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Program in Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA;
* Center of Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA;
Cell Biology, Harvard Medical School, Boston, Massachusetts, USA; and
Department of Pathology, University of Hamburg, D-20246 Hamburg, Germany
1Correspondence: Program in Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Dana 514, Boston MA 02215, USA. E-mail: rkalluri{at}bidmc.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3(IV)NC1. In addition to the well-characterized antibody contribution, a T helper 1 (Th1) response has been suspected as the culprit for glomerular injury. We induced anti-GBM disease in DBA/1, C57BL/6, AKR, and NOD mice with recombinant human 3(IV)NC1 to investigate the involvement of humoral and cellular autoimmunity. DBA/1 mice had crescentic GN 11 wk postimmunization with 3(IV)NC1. C57BL/6 and AKR mice developed a chronic disease course resulting in comparable kidney injury to DBA/1 mice within 6 months. NOD revealed only minor glomerular changes. The rapid course and the severity of the disease in DBA/1 mice can be explained by our immunological findings in their sera and splenocytes: 1) high antibody titers specific for the putative clinically relevant epitope of 3(IV)NC1 with Th1-type isotypes, and 2) a strong proliferative response and high amounts of the inflammatory cytokine IFN-
, secreted by splenocytes stimulated in vitro with 3(IV)NC1, with only low amounts of the anti-inflammatory cytokine IL-10. Our in vivo and in vitro results provide direct evidence that the balance between Th1 and Th2 responses associates with the outcome of anti-GBM disease in mice.—Hopfer, H., Maron, R., Butzmann, U., Helmchen, U., Weiner, H. L., Kalluri, R. The importance of cell-mediated immunity in the course and severity of autoimmune anti-glomerular basement membrane disease in mice.
Key Words: glomerulonephritis • Goodpasture syndrome • 3(IV) type IV collagen • autoimmunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 chain of type IV collagen (3(IV)NC1), one of six genetically distinct chains forming the collagen lattice within basement membranes (1
, 2)
. Its tissue distribution is restricted, with high amounts in the GBM and the alveolar basement membrane of the lungs (2). Both autoantibodies and autoreactive T cells can recognize 3(IV)NC1, but the autoantibodies are considered hallmark of the disease and have been implicated in its pathogenesis (3
4
5)
.
The generation of antigen-specific immunity usually involves T cells as well as antibody-producing B cells; CD4+ T cell differentiation results in two subsets of T cells referred to as T helper 1 (Th1) and T helper 2 (Th2) cells, each characterized by their distinct secreted cytokines. The balance between Th1 and Th2 cells directs the immune response toward different effector mechanisms resulting in different disease outcomes (6)
. As in other organ-specific autoimmune diseases, involvement of a Th1 response has been suggested in anti-GBM disease (7
, 8)
. Macrophages and lymphocytes are a prominent part of the inflammatory infiltrate in the kidneys, and most antibodies found in humans are of the IgG1 isotype (7
, 9)
. These findings relate to a Th-1-like immune response.
Anti-GBM disease can be induced in susceptible mouse and rat strains by immunization with antigen preparations containing 3(IV)NC1 (8
, 10
11
12)
. However, preparation of pure 3(IV)NC1 from tissues requires multiple purification steps and the yield is low. Our initial goal in this study was to establish a strain-dependent anti-GBM disease in mice induced by recombinant 3(IV)NC1 (r3(IV)NC1) produced in mammalian cells, as has been performed in the WKY rat (13)
. Using this model we addressed the following questions: 1) What are the characteristics of the humoral and cellular responses in susceptible vs. resistant mouse strains? 2) How does the initial immune response relate to the development of disease? 3) Is there direct evidence for a Th1 like involvement in anti-GBM disease?
