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The Journal of Immunology, 2006, 176: 1252-1258.
Copyright © 2006 by The American Association of Immunologists

T Cell Epitope Mimicry in Antiglomerular Basement Membrane Disease1

Jon Arends*, Jean Wu*, Jason Borillo*, Luan Troung{dagger}, Cindy Zhou*, Nadarajah Vigneswaran* and Ya-Huan Lou2,*

* Department of Diagnostic Science, Dental Branch, University of Texas Health Science Center, Houston, TX 77030; and {dagger} Department of Pathology, Methodist Hospital, Baylor College of Medicine, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antiglomerular basement membrane (GBM) disease or Goodpasture’s syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4{alpha}3 NC1 (Col4{alpha}3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28–40 of Col4{alpha}3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28–40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28–40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16–25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antiglomerular basement membrane (GBM)3 disease, or Goodpasture’s syndrome, was one of the earliest recognized human autoimmune diseases (1, 2). Clinical symptoms of the disease include glomerulonephritis (GN) and/or pulmonary hemorrhage (1). Historically, the discovery of anti-GBM Ab was a milestone in autoimmune disease research (3). Collagen IV {alpha}3 NC domain (Col4{alpha}3NC) was identified to be the responsible autoantigen (4, 5). Later studies demonstrated that immunization with Col4{alpha}3NC induced anti-GBM GN in animal models (6). However, pulmonary hemorrhage was usually not induced in these same models. Although an association of anti-GBM disease with infections has been suspected, it remains unknown whether autoimmunity to Col4{alpha}3NC is provoked by these infections.

Pathologically, anti-GBM GN has been considered to be a prototypical model for autoantibody-mediated autoimmune diseases (7). However, recent studies, based on either animal models or clinical observations, have shown that T cell mechanisms are potentially the more important mediator of GN (8, 9, 10). The key question for the involvement of T cells in GN pathogenesis has been whether Ag-specific CD4+T cells per se can target renal Ags and initiate glomerular injury. Several early studies on animal models have suggested that T cells or lymphocytes might be required for the development of anti-GBM disease (11). In addition, Col4{alpha}3-specific T cells have been detected in anti-GBM patients (12, 13). To more precisely address this question, we have developed a rat model in which anti-GBM GN is induced by either CD4+ T cells specific to Col4{alpha}3NC1 or by a T cell epitope pCol28–40 of Col4{alpha}3NC1 (14, 15). Importantly, nephritogenic T cell epitope pCol28–40 not only induces severe glomerular injury, but also triggers an Ab response to diverse GBM Ags through B cell epitope spreading (16).

We further characterized this nephritogenic T cell epitope, and our study revealed the 10 aa residues to be the core of the T cell epitope with only three absolutely critical residues (17). The small size and simplicity of this T cell epitope suggest the likelihood of molecular mimicry between the nephritogenic epitope and the microbial peptide. A mounting body of evidence suggests that T cell epitope mimicry of infectious agents may be an important mechanism that leads to the breaking of T cell tolerance (18, 19, 20). Recent studies have shown that T cell epitope mimicry may lead to several human autoimmune diseases, such as multiple sclerosis and myocarditis (18, 19). The role of molecular mimicry, especially at the Ab level, in causing anti-GBM disease or other types of GN, has been previously speculated (21, 22, 23). However, there is a lack of convincing evidence to support this hypothesis, especially in anti-GBM disease.

In our present study we have identified several microbial peptides based on the critical residue motif of the nephritogenic T cell epitope. Three peptides were able to induce modest to severe GN as well as anti-GBM Ab in immunized rats. Interestingly, one of the peptides also induced severe pulmonary hemorrhage. Although the identified microbial peptides do not seem to be associated with human anti-GBM disease, our study raises the possibility of T cell epitope mimicry by microbial peptides in causing human anti-GBM disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Ag preparation and immunization

Peptides were synthesized on an automatic peptide synthesizer, AMS 422 (Gilson) using F-moc chemistry and were purified by reverse phase C18 column on a preparative HPLC (Waters). Purified peptides were analyzed by HPLC for purity and by mass spectrometry for the correct sequence. Peptides, exceeding 95% purity, were dissolved in milli-Q water (Millipore) at a 1 mM concentration and used for immunization or other investigative purposes.

