Airway epithelium in obliterative airway disease Qu, Ning
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Chapter 3
Specific Immune Responses against Airway Epithelial Cells in a Transgenic Mouse Trachea Transplantation Model
for Obliterative Airway Disease
Ning Qu; Aalzen de Haan; Martin C. Harmsen; Frans G.M. Kroese Lou F.M.H. de Leij; Jochum Prop
Transplantation. 2003 Oct 15;76(7):1022-8.
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Abstract
Background
Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies a rejection model of murine trachea transplants is used resulting in obliterative airway disease (OAD) with similarities to human OB. To focus on the role of an immune response specifically against airway epithelium, we transplanted tracheas from transgenic mice expressing human epithelial glycoprotein-2 (hEGP-2) on epithelial cells. We hypothesised that the immune response against the hEGP-2 antigen would result in OAD in the trachea transplants.
Methods
Tracheas from hEGP-2 transgenic and control non-transgenic FVB/N mice were heterotopically transplanted into FVB/N mice and harvested at week 1, 3, 6, and 9. Anti-hEGP-2 antibodies were determined in the recipient blood.
The trachea grafts were analyzed for cellular infiltration, epithelial cell injury, and luminal obliteration.
Results
Recipients of transgenic tracheal grafts gradually developed anti-hEGP-2 antibodies. In the transgenic grafts, the submucosa was infiltrated predominantly by CD4+ T cells. Epithelial cells remained present but showed progressive abnormality. The tracheal lumen showed a mild degree of obliteration. All these changes were absent in non-transgenic FVB/N trachea transplants.
Conclusions
The hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induces specific humoral and cellular immune responses leading to a mild form of OAD. It provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in human lung transplantation.
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Introduction
Obliterative bronchiolitis (OB), one of the most severe complications after lung transplantation, affects 50% of all lung transplant recipients and results in high mortality within 5 years (1,2). Several risk factors have been identified for the development of OB, amongst which episodes of acute rejection are the most prominent. Yet, the pathogenesis of OB remains unclear. As an animal model allowing investigation of human OB after lung transplantation, Hertz and colleagues heterotopically transplanted murine tracheas (3). They found that allogeneic tracheas implanted subcutaneously were rejected with massive cellular infiltration and epithelium loss as opposed to isografts which did not show these changes. By day 21, this lead to obliteration of the lumen of the transplanted trachea by fibroproliferation. This process is called the obliterative airway disease (OAD), and it shares similarities to human OB.
Further studies showed that the rejection of tracheal allografts was mediated by CD4+ and CD8+ T lymphocytes, macrophages and granulocytes, preceding epithelium loss and complete luminal obliteration at day 21 (4-6). In addition to the T cell-driven responses, humoral responses were also found to be involved in OAD (2,7) and the blockade of the complement system could either prevent or inhibit the development of OAD (8).
Clinical histopathologic features of OB suggest that in particular the injury of epithelial cells result from persistent inflammation in small airways leads to ineffective epithelial regeneration and excessive fibroproliferationdue to aberrant tissue repair (9). Also, in animal tracheal transplants, the epithelium is involved in the rejection process leading to OAD (5,10). The effect of airway epithelium injury was investigated by the group of Morris in a rat trachea OAD model. They demonstrated that the loss of airway epithelium either by enzymic removal in isografts, or by transplant rejection in allografts resulted in OAD. The re-seeding of epithelial cells in the isografts (11) or re- growth in allografts (12) largely reduced the level of OAD. These findings suggested that the airway epithelium regulates fibroblast growth and thus protects against fibrotic obliteration of the tracheal transplants. Other studies showed strong sub-mucosal infiltration of immune cells (13), and loss of epithelium preceding the obliteration of the trachea grafts (10,14) supporting that the regulating role of the epithelial cells on fibroblast proliferation may be lost as the result of immune injury.