Our results suggest an equal importance to autoantibodies, T lymphocytes and macrophages in the pathogenesis of anti-GBM disease. We found evidence for the involvement of a Th1-type immune response in the disease in mice and that the balance between the Th1 and Th2 responses determines the outcome of the disease in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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containing the BM40 signal peptide, a Flag tag, and the human 3 or 1(IV)NC1 domain including the last 10 amino acids of the collagenous domain was obtained by PCR modification and subcloning of the original pDS clones (15)
. The chimeric NC1 domain, designated as 311(IV)NC1, was generated by swapping a PvuII fragment containing the signal peptide, Flag tag, and amino-terminal third of 3(IV)NC1 into the 1(IV)NC1 plasmid. The plasmids were transfected into 293EBNA cells (Invitrogen, San Diego, CA, USA) using SuperFect (Qiagen, Chatsworth, CA, USA). Cell lines secreting r-3(IV)NC1 domains into the culture medium were selected by adding 1.5 µg/mL puromycin (Sigma, St. Louis, MO, USA) to the medium [Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 1x penicillin/streptomycin]. DMEM without any additives was added for 2 days; the conditioned medium was collected, run over a FlagM2 agarose column (Sigma), and the bound protein was eluted with 0.1 M glycine pH 3.5. After dialysis against PBS, the proteins were concentrated using Amicon concentrators (Millipore, Bedford, MA, USA), quantified with a BCA assay (Pierce, Rockford, IL, USA), and stored at -80°C until used.
Characterization of recombinant proteins
Proteins were analyzed by SDS-PAGE, Western blot, and ELISA using Goodpasture sera, anti-Flag antibodies, or an 3 chain-specific polyclonal antibody (16)
.
Mice immunizations and disease induction
Male DBA/1J, C57BL/6J, AKR/J, and NOD/LtJ mice (Jackson Laboratories, Bar Harbor, ME, USA) were purchased at 5–6 wk of age. Mice were housed in a pathogen-free facility with free access to food and water. All procedures were approved by the IACUC and conform to the Guiding Principles in the Care and Use of Animals of the American Physiological Society.
Mice were immunized in the hind footpads with 25 µg r-3(IV)NC1 in 50 µL PBS emulsified in an equal volume of Complete Freund Adjuvant containing 50 µg M. tuberculosis H37RA (DIFCO, Detroit, MI, USA). Control mice were immunized with PBS emulsified in Complete Freund Adjuvant. For in vitro experiments, mice were bled and killed 10 days postimmunization and spleens were aseptically collected. Disease induction mice were injected with the same protocol as for the in vitro experiments and boosted three times subcutaneously with 25 µg antigen in Incomplete Freund Adjuvant (DIFCO). Mice were checked regularly for signs of disease. A mouse was considered sick if it lost >10% of its body weight in a week, if it developed noticeable peripheral edema, or if its spontaneous behavior had changed to self-isolation and lethargy. Sick mice were killed during the experiment; the remaining mice were killed at the end points. Serum and organs were collected and processed for immunological and histopathological analysis.
Histology and immunohistochemistry
Kidneys and lungs were fixed in 10% neutral buffered formalin and processed for histology and immunohistochemistry. GBM ruptures, glomerular sclerosis, and crescent formation were assessed in 50 glomeruli per mouse in a blinded fashion in PAS-stained paraffin sections. A GBM rupture was defined by the presence of plasma exudate in the urinary space. Glomerular sclerosis was scored as no sclerosis, <50% sclerosis, or >50% sclerosis. Crescents were counted as either a partial or a circumferential crescent. For immunohistochemistry, paraffin sections were incubated with bacterial protease XXIV (300 µg/mL; Sigma) at 37°C for 15 min, blocked for 20 min in 10% goat or rabbit serum in PBS, and incubated with the primary antibodies at 4°C overnight. Primary antibodies were biotinylated anti-mouse IgG, anti-human CD3, anti-Fibrinogen, anti-Complement C3c (DAKO, Carpinteria, CA, USA), and anti-mouse F4/80 (Accurate Chemical and Scientific Corp., Hicksville, NY, USA). Cross-reactivity of the anti-human CD3 antibodies was tested on spleen tissue by staining the appropriate cells. Detection of the primary antibodies was performed using an ABC-Kit (Vector, Burlingame, CA, USA) or a rat APAAP system (DAKO). The IgG-staining of the GBM was scored in 60 glomeruli per mouse in a blinded fashion as either no staining, weak staining, moderate staining, or strong staining. CD3-positive cells were counted in 20 high-power fields of the kidney cortex in each mouse. F4/80 positivity was assessed in 50 glomeruli of each mouse.
