Introduction

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Lung transplantation has become an increasingly used modality for the treatment of various end-stage pulmonary diseases (1). However, the lung is more prone to rejection than other solid organs, which is likely due to the many immunocompetent cells in the donor lung (1, 2). The immune mechanisms that initiate allograft rejection involve donor lung antigen-presenting cells (APCs), namely macrophages and dendritic cells (DCs), interacting with recipient lymphocytes and leading to upregulated cellular and humoral immunity, which are believed to mediate allograft destruction (1).

The major histocompatibility complex (MHC) antigens are expressed on cells that present antigens to T lymphocytes (4). MHC class I molecules are expressed on all nucleated cells, and MHC class II molecules are primarily expressed on APCs (4). Recognition of the polymorphisms of either donor MHC class I or II by host T lymphocytes initiates alloimmune reactions in vitro (4) and allograft rejection in organs other than lung in vivo (5). Therefore, understanding the contribution of donor MHC molecules in the rejection response may lead to therapies that prevent morbidity and mortality in the lung-transplant patient. However, the specific role of donor MHC molecules in the pathogenesis of lung allograft rejection has not been evaluated. The current study tests the hypothesis that immune responses to allogeneic MHC class I and/or II molecules expressed on donor lung APCs contribute to the immunology and pathology of acute lung allograft rejection.

We have reported a murine model in which the instillation of MHC class I (H-2) and MHC class II (I-a) mismatched bronchoalveolar lavage (BAL) cells (96% macrophages, 1 to 2% DCs) from C57BL/6 mice (I-a^b, H-2^b) induces local interferon (IFN)- and immunoglobulin (Ig) G2a production, and pathology analogous to acute rejection (grade 1) in the lungs of recipient BALB/c mice (I-a^d, H-2^d) (6). This model allows for investigation of the specific roles of donor lung APCs in stimulating local alloimmune responses. Therefore, to examine the specific roles of donor MHC class I and II molecules expressed on APCs in the rejection response, BALB/c mice received instillations of BAL cells from wild-type C57BL/6 mice or C57BL/6 mice that do not express MHC class I, MHC class II, or both MHC class I and II (MHC-deficient) into the lungs weekly for 4 wk. The data show differential roles for donor MHC class I and II molecules in stimulating immunologic and pathologic alterations in recipient lungs.

	Materials and Methods

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Mice

Wild-type female C57BL/6 (I-a^b, H-2^b) and BALB/c (I-a^d, H-2^d) mice, 6 to 8 wk old, were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) (6). Female C57BL/6 mice, 6 to 8 wk old, made deficient in expression of MHC class I (2m /), class II, or both I and II by gene targeting were purchased from Taconic (Germantown, NY) (5, 9, 10). MHC-deficient mice are identical to wild-type mice except for the deleted gene (5, 9, 10). All mice were housed in microisolator cages in the Laboratory Animal Resource Center at the Indiana University School of Medicine in accordance with institutional guidelines (6).

Collection of Donor BAL Cells

Donor BAL cells were obtained by BAL as previously reported (6). In brief, anesthesia was induced in wild-type and MHC-deficient C57BL/6 mice by an intramuscular injection of a mixture of ketamine (80 to 100 mg/kg), acepromazine (8 to 10 mg/kg), and atropine (0.5 mg/kg). After isolation of the trachea by dissection and opening of the thoracic cavity by midline incision, a 20-gauge Teflon catheter was inserted into the trachea and secured by suture. The lungs were lavaged with a total of 20 ml of phosphate-buffered saline (PBS) at 37°C, and cells were isolated from lavaged specimens by centrifugation. BAL cells were resuspended in PBS at a concentration of 1 × 10⁶/ml. Prior studies (6) and preliminary data in the current study confirmed that 96% of donor BAL cells were macrophages and 2% DC.

Instillation of Allogeneic Lung Cells into Recipient Mice

Anesthetized BALB/c mice received the amount of 1.5 × 10⁵ of either wild-type, H-2-deficient (MHC-class I-deficient), 1-a-deficient (MHC class II-deficient), or H-2- and I-a-deficient (MHC class I and II-deficient) BAL cells from C57BL/6 donor mice in 100 µl of sterile PBS (37°C) by nasal insufflation weekly for 4 wk.

Our prior reports demonstrated that nasal insufflation of 100 µl of PBS or 1.5 × 10⁵ autologous (BALB/c) lung cells weekly for 4 wk had no effect on histology, BAL differential cell counts, or cytokine levels in recipient mice (6).

