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Susceptibility for infection-related glomerulopathy depends on non-MHC genes
Kidney International, Vol. 43 (1993), pp. 623—629
Susceptibility for infection-related glomerulopathy depends on non-MHC genes MARIE-LOUISE F. VAN VELTHUYSEN, ALETTA VENINGA, JAN ANTHONIE BRUIJN, EMILE DE HEER, and GERT JAN FLEUREN Laboratory for Pathology, University of Leiden, Leiden, The Netherlands
Susceptibility for infection-related glomerulopathy depends on non-
MHC genes. BALB/c mice infected with T,ypanosoma brucei and treated seven days after inoculation with Diminazene aceturate develop
polyclonal B-cell stimulation, including production of antibodies to known nephritogenic autoantigens and glomerular disease associated with severe albuminuria. To investigate if the susceptibility for glomer-
ular disease in this model is linked to MHC or non-MHC genes, we studied this disease in six mouse strains that were partly congenic for their MHC and partly congenic for their non-MHC genes. The course of
the infection was measured by parasitemia and related to (auto)antibody production, proteinuria and glomerular deposition of immuno-
genetic factors as this may lead to a better understanding of pathogenesis as well as disease inheritance. The glomerular disease described in BALB/c mice induced by Trypanosoma brucei infection is a suitable model to study such relationships, as glomerular disease occurs together with autoantibody production while antibodies reactive with Trypanosomal antigens
do not seem to be involved in the development of the renal disease [5].
To investigate in this model whether the susceptibility for
globulins. The mouse strains could be divided into two groups. The first group consisted of the C57BL/6 (H-2'), C57BL/l0 (H2b) and Bl0.D2 (H-2") strains, which proved to be relatively resistant to infection with Trypanosoma brucei (that is, spontaneous survival >25 days). In sera of these mice antibodies to a broad range of antigens could be found 14 days after inoculation; no proteinuria was observed. The second group consisted of the BALB/c (H-2"), BALB.B (H-2") and DBA/2 (H-2")
developing renal disease and the production of autoantibodies is linked to MHC or non-MHC genes, we studied the development
strains, which were relatively susceptible to the infection. In these
Methods
animals proteinuria occurred and a broad polyclonal B-cell stimulation was seen 42 days after inoculation. No correlation was found between the specificity of circulating antibodies and the occurrence of proteinuria or a glomerular fluorescence pattern. These results indicate that in this model non-MHC genes govern the outcome of the infection as well as the development of polyclonal B-cell stimulation and proteinuria.
Concerning the glomerulopathy, a discrepancy is observed between glomerular immunoglobulin staining patterns and the development of proteinuria. Therefore, an additional pathogenetic role for non-immu-
of renal disease during Trypanosoma brucei infection in six mouse strains either congenic for their MHC or congenic for their background with the previously-described BALB/c mice.
Animals
BALB/c mice were bred in our own facilities. Female BALB.B, C57BL/l0 (BlO), C57BL16 (B6), B10.D2 and DBAI2
mice were purchased from Harlan OLAC (Zeist, the Netherlands). The genetic composition is shown in Table 1.
Parasites
noglobulin humoral factors must be considered.
A stabilate of Trypanosoma brucei (STIB 348c variant A) was
obtained from the Department of Pathology of the BernardNocht-Institut für Schiffs- und Tropenkrankheiten (Hamburg, Germany). This stabilate was expanded after a passage in an irradiated (500 rad) BALB/c mouse. Blood from this animal containing 108 parasites/ml was diluted in glycerol and saline and cryopreserved in batches of 40 d samples.
In general, autoimmune diseases seem to occur more often in genetically predisposed persons [1]. Epidemiologic as well as experimental studies have demonstrated the inheritance patterns to be multigenic. Several reports have demonstrated that certain MHC genes are related to a higher susceptibility for autoimmune diseases. For example HLA-DR2 and HLA-DR3 Inoculation are seen more often in lupus prone patients [21, of whom the HLA-DR2 patients develop glomerular disease more often [3, Cryopreserved blood was diluted in saline, up to 1000 para41. However, neither gene is necessary or sufficient, since many sites per ml. Each animal was inoculated with 0.1 ml (± 100 people carry the haplotype without being affected and many parasites) i.p.
patients do not carry the haplotype at all. Therefore, it is important to relate the genetic composition to possible patho-
Drug administration Diminazene aceturate (Berenil®; Hoechst AG, Frankfurt am
Received for publication July 13, 1992 and in revised form September 28, 1992 Accepted for publication September 29, 1992
Main, Germany) was diluted up to 5 mg/mI in saline and
© 1993 by the International Society of Nephrology
[51.
administered i.p. in a dosage of 40 mg/kg, to achieve a more protracted course of the disease as has been previously shown
623
624
van Velthuvsen et al: Infection-related glomerulopathy
"—" for absence of staining, "+" for staining, and "++" for
Table 1. Mouse strains used Strain
Non-MHC
MHC
BALB/c
BALB BlO B6 DBA BALB BlO
d b b
l0 B6
DBA/2 BALB.B BlO.D2
d
b d
strong staining.
Preparation of antigens and antisera Renal tubular epithelial antigens (RTE), glomerular basement membrane antigens (GBM), DPPIV, and gp330 were prepared
and characterized as described elsewhere [8, 9]. Type IV collagen, antibodies directed against type IV collagen, and
laminin were a gift of Prof. J.-M. Foidart (Dept. of Biology, University of Liege, Belgium). Preparation and characterization of these antigens and antibodies have been described [10, Experimental design 11]. EHS mouse laminin was obtained from Sigma (Sigma Two sets of experiments were performed consecutively ac- Chemical Co. Ltd., Dorset, UK). Dinitrophenol coupled to rat cording to the protocols described [5]. In the first set of serum albumin (DNP-RSA) was provided by Prof. M.R. Daha experiments, seven mice from each of the examined strains (Department of Nephrology, University Hospital of Leiden). were inoculated with Trypanosoma brucei, while five animals ELISA studies from each strain served as controls. In the second set of experiments seven mice of each strain were inoculated with Antibodies to various known nephritogenic antigens and to Trypanosoma brucei and treated seven days after infection with DNP-RSA were measured in sera of control and diseased mice Diminazene aceturate (Tr + Da +). As a control Diminazene to evaluate the degree of polyclonal B-cell stimulation and to aceturate was administered to five animals of each strain (Tr — correlate autoantibody production to the development of renal Da +). Albuminuria was measured weekly. Serum was ob- disease. For the detection of antibodies to RTE, GBM, DPPIV, tained every two weeks. Parasitemia was measured every other day in the first set of experiments, while it was detected weekly in the second experiment set. All animals were sacrificed 42 days after inoculation because, based on earlier observations [5], full blown disease was expected to have developed by then.