A limitation for the investigation of epithelium specific injury in all studies on tracheal allografts is that the alloreactive immune response is not
specifically directed against epithelial cells but against all cell types of the graft tissue, as all the cells express alloantigens. Therefore, it is possible that immune reactivity against cell types other than epithelial cells, for example fibroblasts, also contributes to tissue injury and subsequent fibroproliferation of the trachea.
Transgenic animals expressing neoantigens on specific tissues or cell types may provide new options to investigate the role of epithelium in OAD. In this study, we used transgenic FVB/N mice as donors of tracheas for heterotopic transplantation into non-transgenic syngeneic mice. In these mice, human epithelial glycoprotein-2 (hEGP-2) is expressed specifically on epithelial cells, including trachea epithelial cells, driven by the endogenous specific promoter (15). We hypothesized that the epithelium specific hEGP-2 neoantigen induces an epithelial specific immune responses upon trachea transplantation and the responses cause epithelial injury and the subsequent development of OAD.
Material and methods
Experimental design
Tracheas were transplanted from hEGP-2 transgenic FVB/N mice to non- transgenic FVB/N mice subcutaneously (s.c.) (n=27). As controls, tracheas from non-transgenic FVB/N mice were transplanted s.c. to non-transgenic FVB/N recipients (n=21). Also, as allogeneic controls, MHC fully mismatched C57BL/6 (H-2b) mice tracheas were s.c. transplanted to non-transgenic FVB/N (H-2q) recipients (n=18). About 3 to 11 grafts from each group at each time point were harvested at 1, 3, 6 and 9 weeks after transplantation and were studied for histology by haematoxylin and eosin (H&E) staining and for analyzing cellular infiltration by immunohistochemistry. Serum samples from recipient mice were collected and levels of antibodies against EGP-2 antigen and alloantigens were determined by ELISA and flow cytometry.
Experimental animals
Inbred FVB/N mice transgenic for the hEGP-2 were bred and housed in the Central Animal Facility of Groningen University according to the standard breeding rules for transgenic animals in a conventional condition. Normal FVB/N and C57BL/6 inbred mice were purchased from Harlan (Harlan Zeist,
48
The Netherlands). Animals used in the experiments were between 8-10 weeks of lifetime. All animals received care in compliance with the Dutch regulations and laws. Experimental protocols were approved by the institutional animal ethical review committee.
Heterotopic trachea transplantation
Donor mice (hEGP-2 transgenic and non-transgenic FVB/N or C57BL/6 mice) were anesthetized with halothane and N2O/O2 gas. The mice were then euthanized by abdominal aorta bleeding. Tracheas from these mice were exposed through the anterior midline incision and esophagus, vessels and other surrounding tissues were gently separated. After isolation, the tracheas were transected below the thyroid cartilage and above the bifurcation.
Tracheas were immediately put into cold saline. The tracheal lumen was washed three times with 300µl cold saline by 1ml syringe to remove blood and other fluid inside the lumen. To avoid bending of the trachea after implantation, an orthodontic stainless steel wire (0.036", GAC, New York, USA) was prepared to the same length of the trachea. Both ends of the wire were folded back and made blunt. Tracheas were clipped along with the prepared wires by stainless steel surgical clips (Ligation Clip 316L, Ethicon Endo-Surg. Inc.
Cincinnati, OH 45242-2839, USA) at both ends.
The non-transgenic FVB/N recipients were anesthetized in the same way as the donor mice. After shaving, a small incision was made at the lateral side of the back. By blunt dissection, a subcutaneous pouch was made, the clipped trachea was put into the pouch and the wound was closed by one stitch (6-0 Prolene suture). Each recipient received one trachea graft.
At designated time points the trachea grafts were explanted through an incision immediately outside the grafts tissue. After separating the wires and cutting off the clips, each trachea was cut into two segments: one half was fixed in 10% formalin for H&E staining and the other half was snap frozen in liquid nitrogen for immunohistochemical staining.
