Immunity to collagen V [col(V)] contributes to lung ‘rejection.’ We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well-healed lung grafts have differential susceptibility to anti-col(V)-mediated injury; and expression of the autoimmune cytokines, IL-17 and IL-23, are associated with this process. Adoptive transfer of col(V)-reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post-transplantation. Immunhistochemistry detected col(V) in fresh and well-healed isografts but not native lungs. Hen egg lysozyme-reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL-17 and IL-23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL-17 were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells. These data show that IRI predisposes to anti-col(V)-mediated pathology; col(V)-reactive lymphocytes express IL-17 and IL-23; and anti-col(V)-mediated lung disease is associated with local expression of IL-17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.
We have reported that lung allograft rejection is associated with immunity to a native protein, type V collagen [col(V)] (1–3). Col(V) is a minor collagen, intercalated within type I collagen, a major collagen in the lung (4–6). As such, col(V) is considered as a sequestered antigen in the normal lung and is located in the perivascular and peribronchiolar connective tissues that are the same sites of rejection activity. However, in response to injury, such as ischemia reperfusion injury (IRI) that occurs during the transplantation procedure, potentially antigenic fragments of col(V) could be released locally. These fragments could result in T-cell priming and function as targets of anti-col(V)-mediated disease in lung grafts. Indeed, col(V)-reactive T cells that develop within lung allografts induce ‘rejection-like’ pathology only in isograft, but not in normal lungs (1). Furthermore, our prior study demonstrated that the transplant procedure, itself, was associated with the release of col(V) fragments in bronchoalveolar lavage (BAL) fluid (1). Col(V) fragments were not detected in BAL of any normal rats (1). These data suggest that anti-col(V)-induced lung disease may be a function of the physical state of the lung graft. Fresh isografts may be susceptible, whereas well-healed grafts may be resistant to anti-col(V)-induced pathology. Alternatively, resolution of interstitial remodeling in the post-transplant period may result in altered matrix and ongoing exposure of the lung graft to anti-col(V)-mediated disease.
Many cytokine pathways are associated with autoimmune lung disease and include IL-1β, TNF-α and IL-6 (7–9). However, none of these cytokines have been reported to be active exclusively during autoimmunity. Recently, a new T-cell-derived cytokine pathway has been implicated in the pathogenesis of autoimmune disease (10–17). These cells, termed Th-IL-17 (14), have been reported to be responsible for autoimmunity in the central nervous system (12) and arthritic disease (11). However, the association of IL-17 expression in autoimmune lung disease in general, and col(V)-induced rejection-like pathology in the lung in particular has not been reported previously.
The current study tests the hypothesis that fresh and well-healed lung grafts have differential susceptibility to de novo anti-col(V) immunity, and that this process is associated with altered expression of IL-17. The data show that adoptive transfer of col(V)-reactive cells induced significant rejection-like pathology after transfer into recipients of fresh and well-healed lung grafts. Transcripts for IL-17, were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells, whereas IL-23 (p19) expression tended to be greater in all rats receiving col(V)-reactive cells. Moreover, immunization with col(V) induced expression of IL-17 and IL-23, an inducer of IL-17, in lymph node cells. These data show that prior injury to the lung, such as IRI during transplantation exposes immunogenic col(V) that persists despite histologic resolution of lung injury. In addition, intragraft expression of IL-17 is associated with anti-col(V)-mediated autoimmune lung disease.
Materials and Methods
Pathogen-free, MHC (RT1)-incompatible male rats were used for the study: Wistar Kyoto (WKY, RT1l), Fischer 344 (F344, RT1lv1) rats (250–300 g at the time of transplantation). All rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and housed in the Laboratory Animal Resource Center at Indiana University School of Medicine (Indianapolis, IN) in accordance with institutional guidelines. All studies were approved by the Laboratory Animal Resource Center at Indiana University School of Medicine.
Purified bovine col(V) and purified α1 and α2 chains were a generous gift from David Brand, Ph.D. Isolations procedures for col(V) were reported previously (1–3,18). Hen egg lysozyme (HEL) was purchased from Sigma (St. Louis, MO).