Female Wistar-Kyoto (WKY) rats (4–6 wk of age) were purchased from Harlan Breeders. The rats were maintained in the animal facility at the University of Texas Houston Health Science Center and allowed to acclimate for a minimum of 3 days. Rats were immunized with a peptide (0.125 mmol) emulsified in CFA in one hind footpad and at the base of the tail. Rats immunized with CFA alone or with an irrelevant peptide pZP3 (see Table I) served as controls.


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  		Table I. Synthetic peptides used in the present studya

 
Evaluation of GN and pulmonary hemorrhage

GN was evaluated by proteinuria/albuminuria and renal histopathology. Random urine samples were monitored daily by Multstix (Bayer). Urinary albumin was semiquantitated by 10% SDS-PAGE (2 µl of urine/lane) using BSA as a standard. The experimental animals were killed as indicated. Kidney tissues fixed in Bouin’s solution were used for H&E staining. Glomeruli with cresentic lesion, hypercellularity, and no injury were counted, and the glomerular injury score was calculated as 100 x (number of crescentic glomeruli + 0.5 x number of hypercellular glomeruli)/total glomerular number. A portion of the kidney tissues was snap-frozen in liquid nitrogen for direct immunofluorescence staining. For pulmonary hemorrhage, hemorrhagic spots on the lung surface were observed and pictured when the animals were killed. The severity of pulmonary hemorrhage was graded as follows: 0, normal; 1, <10 hemorrhage spots/loaf with diameter <1 mm; 2, 10–30 spots/loaf with diameter >1 mm; and 3, >30 spot/loaf with diameter >1 mm. Lung tissue was fixed in Bouin’s solution for H&E staining. A portion of the lung tissues was snap-frozen in liquid nitrogen for direct immunofluorescence staining or immunohistochemistry. For immunohistochemistry, a 3-µm frozen section was cut, fixed in cold acetone for 10 min, and preincubated with blocking reagents (Vector Laboratories). The section was incubated with mouse anti-rat CD3 or anti-rat CD11b/c overnight at 4°C (BD Pharmingen), followed by goat anti-mouse IgG-HRP (Southern Biotechnology Associates). The color was developed with 3,3'-diaminobenzidine and counterstained with hematoxylin.

Lymphocyte proliferation assay

Lymphocyte proliferation assay was conducted as previously described (14). Briefly, lymphocytes were prepared from immunized rats, and CD3+ T cells were isolated with a T cell enrichment column (RTCC; R&D Systems). The purity of isolated T cells was determined by flow cytometric analyses (FACSCalibur; BD Biosciences) after staining with anti-CD4-PE and anti-rat IgM/G-FITC Abs (BD Pharmingen). T cells and irradiated syngeneic thymocytes (1/1) were cultured in 96-well plates at 4 x 105 cells/well in 200 ml of complete T cell medium. Peptide (0.3–30 mM) was added to each well in triplicate, and purified protein derivative was used as a positive control. The cells were incubated at 37°C in a humidified, 5% CO2 atmosphere for 72 h, pulsed with [3H]thymidine (0.5 mCi/well) for 18 h (ICN Biomedicals), and harvested onto glass-fiber filters using a semiautomatic cell harvester (Skatron Instruments). The incorporated radioactivity was measured by a liquid scintillation counter (Beckman Coulter). The results were expressed as Dcpm (mean triplicate cpm with Ag minus mean triplicate cpm without Ag) or as a stimulation index (ratio between cpm with and without Ags).