Electron microscopy
Kidney tissue fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) was embedded in araldite, cut with an Ultracut E ultramicrotome (Reichert Jung, Wien, Austria), and stained with uranyl acetate and lead citrate. Specimens were examined with a Zeiss EM 109 electron microscope.
Circulating and GBM-bound anti-3(IV)NC1 antibodies
Anti-3(IV)NC1 antibodies were detected by indirect ELISA as described (8). Sera were preincubated with 5 µg/mL Flag peptide (Sigma) for 3 h at 4°C as a control for antibodies directed against the Flag tag. Secondary antibodies were anti-mouse IgG (Kirkegaard and Perry Laboratory, Gaithersburg, MD, USA), anti-mouse IgG1, IgG2a, and IgG2b (Zymed, San Francisco, CA, USA). Plates were developed with 1 Component TMB Substrate; the reaction was stopped by adding 1 Component Stop Solution (Kirkegaard and Perry) and plates were read at 450 nm in a plate reader (Bio-Rad, Hercules, CA, USA). Analysis of antibodies bound to the GBM was done from snap-frozen kidneys as described previously (17)
.
Proliferation and cytokine production
Spleens were pooled from three mice of each strain 10 days postimmunization. Cells were isolated and red blood cells were removed by lysis. Cells were cultured in round-bottom 96-well plates in serum-free X-Vivo 20 medium (BioWhittaker, Walkersville, MD, USA). For proliferation assays, 5 x 105 cells/well were cultured with 0, 0.5, 5, or 50 µg/mL denatured r-3(IV)NC1 (10 min boiling with immediate cooling on ice) for 72 h. [3H]-Thymidine was added to a final concentration of 1 µCi/well for an additional 12 h. Cells were harvested using a cell harvester (Tomtec, Hamden, CT, USA) and incorporation of [3H]-thymidine was measured using an LKB Betaplate liquid scintillation counter (Wallac, Turku, Finland). Values represent the mean of three wells. For cytokine assays, cells were cultured at a density of 1 x 106 cells/well with 50 µg/mL denatured r-3(IV)NC1. Supernatants were collected at 24 h for IL-2 and IL-4 and 40 h for IL-10 and IFN-. Cytokine ELISA was run as described previously (18). Antibodies and reagents used were from PharMingen (San Diego, CA, USA) and Sigma.
Statistical analysis
Differences between multiple groups were confirmed by the Kruskal-Wallis test and followed by a Mann-Whitney U test to compare two groups with each other. Experiments that did not yield enough independent data points for statistical analysis due to the experimental setup were repeated three times; one representative experiment is shown.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3(IV)NC1, 1(IV)NC1, and 311(IV)NC1 domain were expressed as Flag-tagged recombinant proteins in stably transfected HEK 293 EBNA cells. r-311(IV)NC1 contains the putative clinically relevant immunodominant antibody epitope of 3(IV)NC1 in the framework of the nonreactive 1(IV)NC1 (5). Analysis by SDS-PAGE showed monomeric proteins with a molecular mass of 
28–36 kDa (Fig. 1
A, lanes 1–3). Anti-Flag antibodies recognized all three proteins by both Western blot and ELISA (Fig. 1A
, lane 4, E). Sera of patients with anti-GBM disease recognized both r-3(IV)NC1 and r-311(IV)NC1, but not r-1(IV)NC1 (Fig. 1A
, lane 5, B–D). Reduction of r-3(IV)NC1 resulted in the loss of anti-GBM antibody binding (data not shown). To ensure the chimeric nature of the r-311(IV)NC1, a polyclonal antibody raised against the carboxyl-terminal 36 amino acids of 3(IV)NC1 was used (16). As expected, it recognized r-3(IV)NC1, but binding to r-311(IV)NC1 containing the carboxyl terminus of 1(IV)NC1 was reduced (Fig. 1F
).