Immunization with col(V) or HEL
WKY rats were immunized at the base of the tail with 200 μg of col(V) or HEL (control) emulsified in 200 μL of complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, MI). To boost the initial immunization, 21 days after CFA, rats received an injection of 200 μg of col(V) emulsified in 200 μL of incomplete Freund's adjuvant (IFA) (Difco Laboratories) at the base of the tail. Ten days after boosting, rats were sacrificed, inguinal and mesenteric lymph nodes harvested and individual lymph node lymphocytes isolated. In brief, lymph nodes were mechanically digested by mincing with scissors in media (RPMI 1640, Invitrogen, Grand Island, NY), passed through nylon wool columns to remove large particles, and washed. Any remaining RBC's were lysed with ammonium chloride. Lymph node cells were resuspended in 1% sterile (PBS) prior to adoptive transfer.
Individual lymph node cells isolated from normal rats as well as rats immunized with col(V) and HEL were stained with anti-rat CD3, CD4, CD8, NK cell, CD62L, α4β1, integrin, ICAM-1, VCAM and LFA-1 antibodies (BD Pharmingen, San Diego, CA; and Biolegend, San Diego, CA). All mAbs were added at 0.2–0.5 mg per 1 × 106 cells. Following staining with mAb, cells were washed twice with HBSS containing 0.1% BSA (HBSS/BSA), the cells were then fixed with 1% paraformaldehyde. The cells were resuspended in HBSS/BSA and analyzed using a FACSCalibur cytofluorograph (BD Biosciences, Mountain View, CA).
Immunostaining was performed on 5-μm thick paraffin embedded tissue sections. Preliminary studies utilized sections of rat liver, skin and lung to determine optimal conditions for staining. Negative control stains were utilized in all instances. After dewaxing and hydration slides were rinsed with TBS, and digested by a 5-min incubation in proteinase K (DakoCytomation). After rinsing and incubation in 3% HSO2 for 10 min, slides were incubated with primary rabbit anti-rat col(V) antibodies or control Ab (1:40 dilution) (Chemicon International, Temecula, CA) for 60 min, and rinsed. Antibody detection was performed by a 30-min incubation with biotinylated donkey anti-rabbit antibodies, rinsing then incubation with streptavidin-HRP (Dako) for 30 min and developed by 5-min incubation with DAB (Dako). Slides were then rinsed and counterstained with hematoxylin. Immunohistochemistry using the secondary antibody, alone, were controls for these studies. Tissues were then examined by light microscopy.
Lymph node lymphocytes (3 × 105/well) obtained from either col(V) or HEL immunized WKY rats were plated in U-bottom 96-well microtiter plates (Costar) with 1.5 × 105 irradiated WKY splenocytes (antigen presenting cells [APC]) in 200 μL of complete media. These cells were cultured without or with varying quantities of intact, or α1 or α2 chains of col(V). Eighteen hours prior to the end of a 4-day coculture cells were pulsed with 3H-thymidine. Proliferation was determined from the mean ± SEM counts per minute of 3H-thymidine incorporation in triplicate cultures.
The orthotopic transplantation of left lung isografts (WKY→WKY) was performed as previously reported (1–3,19). All transplantation procedures were performed by S.Y., T.M. and T.I. WKY→WKY isografts do not develop pathologic lesions at any time point post-transplantation (19). Survival exceeded 90% in all transplantation groups. No immunosuppressive therapy was given at any time during the experimental period.
Preliminary studies determined that lymphocytes isolated from inguinal lymph nodes of immunized rats proliferated greatest in response to their respective immunogens compared to lymphocytes isolated from nodes more distal to the immunization site. Accordingly, inguinal node lymphocytes were utilized for all adoptive transfer studies. WKY rats received 2 × 107 lymphocytes from col(V)- or HEL-immunized rats by tail vein injection 24-h prior to transplantation of WKY lungs (time ‘0’), or 30 days postisograft transplantation.
Delayed type hypersensitivity
Delayed type hypersensitivity (DTH) responses were performed as reported previously (16–19). Six days after lung transplantation 15 μg of col(V) and HEL in 30 μL of 1% PBS (diluent) into the right pinnae of recipient rats by s.c. injection using a 30-gauge needle. The left pinnae received an equal volume of diluent and served as the control site. Naive WKY rats were negative controls. The ear thickness was measured with a micrometer caliper (Mitutoyo, Field Tool Supply, Chicago, IL) in a blinded fashion immediately before and 24 h after injection. Ag-specific DTH response was calculated according to the following formula: specific ear swelling = (right ear thickness at 24 h − right ear thickness at 0 h) − (left ear thickness at 24 h − left ear thickness at 0 h) × 10−3 mm. All data are reported as the mean ± S.D. of triplicate measurements.
