INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Advances in immunosuppressive therapy have dramatically improved short-term survival after lung transplantation, which is the sole curable therapy for numerous end-stage pulmonary diseases. However, more than 60% of the recipients develop bronchiolitis obliterans (BO), which is thought to be a major form of chronic rejection, within the first 5 postoperative years, and this disease process severely limits long-term survival (1). BO is histologically characterized by mononuclear cell infiltration, accompanied by an increase in the number of mesenchymal cells and their tissue products in the lamina propria, followed by disappearance of the epithelium and subsequent fibrous occlusion of the airway lumen (2).

Although the pathogenesis of chronic rejection is not fully understood, T-cell-mediated immunity, especially the CD4⁺ cell-related immunological response, is thought to play an important role in this process. It is suggested that major histocompatibility complex (MHC) class II antigens derived from allografts are expressed on the surface of both donor and recipient antigen-presenting cells (APCs), and induce activation and proliferation of CD4⁺ T cells. This activation enables the elaboration of various cytokines that recruit and stimulate other immune cells such as macrophages. Macrophages not only function as APCs and phagocytes, but also are a major source of proinflammatory cytokines and growth factors. In time, these immune responses cause graft injury and ultimately lead to the activation of pathological repair processes that result in fibroproliferation and dysfunction of transplanted organs (3). Many studies have shown that a large number of T cells and macrophages appear in rejected organs, and therefore, migration of these cells to the grafted organ might be one of the important steps in this disease process (4, 5).

RANTES (regulated upon activation, normal T cell expressed and secreted), a member of C-C chemokine family, is a chemoattractant for memory T cells, monocytes, and eosinophils (6, 7). Recent studies have revealed that RANTES is involved in many inflammatory diseases, for example, bronchial asthma, delayed-type hypersensitivity reactions, viral infections, arthritis, chronic eosinophilic pneumonia, and idiopathic interstitial pneumonia (8). Several investigators in the field of transplantation have also demonstrated that RANTES is highly expressed and/or produced in rejected kidneys after transplantation, coronary arteries of transplant-associated accelerated athelosclerosis, allogeneic skin grafts, and bronchoalveolar lavage fluids or biopsy specimens from lung grafts with cytomegalovirus (CMV) pneumonitis or allograft rejection (12). These findings imply that RANTES plays a role in various kinds of disease processes including allorejection, where inflammatory reactions are predominant.

Rodent heterotopic tracheal transplantation models have been utilized for the investigation of BO after lung transplantation because of their histopathological similarities. We previously characterized this model in rats and found that lymphocytic infiltration appears as a precursor to fibrous airway obliteration (FAO) in allografts (16). Neuringer and coworkers also observed a large number of T cells and macrophages in advance of luminal obliteration in a mouse BO model (17). In addition, we have recently demonstrated that RANTES mRNA is up-regulated in the same rat model (18). We speculate that the increased number of lymphocytes and macrophages in allografts is related to the up-regulation of RANTES, and, consequently, contributes to the development of BO.

In the current study, we administered anti-RANTES antibody to rat tracheal allografts to neutralize the action of RANTES. Recombinant RANTES was also infused to isografts to determine if overexpression of RANTES alone could induce FAO. We found that anti-RANTES antibody inhibited CD4⁺ cell migration and prevented the subsequent fibroproliferative process in the allografts. However, exogenous RANTES delivery did not lead to FAO in the isografts. These findings suggest that RANTES plays an important role in the allotransplant-induced obliterative injury in the airway.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Male Brown-Norway and Lewis rats weighing 200 ~ 300 g were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN) and Charles River Canada Inc. (St. Constant, PQ, Canada), respectively. Animal care was provided according to NIH guidelines (Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, Revised 1985), the Guide to the Care and Use of Experimental Animals formulated by the Canadian Council on Animal Care, and the policies stated by the Toronto General Hospital's Animal Care Committee.