Distribution of Instilled BAL Cells in the Airways of Recipient Mice

As previously reported (6), colloidal carbon (100 µl of a 5% saline solution; Pelikan, Hanover, Germany) was instilled by nasal insufflation into the lower respiratory tract of C57BL/6 mice. After a 2-h incubation, the recipient mice underwent BAL and the number of carbon-loaded BAL cells was detected by examination of cytospin preparations using light microscopy. These carbon-loaded BAL cells (1.5 × 10⁵/mouse) were then instilled into the airway of anesthetized BALB/c mice by nasal insufflation. After 2 h, the recipient mice were killed and the thoracic organs were harvested en bloc, fixed by immersion in 4% glutaraldehyde, sectioned, stained with eosin, and examined using light microscopy for the presence of carbon-loaded BAL cells in the alveolar spaces.

To approximate the quantity of cells that reached the alveolar space the following experiments were performed. Carbon-loaded alveolar macrophages (1.5 × 10⁵) were instilled into the lungs of BALB/c mice by nasal insufflation. At 1 h later, the quantity of carbon-loaded macrophages recovered in BAL fluid (BALF) was determined by examination of cytospin preparations by light microscopy. Three separate experiments revealed that an average of 14.3% of 5 × 10⁵ recovered BAL cells were carbon-loaded. Although BAL does not recover all cells present in alveolar space, these data indicate that at least half of the instilled cells reached the alveoli.

Collection of Serum and BAL

At the end of each 4-wk study period for each group, mice were anesthetized with ketamine and acepromazine. The thoracic and abdominal cavities were opened and the mice were exsanguinated by cardiac and inferior vena cava puncture. Serum was collected from centrifuged specimens. After the trachea was dissected and transected, a 20-gauge catheter was inserted into the trachea and the lungs were lavaged with a total of 2.5 ml of sterile PBS. In brief, a 0.5 to 1.0 ml aliquot of PBS was instilled into the trachea and aspirated five times before placement into a specimen container. Cell-free BAL supernatants were obtained by centrifugation of BALF. All serum and BAL supernatants were stored at 80°C until use. Differential cell counts were performed on cytospin preparations of BAL cells.

Quantitation of Cytokines

Enzyme-linked immunosorbent assays (ELISAs) were used to detect IFN-, interleukin (IL)-10, and IL-4 levels in unconcentrated BALF obtained from recipient mice at the completion of each 4-wk study period (6). In brief, all cytokine measurements were performed on Immulon II microtiter plates (Dynatek, Chantilly, VA) by using recombinant cytokine standards and "paired" capture and biotinylated secondary antibodies (all from Pharmingen) per protocol supplied by the manufacturer. Reaction products were read on a Thermomax microtiter plate reader (Molecular Devices, Menlo Park, CA) at 405 nm. Quantitations of values were determined by using the Softmax software program (Molecular Devices). The sensitivity of the assays was 3.12 pg/ml for IL-4, 600 pg/ml for IL-10, and 7.8 U/ml for IFN-.

Total protein in BALF was measured using the Bradford assay. Cytokine concentrations in BAL were adjusted for total protein and reported as adjusted for total protein as shown in RESULTS (6).

IgG Subtype Assays

IgG1 and IgG2a levels were measured in the BALF by ELISA (6, 7). In brief, sheep antimouse IgG1 and IgG2a (The Binding Site, San Diego, CA) diluted to 3 µg/ml in borate-buffered saline (pH 8.2) were adsorbed onto Immulon II microtiter plates (Dynatech) at 37°C for 2 h. The plates were washed six times with 200 µl of PBS per well using a microtiter plate washer (Dynatech) and blotted dry. Standards consisted of purified murine IgG1 and IgG2a (The Binding Site) diluted in PBS containing 0.05% Tween-20 (Sigma, St. Louis, MO) at concentrations of 3.9 to 500 ng/ml. BAL samples were assayed neat and serum samples were diluted 1:100 using 100 µl per well of sample. After a 2-h incubation at 37°C, the washing and blotting procedures were repeated. Sheep antimouse IgG1 or IgG2a conjugated to horseradish peroxidase (The Binding Site) was diluted 1:200 in PBS Tween and applied at 100 µl per well for 2 h at 37°C. The plates were washed and developed by addition of o-phenylenediamine (Sigma). The reaction product was quantitated using a microtiter plate reader (Molecular Devices) reading at 490 nm and the IgG1 and IgG2a concentrations using the "Soft Max" program (Molecular Devices).