Collection of BAL and serum
BAL fluid was obtained from native and transplanted lungs as previously reported (1–3). Cell-free BAL supernatants obtained from centrifuged specimens were stored at −80°C until use. Serum was obtained by centrifugation of blood obtained by cardiac and venous puncture.
Five days post-transplantation, native and transplanted lungs from each group were harvested, fixed, sectioned and stained. Grading for rejection pathology was performed in a blinded fashion by a pulmonary pathologist (O.W.C.) using standard criteria developed by the Lung Rejection Study Group as previously reported (1–3,20).
RNA was extracted from lung, spleen and lymph nodes cells using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Approximately 2 μg of total RNA was reverse transcribed into cDNA using the BioRad iScript kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Amplification mRNA transcripts were performed by using the Qiagen Mastermix kit (Qiagen, Valencia, CA). Transcripts for IL-1β, IL-6 and TNF-α were detected in lung parenchyma and spleen by RT-PCR using the MultiGene-12™ RT-PCR Profiling Kits (SuperArray, Frederick, MD) according to manufacturer's instructions. IL-17 (477 bp), IL-23p19 (645 bp), and IL-12p35 (800 bp) transcripts were identified with the following primers: for IL-17, 5′- ACA GTG AAG GCA GCG GTA CT-3′ (forward) and 5′- GCT CAG AGT CCA GGG TGA AG-3′ (reverse); for IL-23 p19, 5- TCA CAG GGG AGC CTT CTC TA-3 (forward) and 5′- GGC ACT AAG GGC TCA GTC AG-3′ (reverse) and for IL-12p35, 5′- CGG CCA GAG AAA AAT TGA AA-3′ (forward) and 5′- CTA CCA AGG CAC AGG GTC AT-3′(reverse). Amplification was performed as follows: 35 cycles (IL-17 and Il-23p19) or 38 cycles (IL-12p35) under the following conditions: 1 min at 94°C for denaturation, 1 min at 64°C (IL-17 and Il-23p19) or 55°C (IL-12p35) for primer annealing and 1 min at 72°C for primer extension. Reaction products were run on a 1% agarose gel in TAE. Images were analyzed using a ChemiImager 4400 low-light imaging system (Alpha Innotech, San Leandro, CA).
Quantitation of cytokine proteins
IL-1β, IL-6 and TNFα, were measured unconcentrated BAL and serum by enzyme-linked immunosorbent assay (ELISA) using Cytoscreen immunoassay kits (BioSource International, Camarillo, CA) as per manufacture's protocol.
All data are expressed as the mean ± SEM. Differences between groups were determined by analysis of variance. Results were considered statistically different if p-values were <0.05.
Our prior reports demonstrated that lung allograft infiltrating T cells developed strong immunity to col(V) (1). To determine if immunity to col(V) could be induced in the absence of alloimmunity, and if specific immunity varied with specific col(V) alpha chains, WKY rats were immunized with intact col(V) or purified col(V) α1 or α2 chains in CFA and IFA followed by isolation of cells from draining inguinal lymph nodes. Figure 1 shows α1(V) and α2(V) chains each induced dose-dependent proliferation in lymph nodes cells isolated from immunized rats. Notably, the proliferative response to the intact protein was greater than that observed for each alpha chain. Accordingly, all subsequent experiments were performed with intact col(V).
We next determined if adoptive transfer of col(V) or HEL-reactive cells transferred DTH to each antigen. Since IRI may be associated with inflammation that could alter the DTH response, we examined the DTH to each antigen in fresh isografts on the 1st day post-transplantation (day 0), and day 30, a time when all IRI induced inflammation has resolved. Figure 2 shows that normal WKY rats or isograft recipients studied at day 0 or day 30 do not have significant DTH responses to either antigen. However, transfer of col(V) or HEL-reactive cells to isograft recipients on day 0 or day 30 induce vigorous DTH responses to each antigen. Moreover, this response was antigen specific as there was no significant DTH to HEL in rats receiving col(V)-reactive cells, or DTH to col(V) in rats receiving HEL-reactive cells.
