Introduction

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Lung transplantation is used commonly for the treatment of end-stage pulmonary diseases (1). However, acute rejection episodes are a significant cause of morbidity for the transplant patient (1). In addition, repeated acute rejection episodes are the main risk factor for the development of chronic rejection, which is the leading cause of death in lung allograft recipients (1).

Acute rejection episodes are characterized by varying degrees of mononuclear cell infiltrates into the alveolar space and interstitium of the transplanted lung (2). These cells, which are comprised predominantly of lymphocytes (graft infiltrating lymphocytes [GILs]), are believed to be recruited to sites of rejection activity by chemokines (reviewed in Reference 3). In lung diseases other than allograft rejection, the local production of monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cells expressed and secreted (RANTES) have been shown to be responsible for the recruitment of lymphocytes into the lung (4). Although a prior study showed that RANTES was expressed locally during lung allograft rejection (7), there are no studies showing the function of these chemokines in lymphocyte recruitment during the rejection response. Using a rat model of lung allograft rejection (8, 9), the purpose of the current study was to determine (1) whether chemotactic activity for GILs is increased in the allograft during rejection, (2) the cellular source of these chemokines, and (3) the contribution of MCP-1 and RANTES to GIL recruitment.

The data show that chemotaxis for GILs is markedly increased in allograft bronchoalveolar lavage (BAL) fluid (BALF), and that production of MCP-1 and RANTES occurred locally during acute rejection. Chemotaxis studies show that both chemokines were active locally for GIL recruitment during the rejection response. Data also showed that alveolar macrophages (AMs) in the donor lung have a key role in chemotactic activity for GILs, and in the local production of MCP-1 during the rejection response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

Inbred, pathogen-free, major histocompatibility complex (RT1)- incompatible male rats were used for transplantation surgery: F344 (RT1^lv1) and WKY (RT1^l) rats (250 to 300 g). 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.

Transplantation Model

The orthotopic transplantation of left lung allografts was performed as previously reported (8), and used a procedure described by Marck and colleagues (10). In brief, after anesthetizing F344 donor rats with an intramuscular injection of ketamine (40 mg/kg) and xylazine (5 mg/kg), shaving the chest wall, and making a sternotomy incision, the heart and lungs were removed en bloc, and the left lung was resected. Heparinized Lactated Ringer's solution was infused into the pulmonary artery, and the donor lung was wrapped in sterile gauze saturated with saline and placed on ice (4°C) in a sterile beaker until transplantation. The recipient rats (WKY) were anesthetized with a subcutaneous injection of atropine (0.05 mg/kg), followed by an inhalation of 1 to 2% halothane. After the airway was cannulated with a 14-gauge Teflon catheter, the rat was ventilated mechanically with a rodent ventilator (Analytical Specialties Co., St. Louis, MO) with 8.5 ml tidal volume and 70 breaths/min using 100% oxygen and inhalation of 2% isoflurane for maintenance anesthesia. The procedure was performed under a surgical microscope (Micro Tech Instruments, Inc., Colorado Springs, CO). Once a thoracotomy incision was made in the left fourth intercostal space and hemostats were placed on the left pulmonary vessels and bronchus, the left lung was resected. The pulmonary vessels of the donor lung were anastomosed to the recipient by a plastic cuff (developed by one author [Y.S.]) and 7-0 silk sutures (Kono, Chiba, Japan). The donor and recipient bronchi were sutured together with 8-0 Proline sutures (Ethicon, Sommerville, NJ). Immediately after completion of the anastomosis of the bronchus, the hemostat was removed and ventilation was restored. After the left thoracotomy incision was closed over an 18-gauge chest tube utilizing 3-0 silk suture (Ethicon), maintenance anesthesia was discontinued and the animal was allowed to recover. Once spontaneous respirations resumed, the cannula was removed from the airway and the chest tube was removed. The total operating time for harvesting and transplanting the donor lung was approximately 2 h. All transplantation procedures were performed by two authors (Y.S. and K.Y). Transplanted lungs were monitored by serial chest radiographs on the day of transplantation and on Days 1, 4, and 6 after transplantation. The F344  WKY transplant model is associated with the development of mild acute rejection (grade 1) by the end of the first week, moderate to severe rejection (grade 2 to 3) by the end of the second week, and severe rejection (grade 4) by the end of the third week after transplantation (9). Survival exceeded 90% in all transplantation groups. No immunosuppressive therapy was given at any time during the experimental period. In some experiments, AMs were depleted from donor (F344) lungs before transplantation into recipients (WKY).