gp330, laminin, collagen type IV, and DNP ELISA studies were performed as described [8, 12, 13]. In experiment 1, antibodies
were detected in day 14 sera as this was the latest time point sera could be obtained from most mice. In experiment 2, sera obtained 28 and 42 days after inoculation were used. At each time point and of each strain antibodies were measured in four experimental and four control sera in most instances. All sera were tested in two dilutions (1/40 and 1/80, both in duplo). As
Parasitemia Parasites were counted in a smear of tail blood, stained with the extinction for the 1/80 dilution was always much lower than Leishman's stain. The results were expressed as number of for the 1/40 dilution, the results of the 1/40 diluted sera were parasites per 100 erythrocytes. used. Peroxidase-conjugated goat anti-mouse Ig (DAKOPATTS) was applied as second step. As a positive control for Albuminuria the coating procedure of the antigens, we used specific rabbit Mice were held in metabolic cages for 18 hours with free and goat antisera. The positive control for RTE was serum of access to water and food. The albumin concentration in urine mice with chronic graft-versus-host disease known to react with was measured by rocket electrophoresis. these antigens [14]. As a negative control, each serum was Histological methods All kidneys were perfused in vivo with PBS for two minutes,
tested in the same dilutions on a BSA-coated well. In all studies
a standard dilution curve from a reference serum pool of BALB/c mice 42 days after inoculation with parasites was
prior to sacrifice of the animals. For light microscopy, the included. The reaction products were quantified by measuring tissues were fixed in 10% buffered formalin, dehydrated, and extinction at 492 nm with a Titertek Multiscan (Amstelstad by, embedded in Paraplast® (Amsteistad, Amsterdam, The Nether- Zwanenburg, The Netherlands). lands). Sections were cut 5 sm thick and stained with hematoxylin-eosin (H&E) and periodic acid-Schiff reagent (PAS), Statistical analysis according to standard methods. For direct immunofluorescence To analyze the significance of the differences obtained in the studies kidney specimens were snap frozen and stained as various experiments between experimental and control mice an described elsewhere [61. In short, 2 jsm sections were cut in a unpaired Student's t-test was used. The differences with a P cryostat, air dried, and fixed in acetone/ethanol (50/50) for five value <0.05 were considered to be significant. Individual mice minutes and then postfixed in 100% ethanol for 10 minutes were considered to be albuminuric when their albuminuria in 18 before being incubated for one hour with monospecific rabbithours exceeded the mean plus twice the standard deviation of anti-mouse-FITC-conjugated immunoglobulins (Nordic Immu- control mice. nology, Tilburg, The Netherlands) directed against mouse 1gM, IgG, IgG3 and C3. Cross reactivity of the IgG3 antibodies with Results other IgG subclasses was checked by staining kidney specimens Parasitemia and survival of mice with GVH (chronic graft vs. host disease) induced nephritis. In murine GVH-induced nephritis all IgG subclasses Experiment 1. After inoculation with Trypanosoma brucei all except IgG3 are known to be involved [7]. Fluorescence pat- mice, regardless of the strain used, developed a first parasitemic terns were scored in a semiquantitative manner attributing a peak between day 6 and 7. Hereafter the mouse strains could be
625
van Velthuysen et a!: Infection-related glomerulopathy
A
25
B
><
20 U)
15
C',
U)
ci) U)
Cci
o10 5 :
0
A 1
Cci
0
0
30
40
Time, days post-inoculation
Time, days post-inoculation
Fig. 1. Mean value of the percentage of parasites for each strain (N = 7). A. Experiment 1 (Tr + Da —). B. Experiment 2 (Tr + Da +). Symbols
are:(——)BALB/c;(.-i--)B6;(-.-)B1O;(-€-)DBA/2;(x)BALB.B;(+)Bl0.D2.
10000
120 100 ci)
0
1000
E C)
C
E .0 100
0
0
10 20 30 Time, days post-inoculation
40
10 0
10 20 30 Time, days post-inoculation
40
Fig. 2. Experiment 1 (Tr + Da —). A. Percentage of mice from each strain with an albuminuria exceeding the mean plus twice the standard deviation of control mice. Symbols are as in B except: (—+--) BlO; (—+(--) B6. B. Mean of albuminuria in 18 hours (± SEM). Symbols are: BALB/c; ( —i--) B6; (-..) BlO; (-0-) DBA/2; ( >' ) BALB.B; ( >) BlO.D2.
(—)
divided into two groups (Fig. la). In the first group (BALB/c, animals survived until day 42, the end of the observation BALB.B, and DBAI2) the parasitemia rose rapidly and peaked period, except for three B6, two B 10 and four B lO.D2 mice that after day 12. The animals of this group died between day 14 died between 28 and 37 days post-inoculation, respectively. (DBA!2) and day 21 (BALB/c and BALB.B) after inoculation. In the second group of strains (B 10, B6, and Bl0.D2) paraAlbuminuria sitemia rose more slowly after day 12, although in individual Experiment 1. During the first experiment no albuminuria, as mice one or two consecutive parasitemic peaks were seen. These mice died later than those in the first group, between 25 defined above, was measured in B6 and B10.D2 mice except for one mouse of each strain (78 and 134 g/18 hours, respectively). (B6) and 30 (BlO, B10.D2) days after inoculation. Experiment 2. In the second set of experiments all strains The other four strains showed albuminuria (Fig. 2 a and b). showed a similar parasitemia curve (Fig. ib). Although the Experiment 2. Considering the albuminuria of the second height of the parasitemia was different in the individual strains, experiment the strains could be divided in the same two groups these curves shared several characteristics. They all showed a (Fig. 3 a and b) as defined with the parasitemia in experiment 1. parasitemic peak on day 7 as in experiment 1, but now followed BALB/c, BALB.B, and DBAI2 strains developed proteinuria by a slow rise in parasitemia in all strains after day 21. Most according to the criteria mentioned above. However, of the
626
van Velthuysen et al: Infection-related glomerulopathy
A
B
120 C.)
E
0 80
r.