Detection of Alloantibodies

IgG was purified from BALF of recipient mice by passage over protein G columns per manufacturer's protocol (Pharmacia, Piscataway, NJ). IgG antibodies were adjusted to a concentration of 1 µg/ml in media (PBS supplemented with 1% fetal calf serum). Splenocytes (1 × 10⁶) isolated from donor mice (see RESULTS) were incubated with BAL IgG antibodies in media for 1 h on ice. After washing, the cells were stained with fluorescein isothiocyanate (FITC)-labeled goat antimurine IgG antibodies (1 µg/sample, 4°C) (Boehringer Mannheim, Indianapolis, IN) for 30 min followed by analysis on a FACScan flow cytometer (Becton Dickinson, Bedford, MA). FITC-labeled goat IgG antibodies (Boehringer Mannheim) were used as controls for these experiments.

Lung Histology

The thoracic organs of mice were removed en bloc after BAL and were fixed by an intratracheal instillation of 4% glutaraldehyde. Our prior studies (6) demonstrated that the greatest deposition of donor cells occurred in the midlung zones (perihilar distribution). Therefore, four to six cryostat sections were obtained from the perihilar regions of recipient lungs, stained with hematoxylin and eosin, examined under light microscopy, and graded according to the histologic criteria established by the Lung Rejection Study Group (11) as previously reported (6). In addition, all grading of histologic sections was done in blinded fashion by one author (O.W.C.) without prior knowledge of the treatment group (6).

Statistics

The data were initially assessed to confirm normality using a Shapiro-Wilk statistic. Comparisons between groups were analyzed for each of the dependent variables using a one-way analysis of variance with five interventions. For those comparisons demonstrating significance, a post hoc Student-Newman-Keuls was performed to determine differences between interventions. Where multiple comparisons were performed, a Bonferonni correction was applied and the significance level was 0.02. For other comparisons, the level of significance was 0.05.

	Results

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Figure 1 shows the differential cell counts in BALF of recipient BALB/c mice. In the normal BALB/c mouse macrophages comprise 96% and lymphocytes approximately 1 to 2% of BAL cells (6). Compared with normal mice, Figure 1 shows that instillation of autologous BAL cells did not result in alterations in BAL cell types in recipient lungs (6). Similar to our prior studies (6), Figure 1 shows that instillation of allogeneic (C57BL/6) wild-type BAL cells (96% macrophages, 2% DCs) results in a large percentage of lymphocytes in BALF in the recipient lung (P < 0.05 for lymphocytes compared with mice that received autologous cells). The instillation of allogeneic (C57BL/6) BAL cells deficient in MHC class I or II also resulted in a lymphocytic alveolitis that was comparable to differential cell counts in mice that received wild-type cells (P > 0.05 compared with the wild-type group). In contrast, instillation of cells deficient in expression of both MHC class I and II ("MHC-deficient") resulted in a significantly lower percentage of lymphocytes in BALF compared with mice that received wild-type cells, or cells deficient in MHC class I or II, though greater than normal BALB/c mice (P < 0.05). Data showing that lymphocytes comprised up to 40% of recovered BAL cells suggested that would be an increase in the quantity of total cells recovered from mice that received wild-type, MHC class II- deficient, or MHC class I-deficient cells. However, Figure 2 shows that there were no differences in total cell counts in BALF of any recipient mice. The lack of increased cell counts in these groups could be explained by death of the donor macrophages or migration of these cells out of the alveolar space. These data show that instillation of BAL cells expressing either allogeneic MHC class I or II molecules is sufficient to induce a lymphocytic alveolitis in recipient lungs.

View larger version (17K): [in this window] [in a new window]   Figure 1.   BAL differential cell counts. BALB/c mice received 1.5 × 105 BAL cells from wild-type, MHC class I-deficient, MHC class II-deficient, or MHC-deficient C57BL/6 mice into the lungs by nasal insufflation weekly for 4 wk. Filled bars, macrophages; open bars, lymphocytes. At the completion of the experimental period, recipient mice underwent BAL and differential cells counts were determined by using light microscopy to count 300 cells per high-power field on cytospin preparations. Data represent the means ± standard error of the mean (SEM) of five mice in each group (*P < 0.05 compared with normal BALB/c; P > 0.05 compared with wild-type; P < 0.05 compared with normal BALB/c, MHC I-deficient, or MHC II-deficient).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Total cell counts in BALF. BALB/c mice received 1.5 × 105 BAL cells from wild-type, MHC class I-deficient, MHC class II-deficient, or MHC-deficient C57BL/6 mice into the lungs by nasal insufflation weekly for 4 wk. At the completion of the experimental period, recipient mice underwent BAL and total cell counts were determined as described in MATERIALS AND METHODS. Data represent the means ± SEM of five mice in each group.