Depletion of AM

A modification of the "macrophage-suicide" technique was used to deplete AMs in donor lungs before transplantation as previously reported (8). This technique induces apoptosis in AMs (11) and does not induce toxicity in the lung (12). After inducing anesthesia with an intramuscular injection of ketamine (40 mg/kg) and xylazine (5 mg/kg), and cannulating the trachea with a 14-gauge Teflon catheter, 200 ml of liposomes containing dichloromethylene diphosphonate (CL₂MDP-liposomes) were diluted in 600 ml of phosphate-buffered saline (PBS) and instilled intratracheally. (CL₂MDP was a generous gift of Boehringer Mannheim Corp., Mannheim, Germany.) AM depletion was confirmed by quantitating AMs present in cytospin preparations of cells obtained by BAL, and by immunocytochemical analysis of lung sections stained with anti-ED1 antibody, a pan-macrophage marker (Serotec, Oxford, UK) as previously reported (8). Preliminary experiments confirmed our prior report (8), which showed that intratracheal instillation of CL₂MDP-liposomes resulted in > 96% depletion of AMs in recipient lungs by the fourth d after instillation without inducing toxicity in recipient lungs, and that macrophage repopulation with those of the recipient did begin to occur until approximately 9 d after transplantation. All AM-depleted lungs were harvested for transplantation 4 d after liposome treatment.

Collection of BALF

Collection of BALF was performed in ketamine-anesthetized lung transplant recipients 1 wk after transplantation as previously described (8). In brief, after insertion of a catheter into the pulmonary artery and perfusion of the pulmonary circulation with 10 ml PBS (4°C) to remove all blood from the pulmonary vasculature, BAL of native and transplanted lungs was performed by selective cannulation of right and left main-stem bronchi with a 14-gauge catheter secured by suture. While clamping the contralateral bronchus, 5-ml aliquots of sterile PBS were instilled into each main-stem bronchus and aspirated. Cell-free BALF obtained from centrifuged specimens was stored at 70°C until use. BALF differential cell counts were performed by using light microscopy to count 300 cells per high-powered field (hpf) to determine the quantity of AMs, lymphocytes, and polymorphonuclear cells (PMNs) on cytospin preparations.

Isolation of GILs

In brief, 1 wk after transplantation allograft lungs underwent BAL and perfusion of pulmonary vasculature and were resected avoiding all lymph node tissue. After mincing with scissors and enzymatic digestion by stirring lung tissues in a solution of 0.5 mg/ml Collagenase A and 0.06 mg/ml DNAase (both from Boehringer Mannheim, Indianapolis, IN) in complete media (RPMI 1640 [GIBCO, Gaithersburg, MD], 10% fetal calf serum [Hyclone, Logan, UT], 1% penicillin/streptomycin, 1% glutamine, and 0.2% gentamycin [all from GIBCO]) at 37°C for 90 min, mononuclear cells were isolated by centrifuging specimens using a 1.075 and 1.030 Percoll gradient (Pharmacia, Piscataway, NJ). The cells were collected at the interface, washed, and resuspended in complete media (1 × 10⁶/ml). Mononuclear cells were incubated for 1 h at 37°C (5% CO₂) on plastic dishes (Costar, Cambridge, MA) and washed, and nonadherent cells were incubated on nylon wool columns for 60 min at 37°C. Lymphocytes (GILs) were eluted from the columns using warm (37°C) Hanks' balanced salt solution, washed, and resuspended in complete media (1 × 10⁶/ml). In separate experiments, lymphocytes were isolated from lungs of normal F344 and WKY rats using similar techniques.

Chemotaxis Assay

Chemotactic activity in allograft and native lung BALF was determined using a 48-well chemotaxis microchamber (Corning, Aclon, MA). In brief, 25 ml of BAL fluid was placed in the lower chamber. A polycarbonate polyvinylpyrolidone-free membrane (Corning) perforated with 5-mm-diameter pores was placed on top of the lower chamber. The upper chamber was attached, and GILs isolated from untreated allografts were placed into each upper chamber (2.25 × 10⁵ GILs in 50 ml of complete media). After a 3-h incubation at 37°C (5% CO₂), the membrane was removed, stained with Diff-Quik (Biochemical Sciences, Swedesboro, NJ), and examined by light microscopy at ×400 magnification. Migrating cells were quantitated by counting the number of cells embedded in the membrane from each group. In some experiments, the Chemotactic Index for BAL was determined using the formula: (average number of GILs migrating in response to BALF from allograft lungs) divided by (average number of GILs migrating in response to BALF from native lungs).