In our prior studies examining col(V)-reactive cells that developed in lung allograft infiltrating lymphocytes, adoptive transfer of these cells induced rejection-like pathology only in isograft but not in normal lungs (1). These data indicated that the transplantation procedure, itself, which is associated with IRI may render the lung graft susceptible to anti-col(V)-mediated injury. To eliminate the effect of alloimmunity in the disease process, and to use IRI as a model of lung injury, lymph node cells from col(V) or HEL-immunized rats were adoptively transferred into WKY lung isograft recipients at different time points post-transplantation. Our prior studies have demonstrated that IRI resulting from the transplant procedure is manifested by peripobronchiolar and perivascular edema and scant neutrophilic infiltrates. All pathologic lesions begin to resolve 72-h post-transplant (19). In addition, these lungs have normal histology for at least 16 months after the transplant procedure (19).
We next determined if adoptive transfer of col(V) or HEL-reactive cells would induce pathology in isograft and native lungs. Adoptive transfer of col(V)-reactive cells induced perivascular and periobronchiolar mononuclear cell infiltrates in lung isografts (Figure 3). These lesions are identical that seen during clinical and experimental lung allograft rejection and are analogous to ‘mild-to-moderate’ rejection (grade 2–3) pathology as shown in Figure 3. Adoptive transfer of HEL-reactive cells did not induce pathologic lesions in any rats at the same time point (Figure 3). No lesions were seen in the native lungs of any group.
Data showing that adoptive transfer of col(V)-reactive cells induce disease in fresh isograft lungs but not native lungs suggested that the physical condition of the lung may modulate anti-col(V)-mediated disease. Accordingly, a well-healed graft may be less likely to be a risk for col(V)-mediated injury. To answer this question, col(V)-reactive T lymphocytes were adoptively transferred to isograft recipients 30 days post-transplantation, a time when the lung is normal histologically. Interestingly, transfer of col(V)-reactive cells induced extensive perivascular and peribronchiolar mononuclear infiltration identical to moderate-severe acute allograft rejection grade 3–4 (Figure 4). No pathologic lesions were observed in native lungs or lungs of isograft recipients that received HEL-reactive cells at the same time point.
The presence of col(V) and absence of HEL antigens in the lung should be sufficient to explain the differential effects of col(V) and HEL-reactive cells to induce disease. However, another explanation could be related to lymphocyte subsets present in cells used for adoptive transfer. Phenotypic analysis revealed comparable quantities of CD4+ and CD8+ lymphocytes in the lymph node cells isolated from col(V) and HEL-immunized rats (Figure 5). Moreover, CD4+ and CD8+ cells from both groups were similar in expression of CD62L, LFA-1, VCAM, ICAM-1 and α4β1 integrin (Figure 6) that are adhesion molecules reported to be involved in lymphocyte migration to mediastinal lymph node tissues and bronchus associated lymphoid tissue (21). Accordingly, these data suggest that the differential ability of col(V) and HEL-reactive cells to induce lung pathology was not due altered lymphocyte subsets, or differential expression of adhesion molecules utilized for trafficking.
We next determined if the susceptibility of fresh and well-healed isografts to anti-col(V)-reactive T cells was related to injury-induced exposure of col(V). Immunohistochemistry was utilized to localize col(V) in native and isograft lungs of untreated rats at day 0 and day 30. As expected col(V), a sequestered antigen, was not readily detected in the perivascular and peribronchiolar tissues of native lungs. In contrast, it was detected readily in these same tissues isografts at day 0 rats (Figure 7A,B). Notably, not only was col(V) staining detected strongly in the perivascular and peribronchiolar tissues but was also detected in the insterstitium of the alveolar space (Figure 7B). In addition, the sections show active peribronchiolar and perivascular inflammation which is an expected early finding of IRI. Similar to day 0 rats, col(V) was not readily detected in native lungs of day 30 rats (Figure 7C),. Although there was no inflammation in the 30-day isograft lungs, col(V) was readily detected in peribronchiolar and perivascular connective tissues (Figure 7D). Collectively, these data show that IRI induces early and sustained exposure of col(V) in isograft lungs.