60
40
0)
.20 -20
0
10
20
30
40
50
Time, days post-inoculation Time, days post-inoculation Fig. 3. Experiment 2 (Tr + Da +). A. Percentage of mice from each strain with an albuminuria exceeding the mean plus twice the standard deviation of control mice. B. Mean of albuminuria in 18 hours (± sI4). Symbols are: (—•—) BALB/c; (-+—) B6; (-+-) BlO; (-€3—) DBAI2; (X) BALB.B; ( G-) Bl0.D2.
Table 2. Experiment 1, day 14 Strain
BALB/c
ALB D14
Lam
Col
D14
Dl4
+
IgMms++li+ IgMms++li+
Immunofluor.
1gM ms+ + ii +
RTE D14
DPP D14
GP330 D14
DNP
D14
+
—
+
+
+
+
+
+
+
+
+
+
+
+
+
IgG ms++ Ii + BlO
IgG ms++ ii +
GBM
D14
—
+
+
+
+
+
+
+
DBA/2
IgG ms+ 1gM ms++
+
—
—
+
—
—
+
+
BALB.B
IgG ms++ 1gM ms++ ii ++ IgG ms++ ii +
+
+
—
—
—
—
+
+
BlO.D2
1gM ms++ Ii +
—
+
+
+
+
+
+
-I-
B6
IgG ms++
Presence "+" or absence "—" of significant difference in extinction between sera of experimental and control mice in unpaired t-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular basement membrane (GBM), renal epithelial antigens (RTE), DPPIV (DPP), gp330, and DNP. Abbreviations are Immunofluor., immunofluorescence pattern for 1gM and IgG; ms, mesangial, capillary loop; gr, granular; Ii, linear; "+" presence of staining, "+ +" strong staining; albuminuria (ALB) according to Fig. 2.
BALB/c and DBAJ2 strains all experimental mice were albu- B l0.D2 and B 10 mice where IgG3 staining was little less bright minuric, while of the BALB .B strain only five out of seven mice than IgG. became albuminuric (Fig. 3a). Moreover, the height of the Experiment 2. Immunofluorescence of the kidneys of mice albuminuria varied (Fig. 3b). The BlO, B6 and B10.D2 strains from the second experiment (Table 3) all showed mesangial
did not become albuminuric, although two BlO mice had staining for 1gM and IgG. Along the capillary loop 1gM was seen albuminuria values up to 78 ig/l8 hours. Mean value of control BlO mice was 23 g/18 hours (standard 15 tg/l8 hr). The means of the control values of all strains in both experiments ranged between 20 and 50 g/18 hr.
again in a linear pattern in all mice, whereas IgG was seen in a granular pattern in BALB/c mice and in a linear pattern in B6, BALB.B, and B10.D2 mice. IgG3 showed a staining pattern similar to IgG in BALBIc, BlO, B6, and BALB.B mice. While IgG3 staining was less intense than IgG staining in DBAI2 and Immunofluorescence B10.D2 mice. Experiment 1. Immunofluorescence studies of the kidneys of In both experiments the fluorescence patterns were similar all infected mice of experiment 1 (Table 2), showed staining for for all mice within each group. 1gM in the mesangium and along the capillary loop in a linear Polyclonal B-cell stimulation pattern. IgG was detected in the mesangium in these mice and along the capillary wall in a faint linear pattern in BALB/c, All mouse strains showed antibody production to DNP-RSA BALB.B and BlO experimental mice. lgG3 was present in the on all moments measured, except for the B6 strain on day 42. A same distribution pattern and intensity as IgG, except for wide production of antibodies to gp330 was detected as well
627
van Ve/thuysen et a!: Infection-related glomerulopathy Table 3. Experiment 2, day 42
ALB D42
Lam
Cot D42
GBM
RTE
DPP
GP330
DNP
D42
D42
D42
D42
D42
D42
+
+
+
+
+
+
+
+
IgMms++li+
—
+
—
+
-
IgMms++li+
—
—
—
+
+
—
—
+
—
+
+
+
+
+
+
+
+
+
+
+
—
—
—
—
—
+
+
+
Strain
Immunofluor.
BALB/c
1gM ms++ li+ IgG ms++ gr+
BlO
lgG ms++ B6
DBA/2
BALB.B BlO.D2
IgG ms++ li+ 1gM ms++ li+ IgG ms++ 1gM ms++ li+ IgG ms++ Ii+
lgMms++li+
+
IgG ms+ li+ Presence "+" or absence "—" of significant difference in extinction between sera of experimental and control mice in unpaired f-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular basement membrane (GBM), renal epithelial antigens (RTE), DPPIV (DPP), gp330, and DNP. Abbreviations are: Immunofluor., immunofluorescence pattern for 1gM and IgG; ms, mesangial, capillary loop; gr, granular; Ii, linear; "+" presence of staining, "+ +" strong staining; albuminuria (ALB) according to Fig. 3.