Detection of Abs

For detecting Ab to peptides, a previously described ELISA was applied (14, 15, 16). Briefly, plates were coated with 50 ml of peptide in 10 mM carbonate buffer (pH 9.5). Serially diluted serum (100/800) was added to each well in duplicate. The bound rat IgG Abs were detected by HRP-labeled goat anti-rat IgG (1/10,000; Southern Biotechnology Associates) using O-phenoldiamine (0.25 mg/ml) as the substrate. The plates were read on an ELISA reader (Molecular Devices) at 490 nm.

For detecting Abs to GBM, indirect immunofluorescence was conducted. Sera were diluted 1/50 in 3% BSA-PBS and added to frozen sections of normal rat kidney. After 1-h incubation, the sections were incubated with FITC-labeled goat anti-rat IgG or IgM Abs (1/50 dilution; Southern Biotechnology Associates). The sections were viewed by fluorescence microscopy (BH-2; Olympus). SR-13, a rat mAb to GBM and tubular basement membrane, was used as a positive control.

For detection of GBM-bound IgG, IgM, or C3 deposition in the glomerulus, direct immunofluorescence assays were conducted. The kidneys from experimental rats were snap-frozen, and frozen sections were cut. The sections were fixed in cold acetone for 10 min and incubated with FITC-labeled goat anti-rat IgG or IgM Abs (1/50 dilution; see above). In parallel, the sections were incubated with anti-rat C3 Ab labeled with FITC (1/100; ICN Biomedicals).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Searching microbial peptides with the critical residue motif of T cell epitope pCol28–40

Our previous study demonstrated that pCol28–40 is restricted by the rat MHC class II molecule RT-1Bl (17). We also identified 10 aa residues from positions 29–38 to be the minimal core required for T cell epitope activity of pCol28–40 (17). Only the three amino acid residues (positions 31, 33, and 34) were identified as critical (17). Among three critical residues, position 31 is an anchor residue to RT.1Bl, and the other two (positions 33 and 34) contribute to the specificity of the T cell epitope. Besides the three most critical residues for the T cell epitope, positions 30 and 35 are considered to be semicritical, because an alanine substitution at these positions significantly reduced its nephritogenicity.

Based on the critical residue motif (xxtTxNPsxxxx), we searched GenBank for microbial peptide candidates that might mimic the T cell epitope pCol28–40. In the initial search, we identified >300 microbe-derived peptides with the TxNP motif. We further narrowed down to 32 based on two criteria: 1) peptides with T at position 30, S at 35, or both; and 2) peptides derived from microbes associated with human infections. Seven peptides were randomly chosen for synthesis. The synthetic peptides were tested in the present study (Table I). Peptide pHV, which was mistaken to be of HIV origin, was actually derived from type 1 HIV enhancer-binding protein of human.

GN in microbial peptide-immunized rats

The synthetic microbial peptides were first tested for their cross-reactivity with T cell epitope pCol28–40 using two independent pCol28–40-specific T cell lines as probes (Fig. 1A). The T cells of both lines responded to all seven peptides at various levels. None of the seven peptides induced as strong a T cell proliferation as pCol28–40. Among the tested peptides, pCB was the most potent in stimulating pCol28–40-specific T cells. A detailed titration showed that pCB was 10-fold weaker than pCol28–40 in stimulating pCol28–40-specific T cells (Fig. 1B).



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  		FIGURE 1. Cross-reactivity of microbial peptides with nephritogenic T cell epitope pCol28–40. A, Responsiveness of a pCol28–40-specific T cell line ({blacksquare}) or nonspecific T cells ({square}) to microbial peptides; the results are expressed as the incorporation of [3H]thymidine (Dcpm). B, pCol28–40-specific T cells responding to different concentrations of pCol28–40 or pCB. C, T cell responses to pCol28–40 on day 25 or 50 after immunization with microbial peptide pCB or pNM. D, Expansion of the pCol28–40-reactive population among the T cells from pCB-immunized rats after two in vitro stimulations with pCol28–40; as a control, pCol28–40-specific T cells were undetectable in pZP3-immunized rats. T cell responses are expressed as a stimulation index (S.I.).