ELISA assay for anti-hEGP-2 antibody detection
Whole blood samples were collected at different time points after transplantation. Serum hEGP-2 specific antibody levels were detected by ELISA. Briefly, 96-well ELISA plates
were coated overnight with 100 µl/well recombinant soluble hEGP-2 protein (300ng/ml). Serum samples (100µl/well from each sample) in two-fold dilutions incubated in the coated plates for 1.5h at 37oC. Purified mouse-anti- hEGP-2 mAb Moc31 (Purified from hybridoma culture supernatant) (15) at a concentration of 3.5µg/ml was used as the standard. Serum samples from non-transgenic graft recipients were used as negative control. Plates were then washed with PBS/Tween-20 solution and rabbit-anti-mouse IgG peroxidase labeled antibodies (Dako, UK) was applied and incubated for 1h at 37oC. After washing, TBM peroxidase substrate solution was added and plates were stained for 15 min on a shaking bed. The staining reaction was stopped by adding 1M H2SO4 100µl/well and the plates were read on a microplate reader (Emax, Molecular Device.) at 450 nm. Data were analyzed by reader’s software (SoftmaxPro version 1.2.0) and the EGP-2 specific antibodies level related to Moc31 level was calculated.
Flow cytometry assay for alloantibody detection
Alloantibodies directed against donor (C57BL/6) alloantigens were measured in serum from allograft recipients (FVB/N) by analyzing the amount of alloantibodies bound to donor derived spleen T cells by flow cytometry. Serum samples from non-transplanted control mice (normal FVB/N) served as negative control. Serum was de-complemented by incubation at 56oC for 30 min. Serum samples were serially diluted (10 fold) with PBS/FCS (PBS plus 1% FCS) from 1:50 till 1:500,000. Splenocytes from the donor strain (C57BL/6 ) that express alloantigens were harvested and stained with anti- CD3-PE (1:50, Human-anti-Mouse PE labeled, BD Pharmingen) to identify T cells. These cells lack Fc receptors, and as a consequence, do not bind antibodies non-specifically. Splenocytes were then incubated with the diluted serum samples for 20 min on ice, washed and stained with FITC labeled goat-anti-mouse IgG antibodies (1:100, BD Pharmingen) for 20 min on ice.
After another wash, double stained cells (CD3-PE and FITC labeled cells with bound alloantibodies) were detected by flow cytometry (FacsCaliber flow cytometer, Becton Dickinson). After gating on CD3+ T cells, the geometric Mean Fluorescent Intensity (MFI) of the FITC signal was taken. Data was analyzed with WinList software (5.0 version, Verity Company).
Light microscopy and analysis
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Formalin fixed trachea segments were paraffin embedded. Serial cross sections (3µm thick) were cut at 3 levels 50µm apart of each trachea segment, and 3 sections at each level were stained by normal H&E method and evaluated at 50 to 400 magnifications. To analyze the infiltration level of the trachea grafts, a semi-quantitive scoring system was used with the following five grades: 0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration: diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates.
To analyze the obliteration degree of the trachea lumen, the thickness of submucosa (between epithelium and cartilage) was scored using following five grades: 0-no thickening of submucosa, 1-minimal thickening: focal submucosa thickening, 2-mild thickening: submucosa thickening equal to the thickness of the cartilage ring, 3-moderate thickening, submucosa thickening result in a small lumen, 4-severe thickening: total lumen obliteration.
The epithelium was categorized in two types: normal and abnormal based on morphological aspects of epithelial cells, i.e. the differentiation (the presence of ciliated epithelium) and the pseudo-stratification (the presence of pseudostratified epithelium layer). The coverage of the trachea by normal epithelium and abnormal epithelium were scored in percentage.