Table 4. Experiment 2, day 28
5 of 7) of albuminuria in BALB/c and BALB.B mice were not ALB Lam Col GBM RTE DPP GP330 DNP identical during the second experiment (Fig. 3 a and b). The mouse strains with early antibody production to a limited Strain D28 D28 D28 D28 D28 D28 D28 D28 amount of antigens (day 14 of experiment 1) produced antibod— — + + + + + + BALB/c ies to a greater variety of antigens later in the disease (day 42 of — — — — + + i+ BlO — — — — — + B6 + + experiment 2). This phase was associated with proteinuria (Fig. — — — — + + + + DBA/2 3, Table 3). However, the polyclonal stimulation itself was no — — — — + + + + BALB.B clue to the occurrence of the proteinuria, because, except for — — — — — + + + BlO.D2 BlO mice, none of the mouse strains in which antibodies could Presence "+" or absence —" of significant difference in extinction be detected to all antigens tested on day 14 of experiment 1, between sera of experimental and control mice in unpaired f-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular developed proteinuria in the first nor in the second experimental basement membrane (GBM), renal epithelial antigens (RTE), DPPIV protocol (Tables 2 and 3). (DPP), gp330, and DNP, 28 days after inoculation. Albuminuria (ALB) No direct relationship was seen between the antibodies to a is according to Fig. 2. specific antigen and the occurrence of proteinuria (Tables 2, 3 and 4) nor was there a specific glomerular fluorescence pattern. Although in the first set of experiments the presence of IgG (Tables 2 and 3). Considering the antibody production patterns along the capillary loop seemed to be related to proteinuria, this the mouse strains could be divided in the same two groups as was not the case in the second set of experiments. Detectable described in association with proteinuria and parasitemia antibodies to laminin, collagen type IV or GBM did not coincide (BALB/c, BALB.B vs. B6, BlO, Bl0.D2). The first showed antibodies to all antigens measured on day 42 of the second with a linear fluorescence pattern as might have been expected
from previous studies [15]. Nor could antibodies to RTE, experiment (Table 3) while the others showed, in contrast, DPPIV, and gp330 account for a granular fluorescence pattern, antibodies to all antigens measured on day 14 of the first experiment (Table 2). Sera of experiment 2 day 28 showed an as was expected from the literature [9, 16, 17]. However, in a intermediate pattern (Table 4). DBA/2 mice were an exception, recent study Cavalot et al [18] showed that antibodies to laminin showing an intermediate antibody production pattern on all and collagen type IV, due to polyclonal B-cell activation, could be present in the circulation without binding to the glomerular moments measured. capillary wall, even after isolated kidney perfusion with serum Discussion
containing these antibodies. The lack of binding of these
In this paper the role of MHC and non-MHC in the induction of glomerular disease during experimental Trypanosoma brucei infection was investigated. The six mouse strains studied could be divided into two groups according to their reaction patterns following infection with Trypanosoma brucei (B 10, B6, B 10.D2
antibodies was not explained. Therefore we can only speculate that in this model, the lack of binding is due to either the low
titer of antibodies present or to a lack of exposure of the
corresponding epitopes. We looked in more detail for IgG3 since this isotype was vs. BALB/c, BALB.B and DBA/2), concerning parasitemia, reported to be nephritogenic [19, 20]; we observed a prominent proteinuria, and antibody production. This indicates a role for binding of IgG3 in the glomeruli of Trypanosoma brucei inthe background genes in immunoregulation, since the groups fected BALB/c mice [5]. No clear differences of IgG3 staining corresponded with the non-MHC (BALB/c, BALB.B vs. BlO patterns were seen between strains. In all strains the IgG3 and B lO.D2) and not with the MHC genes (Table 1). However, staining pattern was similar to that of IgG. If the proteinuria in both experiments and in all strains could if the mice had a BALB background, some influence of MHC genes was seen, because the height and the incidence (7 of 7 vs. be explained by a similar pathogenetic mechanism, neither an
628
van Velthuysen et a!: Infection-related glomerulopathy
References
immune-complex mediated, nor a polyclonal B-cell stimulationrelated pathogenesis could be supported in this model, although
antibodies to a nephritogenic antigen not tested might still be responsible.
The absence of albuminuria in the B6, BlO and Bl0.D2
strains during the second experiment could not be explained by the fact that these mice were more severely diseased, as neither their clinical behavior nor their parasitemia were significantly different from those of the other strains. Moreover, the clinical status of the mice in the first experiment was even worse, while
this did not prevent these mice from developing proteinuria. However, the cause of death of these mice remains uncertain. It is more likely that a different factor than immunoglobulins is responsible for the proteinuna in this model. This is a new point of view in research for the pathogenetic mechanisms of immunologically mediated glomerulonephritis, although the role of several humoral factors in autoimmune diseases is under current study [3, 21—251.
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Acknowledgments The authors thank: Professor Ph.J. Hoedemaeker for critically reading the manuscript; J. van der Velde for constructive statistical advice; C. van Osnabrugge-Bondon and A. Mayen for technical assistance; J.
van der Ploeg for taking care of the animals; E. van Leer for the donation of sera; and Professor J.H.H. Ehrich for the donation of trypanosomes.
Reprint requests to Marie-Louise van Velthuysen, M.D., University of Leiden, P.O. Box 9603, 2300 RC Leiden, The Netherlands.