Donor and Recipient Operations

Heterotopic tracheal transplantation was performed as previously described (16, 18, 19). Briefly, following pentobarbital sodium (80 mg/ kg, intraperitoneally) administration, tracheas of Brown-Norway rats were excised from the larynx to the carina via median neck incisions. The tracheal graft was divided into two segments with approximately nine cartilaginous rings each. In the treatment groups, the tubing tip of a miniosmotic pump (Alzet model 1007D or 2002; Alza Corp., Palo Alto, CA) was fixed to the edge of the graft with an 8-0 Proline (ETHICON, Inc., Somerville, NJ) to prevent possible interference of luminal obliteration by the tubing itself. The graft was soaked in saline prior to the transplantation.

Recipients (Brown-Norway [isograft] or Lewis rat [allograft]) were anesthetized with an intraperitoneal injection of acepromazine (2.5 mg/kg) and ketamine (75 mg/kg). Cefazolin sodium (5 mg) was administered subcutaneously. Two subcutaneous pouches were made on the back of the recipient. Each graft with or without the pump was placed into a subcutaneous pouch. The skin incisions were closed with 5-0 Vicryl sutures (ETHICON, Inc.).

The tracheal graft was removed on the prescheduled posttransplant days. The middle one-third of the tracheal segment was fixed with 10% buffered formalin. The other portions were rapidly frozen with liquid nitrogen and then stored at  80° C.

Anti-rat RANTES Antibody and Recombinant Rat RANTES

Five hundred micrograms of polyclonal anti-rat RANTES antibody (Autogen Bioclear UK Ltd., Mile Elm, Wiltshire, UK) or nonspecific rabbit immunoglobulin G (IgG) (Sigma-Aldrich Canada LTD., Oakville, ON, Canada) was reconstituted in 500 µl of saline and infused continuously to allograft tracheas at a rate of 12 µl/d for 7 or 14 d using a miniosmotic pump to allografts. Model 1007D was used for 7-d infusion and model 2002 for 14 d.

Recombinant rat RANTES (1 µg) (Biosource International, Camarillo, CA) was dissolved in 1 ml of phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA). This RANTES solution or vehicle (PBS with 0.1% BSA) was also delivered at 12 µl/d, for either 7 or 28 d to isografts. In the 28-d infusion study, the pump (model 2002) was replaced on Day 14.

Experimental Groups

All recipients were transplanted with either a tracheal isograft or allograft. The allograft study group was divided into three subgroups as follows. Co-A (n = 5): allotransplant controls without infusion; IgG (n = 5): allotransplant with nonspecific rabbit IgG infusion; Ab (n = 5): allotransplant with anti-RANTES antibody infusion (Figure 1). Grafts were locally infused with designated solutions for 7 or 14 d except for those in the Co-A subgroup, and then harvested on either Day 7 or Day 21, respectively.

View larger version (28K): [in this window] [in a new window]   Figure 1.   Experimental protocols. Tx = transplantation.

Similarly, three subgroups were designed in the isograft study group. Co-I (n = 4): isotransplant controls without infusion; Ve (n = 4): isotransplant with vehicle infusion; Ra (n = 4): isotransplant with RANTES infusion. Grafts in this group were removed on Day 7 or Day 28 after tracheal transplantation, and the infusion continued until the day of tracheal removal.

RANTES Enzyme-linked Immunosorbent Assay (ELISA)

To determine RANTES stability at body temperature, recombinant rat RANTES (1 µg/ml in PBS with 0.1% BSA) was put into a microcentrifuge tube and kept at 37° C in an incubator for the designated period. Immunoreactive amounts of RANTES were measured using an ELISA kit (Biosource International, Camarillo, CA). The concentrations of RANTES in minipumps before and after delivery were also measured by ELISA.