 
The seven peptides were used to immunize WKY rats. All peptides induced significant proteinuria in the immunized rats (Fig. 2A). However, proteinuria development patterns were different among the peptides. The rats immunized with peptides pPV, pLIA, and pNM showed low, but significant, proteinuria (250–1000 mg/dl); proteinuria lasted for 2 wk and declined to a normal level. In pHV-immunized rats, significant proteinuria was detected 15–25 days after immunization and lasted for next 2 wk, with a range of 650-2300 mg/dl at the peak; the proteinuria level gradually decreased to an almost normal level (after 40 days). Immunization with pSP led to proteinuria with individual variations; a few developed severe proteinuria, but others did not. However, in pCB- and pNV-immunized rats, proteinuria persisted and progressed. In 1/6 of them, proteinuria level even reached to a comparable range of those in pCol28–40-immunized rats after 25 days (>3000 mg/dl). Significant proteinuria suggested glomerular injury in these rats (Fig. 2A).



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  		FIGURE 2. Proteinuria and glomerular injury in WKY rats immunized with different microbial peptides. A, Albuminuria in the immunized rats on day 25 ({square}) and 45 ({blacksquare}; n = 5 for each group). B, GN, expressed as the glomerular injury score, in immunized rats. Each circle represents an individual rat; the horizontal bars show the average of each group. the number of animals for each group is shown at the bottom. C, Kidney sections stained with H&E show glomerular injury in the rats corresponding to different glomerular injury scores, as indicated by the brackets.

 
Renal pathology of the immunized rats was determined 40–50 days after immunization (Fig. 2B). Although pHV induced a high level of transient proteinuria, all immunized rats except one showed very mild glomerular damage. Similarly, only minor glomerular injury was observed in the rats immunized with pPV, pLIA, and pNM (Fig. 2, B and C). Those minor pathological changes included infiltration of leukocytes in both glomeruli and the interstitium, and significantly enlarged glomerular tufts. In pSP-immunized rats, modest glomerular injury was seen in 2 of 12 rats. Peptide pNV induced modest GN in 16% of the rats despite development of a high level of proteinuria in these rats. However, pCB induced modest to severe glomerular injury in 25% of the rats with a similar pathological feature induced by pCol28–40. The lesions included cellular or fibrotic crescent lesion (Fig. 2, B and C). All three peptides (pCB, pNV, and pSP) that induced significant glomerular injury showed a higher level cross-reactivity with T cell epitope pCol28–40 than the other three (Fig. 1A).

Comparison of T cell response in pCB- and pNM-immunized rats

The T cell response in pCB-immunized rats was determined in detail. Peptide pNM, which was minimally nephritogenic, was used as a control. T cells isolated from pNM-immunized rats on day 25 responded to pNM and, at a much lower level, to pCol28–40 (Fig. 1C). However, T cell reactivity to pCol28–40 decreased with time. Thus, T cells isolated on day 50 no longer responded to pCol28–40 (Fig. 1C). T cells isolated from pCB-immunized rats responded to both pCB and pCol28–40 at an early stage (day 25; Fig. 1C). However, unlike in pNM-immunized rats, the T cells of pCB-immunized rats were able to respond to pCol28–40 even on day 50 (Fig. 1C).

The presence of pCol28–40-specific T cells in pCB-immunized rats was confirmed by in vitro T cell expansion experiments. T cells isolated from pCB-immunized rats were stimulated in vitro by pCol28–40. T cells from rats immunized with pZP3, which was an irrelevant peptide, were used as a control. After two cycles of stimulation with pCol28–40, T cells from pCB-immunized rats responded to pCol28–40 at a higher level (Fig. 1D). In contrast, T cells from pZP3-immunized rats showed no reactivity to pCol28–40 after in vitro stimulation. Thus, immunization with pCB generated a T cell population that reacted to pCol28–40.