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IgG3 production in MRL/lpr mice is responsible for development of lupus nephritis. J immunol 147:515—519, 1991 21. NAIKI M, SUEHIRO S, IMA! Y, OSAWA T: Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice. mt j Immunopharmacol 11:663—671, 1989 22. BARAN D, LANTZ 0, DOSQUET P, SFAKSI A, DRUET F: Interleukin-2 production in Brown-Norway rats with HgCI2-induced autoimmune disease: Paradoxical in vivo versus in vitro findings. Clin Exp immunol 73:401—405, 1988 23. TER BORG EJ, HORST G, LIMBURG PC, KALLENBERG CGM:
Changes in plasma levels of interleukin-2 receptor in relation to disease exacerbations and levels of anti-dsDNA and complement in systemic lupus erythematosus. Gun Exp Immunol 82:21—26, 1990 24. HISHIKAWA T, TOKANO Y, SEKIGAWA I, ANDO S, TAKASAKI Y,
HASIuMOTO H, HIROSE S, OKUMURA K, ABE M, Smiw T: HLA-DP T cells and deficient interleukin-2 production in patients with systemic lupus erythematosus. Clin Immunol Immunopathol 55:285—296, 1990 25. MIYAGI J, MINATO N, SUMIYA M, KASAHARA T, KANO S: Two
629
types of antibodies inhibiting interleukin-2 production by normal lymphocytes in patients with systemic lupus erythematosus. Art hr Rheum 32:1356—1364, 1989 26. DE GEE ALW, LEVINE RF, MANSFIELD JM: Genetics of resistance
to African trypanosomes VI. Heredity of resistance and variable surface glycoprotein-specific immune responses. J Immunol 140: 283—288, 1988
27. SEED JR, SECHELSKI JB: African trypanosomes: Inheritance of factors involved in resistance. Exp Parasitol 69:1—8, 1989 28. SILEGHEM M, DAIUI A, DE BAETSELIER P: In vitro simulation of
immunosuppression caused by Trypanosoma brucei. Immunology 73:246—248, 1991
29. ROELANTS GE: Natural resistance to African trypanosomiasis. Parasite immunol 8:1—10, 1986 30. MoRRIsoN WI, MURRAY M: The role of humoral immune responses in determining susceptibility of AU and C57BL/6 mice to infection with Trypanosoma congolense. Parasite Immunol 7:63—79, 1985 31. SILEGHEM M, HAMERS R, DE BAETSELIER P: Active suppression of
interleukin 2 secretion in mice infected with Trypanosoma brucei AnTat 1.1.E. Parasite Immunol 8:641—649, 1986
Susceptibility for infection-related glomerulopathy depends on non-MHC genes MARIE-LOUISE F. VAN VELTHUYSEN, ALETTA VENINGA, JAN ANTHONIE BRUIJN, EMILE DE HEER, and GERT JAN FLEUREN Laboratory for Pathology, University of Leiden, Leiden, The Netherlands
Susceptibility for infection-related glomerulopathy depends on non-
MHC genes. BALB/c mice infected with T,ypanosoma brucei and treated seven days after inoculation with Diminazene aceturate develop
polyclonal B-cell stimulation, including production of antibodies to known nephritogenic autoantigens and glomerular disease associated with severe albuminuria. To investigate if the susceptibility for glomer-
ular disease in this model is linked to MHC or non-MHC genes, we studied this disease in six mouse strains that were partly congenic for their MHC and partly congenic for their non-MHC genes. The course of
the infection was measured by parasitemia and related to (auto)antibody production, proteinuria and glomerular deposition of immuno-
genetic factors as this may lead to a better understanding of pathogenesis as well as disease inheritance. The glomerular disease described in BALB/c mice induced by Trypanosoma brucei infection is a suitable model to study such relationships, as glomerular disease occurs together with autoantibody production while antibodies reactive with Trypanosomal antigens
do not seem to be involved in the development of the renal disease [5].
To investigate in this model whether the susceptibility for
globulins. The mouse strains could be divided into two groups. The first group consisted of the C57BL/6 (H-2'), C57BL/l0 (H2b) and Bl0.D2 (H-2") strains, which proved to be relatively resistant to infection with Trypanosoma brucei (that is, spontaneous survival >25 days). In sera of these mice antibodies to a broad range of antigens could be found 14 days after inoculation; no proteinuria was observed. The second group consisted of the BALB/c (H-2"), BALB.B (H-2") and DBA/2 (H-2")
developing renal disease and the production of autoantibodies is linked to MHC or non-MHC genes, we studied the development
strains, which were relatively susceptible to the infection. In these
Methods
animals proteinuria occurred and a broad polyclonal B-cell stimulation was seen 42 days after inoculation. No correlation was found between the specificity of circulating antibodies and the occurrence of proteinuria or a glomerular fluorescence pattern. These results indicate that in this model non-MHC genes govern the outcome of the infection as well as the development of polyclonal B-cell stimulation and proteinuria.
Concerning the glomerulopathy, a discrepancy is observed between glomerular immunoglobulin staining patterns and the development of proteinuria. Therefore, an additional pathogenetic role for non-immu-
of renal disease during Trypanosoma brucei infection in six mouse strains either congenic for their MHC or congenic for their background with the previously-described BALB/c mice.
Animals
BALB/c mice were bred in our own facilities. Female BALB.B, C57BL/l0 (BlO), C57BL16 (B6), B10.D2 and DBAI2
mice were purchased from Harlan OLAC (Zeist, the Netherlands). The genetic composition is shown in Table 1.
Parasites
noglobulin humoral factors must be considered.
A stabilate of Trypanosoma brucei (STIB 348c variant A) was
obtained from the Department of Pathology of the BernardNocht-Institut für Schiffs- und Tropenkrankheiten (Hamburg, Germany). This stabilate was expanded after a passage in an irradiated (500 rad) BALB/c mouse. Blood from this animal containing 108 parasites/ml was diluted in glycerol and saline and cryopreserved in batches of 40 d samples.
In general, autoimmune diseases seem to occur more often in genetically predisposed persons [1]. Epidemiologic as well as experimental studies have demonstrated the inheritance patterns to be multigenic. Several reports have demonstrated that certain MHC genes are related to a higher susceptibility for autoimmune diseases. For example HLA-DR2 and HLA-DR3 Inoculation are seen more often in lupus prone patients [21, of whom the HLA-DR2 patients develop glomerular disease more often [3, Cryopreserved blood was diluted in saline, up to 1000 para41. However, neither gene is necessary or sufficient, since many sites per ml. Each animal was inoculated with 0.1 ml (± 100 people carry the haplotype without being affected and many parasites) i.p.
patients do not carry the haplotype at all. Therefore, it is important to relate the genetic composition to possible patho-
Drug administration Diminazene aceturate (Berenil®; Hoechst AG, Frankfurt am
Received for publication July 13, 1992 and in revised form September 28, 1992 Accepted for publication September 29, 1992
Main, Germany) was diluted up to 5 mg/mI in saline and
© 1993 by the International Society of Nephrology
[51.
administered i.p. in a dosage of 40 mg/kg, to achieve a more protracted course of the disease as has been previously shown
623
624
van Velthuvsen et al: Infection-related glomerulopathy
"—" for absence of staining, "+" for staining, and "++" for
Table 1. Mouse strains used Strain
Non-MHC
MHC
BALB/c
BALB BlO B6 DBA BALB BlO
d b b
l0 B6
DBA/2 BALB.B BlO.D2
d
b d
strong staining.