Our prior studies have shown that instillation of allogeneic BAL cells from C57BL/6 mice induces the production of IFN- in recipient lungs (6). To determine the role of MHC antigens on the donor cells in stimulating local IFN- production, we next instilled C57BL/6 BAL cells deficient in MHC class I, II, or both into lungs of BALB/c mice. Figure 3 shows that compared with wild-type cells, instillation of cells deficient in MHC class I or II induces significantly less IFN- production in BALF of recipient mice (P < 0.05 compared with wild-type). In contrast, instillation of MHC-deficient BAL cells did not induce the production of IFN- when instilled into the lung (Figure 3). Similar to prior reports (6), IFN- was not detected in BALF of normal BALB/c mice or BALB/c mice that received four weekly instillations of autologous BAL cells. IL-4 or IL-10 was not detected in BALF of any mice. Collectively, these data show that local production of IFN- is dependent on the instillation of donor cells expressing either allogeneic MHC class I or II.

View larger version (19K): [in this window] [in a new window]   Figure 3.   IFN- levels in BALF. BALB/c mice received 1.5 × 105 BAL cells from wild-type, MHC class I-deficient, MHC class II- deficient, or MHC-deficient C57BL/6 mice into the lungs by nasal insufflation weekly for 4 wk. At the completion of the experimental period, recipient mice underwent BAL, and IFN- levels were determined by ELISA. All values are reported as adjusted for total protein. Data represent the means ± SEM of five mice in each group (*P < 0.05 compared with wild-type).

Our prior studies have shown that instilling allogeneic MHC-sufficient BAL cells into recipient murine lungs induces the selective production of IgG2a locally (6, 7). We next determined the role of MHC antigens expressed on donor lung BAL cells in stimulating local IgG2a production. In the normal murine lung, IgG2a is present at very low levels relative to the predominant IgG subclass in the lower respiratory tract, IgG1 (6, 7). Therefore, under normal conditions, the IgG2a/IgG1 ratio in BALF is << 1. Consistent with our prior reports (6, 7), Figure 4 shows that instillation of allogeneic, wild-type C57BL/6 BAL cells weekly for 4 wk induces the selective production of IgG2a in recipient murine lungs such that the IgG2a/IgG1 ratio is markedly increased and > 1. In contrast, instillation of cells deficient in MHC class I or II resulted in a significant reduction in the IgG2a/IgG1 ratio compared with that observed after instillation of wild-type cells (P < 0.05 for either MHC I- or II-deficient) (Figure 4). However, although the IgG2a/IgG1 ratio was deduced it was still > 1 (Figure 4). The reduction in the ratio was not due to an increase in IgG1 levels, but due to a decreased quantity of IgG2a in BALF (data not shown). Interestingly, instillation of MHC-deficient cells did not induce upregulated IgG2a production locally, therefore resulted in an IgG2a/ IgG1 ratio similar to normal mice, and therefore was significantly less than that induced by wild-type or class I- or class II-deficient cells (P < 0.05, Figure 4). These data show that either MHC class I or II on donor lung APCs is capable of stimulating local IgG2a production, though less than that induced by wild-type cells.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Ratio of IgG2a/IgG1 in BALF. BALB/c mice received 1.5 × 105 BAL cells from wild-type, MHC class 1-deficient, MHC class II-deficient, or MHC-deficient C57BL/6 mice into the lungs by nasal insufflation weekly for 4 wk. At the completion of the experimental period, recipient mice underwent BAL and IgG2a, and IgG1 levels were determined by ELISA. The IgG2a/IgG1 ratio is reported as described in MATERIALS AND METHODS. Data represent the means ± SEM of five mice in each group (*P < 0.05 compared with wild-type; P < 0.05 compared with wild-type, MHC I-, or MHC II-deficient).