Nephritogenic microbial peptides induce anti-GBM Ab

The cross-reactivity of B cell epitopes between pCol28–40 and microbial peptides was investigated using purified circulating pCol28–40-specific Ab as a probe. Purified pCol28–40-specific Ab failed to respond to any of the seven peptides (Fig. 3A). Thus, there was no B cell epitope cross-reactivity between pCol28–40 and these peptides. Next, circulating Ab in the rats immunized with seven synthetic peptides was determined by ELISA. Without any exception, all seven peptides elicited circulating Ab against themselves (data not shown). However, these Abs showed little or no cross-reactivity with pCol28–40 (Fig. 3B). We next observed binding of IgG Ab to GBM using direct immunofluorescence. Linear binding of IgG Ab to endogenous GBM were found in four of nine rats with modest to severe GN of either pCB- or pNV-immunized rats (Fig. 3C). However, no circulating anti-GBM Ab was found in those rats using either ELISA or indirect immunofluorescence (data not shown).



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  		FIGURE 3. Ab responses in WKY rats immunized with microbial peptides. A, The B cell epitope cross-reactivity between microbial peptides and pCol28–40; Ab reactivity was determined by ELISA using pCol28–40-specific Ab as probe. B, Circulating Ab activity to pCol28–40 from rats immunized with different peptides, as indicated; the serum dilution was 1/200. C, Direct immunofluorescent staining shows intense linear GBM-bound IgG in a pCB-immunized rat.

 
Pulmonary hemorrhage in pCB-immunized rats

Although the T cell epitope pCol28–40 induces extremely severe GN, pulmonary hemorrhage, which is another important clinical feature of Goodpasture’s syndrome, was not observed (Fig. 4). Interestingly, half (six of 12) of rats immunized with pCB developed significant pulmonary hemorrhage (Fig. 4, C and E). Three of them could be described as severe, and the hemorrhagic area exceeded 10% of the entire lung surface (Fig. 4C). Histologically, severe interstitial hemorrhage and mild inflammation were present in large areas of lung tissue of pCB-immunized rats. Higher magnification revealed RBC in both alveolar walls and alveolar lumens, causing distortion of the lung tissue structure (Fig. 4E). Immunohistochemistry on the lung tissue of pCB-immunized rats showed that the majority of infiltrating leukocytes were T cells (Fig. 4, F and G). The second most abundant were CD11+ cells, probably macrophages. Morphological observation failed to detect a significant number of neutrophils. Infiltration of T cells and CD11+ cells was not observed in CFA controls. Among pCB-immunized rats, no correlation between the severity of hemorrhage and glomerular injury was observed (Fig. 5). For example, one rat without significant glomerular injury developed severe pulmonary hemorrhage. Finally, a direct immunofluorescent study failed to detect Ab (IgG or IgM) binding to alveolar basement membrane in all experimental rats, including those with severe pulmonary hemorrhage (data not shown).



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  		FIGURE 4. Pulmonary hemorrhage in WKY rats immunized with pCB. A–C, Pictures show lungs from CFA- (A), pCol28–40- (B), or pCB-immunized (C) rats. D, Lung section (H&E stain) from a pCol28–40-immunized rat shows an absence of hemorrhage; rare inflammatory cells are noted in the alveolar walls. E, Lung section (H&E stain) from a pCB-immunized rat shows severe interstitial hemorrhage and mild inflammation in large areas of lung tissue (arrows); a higher magnification (inset) shows RBC in both alveolar walls and alveolar lumens, causing a distortion of the lung tissue structure. F and G, Immunoperoxidase staining for CD3; numerous CD3+ T cells are present around alveoli in pCB-immunized rats (G), but not in the CFA control (F). Inset in G, Higher magnifications show CD3+ T cells surrounding alveoli (x200 and x400).