Preparation of antigens and antisera Renal tubular epithelial antigens (RTE), glomerular basement membrane antigens (GBM), DPPIV, and gp330 were prepared
and characterized as described elsewhere [8, 9]. Type IV collagen, antibodies directed against type IV collagen, and
laminin were a gift of Prof. J.-M. Foidart (Dept. of Biology, University of Liege, Belgium). Preparation and characterization of these antigens and antibodies have been described [10, Experimental design 11]. EHS mouse laminin was obtained from Sigma (Sigma Two sets of experiments were performed consecutively ac- Chemical Co. Ltd., Dorset, UK). Dinitrophenol coupled to rat cording to the protocols described [5]. In the first set of serum albumin (DNP-RSA) was provided by Prof. M.R. Daha experiments, seven mice from each of the examined strains (Department of Nephrology, University Hospital of Leiden). were inoculated with Trypanosoma brucei, while five animals ELISA studies from each strain served as controls. In the second set of experiments seven mice of each strain were inoculated with Antibodies to various known nephritogenic antigens and to Trypanosoma brucei and treated seven days after infection with DNP-RSA were measured in sera of control and diseased mice Diminazene aceturate (Tr + Da +). As a control Diminazene to evaluate the degree of polyclonal B-cell stimulation and to aceturate was administered to five animals of each strain (Tr — correlate autoantibody production to the development of renal Da +). Albuminuria was measured weekly. Serum was ob- disease. For the detection of antibodies to RTE, GBM, DPPIV, tained every two weeks. Parasitemia was measured every other day in the first set of experiments, while it was detected weekly in the second experiment set. All animals were sacrificed 42 days after inoculation because, based on earlier observations [5], full blown disease was expected to have developed by then.
gp330, laminin, collagen type IV, and DNP ELISA studies were performed as described [8, 12, 13]. In experiment 1, antibodies
were detected in day 14 sera as this was the latest time point sera could be obtained from most mice. In experiment 2, sera obtained 28 and 42 days after inoculation were used. At each time point and of each strain antibodies were measured in four experimental and four control sera in most instances. All sera were tested in two dilutions (1/40 and 1/80, both in duplo). As
Parasitemia Parasites were counted in a smear of tail blood, stained with the extinction for the 1/80 dilution was always much lower than Leishman's stain. The results were expressed as number of for the 1/40 dilution, the results of the 1/40 diluted sera were parasites per 100 erythrocytes. used. Peroxidase-conjugated goat anti-mouse Ig (DAKOPATTS) was applied as second step. As a positive control for Albuminuria the coating procedure of the antigens, we used specific rabbit Mice were held in metabolic cages for 18 hours with free and goat antisera. The positive control for RTE was serum of access to water and food. The albumin concentration in urine mice with chronic graft-versus-host disease known to react with was measured by rocket electrophoresis. these antigens [14]. As a negative control, each serum was Histological methods All kidneys were perfused in vivo with PBS for two minutes,
tested in the same dilutions on a BSA-coated well. In all studies
a standard dilution curve from a reference serum pool of BALB/c mice 42 days after inoculation with parasites was
prior to sacrifice of the animals. For light microscopy, the included. The reaction products were quantified by measuring tissues were fixed in 10% buffered formalin, dehydrated, and extinction at 492 nm with a Titertek Multiscan (Amstelstad by, embedded in Paraplast® (Amsteistad, Amsterdam, The Nether- Zwanenburg, The Netherlands). lands). Sections were cut 5 sm thick and stained with hematoxylin-eosin (H&E) and periodic acid-Schiff reagent (PAS), Statistical analysis according to standard methods. For direct immunofluorescence To analyze the significance of the differences obtained in the studies kidney specimens were snap frozen and stained as various experiments between experimental and control mice an described elsewhere [61. In short, 2 jsm sections were cut in a unpaired Student's t-test was used. The differences with a P cryostat, air dried, and fixed in acetone/ethanol (50/50) for five value <0.05 were considered to be significant. Individual mice minutes and then postfixed in 100% ethanol for 10 minutes were considered to be albuminuric when their albuminuria in 18 before being incubated for one hour with monospecific rabbithours exceeded the mean plus twice the standard deviation of anti-mouse-FITC-conjugated immunoglobulins (Nordic Immu- control mice. nology, Tilburg, The Netherlands) directed against mouse 1gM, IgG, IgG3 and C3. Cross reactivity of the IgG3 antibodies with Results other IgG subclasses was checked by staining kidney specimens Parasitemia and survival of mice with GVH (chronic graft vs. host disease) induced nephritis. In murine GVH-induced nephritis all IgG subclasses Experiment 1. After inoculation with Trypanosoma brucei all except IgG3 are known to be involved [7]. Fluorescence pat- mice, regardless of the strain used, developed a first parasitemic terns were scored in a semiquantitative manner attributing a peak between day 6 and 7. Hereafter the mouse strains could be
625
van Velthuysen et a!: Infection-related glomerulopathy
A
25
B
><
20 U)
15
C',
U)
ci) U)
Cci
o10 5 :
0
A 1
Cci
0
0
30
40
Time, days post-inoculation
Time, days post-inoculation
Fig. 1. Mean value of the percentage of parasites for each strain (N = 7). A. Experiment 1 (Tr + Da —). B. Experiment 2 (Tr + Da +). Symbols
are:(——)BALB/c;(.-i--)B6;(-.-)B1O;(-€-)DBA/2;(x)BALB.B;(+)Bl0.D2.