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Anti-GBM disease in mice
Of the four mouse strains investigated, only DBA/1 developed clinical signs of end-stage renal disease within the third month after immunization (Table 1
). Histological analysis of DBA/1 kidneys revealed a necrotizing GN with evidence of fresh GBM ruptures in 9%, segmental or complete glomerular sclerosis in 40%, and crescent formation in 27% of the glomeruli (see Fig. 2B
, Fig. 3A-C
, Fig. 4A,G-I
). Extensive tubulointerstitial damage was present (Fig. 2
B). In contrast, kidneys of C57BL/6 mice showed considerably less severe pathological changes. Although 6% of the glomeruli had fresh GBM ruptures, only 4% of the glomeruli had developed crescents, 2% sclerosis, and there was no tubulointerstitial disease (Fig. 3
B, C). Glomeruli of AKR mice showed minor glomerular abnormalities and kidneys of NOD mice looked normal 11 wk postimmunization (Fig. 2D, E
, Fig. 3A-C
).
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C57BL/6, AKR, and NOD mice were followed for >6 months after immunization. Two of the C57BL/6 developed clinical signs of anti-GBM GN, one of them died of severe lung hemorrhage after 5.5 months (Table 1
, Fig. 2F
). Kidneys of C57BL/6 and AKR mice killed after 6 months had crescents and/or sclerosis in 30% of the glomeruli with tubulointerstitial disease (Fig. 2G, H
, Fig. 3B, C
). Glomeruli of NOD mice still showed only little pathology with <5% crescents and/or sclerosis (Fig. 2I
, Fig. 3B, C
, and data not shown).
Immunohistochemical staining for IgG gave a linear/granular pattern in DBA/1 and C57BL/6, a mainly granular pattern in AKR, and an incomplete linear/granular pattern in NOD mice (Fig. 4
A,D–F). Transmission electron microscopy showed subepithelial immune deposits in the GBM of DBA/1 mice 11 wk after immunization and in C57BL/6 and AKR mice 6 months after immunization (Fig. 4B, C
, and data not shown). Samples from earlier time points showed markedly fewer immune deposits (data not shown). The intensity of the IgG staining was scored for both the 11 wk and the 6 months experiments. After 11 wk, 90% of the glomeruli of DBA/1 mice showed a strong staining, compared with 44% in C57BL/6, 18% in AKR, and 2% in NOD mice (Fig. 3C
). At the 6 months point, the staining intensity of C57BL/6 and AKR mice was similar to that seen in DBA/1 mice after 11 wk.
Fibrin was detected in the crescents and the mesangial matrix of mice with crescentic GN (Fig. 4G
) and complement C3 was observed in a granular pattern along the GBM, whereas the staining for complement C1q was negative (data not shown).
Antibody response to r-3(IV)NC1
In an initial experiment, we verified the dose dependency of the antibody response. At day 10, C57BL/6 mice immunized with 125 µg showed a slightly higher antibody titer in the serum than mice immunized with 25 µg. A 5 µg immunized group mounted a much lower antibody titer than either the 125 µg and 25 µg immunized groups (Fig. 5
A).