 


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  		FIGURE 5. Analysis of the correlation between pulmonary hemorrhage and the severity of GN in pCB-immunized WKY rats (A) or pCol28–40-immunized rats (B). Each symbol represents an individual animal; see Materials and Methods for pulmonary hemorrhage grading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study demonstrated that microbial peptides with a similar critical residue motif of nephritogenic T cell epitope pCol28–40 induced GN. Importantly, the nephritogenic microbial peptides also elicited an anti-GBM Ab response. The anti-GBM Ab was not related to the microbial peptides, because none of the peptides even showed cross-reactivity with wild-type peptide pCol28–40. We believe that anti-GBM Ab was the result of B cell epitope spreading, as we have reported previously (16). Thus, a single nephritogenic microbial peptide was sufficient to initiate a clinical spectrum of anti-GBM GN, which includes proteinuria, glomerular injury, and the anti-GBM Ab. The association of Goodpasture’s syndrome with infections has also been described (24). In addition, molecular mimicry as a cause of this disease has been proposed (23). Thus, the significance of the present study is that we have provided the first experimental evidence to support molecular mimicry as a potential cause of anti-GBM disease, although the nephritogenic microbial peptides used in the present study seem to be irrelevant to anti-GBM disease in humans. We will continue to test more microbial peptides or proteins that have been known to be involved in human infections. Our study did not directly answer the question of whether molecular mimicry is truly a cause of human Goodpasture’s syndrome or anti-GBM disease. However, we believe that the present study was a necessary first step for our future investigation of possible molecular mimicry during infection as a cause of human anti-GBM disease.

It is clear that T cell epitope mimicry was the mechanism for the microbial peptides to induce anti-GBM disease in our model. This conclusion is supported by the following results. First, the microbial peptides were selected based on the critical residue motif of T cell epitope in pCol28–40. As expected, those peptides showed cross-reactivity with pCol28–40 at different levels. There was a positive correlation between the level of cross-reactivity of the peptides with pCol28–40 and their nephritogenicity (Figs. 1 and 2). As an example, pCB, which showed the highest cross-reactivity with pCol28–40, is the most nephritogenic. Second, the T cells from rats immunized with nephritogenic microbial peptides were able to respond to pCol28–40. Furthermore, the T cell population reactive to pCol28–40 in pCB-immunized rats in the presence of pCol28–40 was observed. Third, none of the tested microbial peptides elicited Ab, which cross-reacts with pCo28–40, although each peptide induced Ab specific to itself. This result ruled out any potential involvement of the B cell epitope in the microbial peptide in pathogenesis of GN. In addition, we have repeatedly demonstrated that the B cell epitope in pCol28–40 is peptide specific and unrelated to anti-GBM Abs or GN (15, 16, 17).

We have concluded that the mimicry of the T cell epitope pCol28–40 by microbial peptides induces anti-GBM GN in our model. Despite this conclusion, several critical questions still need to be addressed. First, why do some peptides, which were also selected based on the identical motif, fail to induce the disease, and why does a nephritogenic microbial peptide induce severe disease in only a relatively small percentage of immunized rats? There are several possibilities. The contributions of other residues to the specificity of the T cell epitope may need to be reconsidered. It is probably important to determine whether nonconservative substitution of those residues would lead to an altered specificity of the T cell epitope. This phenomenon has been well documented in many previous studies (25). In some cases, a subtle alteration of T cell epitope specificity may lead to partial activation, which leads to T cell anergy rather than T cell activation (26). Another possibility is that the peptide may nest several overlapped T cell epitopes, leading to the activation of diverse T cell populations with different specificities (27); if the TCR repertoire gradually skewed toward a nonpathogenic microbial peptide-specific epitope as the result of competition between nest epitopes, the general T cell population will no longer or only transiently cause glomerular injury. This probably explains why some peptides induced only a transient proteinuria. Another important question to be addressed is whether the proteins from which the microbial peptides are derived are able to induce the disease? Or, even more important, does infection of the microbe lead to the disease? Currently, we are planning to test the nephritogenicity of the microbial proteins.