10000
120 100 ci)
0
1000
E C)
C
E .0 100
0
0
10 20 30 Time, days post-inoculation
40
10 0
10 20 30 Time, days post-inoculation
40
Fig. 2. Experiment 1 (Tr + Da —). A. Percentage of mice from each strain with an albuminuria exceeding the mean plus twice the standard deviation of control mice. Symbols are as in B except: (—+--) BlO; (—+(--) B6. B. Mean of albuminuria in 18 hours (± SEM). Symbols are: BALB/c; ( —i--) B6; (-..) BlO; (-0-) DBA/2; ( >' ) BALB.B; ( >) BlO.D2.
(—)
divided into two groups (Fig. la). In the first group (BALB/c, animals survived until day 42, the end of the observation BALB.B, and DBAI2) the parasitemia rose rapidly and peaked period, except for three B6, two B 10 and four B lO.D2 mice that after day 12. The animals of this group died between day 14 died between 28 and 37 days post-inoculation, respectively. (DBA!2) and day 21 (BALB/c and BALB.B) after inoculation. In the second group of strains (B 10, B6, and Bl0.D2) paraAlbuminuria sitemia rose more slowly after day 12, although in individual Experiment 1. During the first experiment no albuminuria, as mice one or two consecutive parasitemic peaks were seen. These mice died later than those in the first group, between 25 defined above, was measured in B6 and B10.D2 mice except for one mouse of each strain (78 and 134 g/18 hours, respectively). (B6) and 30 (BlO, B10.D2) days after inoculation. Experiment 2. In the second set of experiments all strains The other four strains showed albuminuria (Fig. 2 a and b). showed a similar parasitemia curve (Fig. ib). Although the Experiment 2. Considering the albuminuria of the second height of the parasitemia was different in the individual strains, experiment the strains could be divided in the same two groups these curves shared several characteristics. They all showed a (Fig. 3 a and b) as defined with the parasitemia in experiment 1. parasitemic peak on day 7 as in experiment 1, but now followed BALB/c, BALB.B, and DBAI2 strains developed proteinuria by a slow rise in parasitemia in all strains after day 21. Most according to the criteria mentioned above. However, of the
626
van Velthuysen et al: Infection-related glomerulopathy
A
B
120 C.)
E
0 80
r.
60
40
0)
.20 -20
0
10
20
30
40
50
Time, days post-inoculation Time, days post-inoculation Fig. 3. Experiment 2 (Tr + Da +). A. Percentage of mice from each strain with an albuminuria exceeding the mean plus twice the standard deviation of control mice. B. Mean of albuminuria in 18 hours (± sI4). Symbols are: (—•—) BALB/c; (-+—) B6; (-+-) BlO; (-€3—) DBAI2; (X) BALB.B; ( G-) Bl0.D2.
Table 2. Experiment 1, day 14 Strain
BALB/c
ALB D14
Lam
Col
D14
Dl4
+
IgMms++li+ IgMms++li+
Immunofluor.
1gM ms+ + ii +
RTE D14
DPP D14
GP330 D14
DNP
D14
+
—
+
+
+
+
+
+
+
+
+
+
+
+
+
IgG ms++ Ii + BlO
IgG ms++ ii +
GBM
D14
—
+
+
+
+
+
+
+
DBA/2
IgG ms+ 1gM ms++
+
—
—
+
—
—
+
+
BALB.B
IgG ms++ 1gM ms++ ii ++ IgG ms++ ii +
+
+
—
—
—
—
+
+
BlO.D2
1gM ms++ Ii +
—
+
+
+
+
+
+
-I-
B6
IgG ms++
Presence "+" or absence "—" of significant difference in extinction between sera of experimental and control mice in unpaired t-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular basement membrane (GBM), renal epithelial antigens (RTE), DPPIV (DPP), gp330, and DNP. Abbreviations are Immunofluor., immunofluorescence pattern for 1gM and IgG; ms, mesangial, capillary loop; gr, granular; Ii, linear; "+" presence of staining, "+ +" strong staining; albuminuria (ALB) according to Fig. 2.
BALB/c and DBAJ2 strains all experimental mice were albu- B l0.D2 and B 10 mice where IgG3 staining was little less bright minuric, while of the BALB .B strain only five out of seven mice than IgG. became albuminuric (Fig. 3a). Moreover, the height of the Experiment 2. Immunofluorescence of the kidneys of mice albuminuria varied (Fig. 3b). The BlO, B6 and B10.D2 strains from the second experiment (Table 3) all showed mesangial
did not become albuminuric, although two BlO mice had staining for 1gM and IgG. Along the capillary loop 1gM was seen albuminuria values up to 78 ig/l8 hours. Mean value of control BlO mice was 23 g/18 hours (standard 15 tg/l8 hr). The means of the control values of all strains in both experiments ranged between 20 and 50 g/18 hr.
again in a linear pattern in all mice, whereas IgG was seen in a granular pattern in BALB/c mice and in a linear pattern in B6, BALB.B, and B10.D2 mice. IgG3 showed a staining pattern similar to IgG in BALBIc, BlO, B6, and BALB.B mice. While IgG3 staining was less intense than IgG staining in DBAI2 and Immunofluorescence B10.D2 mice. Experiment 1. Immunofluorescence studies of the kidneys of In both experiments the fluorescence patterns were similar all infected mice of experiment 1 (Table 2), showed staining for for all mice within each group. 1gM in the mesangium and along the capillary loop in a linear Polyclonal B-cell stimulation pattern. IgG was detected in the mesangium in these mice and along the capillary wall in a faint linear pattern in BALB/c, All mouse strains showed antibody production to DNP-RSA BALB.B and BlO experimental mice. lgG3 was present in the on all moments measured, except for the B6 strain on day 42. A same distribution pattern and intensity as IgG, except for wide production of antibodies to gp330 was detected as well
627
van Ve/thuysen et a!: Infection-related glomerulopathy Table 3. Experiment 2, day 42
ALB D42
Lam
Cot D42
GBM
RTE
DPP
GP330
DNP
D42
D42
D42
D42
D42
D42
+
+
+
+
+
+
+
+
IgMms++li+
—
+
—
+
-
IgMms++li+
—
—
—
+
+
—
—
+
—
+
+
+
+
+
+
+
+
+
+
+
—
—
—
—
—
+
+
+
Strain
Immunofluor.