Immunohistochemistry

Frozen tracheal specimens were embedded in O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA), cut to 5-µm sections, placed on poly-L-lysine-coated slides, air dried for 2 h at room temperature, and fixed with acetone for 15 min. After blocking with Protein Block Serum-Free solution (DAKO Diagnostic Canada Inc., Mississauga, ON, Canada), the sections were incubated with diluted polyclonal anti-RANTES (1:50) for 2 h or monoclonal anti-rat CD4 (1:250) (Pharmingen Canada, Mississauga, ON, Canada) for 30 min. The secondary antibody and alkaline phosphatase conjugation steps were performed according to the instruction of LSAB 2 Alkaline Phosphatase Kit for Rat Specimens (DAKO Diagnostic Canada Inc.). The color reaction was developed with the addition of Fast Red Substrate System (DAKO Diagnostic Canada Inc.) including an endogenous alkaline phosphatase activity blocker (Levamisole; DAKO Diagnostic Canada Inc.). Finally, slides were counterstained with hematoxylin. Slides were also incubated with PBS containing 0.1% BSA and isotype-specific IgG from rabbit or mouse as negative controls.

Morphometry

The formalin-soaked middle portion of the tracheal segment was sliced in 4-µm slices for hematoxylin and eosin or elastic trichrome staining after paraffin embedding. Sections were utilized for computerized morphometry as described by Reichenspurner and coworkers (20). Briefly, images of the section taken with a microscope (Nikon Labophot; Nikon Inc., Melville, NY) and an attached video camera (Pulnix model TMC-7I; Pulnix America Inc., Sunnyvale, CA) were transferred to a C-imaging 1280 morphometric system (Compix Inc., Cranberry Township, PA). The luminal circumference, the epithelial lining, the margin of patent lumen, and the margin within the cartilage were traced with manual drawing and each length or area was calculated by the computer system. The percentage epithelial loss was expressed as (1  the length of epithelial lining/the length of luminal circumference) × 100%. The percentage luminal obliteration was expressed as (1  the area of patent lumen/the area of inside cartilage) × 100%.

Quantification of CD4⁺ Cells

CD4⁺ cells were quantified as the total number of positively stained cells in the epithelial and subepithelial spaces in eight high-power fields around the tracheal section. Each field was chosen at 0, 45, 90, 135, 180, 225, 270, and 360° starting from the center of membranous portion. The picture was captured with the combination of a microscope (Nikon E 1000, Nikon Inc.), a digital video camera (DEI-TD 750, Optronics, Goleta, CA), and Scion Image version 1.6^a (Scion; Frederick, MD). The counting was performed by two investigators in a blinded fashion.

Statistical Analysis

Data are expressed as mean values ± standard deviation of the means. One-way ANOVA or Kruskal-Wallis test (if normality tests failed) was used for analysis of differences between groups. Multipair comparisons were performed using Student-Newman-Keuls test. Data are considered statistically significant if p values are less than 0.05. All analyses were performed using SigmaStat version 1.0 statistical software (Jandel Scientific, San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

RANTES Protein Expression in Grafted Tracheal Tissue

Using semiquantitative RT-PCR, we have demonstrated that RANTES mRNA was expressed in both rat tracheal allografts and isografts, and it was persistently up-regulated in allografts (18). In this study, we first determined the expression of RANTES protein and its localization in graft tissues by immunohistochemistry. RANTES protein was observed in both allograft (Day 7 and Day 21) and isograft sections (Day 7 and Day 28). RANTES positive staining was found around infiltrating mononuclear cells as determined by their shape and localization. Epithelial cells were also stained. Photographs of Day 7 control allograft and Day 7 control isograft sections are presented in Figures 2A and 2B, respectively. When the first antibody was replaced with nonimmune rabbit IgG, no staining was observed (Figure 2C).

View larger version (91K): [in this window] [in a new window]   Figure 2.   RANTES immunohistochemistry staining. Positive staining was found around mononuclear cells (small arrows) and epithelial cells (large arrow) in the allograft (A) and isograft (B) on Day 7 (original magnification: ×400). When the Day 7 allotracheal section was incubated with isotype-specific IgG (negative control), no positive staining was found (C ) (original magnification: ×400).