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Next, the time course of antibody production was measured using 25 µg r-3(IV)NC1 in CFA for immunization and boosting three times. DBA/1 and C57BL/6 mice mounted a strong antibody response to the primary immunization as measured by indirect ELISA (Fig. 5B
). However, AKR and NOD mice built up their titers after the booster injections, eventually reaching plateau levels similar to DBA/1 and C57BL/6. Reactivity of mouse sera to r-3(IV)NC1, r-311(IV)NC1, and r-1(IV)NC1 was tested by ELISA to check the specificity of the circulating antibodies (Fig. 5C
). None of the sera showed specific binding to the highly homologous 1(IV)NC1 compared with CFA control sera; however, all sera reacted with the chimeric 311(IV)NC1, indicating that a substantial portion of the circulating 3(IV) antibodies is directed against the putative clinically relevant amino-terminal part of the 3(IV)NC1 domain.
Analysis of the isotype pattern demonstrated a Th1-like antibody response in DBA/1 and AKR mice. High titers of IgG2a and low titers of IgG1 and IgG2b were evident in these two strains. In comparison, C57BL/6 and NOD mice produced Th2-type antibodies of the IgG1 and IgG2b isotypes (Fig. 6
A, B). IgG2a was undetectable in both strains because these mice lack the IgG2a allele (19). To investigate the composition and the specificity of the GBM-bound antibodies, IgG were eluted from homogenized kidneys and tested by ELISA. Their isotype pattern and their antigen specificity closely resembled the pattern seen in the serum (Fig. 6C
and data not shown).
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T cell response to r-a3(IV)NC1
To assess the contribution of the cellular immune response, we checked the in vitro proliferation and cytokine patterns of spleen cells 10 days postimmunization. Splenocytes from DBA/1 mice stimulated in vitro with r-3(IV)NC1 incorporated 3- to 4-fold more [3H]-thymidine than cells of C57BL/6 mice and 10-fold more than cells of AKR and NOD mice (Fig. 7
A). DBA/1 and C57BL/6 spleen cells secreted high levels of IL-2 (100–300 pg/mL) and IFN- (8–10 ng/mL) upon in vitro stimulation. In contrast, IL-2 was not detectable and IFN- secretion was lower (4–6 ng/mL) in splenocytes from AKR and NOD mice (Fig. 7B, C
). The anti-inflammatory cytokine IL-10, on the other hand, was secreted in a high concentration from C57BL/6 spleen cells (800 pg/mL); less secretion was found in AKR and NOD splenocytes (300–400 pg/mL) and even less IL-10 (100 pg/mL) was detected in the supernatants from DBA/1 spleen cells (Fig. 7D
). IL-4 was not detected in any of the samples (data not shown).
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We also analyzed effector cells in the kidneys by immunohistochemistry. The kidney cortex of DBA/1 mice contained threefold more CD3-positive T lymphocytes than the other mouse strains or controls (Fig. 4H
, Fig. 7E
). Macrophages, identified by F4/80-positive staining, were detected in almost 90% of the glomeruli of DBA/1 mice but in <10% of the glomeruli of the other strains (Fig. 4I
, Fig. 7F
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Investigators have only started to look at the relevance of cell-mediated immune responses in anti-GBM disease, and some of the studies speculate that a cell-mediated Th1-like autoimmune reaction and not antibodies is the culprit for the rapidly progressive GN (8
, 20
, 21)
. The main obstacle that hampered the analysis of the relevance and contribution of antibodies and especially T lymphocytes in animal models of anti-GBM disease has been the preparation of large amounts of pure autoantigen. r-3(IV)NC1 expressed in a mammalian expression system enabled us to study both antibody and T cell responses in a mouse model of anti-GBM GN.
A key property of the antibody epitope is its dependency on the conformation of the antigen, which is due to several intramolecular disulfide bonds (22)
. The r-3(IV)NC1 antigen used retains this antibody binding characteristic as shown by Western blot and ELISA. Similarly expressed r-3(IV)NC1 protein has been used successfully to identify and fine-map the antibody epitope relevant for the human disease (5
, 23)
.