BALB/c
1gM ms++ li+ IgG ms++ gr+
BlO
lgG ms++ B6
DBA/2
BALB.B BlO.D2
IgG ms++ li+ 1gM ms++ li+ IgG ms++ 1gM ms++ li+ IgG ms++ Ii+
lgMms++li+
+
IgG ms+ li+ Presence "+" or absence "—" of significant difference in extinction between sera of experimental and control mice in unpaired f-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular basement membrane (GBM), renal epithelial antigens (RTE), DPPIV (DPP), gp330, and DNP. Abbreviations are: Immunofluor., immunofluorescence pattern for 1gM and IgG; ms, mesangial, capillary loop; gr, granular; Ii, linear; "+" presence of staining, "+ +" strong staining; albuminuria (ALB) according to Fig. 3.
Table 4. Experiment 2, day 28
5 of 7) of albuminuria in BALB/c and BALB.B mice were not ALB Lam Col GBM RTE DPP GP330 DNP identical during the second experiment (Fig. 3 a and b). The mouse strains with early antibody production to a limited Strain D28 D28 D28 D28 D28 D28 D28 D28 amount of antigens (day 14 of experiment 1) produced antibod— — + + + + + + BALB/c ies to a greater variety of antigens later in the disease (day 42 of — — — — + + i+ BlO — — — — — + B6 + + experiment 2). This phase was associated with proteinuria (Fig. — — — — + + + + DBA/2 3, Table 3). However, the polyclonal stimulation itself was no — — — — + + + + BALB.B clue to the occurrence of the proteinuria, because, except for — — — — — + + + BlO.D2 BlO mice, none of the mouse strains in which antibodies could Presence "+" or absence —" of significant difference in extinction be detected to all antigens tested on day 14 of experiment 1, between sera of experimental and control mice in unpaired f-test, for antibodies to laminin (LAM), collagen type IV (Col), glomerular developed proteinuria in the first nor in the second experimental basement membrane (GBM), renal epithelial antigens (RTE), DPPIV protocol (Tables 2 and 3). (DPP), gp330, and DNP, 28 days after inoculation. Albuminuria (ALB) No direct relationship was seen between the antibodies to a is according to Fig. 2. specific antigen and the occurrence of proteinuria (Tables 2, 3 and 4) nor was there a specific glomerular fluorescence pattern. Although in the first set of experiments the presence of IgG (Tables 2 and 3). Considering the antibody production patterns along the capillary loop seemed to be related to proteinuria, this the mouse strains could be divided in the same two groups as was not the case in the second set of experiments. Detectable described in association with proteinuria and parasitemia antibodies to laminin, collagen type IV or GBM did not coincide (BALB/c, BALB.B vs. B6, BlO, Bl0.D2). The first showed antibodies to all antigens measured on day 42 of the second with a linear fluorescence pattern as might have been expected
from previous studies [15]. Nor could antibodies to RTE, experiment (Table 3) while the others showed, in contrast, DPPIV, and gp330 account for a granular fluorescence pattern, antibodies to all antigens measured on day 14 of the first experiment (Table 2). Sera of experiment 2 day 28 showed an as was expected from the literature [9, 16, 17]. However, in a intermediate pattern (Table 4). DBA/2 mice were an exception, recent study Cavalot et al [18] showed that antibodies to laminin showing an intermediate antibody production pattern on all and collagen type IV, due to polyclonal B-cell activation, could be present in the circulation without binding to the glomerular moments measured. capillary wall, even after isolated kidney perfusion with serum Discussion
containing these antibodies. The lack of binding of these
In this paper the role of MHC and non-MHC in the induction of glomerular disease during experimental Trypanosoma brucei infection was investigated. The six mouse strains studied could be divided into two groups according to their reaction patterns following infection with Trypanosoma brucei (B 10, B6, B 10.D2
antibodies was not explained. Therefore we can only speculate that in this model, the lack of binding is due to either the low
titer of antibodies present or to a lack of exposure of the
corresponding epitopes. We looked in more detail for IgG3 since this isotype was vs. BALB/c, BALB.B and DBA/2), concerning parasitemia, reported to be nephritogenic [19, 20]; we observed a prominent proteinuria, and antibody production. This indicates a role for binding of IgG3 in the glomeruli of Trypanosoma brucei inthe background genes in immunoregulation, since the groups fected BALB/c mice [5]. No clear differences of IgG3 staining corresponded with the non-MHC (BALB/c, BALB.B vs. BlO patterns were seen between strains. In all strains the IgG3 and B lO.D2) and not with the MHC genes (Table 1). However, staining pattern was similar to that of IgG. If the proteinuria in both experiments and in all strains could if the mice had a BALB background, some influence of MHC genes was seen, because the height and the incidence (7 of 7 vs. be explained by a similar pathogenetic mechanism, neither an
628
van Velthuysen et a!: Infection-related glomerulopathy
References
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The absence of albuminuria in the B6, BlO and Bl0.D2
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this did not prevent these mice from developing proteinuria. However, the cause of death of these mice remains uncertain. It is more likely that a different factor than immunoglobulins is responsible for the proteinuna in this model. This is a new point of view in research for the pathogenetic mechanisms of immunologically mediated glomerulonephritis, although the role of several humoral factors in autoimmune diseases is under current study [3, 21—251.
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Acknowledgments The authors thank: Professor Ph.J. Hoedemaeker for critically reading the manuscript; J. van der Velde for constructive statistical advice; C. van Osnabrugge-Bondon and A. Mayen for technical assistance; J.
van der Ploeg for taking care of the animals; E. van Leer for the donation of sera; and Professor J.H.H. Ehrich for the donation of trypanosomes.
Reprint requests to Marie-Louise van Velthuysen, M.D., University of Leiden, P.O. Box 9603, 2300 RC Leiden, The Netherlands.
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