To determine the role of MCP-1 and RANTES in BALF in GIL recruitment, rabbit antirat MCP-1, RANTES, or isotype- and species-matched control antibodies (5 µg/ml) (Peprotech, Inc., Rocky Hill, NJ) were added to the bottom wells of chemotaxis chambers in some experiments.

Chemokine Enzyme-Linked Immunosorbent Assay

MCP-1 and RANTES were measured in BALF of native and allograft lungs. In brief, 100 µl of BALF or serial dilutions of MCP-1 or RANTES standards (Peprotech) in PBS-5% Tween (Sigma, St. Louis) were incubated in 96-well microtiter plates overnight at 4°C. After washing in PBS-5% Tween, plates were coated with either rabbit antirat MCP-1 or RANTES immunoglobulin (Ig) G antibodies (Peprotech) (3 µg/ml in PBS-5%Tween), incubated overnight at 4°C, washed, and incubated overnight with goat antirabbit biotinylated IgG antibody (1 µg/ml) (Peprotech). After washing, the plates were developed by the addition of avidin-peroxidase and reaction products were read on a microtiter plate reader. The sensitivities of assays were 14 and 190 pg/ml for MCP-1 and RANTES, respectively.

Immunohistochemistry for Detection of Intracellular Chemokines

Localization of MCP-1 and RANTES was performed on F344 lung allografts 1 wk after transplantation into WKY recipients and normal F344 lungs by immunohistochemistry. In brief, after allograft recipients or normal rats were killed, the thoracic organs were harvested, lungs were fixed by an intratracheal instillation of 4% glutaraldehyde, and sections were made from paraffin- embedded tissues. Tissue sections were dewaxed by immersion in xylene, rehydrated by immersion in ethanol, fixed with acetone, and then washed in Tris-buffered saline (TBS). After blocking with rabbit serum, sections were incubated with biotinylated rabbit antirat MCP-1, rabbit antirat RANTES, or rabbit IgG (control) antibodies (all from Peprotech), then washed. Sections were developed by addition of streptavidin alkaline phosphatase (Kirkegaard and Perry, Gaithersburg, MD) followed by addition of substrate (Kirkegaard and Perry). After a 20-min incubation, the slides were washed in TBS, counterstained with hematoxylin, coverslipped, and examined by light microscopy.

Immunocytochemistry

Immunocytochemistry was used to determine the quantity and percentages of CD4+ and CD8+ T lymphocytes, and natural killer (NK) cells present in GILs (8). CD4+ and CD8+ T lymphocytes and NK cells were identified by mouse antirat CD4+ (W3/25), mouse antirat CD8+ (OX8) (both from Serotec), and mouse antirat NK monoclonal antibodies (10/78) (PharMingen, San Diego, CA), respectively. In brief, cytospin preparations of GILs from transplanted lungs in each group were incubated with mouse antirat antibodies (5 µg/ml) for 30 min and washed. After a 30-min incubation with biotinylated goat antimouse antibodies (diluted 1/150 in PBS) (Kirkegaard and Perry), specimens were incubated with streptavidin-peroxidase (100 µl) (Kirkegaard and Perry) for 30 min and washed, followed by a 20-min incubation with substrate (5% New Fuschin in 2 M HCl) (Merck, West Point, PA) (8). All staining procedures were paired with species- and isotype-matched control antibodies (PharMingen). A total of 300 cells/hpf field were examined on each slide, and the percentage of positive-staining cells was calculated.

Histology

In separate experiments, transplanted lungs from each group were harvested, fixed by an intratracheal instillation of 4% glutaraldehyde, sectioned, stained with hematoxylin and eosin, examined under light microscopy, and graded, according to the histologic criteria established by the Lung Rejection Study Group (13), by a pathologist (author O.W.C.) in a blinded fashion as previously reported (8).