Immunohistochemistry
Frozen tracheas were cut into 5µm thick cross sections, and were immunohistochemically stained by monoclonal antibodies for different markers (4). Briefly, the sections were fixed in acetone for 10 min and then washed 3 times in 0.01M phosphate-buffered saline (PBS). Primary rat antibodies specific for mouse CD5, CD4, CD8 T cells and macrophages [Primary antibodies directed against mouse CD5 (Ly-1), CD4 (L3T4), and CD11b (mac- 1, for macrophages) were from hybridoma culture supernatants, and purified antibodies specific for mouse CD8α (Ly-2) were from BD Pharmingen, San Diego, USA] were applied and incubated for 1h (hybridomas in culture supernatants were not diluted, CD8α was diluted 1:50). The slides were then washed again with PBS. Secondary antibodies (rabbit-anti-rat peroxidase labeled, 1:50 dilution, Dako, UK) plus 3% normal FVB/N mouse serum were applied and incubated for another 1h. After washing in PBS, the slides were colored with AEC (Sigma, 0.5mg in 3.75 ml DMF plus 70ml acetate buffer, pH
=4.9). Endogenous peroxidase was blocked by adding 0.03% H2O2 in the
AEC solution. Slides were counterstained with Mayer’s haemotoxilin (1:10).
Additionally, epithelium was stained by keratin-specific antibody RGE53 (Eurogentec, Parc scientifique du Sart Tilman , 4102 Sering, BE) for epithelium lumen coverage evaluation on 3 selected trachea segments from each time point. The hEGP-2 expression in the transgenic grafts was examined by the anti-hEGP-2 specific monoclonal antibody Moc31 (15).
Trachea infiltrating cell phenotypes were analyzed both inside and outside trachea ring in three sections from each trachea segment (stained by monoclonal antibodies for CD5+, CD4+, CD8+, T cells and macrophage). The positive cells in the grafts were scored, using semi-quantification system with five grades: 0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells. 4-whole tissue dense positive cells. Additionally, from 4 areas each section, CD4+ and CD8+ T cells and macrophages in the same tissue area per microscopic view were counted (400 magnifications) and CD4+/ CD8+ ratios were calculated. All the histology scoring and counting were done by two independent observers.
Results
Immunogenicity of hEGP-2 transgenic trachea grafts
We used hEGP-2 transgenic trachea grafts as a model to study the role of epithelial injury in the development of OAD. We explored whether the transplantation of hEGP-2 transgenic grafts induce epithelial cell specific immune responses by analyzing anti-hEGP-2 antibodies responses and cellular infiltrations.
Antibodies
Anti-hEGP-2 antibodies became detectable in the recipients recieved hEGP-2 tracheas at 3 weeks after transplantation and levels increased sharply between 3 and 6 weeks. (Figure 1A). No anti-hEGP-2 antibodies were detected in the recipients received non-transgenic grafts. This indicated that a hEGP-2 antigen specific humoral response was induced by grafting the hEGP-2 transgenic tracheas. As control, we examined alloantibodies development in C57BL/6 allograft recipients, alloantibodies were present already at 3 weeks and continue to increase at 6 and 9 weeks. (Figure 1B).
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Figure 1.
A. hEGP-2 Antibodies
control 3w 6w 9w
0 2500 5000 7500 10000
week after transplantation
concentrationµg/ml
B. Alloantibodies
control 3w 6w 9w
0 50 100 150 200
week after transplantation
mean fluorescent intensity
Figure 1. Antibody dynamics in recipients of hEGP-2 transgenic and C57BL/6 allogeneic trachea transplants. Anti-hEGP-2 antibodies were detectable in the recipients of hEGP-2 transgenic grafts at 3 weeks after transplantation, and reached high concentrations at 6 and 9 weeks (A). Alloantibodies were present at 3 weeks and continued to increase at 6 and 9 weeks (B). Values represent mean ± standard deviations of four to five animals.