The humoral response to alloantigen may result in the development of alloantibody production. Using IgG antibodies purified from BAL of recipient mice to detect MHC molecules on donor cells by flow cytometry, we next determined whether instillation of allogeneic wild-type C57BL/6 BAL cells induced alloantibody production locally. Figure 5 shows that instillation of wild-type, allogeneic (C57BL/6) BAL cells weekly for 4 wk induced the production of IgG alloantibodies in recipient BALB/c lungs, as evidenced by a 3-fold increase in fluorescence intensity compared with cells stained with IgG antibodies derived from BALF from mice that received autologous cells or control IgG antibodies. To determine the role of MHC antigens on donor cells in local alloantibody synthesis, these studies were repeated using donor cells deficient in MHC class I or II or MHC-deficient cells for instillation into lungs of BALB/c mice. In contrast to all other groups, Table 1 shows that only instillation of allogeneic wild-type BAL induced alloantibody production locally.

View larger version (37K): [in this window] [in a new window]   Figure 5.   Detection of alloantibody production. BALB/c mice received 1.5 × 105 BAL cells from wild-type C57BL/6 mice (Allogeneic) or BALB/c mice (Autologous) into the lungs by nasal insufflation weekly for 4 wk. At the completion of the experimental period, IgG antibodies were purified from BALF of recipient mice and alloantibody detection was performed as described in MATERIALS AND METHODS. Control represents isotype-matched FITC-labeled goat IgG antibodies. Data represent IgG alloantibodies pooled from lungs of five recipient mice in each group.

We next determined whether alloantibodies produced in response to donor BAL cells were anti-MHC class I, II, or both I and II antibodies. IgG purified from BALF of mice that received wild-type BAL cells were used for flow cytometry to detect MHC molecules on donor (C57BL/6) cells deficient in MHC class I or II. Table 2 shows that these IgG antibodies detected alloantigens on donor cells deficient in MHC class I or II. IgG antibodies did not detect any antigens on donor cells deficient in both MHC class I and II (MHC-deficient; Table 2). Antigen-specific antibodies are a very small percentage of the total Ig pool in serum or BAL, and the IgG used for the current studies was comprised of total BALF IgG. Therefore, data in the current study indicate that instillation of allogeneic MHC-sufficient BAL cells induced the production of anti-MHC class I and II IgG alloantibodies that represent a portion of the total pool of IgG in BALF of recipient mice. However, donor cells deficient in either MHC class I or II were unable to elicit alloantibody synthesis.

Our prior studies have shown that instillation of allogeneic BAL cells weekly for 4 wk induces the development of a lymphocytic bronchitis and vasculitis analogous to the histology of grade 1-2 acute lung allograft rejection (6). To determine the role of donor MHC antigens in this process, we next examined the histology of lungs of mice that received four weekly instillations of donor cells deficient in expression of MHC class I, II, or both antigens as compared with instillations of wild-type BAL cells. Similar to our prior studies (6), Figure 6A shows that instillation of allogeneic wild-type MHC-sufficient cells induces mononuclear cell infiltrates in the perivascular and peribronchiolar tissues. The instillation of allogeneic BAL cells deficient in MHC class I or MHC class II induced infiltrates in the perivascular and peribronchiolar tissues of recipient lungs comparable to that observed in response to wild-type-MHC-sufficient cells (data not shown). Figure 6B shows that compared with wild-type cells, instillation of MHC-deficient cells induced less-severe pathologic lesions in the lungs of recipient mice. Our prior reports demonstrated that nasal insufflation of 100 µl of PBS or 1.5 × 10⁵ autologous (BALB/c) lung cells weekly for 4 wk had no effect on histology, BAL differential cell counts, or cytokine levels (6).

View larger version (149K): [in this window] [in a new window]   View larger version (149K): [in this window] [in a new window]   View larger version (146K): [in this window] [in a new window]   Figure 6.   Lung histology in BALB/c mice that received 1.5 × 105 BAL cells from wild-type MHC-sufficient C57BL/6 mice into the lungs by nasal insufflation weekly for 4 wk (A). Photomicrographs show the development of mononuclear cell infiltrates in perivascular and peribronchiolar tissues analogous to grade 1-2 acute lung allograft rejection. (B) shows less-severe cellular infiltrates in lungs of BALB/c mice that received instillations of MHC-deficient BAL cells. (C) shows normal lung histology in lungs of BALB/c mice that received four weekly instillations of autologous BAL cells. Photomicrographs are representative of five to eight mice in each group (original magnification, ×200).