Effect of Anti-RANTES Antibody on the Allograft

To identify the role of RANTES in airway lesions of allografts, neutralizing anti-RANTES antibody was continuously administered to allografts. The effect of the antibody was examined on Day 7 and Day 21, which corresponds to the day of peak lymphocyte infiltration, or the day of complete fibrous obliteration in allografts, respectively (16).

Anti-RANTES antibody reduced the number of infiltrating CD4⁺ mononuclear cells. RANTES up-regulates migration of memory T cells and macrophages that express CD4 molecules on their surfaces (21). To determine the neutralizing effect of anti-RANTES on allografts, CD4⁺ cell infiltration in the epithelial and subepithelial spaces of the transplanted trachea was evaluated by immunohistochemistry staining and morphometric analysis. The monoclonal antibody we used reacts with CD4⁺ T-lymphocytes activated in vitro and in vivo (22, 23). Its specificity and reactivity have been tested by the manufacturer with immunofluorescent staining and flow cytometric analysis, and immunohistochemical staining. For all three groups, CD4⁺ cell counts were prominent on Day 7 and then decreased to approximately half-maximal levels on Day 21. At both time points, the anti-RANTES treatment group showed less than half the number of CD4⁺ cells when compared with the Co-A and IgG groups (p < 0.05) (Figure 3). When the first antibody was replaced with isotype-specific IgG, no positive staining was observed (Figure 3D).

View larger version (100K): [in this window] [in a new window]   Figure 3.   Anti-RANTES antibody reduced infiltration of CD4+ cells in allografts. Representative microphotographs of allografts on Day 7 are presented to demonstrate the infiltration of CD4+ cells (A-D: original magnification: ×400). (A) Allograft control (Co-A); (B) nonimmune IgG (IgG); (C  ) anti-RANTES antibody (Ab). Arrows in the photographs are used to show examples of positive stained cells. During the staining process when the first antibody was replaced with isotype specific IgG, no positive staining was found (D). A lower magnification photograph (×200) from the antibody-treated group is used as an example to show that the higher magnification photographs were taken under the epithelial cell layer (E ). The CD4+ cell counts at Day 7 and Day 21 in the allograft groups are summarized (F  ). The CD4+ cells are expressed as the sum of positive cells in the epithelial and subepithelial spaces in eight high-power fields around the section of the trachea. Each point represents the mean ± SD of five samples. *p < 0.05 versus either the Co-A or IgG group.

Anti-RANTES antibody preserved luminal patency, but not epithelial cell lining. On Day 7, all allograft groups demonstrated mild luminal obliteration that was due to subepithelial thickening and granulation tissue growth in the tracheal lumen. The Co-A and IgG groups developed severe FAO by Day 21, while the Ab group maintained well-preserved luminal patency (Figure 4).

View larger version (100K): [in this window] [in a new window]   Figure 4.   Anti-RANTES antibody prevented the development of luminal obliteration in allografts. Representative elastic trichrome-stained sections of allografts on Day 21 are presented (A: Co-A; B: IgG; C: Ab) (original magnifications: ×100 and ×20 for inset). Luminal obliteration of allografts at Day 7 and Day 21 was quantified by computerized morphometric analysis (D). Each point represents the mean ± SD of five samples. * p < 0.05 versus either the Co-A or IgG group.

The loss of epithelial lining was less than 5% in the Ab group and over 20% in the Co-A and IgG groups on Day 7. In the Ab group, there still maintained a small portion of lumen covered with a flattened metaplastic epithelium until Day 21, whereas the Co-A and IgG groups lost all of the epithelial lining by this time point. These changes, however, did not reach significance between groups (Table 1).

As for histopathological architecture in the subepithelial space, all three groups completely lacked submucosal glands and developed fibroproliferation in this space by Day 21.