Statistics

Individual group means were compared using Student's t test for unpaired data. Data are reported as means ± standard error of the mean (SEM) unless stated otherwise. Differences between groups were considered to be significant where P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Lung allograft rejection is characterized by an influx of mononuclear cells, primarily lymphocytes, into the transplanted lung. To determine whether lymphocytes are increased in number in the allograft during rejection, we determined the number of GILs in F344 lungs 1 wk after transplantation into WKY recipients. Figure 1 shows the total number of lymphocytes present in normal F344 lungs compared with the quantity of GILs present in F344 allografts. The data show that lymphocytes (GILs) are significantly increased in the allograft 1 wk after transplantation (Figure 1; *P < 0.0005).

View larger version (8K): [in this window] [in a new window]   Figure 1.   Total quantity of lymphocytes isolated from lungs of normal F344 rats, and GILs isolated from untreated F344 lungs transplanted into WKY rats. GILs were isolated from allograft lungs 1 wk after transplantation as reported in MATERIALS AND METHODS. Data represent means ± SEM of three normal F344 lungs and six untreated F344 allograft lungs (*P < 0.0005 compared with normal lungs).

The increased quantity of lymphocytes in the allografts suggests that chemoattractant activity was upregulated locally during the rejection response. We then determined chemotactic activity in the allografts by counting the quantity of GILs migrating in response to allograft and native lung BALF. Table 1 shows that compared with the native lung, allograft BALF recruited significantly more GILs (P < 0.0001). These data show that chemotactic activity for lymphocytes is upregulated in lung allografts during acute rejection.

Chemokines are known to be responsible for recruitment of specific types of cells in several disease states (3). MCP-1 and RANTES have been shown to have a role in lymphocyte recruitment in pulmonary diseases other than lung allograft rejection (4). To determine whether the production of MCP-1 and RANTES was upregulated during lung allograft rejection we measured the quantity of these chemokines in F344 lung allografts 1 wk after transplantation into WKY recipients. Figure 2 shows that MCP-1 was undetectable in the native lung. However, MCP-1 levels were upregulated significantly in BALF from untreated allografts during the rejection response (Figure 2). In contrast, Figure 3 shows that low levels of RANTES were detected in the native lung with a slight increase in the allograft lung during the rejection. Due to highly variable levels there was no statistical difference in RANTES levels in the native and transplanted lungs (Figure 3; *P > 0.05). To determine the specific function of MCP-1 and RANTES in lymphocyte recruitment, chemotaxis assays were repeated using allograft BALF in the presence or absence of anti-MCP-1 or anti-RANTES antibodies. Table 2 shows that blockade of MCP-1 in allograft BALF resulted in a significant reduction in GIL chemotaxis (P < 0.0014). Specifically, anti-MCP-1 antibodies reduced chemotaxis for GILs by 47%. Although RANTES production was not increased statistically in allograft BALF during rejection, blockade of RANTES by anti-RANTES antibodies significantly reduced chemotaxis for GILs (P < 0.020), representing a 36% reduction in chemotactic activity compared with allograft BALF alone (Table 2). Anti-MCP-1 and anti- RANTES antibodies had no effect on chemotactic activity for GILs in BALF from native lungs (data not shown).

View larger version (9K): [in this window] [in a new window]   Figure 2.   Levels of MCP-1 in BALF of native and allograft lungs 1 wk after transplantation. MCP-1 was quantitated by ELISA and is reported adjusted for total protein (pg/mg total protein). Data represent means ± SEM of three paired native and untreated allograft lungs. ND, not detected.

View larger version (8K): [in this window] [in a new window]   Figure 3.   Levels of RANTES in BALF of native and allograft lungs 1 wk after transplantation. RANTES was quantitated by ELISA and is reported adjusted for total protein (pg/mg total protein). Data represent means ± SEM of four paired native and allograft lungs (P > 0.05).

MCP-1 and RANTES are produced by several different cell types in the lung, such as lymphocytes, epithelium, and AMs (3). In addition, interactions of these cell types may stimulate synthesis of these chemokines (14). To determine the cellular source of these chemokines, intracellular MCP-1 and RANTES were detected by immunohistochemistry in lung allografts 1 wk after transplantation as described in MATERIALS AND METHODS. Figure 4A shows that only AMs expressed MCP-1 in lung allografts 1 wk after transplantation. Significantly, other mononuclear cells or epithelium in the allografts did not stain for MCP-1. In contrast, Figure 4B shows that RANTES was expressed in AMs and other parenchymal cells in the allograft. However, similar to MCP-1, peribronchiolar mononuclear cell infiltrates did not stain for RANTES. Neither MCP-1 nor RANTES was detectable in normal lung tissue (data not shown).

