and after extensive washing bands were visualizedwith DAB (diaminobenzidine hydrochloride, Sigma)/nickel/imidazole.17
Proteomics
Collagenase-digested F344 GBM preparations were subjected to two-dimensional SDS-PAGE (8%) in duplicate; one of the gels was stained for total protein content using SYPRO Ruby (Bio-Rad Laboratories) whereas the other gel was blotted semidry to polyvinylidene difluoride membranes (as described in "Western Blot Analysis"). Isoelectric focusing was performed on an IPGphor (Amersham Pharmacia Biotech AB, Uppsala, Sweden) using 7-cm Immobiline isoelectric focusing strips of pH range 3 to 10. Before performing the second dimension, strips were treated with dithioerythritol 2,3-dihydroxybutane-1, 4-dithiol (DTE) and iodoacetamide. Spots recognized by the LEW post Tx antibodies were excised from the duplicate gel and digested following the protocol of Shevchenko and colleagues.18 Identification was performed by on-line nano LC-electrospray mass spectrometry on a Q-TOF (Micromass, Manchester, UK) using a 300-µm ID x 5 mm C18-Pepmap trapping column (LC-Packings, Amsterdam, The Netherlands) for clean-up. Tryptic peptides were step-eluted into the mass spectrometer and subjected to MS/MS and their sequences were determined by interactive use of PeptideSearch19 and manual interpretation.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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F344 kidney grafts were stained for the presence of rat
immunoglobulins using the different mouse monoclonal antibodies. Kidney
grafts removed after 7 days showed no immunoglobulin staining. A
prominent rat IgG1 staining was observed in the glomeruli and on the
TBM of F344 grafts removed on days 14 and afterwards and was
impressively more intense at later time points (Figure 1A)
. As the diffuse glomerular staining
did not look like the typical linear pattern of an anti-GBM antibody
deposition, the sections were incubated with a rabbit anti-rat GBM
antiserum followed by a tetramethyl-rhodamine isothiocyanate-conjugated
goat anti-rabbit antiserum (Figure 1B)
. The glomerular staining pattern
was very similar to that of the rat IgG1 staining (Figure 1A)
.
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. Staining of these kidneys using the
polyclonal rabbit anti-rat GBM antiserum resulted in a linear, more
intense GBM-like staining pattern (Figure 1D)
. LEW Recipients of F344 Grafts Develop Microalbuminuria
LEW recipients of F344 grafts excreted on average 8 mg/24 hours of
albumin in their urine from 3 to 4 weeks after Tx (Figure 2)
that persisted during the observed
time period, whereas nonrejecting F344 recipients of LEW grafts
excreted less than 1 mg/24 hours, comparable to normal LEW or F344
rats. In urine of LEW recipients of F344 grafts we were able to detect
rat IgG from 5 weeks after Tx (data not shown).
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F344 kidneys transplanted into non-immunosuppressed LEW recipients
showed changes characteristic of acute rejection that evolved
throughout time to CR. Seven- and 14-day grafts contained prominent
mononuclear cell infiltrates in the interstitium and within dilated
peritubular capillaries. In 30-day grafts significant interstitial
inflammation, mild tubulitis, and vasculitis were observed.
Sixty- and 90-day grafts revealed a progressive increase in
interstitial fibrosis, tubular atrophy, and glomerulosclerosis, as in
previous experiments14
(data not shown). A significant
glomerulitis was present at 7, 14, and 60 days in F344 allografts
removed from LEW recipients but decreased with time to levels
comparable to LEW kidneys removed from F344 recipients (Figure 3A
, P < 0.05).
Grafts removed on days 30 and 60 exhibited pronounced
mesangiolysis (Figure 3A
, P < 0.05).
Glomerulosclerosis increased with time after transplantation and
involved on average 25 to 50% of glomeruli on day 90 (Figure 3A
,
P < 0.05). LEW kidneys removed from F344 recipients
showed only mild glomerulitis, tubulitis, and interstitial inflammation
but did not develop transplant glomerulopathy.