Anti-GBM disease in our model is characterized by clinical signs of renal failure, a necrotizing GN with crescent formation, and the presence of circulating and deposited immunoglobulins specific for the autoantigen, all important features of the human disease (1
, 2)
. The course and extent of anti-GBM disease in mice are clearly strain dependent. DBA/1 mice show an acute and rapid progression of the GN, with many fresh GBM ruptures and extensive reaction expressed by glomerular sclerosis and crescent formation. C57BL/6 and AKR mice have a more chronic course. Although C57BL/6 mice reveal a similar amount of GBM ruptures 11 wk postimmunization, they have significantly fewer reactive changes than DBA/1. NOD mice, which spontaneously develop autoimmune diabetes, show very little evidence of anti-GBM disease even 6 months after immunization. Although we have observed lung bleeding in only one C57BL/6 mouse, immunization of FcRIIB-deficient mice on a C57BL/6 genetic background with bovine type IV collagen results in alveolar hemorrhage and crescentic GN, suggesting the inhibitory Ig Fc receptor IIB as a susceptibility factor, especially for the lung injury (12)
.
Our study provides evidence for the importance of both autoantibodies and cell-mediated immunity during the induction and effector phases of the autoimmune response. Factors implicated in the activation of B and T lymphocytes are antigen dose, availability of costimulation, and a disease-susceptible genetic background (24
25
26
27)
. We have looked at the extent of the antibody response only with regard to the antigen dose in this study, but others have shown dose-dependent disease induction in WKY rats (17)
. In this model blockade of the CD28-B7 costimulatory pathway by CTLA4-Ig, protein ameliorates the disease (20
, 28)
. Anti-GBM disease in humans is strongly linked to HLA DRB1*1501 (29)
and is linked to MHC class II genes in the mouse (8)
. On the other hand, the MHC background does not seem important in the rat model (30)
, but other susceptibility genes such as the already mentioned Fc receptors may be relevant. DBA/1 mice are susceptible to collagen-induced arthritis, an autoimmune disease involving autoantibodies as well as autoreactive T cells in its pathogenesis (31)
. As anti-GBM GN can be induced with a chain of type IV collagen, it may well be that DBA/1 mice have the genetic susceptibility to autoimmune diseases connected to collagen or collagen-related antigens.
The rapid course and the severity of anti-GBM disease in DBA/1 mice can be explained by our immunological findings. These mice show a strong Th1-type antibody response to the initial immunization and a high specificity of the circulating and the GBM-bound antibodies for the clinically relevant amino-terminal part of the 3(IV)NC1 domain. Our study demonstrates that mice can launch a strong Th1 response after immunization with r-3(IV)NC1 and that not only the amount of IFN- determines the course of the disease, but the balance between Th1- and Th2-like cytokines, may contribute as well to disease development. Splenocytes from DBA/1 mice secreted about as much Th1 cytokines (IL-2, IFN-) as splenocytes from C57BL/6 mice upon in vitro stimulation with r-3(IV)NC1. However the secretion of IL-10, a regulatory anti-inflammatory cytokine, was much higher in C57BL/6 than in DBA/1 mice. The presence of IL-10 may have skewed the immune response toward a Th2 pathway in C57BL/6, which is indicated by less proliferation and higher amounts of IgG1 and IgG2b compared with DBA/1, resulting in a change in the severity of disease development in C57BL/6 mice. AKR and NOD mice launched a lower overall immune response but differ from each other in their antibody isotype pattern, which may explain why anti-GBM disease develops in AKR mice after 6 months but not in NOD. According to the recently proposed linear differentiation model of T cell activation, priming of a large number of Th1 lymphocytes depends on a continuous supply of IL-12 secreted by dendritic cells. As IL-12 production is exhausted, conditions for Th2 differentiation will develop (32)
. The balance between Th1 and Th2 cells will not only result in a different set of T effector and memory cells, but will also affect the differentiation of B cells to antibody-secreting plasma cells. In mice IFN- is necessary for the generation of IgG2a (33)
, whereas relocation of a subset of activated Th2 cells to T cell zones near B cell follicles delivers help for the production of IgG1 and IgG2b (34)
.