One of the important findings in our current study is that a nephritogenic microbial peptide, pCB, induced not only GN, but also severe pulmonary hemorrhage. There are numerous animal models for anti-GBM disease. However, pulmonary hemorrhage is much less common in these models. For example, the potent nephritogenic T cell epitope pCol28–40 of Col4{alpha}3 in our model fails to induce pulmonary hemorrhage (16). An obvious question that remains is why a microbial peptide, which is much less potent in the induction of glomerular injury, causes pulmonary hemorrhage? A recent study reported pulmonary hemorrhage induced by the anti-GBM Ab (28). However, in our model anti-GBM Ab is unlikely to be a causative reagent of pulmonary hemorrhage. First, we did not detect anti-GBM Ab binding to alveolar basement membrane. Second, pCol28–40 induces a much stronger anti-GBM Ab response, yet it fails to induce pulmonary hemorrhage (17). Thus, it seems that a T cell mechanism may be responsible for pulmonary hemorrhage, at least in our model. Subtle variations in T cell epitope specificity or different presentations by APCs due to nest-overlapped epitopes between two tissue sites may explain the different pathogenesis caused by the two peptides. In fact, a different presentation of the same autoantigen at different tissues has been reported. For example, despite GAD expression in several locations (such as brain and kidney) (29), GAD-specific T cells only attack the {beta}-islet, causing diabetes. It will be our next step to investigate whether Ag processing and presentation are different in the two locations.

As a summary of both our present and previous studies, we are now able to propose a hypothetic mechanism by which an infection leads to a full clinical spectrum of anti-GBM disease or Goodpasture’s syndrome through a single T cell epitope mimicry (Fig. 6). The activation of nephritogenic T cells by a mimicking peptide of microbial origin not only leads to glomerular injury, but also triggers a diversified anti-GBM Ab response. In addition, a subtle variation in T cell epitope mimicry may result in pulmonary injury and/or glomerular injury. However, it needs to be emphasized that all our observations or discoveries have been made in our animal model, and the microbial peptides may not be related to actual human anti-GBM diseases. Thus, it remains an open question whether T cell epitope mimicry is the real cause of Goodpasture’s syndrome or human anti-GBM disease. This question may be true for other human autoimmune diseases as well. Although an increasing number of autoimmune diseases have been suspected to be caused by T cell epitope mimicry (30, 31, 32), the nature of T cell epitope mimicry seems more complicated than we had thought (20). It remains controversial whether the T cell epitope can indeed lead to autoimmune disease. We believe, however, that it is worthwhile to test our hypothesis in the human disease. The first step will be a detailed analysis of the T cell response or the detection of Ag-specific T cells in injured glomeruli. Fortunately, several groups of investigators have been exploring the T cell response to Goodpasture’s autoantigen, and several T cell epitopes have already been identified (12, 13, 33, 34). Although it is still questionable whether the identified human T cell epitopes are truly pathogenic, the identified epitopes could be used for searching for potential viral or bacterial mimicking peptides.



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  		FIGURE 6. The schematic diagram illustrates the hypothetic mechanism by which a single T cell epitope mimicry during the infections induces a full spectrum of clinical manifestations ({cjs2108}) of anti-GBM GN in a certain population with specific genetic backgrounds.

 

   Acknowledgments
 
We thank Dr. Y. Sado (Okayama University, Okayama, Japan) for providing mAbs to GBM. Histology supports were provided by the Histology Core Laboratory, Dental Branch, University of Texas Health Science Center (Houston, TX).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1DK60029 (to Y.H.L.) and internal research support from the Dental Branch, University of Texas Houston Health Science Center (to Y.H.L.). Back

2 Address correspondence and reprint requests to Dr. Yahuan Lou, Department of Diagnostic Science, Dental Branch, University of Texas Houston Health Science Center, Houston, TX 77030. E-mail address: yahuan.lou{at}uth.tmc.edu Back

3 Abbreviations used in this paper: GBM, glomerular basement membrane; Col4{alpha}3NC1, noncollagen domain of collagen type IV {alpha}3 chain; GN, glomerulonephritis; WKY, Wistar-Kyoto rat. Back

Received for publication July 11, 2005. Accepted for publication October 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Couser, W. G.. 1999. Glomerulonephritis. Lancet 353: 1509-1515. [Medline]
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