Infiltration
Cellular infiltrations were observed in the hEGP-2 tracheas mainly in the submucosa inside the trachea ring as demonstrated by H&E staining (Figure 2). The grading of the infiltration increased from minimal to mild cellular
infiltration inside trachea ring at 1 week to mild to moderate level at 3, 6, 9 week after transplantation (Table 1). The infiltration outside the trachea was minimal and there was no clear increase with time. Analysis of the cellular infiltration by immunohistochemistry showed that CD5+, CD4+ and CD8+ T cells appeared at 1 week post-transplantation reached a plateau at 3, 6 weeks and decreased at 9 weeks inside the trachea rings (Table 2). CD4+ T cells were dominant in numbers compared to CD8+ T cells at the same infiltrating tissue area, with CD4+/ CD8+ ratio between 2.7 to 3.1 at different time points (Table 3). Fewer T cells were observed outside the trachea ring and their numbers did not increase with time. This was accordant to the H&E stained findings. Macrophages appeared in the hEGP-2 grafts at 1 week, peaked at 3 and 6 weeks, started to decrease at 9 weeks (Table 3). In control non- transgenic FVB/N grafts, only minimal infiltration was found inside and outside
Figure 2.
Figure 2. Histology of hEGP-2 transgenic, non-transgenic FVB/N and allogeneic C57BL/6 tracheas at 3 weeks after transplantation. Transgenic grafts showed moderate cellular infiltrations in the submucosa. Few infiltrating cells were observed outside the trachea cartilage ring (A). The epithelium was partly flattened (D) and luminal obliteration was mild (A). Non-transgenic grafts showed minimal cellular infiltrations inside and outside the tracheal cartilage ring (B). Well differentiated normal epithelium was observed (E), and no obliteration of the lumen (B). Allogeneic grafts showed minimal cellular infiltrations both inside and outside trachea cartilage ring (C). No epithelium was left and the lumen was totally obliterated (F,C).
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Table 1. Cellular infiltrationa inside and outside trachea cartilage rings (H&E)
EGP-2 grafts FVB/N grafts C57BL/6 grafts
infiltration infiltration infiltration n Median(Range)c n Median(Range)c n Median(Range)c Inb 1w 5 1.25 (1-2) 5 1 (0-2) 3 2 (1-2.5)
3w 11 2.5 (1-4) 9 1 (0-1) 8 1.1 (1-2.5) 6w 7 2.75 (1-4) 4 1 (1-1) 5 0.75 (0.5-1) 9w 3 2.5 (1.5-3) 2 1 (0.5-1.5) 2 0 (0-0) Outc 1w 5 1 (0-1) 5 1.5 (0-2) 3 2.5 (1-3)
3w 11 1 (0-2.5) 9 0 (0-0.5) 8 2 (1-2.5) 6w 6 0.5 (0-1) 4 0 (0-2) 5 0.9 (0.5-2.5) 9w 2 1 (0.5-1.5) 2 0 (0-0) 2 0.5 (0.5-0.5)
aSoring standard:
0-no infiltration, 1-minimal infiltration: scattered or diffuse cellular infiltrates, 2-mild infiltration:
diffuse cellular infiltrates with one area dense infiltrate, 3-moderate infiltration: more than one area infiltrates, 4-severe infiltration: a thick layer of dense cellular infiltrates;
binside trachea cartilage ring;
coutside trachea cartilage ring;
the trachea rings, and this did not increase with time (Table 1) supporting that the response against transgenic grafts was hEGP-2 specific and was not caused by transplantation injury.
In the allograft controls, an acute cellular infiltration was observed with a mild to moderate cellular infiltration at 1 week, decreased thereafter and disappeared by 9 weeks (Table 1). Interestingly, the localization of the infiltration in allografts differed from that in the hEGP-2 transgenic grafts: in the allografts, the infiltration was more observed outside trachea while in the hEGP-2 grafts the infiltration was mainly observed within the submucosa inside the trachea (Table 1). Also, CD5+, CD4+ and CD8+ cellular infiltration was predominantly localized outside of the trachea ring at 1, 3, 6 weeks (Table 2) in the allografts. In contrast to the transgenic grafts, CD4+ T cells in the allografts were outnumbered by CD8+ T cells at 1 and 3 weeks with a CD4+/CD8+ ratio <0.6 (Table 3). Macrophages were observed in high numbers at 1, 3, 6 weeks after transplantation (Table 3).