Kidney injury develops weeks after the immunizations, although deposition of the autoantibodies in the GBM occurs much earlier. In DBA/1 mice, weak to moderate staining of IgG bound to the GBM is already present in most glomeruli 6 wk after the first immunization (unpublished results). The antibody epitope in human disease is cryptic and exposed only after denaturation (22)
. Sera from patients bind well to purified denatured rat GBM, but in vivo antibody binding to the GBM of rat kidneys has been documented only after perfusion with hydrogen peroxide to expose the epitope before perfusion with the antibodies (35)
. Therefore, the cryptic nature of the epitope may explain the delay between antibody production, deposition, and the beginning of the disease in our mouse model. Our data show that deposition correlates well with glomerular sclerosis and crescent formation, indicating that the amount of GBM-bound antibodies is relevant for the disease. Although not typical, the presence of subepithelial immune deposits within the GBM has been documented in several cases of human anti-GBM GN (36)
. WKY rats immunized with collagenase-solubilized sheep GBM but not rat GBM showed a similar linear/granular pattern, suggesting that the slight differences in the autoantigen between species may result in in situ formation of larger immune complexes than normally observed in the disease (11
, 37)
.
The numbers of CD3-positive T lymphocytes and F4/80-positive cells detected in the diseased DBA/1 mice are significantly higher than those in other immunized strains. Those findings suggest a rapid and vigorous cellular immune response due to antigen-specific mechanisms, as can be seen in a delayed-type hypersensitivity reaction. A similar pattern has been documented in the WKY rat model. Lymphocytes and macrophages are decreased as a result of treatment with antibodies to ICAM-1 and LFA (38)
, disruption of the costimulatory pathways (20
, 28)
, or induction of oral tolerance (21)
, all targeting the cellular immune response. Finally, r-3(IV)NC1 produced in Escherichia coli causes GN in WKY rats but not in SJL or Balb/c mice. This bacterial antigen lacks the B cell epitope of anti-GBM disease due to the absence of disulfide bonds. Since there is no correlation between deposited IgG and GN in this study, antigen-specific T cell responses are thought to be responsible for the development of kidney disease (39)
.
Our data as well as other published data may help in establishing a revised model for the pathogenesis of anti-GBM disease. Initially a strong Th1 response to the autoantigen is launched with only a weak accompanying Th2 response. The binding of autoantibodies to the GBM is favored by the exposure of the cryptic epitope, and high amounts of bound antibodies result in GBM ruptures that set off the glomerular injury. Antigen-specific Th1 effector/memory cells are attracted to the site of inflammation, resulting in a strong delayed-type hypersensitivity reaction that may be responsible for the majority of the kidney damage.
This working hypothesis may provide the framework for future investigations and therapeutic approaches targeting the mechanisms involved in translation of the autoimmune response to glomerular injury in anti-GBM GN.
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ACKNOWLEDGMENTS |
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Received for publication August 1, 2002. Accepted for publication January 7, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. Reynolds, E. I. Prodromidi, J. K. Juggapah, D. S. Abbott, K. A. Holthaus, R. Kalluri, and C. D. Pusey Nasal Administration of Recombinant Rat {alpha}3(IV)NC1 Prevents the Development of Experimental Autoimmune Glomerulonephritis in the WKY Rat J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1350 - 1359. [Abstract] [Full Text] [PDF]
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A. R. Kitching, A. L. Turner, T. Semple, M. Li, K. L. Edgtton, G. R. Wilson, J. R. Timoshanko, B. G. Hudson, and S. R. Holdsworth Experimental Autoimmune Anti-Glomerular Basement Membrane Glomerulonephritis: A Protective Role for IFN-{gamma} J. Am. Soc. Nephrol., July 1, 2004; 15(7): 1764 - 1774. [Abstract] [Full Text] [PDF]
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