View larger version (137K): [in this window] [in a new window]   View larger version (134K): [in this window] [in a new window]   View larger version (144K): [in this window] [in a new window]   Figure 4.   Immunohistochemical localization of MCP-1 and RANTES in F344 lung allografts 1 wk after transplantation into WKY recipients. Intracellular staining for MCP-1 and RANTES was performed on tissue sections of lung allografts as described in MATERIALS AND METHODS. Data represent the staining for chemokines in sequential tissue sections of the same lung allograft. MCP-1 (A) is identified as dark blue inclusions within cells that have morphology of AMs (see arrows). RANTES (B) is identified as dark blue intracellular inclusions within different cell types including AMs (arrows). (C ) represents staining the tissue section with isotype control antibodies. Photomicrographs representative of five separate allograft lungs harvested 1 wk after transplantation (original magnification: ×400).

AMs in the donor lung appeared to be the major source of MCP-1, and this cytokine contributed to GIL recruitment. RANTES was produced by other cell types in the lung, including AMs, and both MCP-1 and RANTES contributed to GIL recruitment. Therefore, if AMs are the major source of MCP-1, then depletion of donor lung AMs should result in reduced local levels of MCP-1 and diminished chemotaxis for GILs. Conversely, depletion of AMs should have no effect on local levels of RANTES. The "macrophage-suicide" technique (8, 12) was used to deplete AMs from F344 lung allografts before transplantation into WKY recipients. Similar to our prior report (8), the "macrophage-suicide" technique depleted > 96% of AMs (see MATERIALS AND METHODS; data not shown). To determine whether AM depletion resulted in diminished chemotactic activity in allograft BALF, chemotactic activity was determined in BALF obtained from AM-depleted allografts compared with the native lung and reported as the Chemotactic Index described in MATERIALS AND METHODS. The Chemotactic Index for untreated F344 lung allografts compared with native lungs is shown in Figure 5 (the Chemotactic Index for untreated allografts is derived from GIL migration data for control allografts compared with native lung shown in Table 1). Figure 5 shows that compared with untreated allografts, depletion of AMs before transplantation resulted in a significant reduction in the Chemotactic Index (Figure 5; *P < 0.0001). We then determined whether depletion of donor AM in F344 lung allografts before transplantation into WKY recipients resulted in lower levels of MCP-1 locally during lung allograft rejection. Figure 6 shows the levels of MCP-1 in BALF from untreated and AM-depleted allografts. Depletion of AMs abrogated local production of MCP-1, resulting in an 86% reduction in MCP-1 levels in allograft BALF, consistent with immunohistochemical data showing that AMs were the major source of this chemokine (Figure 6; *P < 0.025). As expected, Figure 7 shows that depletion of AMs had no effect on local levels of RANTES (*P > 0.05).

View larger version (9K): [in this window] [in a new window]   Figure 5.   Chemotactic Index in BALF of untreated and AM- depleted F344 lung allografts 1 wk after transplantation into WKY recipients. Chemotactic Index was determined as described in MATERIALS AND METHODS. Data represent means ± SEM of six untreated and five AM-depleted allografts (*P < 0.0001 compared with untreated allografts).

View larger version (9K): [in this window] [in a new window]   Figure 6.   Effect of AM depletion on MCP-1 levels in BALF. AM depletion of donor lungs was as described in MATERIALS AND METHODS. Data show levels of MCP-1 in untreated and AM-depleted F344 allograft lungs 1 wk after transplantation into WKY recipients. Data shown for untreated allografts are the same as shown in Figure 2. MCP-1 was quantitated in BALF by ELISA and is reported adjusted for total protein (pg/mg total protein). Data represent means ± SEM of three untreated allograft and four AM-depleted allograft lungs (*P < 0.025 compared with untreated allograft lungs).

View larger version (8K): [in this window] [in a new window]   Figure 7.   Effect of AM depletion on RANTES levels in BALF. AM depletion of donor lungs was as described in MATERIALS AND METHODS. Data show levels of RANTES in untreated and AM-depleted F344 allograft lungs 1 wk after transplantation into WKY recipients. Data shown for untreated allografts are the same as shown in Figure 3. RANTES was quantitated in BALF by ELISA and is reported adjusted for total protein (pg/mg total protein). Data represent means ± SEM of three untreated allograft and four AM-depleted allograft lungs (*P > 0.05 compared with untreated allograft lungs).