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. Duplication of the GBM, with interposition of electron-lucent
material (Figure 3C)
was evident from ultrastructural examination of
F344 grafts removed from LEW recipients 30 days after Tx; the
morphology of podocytes and endothelium appeared normal. IgG1 Anti-GBM Antibodies in Sera of LEW Recipients of F344 Kidney Grafts
Because rat IgG1 antibodies were bound to the glomeruli of
rejecting kidneys and the GBM appeared severely damaged by
ultrastructural examination we investigated if an IgG1 antibody
response against the GBM was present in the LEW recipients of F344
grafts. To test for anti-GBM reactivity in serum of transplanted rats
an anti-GBM ELISA was developed. In this ELISA, we coated
collagenase-digested F344 or LEW GBM to the plates, incubated the wells
with post Tx sera and subsequently stained for immunoglobulin binding.
Serum samples collected weekly after Tx were tested. LEW recipient rats
produced variable amounts of antibodies that were strongly reactive
with F344 GBM from 3 weeks after Tx up to 100 days (Figure 4A)
. The antibodies reactive with F344
GBM were of the IgG1 isotype. These IgG1 antibodies were specific for
F344 GBM, and were not reactive with LEW GBM (Figure 4B)
. When the
ELISA was performed using light chain-specific antibodies similar
results were obtained (data not shown). The reverse combination, F344
recipients of LEW grafts, did not produce detectable IgG1 (Figure 4, A and B)
or other light chain containing antibodies that were
reactive with F344 or LEW GBM (not shown).
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. Only a marginal,
but nonsignificant, binding of IgM antibodies to F344 GBM was observed.
Normal LEW serum did not contain antibodies reactive with either F344
or LEW GBM.
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Elution of Kidney-Bound Antibodies Yielded IgG1 Antibodies Reactive with F344 GBM Preparations
To further confirm that IgG1 antibodies deposited in glomeruli of
chronically rejecting F344 kidneys were indeed reactive to the GBM,
isolated glomeruli were subjected to acid elution. Only eluates
prepared from PBS-perfused Tx F344 kidneys contained IgG1 antibodies
that bound in the ELISA coated with F344 GBM only (Figure 6)
. In contrast, eluates obtained from
LEW kidneys removed from F344 recipients and eluates harvested from
normal F344 and LEW kidneys did not contain any detectable anti-GBM
antibodies. Finally, antibodies eluted from rejecting F344 grafts
reacted only with F344 GBM preparations and not with LEW GBM (Figure 6)
. Eluates were negative for IgM and IgG2a binding.
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LEW recipients of F344 grafts treated with low-dose CsA to prevent
acute rejection did not produce microalbuminuria (<1 mg/24 hours;
Figure 7A
) and the histology of these
kidneys showed only mild interstitial inflammation. CsA treatment
abolished production of kidney-bound (data not shown) or circulating
(Figure 7B)
anti-GBM antibodies, whereas the total amount of
circulating antibodies is comparable to normal rats (data not shown).
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To further analyze the F344 GBM antigens recognized by LEW IgG1
antibodies, Western blot analysis was performed using
collagenase-digested GBM preparations. When the collagenase-digested
GBM preparations were electrophoresed under nonreducing conditions,
F344 and LEW GBM showed similar protein bands in the Coomassie-stained
blot (Figure 8)
. After blotting and
incubation with LEW anti-F344 sera after Tx (day 60) several bands were
observed in F344, but not in LEW GBM (Figure 8)
. These bands were not
detected using control F344 anti-LEW sera after Tx or using normal LEW
or F344 rat sera. The proteins recognized in collagenase-digested F344
GBM preparations by posttransplant LEW sera are 
120, 165, and >>200
kd.
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1 Chain of Collagen Type VI Together with the 5 Chain of
Collagen Type IV
Analysis of collagenase-digested F344 GBM preparations on
two-dimensional SDS-PAGE resulted in a strongly reactive spot of 120
kd (Figure 9A
, spot 1)
containing the peptides: VDSYGGFLR, GMVFGIPDGVLELVPQR,
and LSFDQPSDFK that were unique for perlecan. A weak spot of 40 kd
(Figure 9A
, spot 2)contained the peptides: VPNYQALLR,
VAVVQYSGQGQQQPGR, and GVLYQTVSR unique for the 1 chain of collagen
type VI and peptide GQSIQPFISR unique for the 5 chain of collagen
type IV. Thus, one spot contained the heparan sulfate
proteoglycan (HSPG) perlecan and the other contained peptides
corresponding to both collagen 1 type VI and 5type IV.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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1 chain of collagen type VI in association
with the 5 chain of collagen type IV are deposited along the GBM of
F344 kidney allografts. Transient treatment with CsA prevented both
acute and CR as well as IgG1 anti-GBM antibody production by LEW
recipients. In the F344 to LEW renal allograft model proteinuria is present, one of the characteristics of CR. This urinary protein leakage is considered to be of glomerular origin, because we detected large molecular weight proteins (ie, IgG) in urine (data not shown), suggesting that the glomerular filtration barrier is damaged. Electron microscopic analysis of F344 kidney grafts revealed an intact podocyte architecture, but a duplicated GBM. This suggests indeed that the GBM is one of the main structural targets of the immune response during CR.