Inflammatory disorders, including those involving the lung have been associated with production of proinflammatory cytokines such as IL-6, IL-1β, TNF-α and IL-17. Therefore, we next determined if isograft lung pathology induced by transfer of col(V)-reactive cells was associated with local expression of these cytokines. Gene transcripts for IL-6, IL-1β and TNF-α were not altered either locally (isograft) or systemically (spleen) in untreated rats or those that received col(V)-reactive cells compared to normal rats (data not shown). Whereas TNF-α and IL-6 protein were not detected, col(V)-reactive cells strongly induced IL-1β in BAL fluid of isografts 5 days after adoptive transfer (p < 0.05) Figure 8A) with a trend to higher levels in serum (Figure 8B). Adoptive transfer of HEL-reactive cells had no effect on these cytokine levels (data not shown).
Recent studies suggest a distinct role for IL-17 in autoimmune diseases (10–17). Since IL-17 is induced by IL-23, then we next determined if both cytokines were induced locally and systemically. IL-23 is a heterodimer comprised a unique p19 subunit and a p40 subunit shared with IL-12. Therefore, to discern expression of IL-23 and IL-12 we determined transcripts unique to each cytokine-p19 for IL-23 and p35 for IL-12. Transcripts for IL-23 (p19) and IL-12 (p35) were not increased in the spleens of any experimental groups (data not shown). However, Figure 9A shows a trend towards higher IL-23p19 expression in isograft lungs of rats that received col(V)-reactive cells at all time points compared to other groups. In contrast, transcripts for IL-17 were upregulated significantly in fresh isografts, i.e. 5 days after adoptive transfer and lung transplantation (Figure 9B). Notably, transfer of col(V)-reactive cells to recipients of well-healed isografts (30 days post-transplant) did not induce up-regulation of IL-17 or IL-23. Transfer of HEL cells did not induce local or systemic expression of any cytokines studied.
We next determined if the transferred cells were a potential source of IL-17 and IL-23. To answer this question, IL-17 and IL-23 (p19) transcript expression were determined in lymph node cells utilized for adoptive transfer that were isolated from immunized rats. Figure 10 shows immunization with col(V), but not HEL, significantly induced IL-17 expression (p < 0.01), and a trend toward greater IL-23 expression in the same cells (Figure 10).
The key findings from the current study are that de novo cellular immunity to a native collagen, col(V), is able to mediate rejection-like pathology in lung grafts in the absence of alloimmunity. In addition, the physical state of the lung determines susceptibility to anti-col(V)-mediated immunity, but even well-healed lungs remain very susceptible to anti-col(V)-mediated injury. Furthermore, intragraft expression of IL-17 is associated with anti-col(V)-mediated autoimmune lung disease in fresh lung grafts.
We have previously reported that lung allograft rejection is strongly associated with cellular immunity to col(V) (1–3). Using an experimental approach similar to the current study, our prior report demonstrated that adoptive transfer of lung allograft infiltrating T cells reactive to col(V) induced rejection-like pathology in the lung (1). However, in the current study col(V)-reactive T cells were developed de novo after immunization with col(V) and the pathogenecity of the cells was demonstrated by their ability to induce mononuclear cell infiltration in lung isogafts. Immunization with col(V), itself, did not induce pathologic lesions in the lungs of immunized rats (data not shown) likely because prior lung injury is required for anti-col(V)-induced pathology. Similar to the prior study, col(V)-reactive T cells did not induce pathologic lesions in lungs of normal rats, but only in lung isografts.
The inability of col(V)-reactive T cells to induce disease in lungs of normal rats could be due to the location of col(V). In the lung, col(V) is a sequestered antigen, intercalated within fibrils of col(I), the major pulmonary collagen (4). As such, immunity to col(V) or recognition of col(V) as an antigen may only occur after lung injury such as that occurring during IRI following lung transplantation. In turn, the injury response may induce the activity of metalloproteinases (MMPs) able to degrade col(V) resulting in release of potential antigenic col(V) fragments. Indeed, we reported that lung allograft rejection in rats is associated with intragraft expression of MMP2 and MMP9 that are capable of degrading col(V) (22). Therefore, it is interesting to speculate that modalities which block MMP activity during IRI ischemia-reperfusion or other forms of lung injury may abrogate the development of immunity to col(V) post-lung transplantation.