Table 2. Phenotypes of T cells and infiltration levelsa inside and outside trachea (IHC)
C57BL/6 grafts
CD5 CD4 CD8 n Median(Range) Median(Range) Median(Range) 1w 4 0.5 (0-1) 0.8 (0-1.5) 0 (0-1) 3w 5 2.25 (1-2.5) 1 (0-2) 1.5 (1-2) 6w 5 1 (0-1) 0.5 (0-2.5) 0 (0-1) in
9w 2 0 (0-0) 0.5 (0-1) 0.8 (0.5-1) 1w 3 2 (2-3) 2.5 (1-2.5) 2 (1-2.5) 3w 5 1.5 (0-2.5) 1 (0-2.5) 1.5 (1-3) 6w 5 2 (1-2) 1.5 (0-2) 0.5 (0-1) outc
9w 1 0 (-) - 0.5 (-)
aScoring standard:
0-no positive cells, 1-scattered positive cells, 2-small clusters or one area with dense positive cells, 3-more than one area or a large area plus clusters of positive cells. 4-whole tissue dense positive cells;
binside trachea cartilage ring;
coutside trachea cartilage ring;
Table 3. CD4/CD8 ratios and macrophage numbers
EGP-2 grafts (n=5), mean C57BL/6 grafts (n=4), mean
week CD4 CD8 CD4/CD8a MΦ CD4 CD8 CD4/CD8a MΦ
1w 34.2 12.8 2.7 51.8 41 100.8 0.41 69.4
3w 186.7 61.75 3 191.5 28.25 47.3 0.60 158
6w 162 51.5 3.1 194.5 75 36 2.1 83
9w 39 13.5 2.8 74 - - - -
aThe ratios were calculated based on the positive cell numbers found in the same tissue area positive for CD4 and CD8 T cells infiltrations;
EGP-2 grafts
CD5 CD4 CD8 n Median(Range) Median(Range) Median(Range) 1w 5 1.5 (0-2) 2 (1-3) 0 ( 0-1) 3w 6 2 (1-3) 3 (2-3.5) 1 (0.5-1) 6w 5 2 (0-3) 3 (1-3) 1 (0-1) inb
9w 3 1 (1-2) 1.5 (1-2) 0.5 (0-1) 1w 5 1 (0-2) 0.5 (0.5-1) 0.5 (0-1) 3w 3 1 (0-2.5) 2 (1-2) 0.5 (0-1) 6w 5 1 (0-1) 0.5 (0-3) 0 (0-2.5) outc
9w 2 1.5 (1-2) 2 (1-2) 0 (0-1.5)
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Epithelial cells abnormality and luminal obliteration
After transplantation, the epithelium of hEGP-2 transgenic grafts was present, but it was partially abnormal: a thin layer of flattened non-ciliated cells (Figure 3A). This remained unchanged up to 6 weeks. At 9 weeks the percentage of abnormal epithelial cells increased significantly while the percentage of epithelium coverage decreased slightly, indicating a progressing epithelial injury (Figure 3A). Expression of hEGP-2 in the transgenic graft epithelium was checked by hEGP-2 specific Moc31 antibodies and both normal and abnormal epithelium expressed hEGP-2 at all time points. The keratin specific staining indicated that the cells lining the lumen after transplantation in the grafts were of epithelial origin (data not shown). Obliteration grading showed a slight thickening of the submucosa from minimal at 1 week to mild level at 9 week (Figure 3B).
In non-transgenic control trachea grafts, the epithelium showed a well ciliated and pseudostratified appearance (Figure 3A). In parallel with the normal epithelium, no obliteration of the lumen was detectable in non- transgenic trachea grafts (Figure 3B). In the allograft controls, the loss of epithelium and significant epithelium abnormality were already observed at 1 week post-transplantation (Figure 3A). The epithelium was totally absent and the lumen was occluded from 3 week and onwards. (Figure 3A,B).