Effect of Exogenous RANTES on the Isograft

To determine whether RANTES alone has the ability to induce FAO, isografts were administered exogenous RANTES. In this study section, 7 or 28 d was chosen as the RANTES-infusion period. Our previous observation demonstrated that isografts regenerate almost normal tracheal structure within 7 d (16). In a preliminary study, isografts given RANTES or vehicle solution for 14 d did not develop FAO on Day 21. Instead, the epithelial lining was regenerated on most of the luminal surface. To ensure the effect of exogenous RANTES on isografts, we decided to administer RANTES continuously for a longer period of 28 d.

RANTES was stable at 37° C for 14 d. Before performing RANTES infusion studies, RANTES stability was examined at 37° C. The immunoreactivity of diluted recombinant rat RANTES was maintained up to 14 d (data not shown). We also measured RANTES concentrations in minipumps before and after 14-d delivery in vivo, which remained at the same level (data not shown).

RANTES led to the accumulation of CD4⁺ infiltrating mononuclear cells. CD4⁺ cells in isografts were quantified to define whether exogenous RANTES enhances CD4⁺ mononuclear cell migration. On Day 7, the Co-I group regenerated almost normal tracheal architecture while both the Ve and Ra groups exhibited ischemic necrosis and inflammatory changes. A larger number of CD4⁺ cells were observed in the Co-I group than in either the Ve or Ra group (p < 0.05). However, the CD4⁺ cells in the Co-I group were diffusely distributed in both membranous (Figure 5A) and cartilage (Figure 5C) portions, whereas those in the Ve and Ra groups were primarily localized to the membranous portion and the intraluminal granulation tissue (Figure 5B), but very few in the cartilage portion (Figure 5D). On Day 28, the number of CD4⁺ cells in the Ra group increased sharply (Figure 5G) and was found to be double that of either the Co-I (Figure 5E) or Ve (Figure 5F) group (p < 0.05) (Figure 5).

View larger version (111K): [in this window] [in a new window]   Figure 5.   Recombinant RANTES-modulated CD4+ cell migration in isografts. Representative microphotographs of CD4 immunohistochemistry staining of isografts on Day 7 and Day 28 are presented to show the CD4+ cell distribution. At Day 7, the isograft control (Co-I) group showed diffusely distributed CD4+ cells in both the membranous portion (A) and the cartilaginous portion (C ). The RANTES-treated (Ra) group had dense CD4+ cell infiltration in the membranous portion and in the granulation tissue grown in the lumen (B), but not in the cartilaginous portion (D). At Day 28, CD4+ cells were more in the Ra group (G) compared with the Co-I (E ) and the vehicle infusion (Ve) group (F ) (original magnifications: ×100 for A and B; ×400 for C-G). An inset is presented to show the cartilage and membranous fractions of the tracheal sections. The CD4+ cell counts at Day 7 and Day 28 in the isograft groups are summarized (H ). Each point represents the mean ± SD of 4 samples. * p < 0.05 versus either the Ve or Ra group. ** p < 0.05 versus either the Co-I or Ve group.

RANTES did not promote FAO but induced mild subepithelial thickness. The Co-I group regenerated complete epithelial coverage with minimum luminal obliteration on Day 7. In contrast, the Ve and Ra groups showed moderate epithelial lining loss (p < 0.05) and partial airway narrowing, which was caused by subepithelial thickness and granulomatous plugging. The Ra group showed significantly increased luminal obliteration than the other isograft groups at this stage, implying that exogenous RANTES accelerated these early stenotic changes. On Day 28, granulomatous changes in the Ra and Ve groups disappeared. Luminal obliteration of the Ra group, however, was still significantly higher than that of either the Co-I or Ve group (Figure 6). This change in the Ra group was restricted to the subepithelial space and did not involve the generation of fibrous tissue within the lumen. Other than the increased subepithelial thickness, the Ra group showed almost normal tracheal architecture, as did the other isograft groups (Figure 6 and Table 1).