The current study shows that a single adoptive transfer of col(V)-reactive cells induces perivascular mononuclear infiltrates in lung isografts analogous to mild-to-moderate lung allograft rejection. In our prior report examining alloimmune-induced autoimmunity to col(V), we demonstrated col(V) fragments were readily detected in isograft BAL fluid in an early period post-transplantation. As such, IRI which is an inevitable consequence of transplantation surgery is involved in exposure of col(V). However, we have shown that this injury pattern, characterized by mild perivascular and peribronchial edema, begins to resolve by the 3rd post-opoterative day. By day 7, the isografts are normal and maintain that condition for at least 16 months post-transplantation. A fundamental question then becomes why the well-healed grafts remain susceptible to anti-col(V)-induce pathology. We hypothesized that healing of the perivascular and peribronchiolar tissues that are injured in response to IRI may result in disorganized matrix such that col(V) epitopes remain exposed and no longer sequestered. Adoptive transfer of col(V)-reactive cells could easily recognize their antigen and, in turn, mediate lung pathology. Indeed, immunohistochemical studies in Figure 7 confirm that IRI results in prolonged exposure of col(V) in the lung insterstitium.
It is also important to note the striking similarity of anti-col(V)-induced lung pathology to that observed in acute lung allograft rejection. Perivascular and peribronchiolar monluclear cell infiltrates are present in both lesions. We have reported that clinical lung allograft rejection is associated with anti-col(V) immunity (Burlingham and Wilkes, submitted). Therefore, it is interesting to speculate that the lesions consistent with acute lung allograft rejection may in part represent autoimmunity to col(V). The interplay of alloimmunity and autoimmunity is supported further by a recent report from Chalasani et al. (23) demonstrating that col(V) gene expression was highly associated with the ability of alloreactive T cells to induce cardiac allograft rejection. These data also suggest that autoimmunity to col(V) may not be limited to pulmonary diseases.
Many pulmonary inflammatory disorders, including autoimmune-related lung disease, have been associated with up-regulation of pro-inflammatory cytokines. This is consistent with data in the current study showing that perivascular and peribronchiolar mononuclear cell infiltrates induced by col(V)-reactive lymphocytes were associated with increased expression of IL-1β. However, very recent reports specifically link two cytokines, IL-17 and IL-23, to the development of autoimmune disease (10–17). IL-23 is a heterodimeric cytokine composed of a unique p19 subunit, and a p40 subunit shared with IL-12. In contrast to IL-12 that induces Th1 cell development, IL-23 induces growth of pathogenic CD4+ T-cell population characterized by the production of a few cytokines, most notably IL-17. These cells, termed Th IL-17 by Langrish et al. (14), have been implicated in pathogenesis of autoimmune diseases of the central nervous system and arthritis in rodents (11,12). In the current study adoptive transfer of col(V), but not HEL, reactive cells induced a trend in toward higher local (isograft) expression of IL-23. This trend was specific for IL-23 and did not involve IL-12 as only p19 was increased without alteration in the IL-12-specific p35 subunit. However, only IL-17 was increased significantly in fresh isografts post-transfer of col(V)-reactive cells. Interestingly, transfer of these cells to well-healed isografts, although inducing lung pathology, did not induce increased IL-17. The difference in IL-17 expression between fresh and well-healed grafts may be due to alterations in other cytokines that could induce IL-17 in the early transplant period. Data shown in Figure 10 suggest that a potential source of IL-17 and IL-23 could be the cells utilized for immunization. It is interesting to observe that an autoantigen, col(V), and not an irrelevant antigen such as HEL, induce expression of IL-17 and IL-23 transcripts in lymph node cells. These studies are limited by the current lack of commercially available reagents to examine IL-17 and IL-23 protein expression in rats. Future studies will determine the specific cell type producing these cytokines and their specific role in the pathogenesis of col(V)-mediated lung disease.
In summary, we hypothesize that immunity to col(V) may be a final common pathway to the development of autoimmune and alloimmune lung disease. In addition, IL-17 and IL-23, cytokines recently linked to autoimmune diseases in nonpulmonary tissues, are also induced in response to anti-col(V)-mediated lung disease. In addition, these studies could also help to explain how different forms of lung injury, such as infection, could trigger ‘rejection’ in lung allografts. For example, remodeling induced by inflammation and subsequent healing could result in prolonged exposure of col(V) epitopes. Accordingly, therapies that block the remodeling process could impact the clinical course of autoimmune and alloimmune pulmonary diseases post-lung transplantation.