Data showing diminished chemotactic responses and reduced chemokine levels in AM-depleted allografts suggest that fewer GILs would be present in AM-depleted compared with untreated allografts during acute rejection. Therefore, we next determined the quantity of GILs present in untreated and AM-depleted F344 lung allografts. Figure 8 shows that significantly fewer GILs were recovered from AM-depleted compared with untreated allografts (**P < 0.0059). Immunocytochemistry was then used to determine the phenotype of the cells present in the GILs. In the normal rat lung, CD4+ are the predominant T lymphocytes. In contrast, allograft rejection is associated with an influx of CD8+ T lymphocytes. Indeed, Figure 9 shows that CD8+ T lymphocytes comprise the majority of cells present in GILs during acute rejection. In contrast, depletion of AMs before transplantation results in significantly fewer CD8+ GILs in F344 allografts 1 wk after transplantation into WKY recipients (Figure 9; *P < 0.027).

View larger version (8K): [in this window] [in a new window]   Figure 8.   Total quantity of GILs isolated from untreated and AM-depleted F344 lungs transplanted into WKY rats. GILs were isolated from allograft lungs 1 wk after transplantation as reported in MATERIALS AND METHODS. Data shown for untreated allografts are the same as that shown in Figure 1, which represents the mean ± SEM of six untreated F344 allografts. Data also represent the mean ± SEM of six AM-depleted allografts (*P < 0.0059 compared with untreated allografts).

View larger version (11K): [in this window] [in a new window]   Figure 9.   GIL differential cell counts for CD4+ and CD8+ T lymphocytes and NK cells in GILs of untreated and AM-depleted F344 allograft lungs 1 wk after transplantation into WKY recipients. CD4+ and CD8+ T lymphocytes and NK cells were identified by immunocytochemistry as described in MATERIALS AND METHODS and represent the number of positively staining cells identified by counting 300 cells on cytospin preparations. Data represent means ± SEM of six untreated and five AM-depleted allografts (*P < 0.027 compared with CD8+ cells in untreated allografts).

Because lung allograft rejection is characterized by varying amounts of perivascular and peribronchiolar lymphocytic infiltrates, and AM depletion resulted in fewer GILs in the allograft, we then determined whether AM depletion affected the severity of rejection pathology. Compared with untreated allografts (Figure 10A), Figure 10B shows that AM depletion resulted in less-severe rejection pathology at 1 wk after transplantation.

View larger version (73K): [in this window] [in a new window]   Figure 10.   Pathology of untreated (A) and AM-depleted (B) F344 lung allografts 1 wk after transplantation into WKY recipients. AM depletion (B) resulted in fewer perivascular and peribronchiolar infiltrates and less severe rejection pathology compared with untreated allografts (A). Photomicrographs are representative of six untreated and eight AM-depleted allografts (original magnification: ×100).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Lung allograft rejection is associated with varying degrees of perivascular and peribronchiolar cellular infiltrates which are comprised primarily of T lymphocytes. In pulmonary disorders other than allograft rejection, lymphocyte recruitment is directed by several different chemokines. However, chemotactic activity for GILs, the specific role of chemokines in the recruitment of GILs, and the potential cellular source of these chemokines in lung allograft rejection have not been reported previously. The current study shows that chemotactic activity for GILs is upregulated during lung allograft rejection, and that MCP-1 and RANTES contribute to GIL recruitment. In addition, donor lung AMs are the source of MCP-1 that are produced during acute rejection and therefore have a key role in the recruitment of GILs during the rejection response.