View larger version (82K): [in this window] [in a new window]   Figure 6.   Recombinant RANTES did not induce FAO in isografts. Representative microphotographs of hematoxylin/eosin-stained sections of isografts on Day 28 are presented. All isograft groups regenerated respiratory epithelium (A: Co-I; B: Ve; C: Ra) (original magnification: ×200). Luminal obliteration of isografts at Day 7 and Day 28 is shown (D). Each point represents the mean ± SD of four samples. * p < 0.05 versus either the Co-I or the Ve group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Since the heterotopic tracheal transplant murine model was reported by Hertz and coworkers in 1993 (24), many studies have used this model or a similar rodent model to investigate the pathogenesis of BO after lung transplantation. For example, we have previously shown that rat tracheal isografts are able to regenerate epithelium and retain luminal patency, whereas allografts become obliterated by fibrous tissue following epithelial destruction and lymphocytic infiltration (16). These results suggest that the FAO lesion in allografts is related to the immunological mismatch. Kelly and coworkers (25) and Neuringer and coworkers (17) reported that tracheal allografts placed in wild type recipients showed severe T-cell infiltration prior to FAO. However, those in severe combined immunodeficient (SCID) mice did not develop FAO but regenerated normal tracheal structure (17, 23). We have then demonstrated that manipulation of the immune response can protect against the development of FAO. Using adenovirus-mediated gene transfection of interleukin 10 (IL-10), which down-regulates various T-cell and antigen-presenting cell (APC) functions, we were able to prevent the development of FAO in tracheal allografts (19). These studies imply that the T-cell-mediated immune response is crucial in the process of FAO in the rodent heterotopic tracheal transplant model, similar to other forms of allorejection.

T-cell immunity is regulated by various cytokines and chemokines. RANTES, a C-C chemokine, is a major chemoattractant for T cells, eosinophils, and macrophages. Unlike other chemokines, it also acts as an activator of T cells (26). RANTES, therefore, might have an important role in the development of T-cell-mediated immunity by facilitating migration and activation of immune cells. Moreover, the involvement of RANTES in T-cell immunity implicates a role in allorejection. Indeed, several clinical and animal studies have shown up-regulation of RANTES mRNA and/or protein in rejected transplant organs (12, 27). We recently demonstrated that mRNA levels of Th1 cytokines (IL-2 and interferon- [IFN-]) and C-C chemokines (MCP-1 and RANTES) were highly and persistently expressed in the tracheal allograft (18). This suggests that alloimmune responses in this model are ongoing at least until the time that FAO is almost completed (21 d after transplant). Based on these results and reasoning, we have focused on the function of RANTES in the evolution of FAO in the rat heterotopic tracheal transplant model.

In the allograft study, we found that both control and nonspecific IgG-infused groups showed a marked number of CD4⁺ infiltrating mononuclear cells (CD4⁺ T cells and macrophages), moderate luminal obliteration, and mild epithelial loss on Day 7. The number of CD4⁺ cells in these groups was reduced by half on Day 21 while the graft developed severe FAO and lost the entire epithelial lining. In contrast, the anti-RANTES treatment group had less than 50% of the CD4⁺ cell counts compared with the other allograft groups at both time points, and preserved luminal patency until Day 21. These results suggest that locally administered anti-RANTES antibody inhibits the migration of CD4⁺ cells into allografts and extends the period of airway opening.