Figure 3.
A.Epithelium coverage and abnormality
1 3 6 9 1 3 6 9 1 3 6 9
0 50
100 ep-coverage
abnormal
EGP2 FVB/N C57BL/6
week after transplantation trachea lumen epithelium coverage %
B.Luminal obliteration
0 1 2 3 4 5
1 3 6 9 1 3 6 9 1 3 6 9
week after transplantation
EGP-2 FVB/N C57BL/6
luminal obliteration score (0-4)
Figure 3. Epithelium coverage, abnormality and luminal obliteration of trachea transplants.
Epithelium was present almost 100% in hEGP-2 transgenic grafts. The appearance of the epithelium was less than 50% abnormal during the first 6 weeks. At 9 weeks, most of the epithelium has become abnormal (A). Obliteration grading in hEGP-2 grafts showed the thickening of the submucosa from minimal level at 1 week to mild level at 9 week (B). In the FVB/N trachea grafts, the epithelium showed normal, well ciliated and pseudostratified appearance (A) and no obvious obliteration of the lumen was observed (B). In the C57BL/6
58
allografted tracheas, the loss of epithelium was already observed at 1 week (A) and the remaining epithelium was abnormal. The tracheal lumen was occluded within 3 week (B).
Error bars in Figure 3A indicated the standard error of the mean of three to five experiments.
Values in Figure 3B represent the median and range of three to five experiments.
Discussion
In this study we transplanted tracheas from transgenic FVB/N mice, in which expression of the hEGP-2 neoantigen is restricted to epithelial cells. The tracheas were transplanted to non-transgenic FVB/N mice to study the role of immune injury to airway epithelial cells in the development of OAD. Our data show a strong immune response against epithelial hEGP-2 antigens, as demonstrated by high levels of hEGP-2 specific antibodies and heavy cellular infiltrations, leading to epithelial cell abnormality and partial luminal obliteration in the transplantated tracheas.
The presence of anti-hEGP-2 antibodies prove that antigens expressed on tracheal epithelial cells can induce an epithelial specific immune response.
The potential role of the antibodies in the development of OAD depends on their functional capabilities. Therefore, we performed a pilot study testing the cytotoxicity of hEGP-2 antibody positive serum in a complement-dependent cell cytotoxicity assay. We found that the hEGP-2 Ab positive serum (dilution 1: 10) was capable to lyse up to 50% of a hEGP-2 expressing cell line (data not shown). A control hEGP-2 negative cell line was not lysed under the same conditions supports that the antibodies induced by the transgenic trachea transplant have cytotoxic-capabilities. Comparing the antibody responses after transgenic and allogeneic trachea transplantation, we noticed that the antibody level increased more sharply in the hEGP-2 transgenic recipients during 3 to 6 weeks (Figure 1A,B) while alloantibody levels showed a steady increase. This may indicates a late-coming development of hEGP-2 antibodies. Despite a later appearance of anti-hEGP-2 antibodies, their cytotoxic characteristics may contribute to the graft injury, as in clinical studies where the development of alloantibodies correlated to the occurrence of OB (16-18).