Transplant glomerulopathy develops in 5 to 10% of human renal
transplants, in 20% of patients with CR and is associated with
proteinuria and poor graft survival.3,4
Its pathogenesis
is unknown, but it is thought to be related to glomerular endothelial
cell injury, as described for transplant glomerulitis and
antibody-mediated rejection.20-22
However, by electron
microscopy the endothelium does not always appear to be damaged, as is
the case in our rat renal allograft model.
Perlecan is essential for maintaining the integrity of basement membranes (BM).23 Injection of rats with monoclonal antibodies against HSPG resulted in proteinuria and BM thickening,24,25 suggesting that antibodies against the HSPG perlecan may contribute to the proteinuria observed in our model for CR. BM alterations, duplications, and abnormal matrix depositions including collagen type VI and HSPG have been associated with various (chronic) human diseases involving BM changes and proteinuria.26 Increased expression of perlecan has been described in various kidney diseases including diabetic nephropathy,27,28 membranous nephropathy,29,30 minimal change nephrotic syndrome,31 and diffuse mesangial sclerosis.32 In addition, accumulation of collagen VI has also been described in various diseases including diabetic nephropathy,33 diffuse mesangial sclerosis,32 and membranous glomerulonephritis.34
Perlecan and 1(VI)/5(IV) collagen are both located on the
endothelial side of the GBM, limited to focal
accumulations,31
and to the mesangial
matrix.31,35-37
In addition, type VI collagen is produced
by endothelial cells.38
Together this suggests that
initial antibody-mediated GBM damage can occur independent of
endothelial cell injury, but may in the long term affect endothelial
cell architecture and/or function as observed in patient
specimens.39
In the F344 kidney grafts, IgG1 staining along the GBM appeared
nonlinear in contrast to classical anti-GBM antibody disease in which
anti-GBM antibody deposits are linear. Our observed nonlinear staining
pattern may be explained by limited focal accumulations of perlecan and
1(VI)/5(IV) collagen. Staining kidney sections with an antibody
directed against the heparan sulfate side chain of HSPG has previously
also resulted in a nonlinear pattern, again supporting focal
accumulation sites of perlecan.40
Furthermore, staining
for collagen type IV would normally also result in a linear GBM
staining, in contrast to the F344 kidney allografts exhibiting a
nonlinear staining. However, in the proteomics experiment we found
collagen type IV in association with collagen type VI, suggesting that
the LEW antibodies are directed against the associated proteins rather
than the individual proteins, thereby possibly explaining the observed
localization.
A strong interaction of the 1 chain of collagen VI with the carboxyl
terminal globular domain of type IV collagen has been
described36
and it has been suggested that this collagen
interaction anchors endothelial BMs to the extracellular matrix. The
identification of both 1(VI) and 5(IV) collagen in one spot of
the two-dimensional gel suggests the presence of an interaction between
those two collagen molecules in renal BMs. The size of the bands on
SDS-PAGE for both collagen and perlecan is smaller than expected on
basis of molecular weights described in literature being 150 to 160 and
467 kd, respectively. However, we have used GBM preparations obtained
after various isolation steps, including sonification, which might
contribute to partial degradation of the large extracellular matrix
proteins. It has been reported that perlecan is fragmented during
isolation into smaller proteolytic fragments with different sizes,
including fragments of 95 to 130, 150, and 250 kd.41
The
peptide sequences obtained by proteomics are all derived from domains
II and III of the perlecan protein, suggesting that only a small part
of the perlecan molecule was represented in the identified protein
spot.