However, as for the epithelial lining, anti-RANTES antibody did not prevent the destruction of the epithelium. Several studies have indicated a relevant role of epithelial cells as APCs in airway allorejection. The bronchiolar epithelium in the human transplanted lung with BO has been reported to show increased level of MHC class II (HLA-DR and HLA-DP) antigens (30). Rat allotracheas showed markedly enhanced expression of epithelial MHC class II antigens and proliferation of T cells and macrophages in the early phase after transplantation (31). Immunosuppression by cyclosporine A (CsA), which blocks T-cell functions, especially IL-2 production, down-regulated MHC class II expression on the epithelial cell surface, and inhibited the development of FAO in the allograft in a dose-dependent manner. The allotrachea treated with sufficient CsA preserved not only luminal patency but also the respiratory epithelium (31). The fact that the administration of anti-RANTES antibody to allografts did not maintain the epithelial lining in this study implies that anti-RANTES antibody does not block MHC-class II antigen expression on epithelial cells, and therefore it is not sufficient to control the entire alloimmune responses. In other words, RANTES probably acts as a cofactor in the complicated process of allorejection. The combination of anti-RANTES and other immunosuppressive treatments may prove to be an effective preventive strategy or therapy for chronic rejection. Current immunosuppressive drugs have significant toxicities such as the renal toxicity of CsA and the side effects of steroids. Anti-RANTES antibody administered in the grafted organ might inhibit the T-cell and macrophage migration and thus allow for a reduction in dose of the more toxic immunosuppressive agents.

In the isograft study, recombinant rat RANTES was infused until Day 28 in an attempt to enhance the effect of RANTES. Of particular interest, a more intense CD4⁺ cell infiltrate was found in the control group than in the other treatment isograft groups on Day 7. This might be due to easier access of CD4⁺ cells to the graft since the control isograft had recovered from the ischemic insult and had reconstituted vascularization by this time point. In contrast, the treatment groups still demonstrated ischemic necrosis and therefore did not develop adequate revascularization. Consequently, most immune cells likely originated in the immediate surrounding area. The prolonged ischemic phase seen in the treatment groups might have been due to the infusion pump and extended surgery rather than RANTES. Increased granulation tissue with intensive CD4⁺ cell infiltration, which indicates the enhanced inflammatory response, in the RANTES-infused trachea was presumably induced by recombinant RANTES to some extent. Although RANTES induced CD4⁺ cell migration on Day 28, all three groups regenerated their ciliated respiratory epithelium and showed no evidence of FAO. It appears that RANTES, unlike platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF) (32), does not promote FAO in isografts by itself; rather, it requires other triggers or stimulants to the lesion. Another possibility is that the effect of RANTES is dose dependent. With the dose we delivered, RANTES increased CD4⁺ cells accumulation, but this dose may not be sufficient enough to induce fibrotic airway obliteration. Our observation that the RANTES-infused isograft showed more luminal narrowing implies that a RANTES-enhanced nonalloimmune inflammatory response encourages a different type of airway stenosis (thickness of subepithelial space) from FAO.

Although the rat heterotopic tracheal transplant model does not reproduce the exact lesion or clinical manifestation of human posttransplant BO, it continues to prove useful for the study of the pathogenesis of this serious complication. It in fact closely mimics the characteristic pathological human features. In this allograft study, we used completely MHC mismatched inbred strains and did not administer any immunosuppressive therapy to the recipients. This is unlike the situation in clinical transplantation, and probably accelerates the development of FAO. Furthermore, in clinical transplantation, various factors could affect the incidence of chronic rejection other than alloimmune responses. However, since recent clinical studies revealed RANTES up-regulation and T-cell and macrophage infiltration of rejected organs, including grafted lung, the results from our study are likely to be applicable to human BO (12, 13, 15).

In summary, we have demonstrated that neutralization of RANTES by local infusion of anti-RANTES antibody decreased the number of infiltrating CD4⁺ mononuclear cells and inhibited the development of FAO in tracheal allografts. We also showed that continuous administration of recombinant RANTES did not induce FAO in isografts, although it enhanced migration of CD4⁺ cells. These findings implicate a role for RANTES as an enhancer of CD4⁺ T-cell and macrophage migration, which contributes to the evolution of FAO. Therefore, RANTES appears to act in concert with other factors to promote allorejection. The local administration of anti-RANTES (e.g., via inhalation) might reduce the required doses of other immunosuppressive agents and may be used as part of a therapeutic strategy to prevent or treat bronchiolitis obliterans in lung transplantation.