The expression of chemokines in allograft rejection has been well documented in organs other than the lung (15- 18). For example, MCP-1, RANTES, and lymphotactin are expressed early during renal allograft rejection (15); MCP-1 is induced in cardiac allograft rejection (16); and monokine induced by interferon- (Mig) is produced during rejection of skin allografts (18). However, few studies have examined the functional role of chemokines in allograft rejection. Mig has been shown to have a key role in lymphocyte recruitment during skin allograft rejection (18), and a recent study data showed that CCR1, the receptor for RANTES, MCP-1 (and other MCPs), and macrophage inflammatory protein (MIP)-1, has a key role in cardiac allograft rejection (19). In contrast, only RANTES and interleukin (IL)-8 have been reported to be expressed during acute and chronic lung allograft rejection, respectively (7, 20). Before the current study, IL-8, which recruits PMNs, is the only chemokine shown to have a specific function during rejection in the transplanted lung (20). In the current study the production of MCP-1 and RANTES occurred during the rejection response, and both of these chemokines were functional in recruitment of GILs. The difference between the level of RANTES and its function could be explained by the possibility that extremely low levels of RANTES are required to induce GIL recruitment, and that these levels may not be readily detected by enzyme-linked immunosorbent assay (ELISA). These data and a prior report (20) clearly demonstrate a role of MCP-1, RANTES, and IL-8 in the pathogenesis of lung allograft rejection.

Multiple chemokines are reported to be important in T-lymphocyte recruitment in different disease states (3). In addition, depending on the type of inflammatory response (i.e., infection or alloimmune activity), the types of chemokines expressed may vary relative to the progression of disease activity (6, 15, 21). Indeed, in ongoing studies using our rat lung transplant model, MCP-1 was undetectable in allograft BALF several wk after transplantation despite progression of rejection activity and lymphocyte infiltration into the allograft (data not shown). These data suggest that other chemokines, such as MIP-1 and/or MIP-2 (16) or others, were operative. Indeed, in data not shown, MIP-1 production was upregulated locally 1 wk after transplantation. Because MIP-1 has been reported to be chemotactic for lymphocytes in other studies (16), the activity of this chemokine could explain why anti- MCP-1 and anti-RANTES antibodies did not completely abrogate GIL chemotaxis in the current study. These data also indicate that because there are several chemokines which have redundant functions and may utilize the same receptors, it is unlikely that there is one dominant chemokine operative in GIL recruitment at a given time during lung allograft rejection.

Although MCP-1 and RANTES are made by several different types of cells, AMs are believed to be a major source of MCP-1 and RANTES in pulmonary diseases (4- 6). For example, Lukacs and colleagues (4) reported that AM-derived MCP-1 recruited T lymphocytes during allergic airway inflammation in rats. In addition, AM-derived MCP-1 recruited T-lymphocytes during cryptococcal pneumonia (6), and Monti and colleagues (7) reported that lung macrophages express RANTES during lung allograft rejection in humans. Alternatively, other cells such as epithelium (14, 22) could produce these chemokines alone or in response to AMs activated by local alloimmune responses. However, data in the current study show that donor lung AMs are the key source of MCP-1 early in the rejection response, and that AMs and other parenchymal cells in the lung contribute to the production of RANTES. In the rat model of lung transplantation, donor lung AMs are eliminated from the allograft by the second wk after transplantation (23). Our ongoing studies show that MCP-1 and RANTES levels decline progressively and are not readily detected in the allograft by the third wk after transplantation (data not shown). Interestingly, macrophages produce MCP-1 in response to interactions with T lymphocytes in mixed leukocyte reactions (24), and interactions of donor lung AMs with host T lymphocytes are involved in initiating lung allograft rejection (25, 26). Collectively, the data strongly suggest that donor lung AMs are a major source of MCP-1 during lung allograft rejection. Indeed, the findings in the current study are consistent with a recent report from Torres and colleagues showing that depletion of conjunctival macrophages abrogated local MCP-1 production during corneal allograft rejection (27). In addition, these data are consistent with a prior report which showed that AMs may be one of the sources of RANTES during lung allograft rejection in humans (7).

GILs, which are primarily T lymphocytes, are believed to have a key role in the pathogenesis of lung allograft rejection. Indeed, T lymphocytes are the target of the immunosuppressive agents used to prevent and treat lung allograft rejection (1). Therefore, therapeutic interventions that could limit lymphocyte recruitment into the allograft may prevent or downregulate rejection episodes. Data in the current study showed that MCP-1 and RANTES were active in GIL recruitment, and that depletion of AMs abrogated MCP-1 production and chemotaxis for GILs. Additionally, diminished chemotactic responses were associated with less-severe rejection pathology. These studies suggest that further analysis of chemokines operative during various stages of lung allograft rejection may identify new targets for therapeutic intervention that could downregulate rejection episodes and therefore prolong the lives of lung allograft recipients.