In parallel, the pattern of cellular infiltration in the hEGP-2 transgenic tracheas is consistent with a specific immune response against epithelial cells:
the infiltrates are mainly located within the submucosa of the trachea near the hEGP-2 antigen expressing epithelium. It can be excluded that this infiltration is caused by non-specific factors related to the transplantation procedure
because the infiltration was absent in the control non-transgenic trachea transplants (Table 1). Furthermore, the infiltration in allografts is more diffusely spread throughout the tracheal tissue, suggesting targeting towards the alloantigens expressed on all cells. It is remarkable that the proportion of CD8+ was low within the infiltrating cells in the transgenic transplants. This phenomenon was particularly obvious when comparing the CD4+/CD8+ ratios in the transgenic tracheas with those in the allogeneic tracheas, where our CD4+/CD8+ ratios were in agreement with values described in the literature (4). The low proportion of CD8+ T cells in the hEGP-2 transgenic tracheas may be due to the different pathway of recognition of the hEGP-2 neoantigen compared to alloantigens. In allograft rejection, it is well documented that significant numbers of alloreact CD4+ and CD8+ T cells pre-exist in recipients are able to directly recognize alloantigens (19). In our transgenic situation, such pre-existing antigen-reactive T cells directly recognizing the hEGP-2 antigen are lacking. Thus, CD4+ and CD8+ T cells in the transgenic situation need to be activated de novo via an indirect antigen recognition pathway, involving processing of exogenous and endogenous neoantigen and presentation of antigen-derived peptides in MHC class II and I molecules, respectively. MHC class II-restricted antigen presentation of exogenously derived antigen, such as hEGP-2 from necrotic or apoptotic epithelial cells most likely depends on presentation of the hEGP-2 by professional antigen presenting cells (APCs) and activate mainly CD4+ T cells. MHC class I- restricted antigen presentation of endogenous hEGP-2 is likely to depend on hEGP-2 expressing epithelial cells. Being 'non-professional' APC, these cells may not have the capacity of inducing an MHC class I-restricted CD8 T cell response. This may explain the predominance of CD4+ T cells amongst the infiltrating cells in the transgenic trachea transplants. Although it has also been shown that both CD4+ and CD8+ T cells could contribute to the development of OAD (20,21) the low proportion of CD8+ T cells in transgenic grafts may indicate that a cytotoxic T cell response is weak.
The development of OAD with epithelial injury and luminal obliteration in the transgenic transplants is in line with the moderate epithelial cells specific immune responses that we found in the tracheas. The epithelium of transgenic tracheas exhibited a significant degree of abnormality, but this became apparent as late as 9 weeks after transplantation (Figure 3A). In addition, the epithelial cells were still of donor origin as they continued to express the hEGP-2 antigen after transplantation. This is in contrast to the allogeneic tracheas, where epithelial cells were largely abnormal at 1 week
60
and were virtually absent by 3 weeks after transplantation. The obliteration of the tracheal lumen inversely correlated with the presence of epithelium: only mild obliteration in the transgenic tracheas and complete obliteration in the allogeneic tracheas. Several studies have shown that airway epithelial cell can inhibit proliferation of fibroblast, both in vitro (22,23) and in vivo (24,25). In rat trachea transplants, epithelial cells were capable to inhibit luminal obliteration after transplantation. This was shown in trachea transplants that were made devoid of epithelium by exogenous methods (11). Upon transplantation these tracheas obliterated quickly, unless epithelium was allowed to re-grow either from adjacent recipient tissue (12) or from co-transplanted donor tissue (11).
The mild obliteration in our transgenic tracheas, however, does not seem to result mainly from fibroproliferation but rather reflect the volume of infiltrating cells in the submucosa. This is consistent with the possible inhibiting role of the presence of partial abnormal epithelium in the lumen on fibroproliferation.
We have shown that transplantation of hEGP-2 transgenic tracheas induces an epithelial cell specific immune response composed of both antibodies and T cell reactivity. This response causes epithelium abnormality and partial airway obliteration. In future studies the epithelial cell specific immune response can be intensified, because hEGP-2 antibodies appeared late and CD8+ T cells proportion remained low in the submucosal infiltrates and the enhancement could be done by pre-transplant induction of anti-hEGP- 2 antibodies and hEGP-2 specific cytotoxic T cells through immunization with hEGP-2. We conclude that the transplantation of hEGP-2 transgenic tracheas provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in lung transplanted patients.
Acknowledgement
The authors thank Henk van de Molen for the assistance in the animal experiments; Dr. Wijnand Helfrich for supplying the purified hEGP-2 protein and Dr. Yijin Ren, from Orthodontic Department of University Medical Center of Nijmegen, The Netherlands for helping with the orthodontic wires.
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