Antibody deposition along the TBM,42 the GBM,43 and in glomeruli44,45 have been observed in human renal allografts, but so far no follow-up information is available on clinical outcome. In our model, IgG1 and IgG2a antibody binding to TBM antigens was observed in combination with glomerular IgG1 deposition. We cannot exclude that sharing of antigens between TBM and GBM30 causing IgG1 deposits in both compartments. Perlecan and collagen VI are known to be expressed in most renal BMs, including the TBM.30,35,46
The induction of antibodies against perlecan or 1(VI)/5(IV)
collagen might be the result of the formation of new epitopes exposed
in the GBM after injury. Alternatively, antibodies might be induced as
a result of (genetic) differences in perlecan or 1(VI)/5(IV)
collagen between donor and recipient. Alternative splicing of human
collagen type VI has been reported47
and restriction
fragment polymorphisms are described for perlecan.48,49
Finally, heparan sulfate side chains of perlecan might be altered by
transplantation-related processes, such as ischemia/reperfusion injury
resulting in generation of reactive oxygen species.27
The
question remains unanswered as to what the difference is between the
molecules of the donor and recipient that ultimately result in antibody
production. Once antibodies are formed they may play a role in
maintaining the GBM abnormalities and thereby result in urinary protein
loss. Binding of antibodies results in activation of the complement
system and may subsequently induce the release of inflammatory
mediators and recruitment of inflammatory cells leading to graft
rejection. In the F344 to LEW renal allograft model we were able to
detect C3 and C5b-9 in the glomeruli and on the TBM (data not shown) of
rejecting F344 grafts removed from LEW recipients, indicating that the
complement activation cascade is involved.
LEW recipients of F344 grafts were transiently treated with low-dose CsA to investigate the requirement of acute rejection episodes for antibody production and disease development. The total amount of antibodies in serum was comparable to normal rat serum. Because the CsA-treated rats did not develop disease, whereas all other LEW recipients of F344 grafts did, we hypothesize that acute rejection is the initial event and is a prerequisite for CR. Alternatively or in addition, CsA treatment might have induced tolerance for the GBM antigens. LEW post Tx sera bound to the GBM and TBM of normal F344 kidney sections in vitro (data not shown). In addition, preliminary data show that LEW post Tx antibodies bind to kidneys of unilaterally nephrectomized F344 rats on intra-arterial injection, suggesting that the epitopes of perlecan and collagen are not induced as a consequence of CR-mediated changes.11 Therefore, we hypothesize that the local intragraft immune activation status of the recipient, which is associated with acute rejection, is required for production of IgG1 antibodies directed against the GBM. This antibody response could then conceivably contribute to the pathogenesis of CR. Thus, LEW recipients of F344 kidney allografts produce antibodies against the novel epitope whereas F344 recipients that encounter LEW antigens do not produce antibodies. It is not known what differences exist between F344 and LEW recipients, but there might be a difference in cytokine production during the initial phase of rejection that results in antibody production only in LEW recipients. This cytokine production pattern might be altered after CsA treatment abolishing formation of damaging antibodies recognizing graft antigens.
Th2 cytokines are known to skew the humoral response into production of IgG1 antibodies.5 This would be consistent with the hypothesis that activated CD4+ Th2 cells late after transplantation play the most critical role in the initiation and/or maintenance of chronic allograft rejection.5 Type 2 cytokines stimulate antibody production, are associated with the regulation of some of the effector mechanisms of CR, and may influence the disease process directly through the regulation of matrix metabolism involved in tissue restructuring.50
In conclusion, we found circulating and kidney graft-bound IgG1
antibodies against the GBM in LEW recipients of F344 grafts undergoing
CR, recognizing perlecan and 1(VI)/5(IV) collagen of the GBM. The
F344 grafts showed histological signs of transplant glomerulopathy,
including the characteristic BM duplications. Long-term surviving LEW
grafts in F344 recipients or F344 grafts in LEW recipients treated with
low-dose CsA showed no signs of CR and no production of anti-GBM
antibodies was detected.
We conclude that IgG1 antibodies recognizing perlecan and
1(VI)/5(IV) collagen play a crucial role in the pathogenesis of
transplant glomerulopathy observed during CR in rats.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the Dutch Kidney Foundation (grant no. C98.1783).
Accepted for publication January 2, 2002.
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