	Discussion

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The pathology and immunology of acute lung allograft rejection is believed to be initiated by recipient T lymphocytes interacting with MHC class I and/or II molecules expressed on donor lung APCs, namely, macrophages and DCs (1, 6). To determine the role of MHC antigens expressed on these cells in local alloimmune responses, the current study used allogeneic BAL cells (96% macrophages, 2% DCs) from wild-type mice and mice deficient in MHC class I, II, or both MHC antigens for instillation into recipient murine lungs. The data show that both donor MHC class I and II antigens contribute to local T-helper (Th) 1 (IFN-) responses, and that cells that do not express functional MHC antigens are unable to induce local IFN- production. Similarly, MHC class I and II antigens contribute to stimulating local IgG2a production. Instillation of allogeneic wild-type BAL cells induces the production of both anti-MHC class I and II alloantibodies locally, but alloantibody synthesis is dependent on expression of both donor MHC class I and II antigens. Finally, expression of MHC class I or II on donor cells is sufficient to induce the pathology of acute lung allograft rejection comparable to that induced by wild-type cells.

IFN- is believed to be important in allograft rejection because of its key role in upregulating MHC expression, inducing cytotoxic lymphocyte activity, and stimulation of alloantibody production (12, 13). Using the same donor- host combinations as the current study, our prior reports (6) demonstrated that instillation of allogeneic lung APCs induces the production of IFN- production in recipient lungs. Data in the current study extend these observations by showing that donor-derived MHC class I and II molecules both contribute to stimulating local IFN- synthesis, and that the expression of either MHC class I or II on donor cells is required to induce IFN- production locally. Our prior study has shown that allogeneic lung DCs, and not macrophages, were responsible for inducing IFN- synthesis in the lung in vivo. Using a different experimental system, Sornasse and colleagues (14) also showed that murine DCs induced IFN- production when injected into mice. Therefore, data in the current study suggest that MHC class I or II expressed on donor lung DCs, and not macrophages, likely have key roles in stimulating local IFN- production during lung allograft rejection.

Specific cytokines stimulate certain IgG subclasses in mice (15). The Th1 cytokine, IFN-, induces IgG2a production (15), and IL-4 stimulates IgG1 synthesis (15). Therefore, data in the current study showing that donor cells deficient in MHC class I or II expression resulted in diminished IgG2a production locally compared with wild-type cells are consistent with data showing that these cells induce less IFN- synthesis relative to wild-type donor cells when instilled into the lung. Further, data showing that donor lung cells that lacked functional MHC class I and II antigens (MHC-deficient) did not induce excess IgG2a production locally are consistent with the data showing an inability of these cells to induce IFN- synthesis. However, the specific mechanism of local IgG2a production induced by allogeneic MHC class I and II remains to be determined.

Prior reports in allografts other than the lung have shown that alloantibody production may occur during the rejection response, and the specific role of donor MHC antigens in this process (16, 17). For example, Coffman and associates (17) examined alloantibody production in response to MHC-sufficient and MHC class I-deficient renal allografts. The data showed that renal allografts from MHC-sufficient mice induced anti-MHC class I and II alloantibodies. In addition, MHC class I-deficient renal allografts transplanted into fully allogeneic mice induced MHC class II, and not class I, alloantibodies (17). In contrast to these studies, the production of alloantibodies in the lung has not been reported previously. The current study showed that instillation of MHC-sufficient cells induced anti-MHC class I and class II alloantibody production in recipient lungs. An unexpected finding in the current study is data showing the requirement for both donor MHC class I and II antigens to stimulate alloantibody production. Inasmuch as IFN- has been reported to have a key role in stimulating alloantibody synthesis (13), the data showing impaired IFN- levels locally in response to cells deficient in MHC class I or II antigens could explain the inability of these cells to induce alloantibody production. However, the specific mechanism of alloantibody production in response to MHC-sufficient and -deficient cells is unclear and will be examined in future studies.

Despite recent advances, allograft rejection remains the greatest impediment to the long-term survival of the lung-transplant recipient (1). In experimental models in allografts other than the lung, several strategies, including masking donor MHC antigens, have been proposed to prevent rejection episodes (18). Data in the current study showing diminished cellular, humoral, and pathologic responses in lungs of mice that received MHC-deficient cells suggest that techniques that minimize expression of functional donor MHC antigens may be effective in downregulating rejection episodes in lung allograft recipients. The current data highlight the importance of donor MHC molecules in stimulating the rejection response. Chronic rejection is the leading cause of death in lung-transplant recipients, resulting in a five-year survival rate of less than 50% (1). Repeated acute rejection episodes are the main risk factor for chronic rejection. Because disparities between MHC antigens of donor and recipient are key to inducing acute rejection, the current data are consistent with other studies showing that attempts to minimize MHC mismatches should prevent rejection episodes (19, 20) and possibly prolong the life of lung allograft patients.