Published ahead of print on April 1, 2004, doi:10.1165/rcmb.2003-0440OC
© 2004 American Thoracic Society DOI: 10.1165/rcmb.2003-0440OC Induction of Mast Cell Activation and CC Chemokine Responses in Remodeling Tracheal AllograftsCardiovascular Research Institute, and Departments of Medicine, Surgery, and Pathology, University of California, San Francisco, California Address correspondence to: Dr. Kenneth C. Fang, Box 0911, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0911. E-mail: kfang{at}itsa.ucsf.edu
Activated mast cells release stored and newly synthesized mediators that influence the caliber and responsiveness of inflamed airways. In this work, we show that alloimmune-mediated mechanisms induce mast cell activation and expression of CC chemokines in remodeling rat tracheal allografts. Decreased expression of rat mast cell protease (RMCP) I and II, in concert with tryptase release in tracheal allografts, identified degranulation of stored serine proteases as an early mast cell response to allotransplantation. Transient upregulation of c-Kit expression occurred in a synchronous manner, suggesting that c-Kit receptor signaling controls mast cell responses. Increased expression of CC chemokine ligand (CCL) 2 and CCL3 by RMCP I–positive cells identified mast cells as epithelial and mesenchymal sources of chemoattractant chemokines in allograft airways. Cyclosporin A immunosuppression both attenuated and delayed these changes in mast cell phenotypes. Incubation of rat basophil leukemia 2H3 cells with CCL2 or CCL3 decreased surface c-Kit expression, an effect blocked by protease inhibitors. By controlling surface receptor availability, CC chemokines may regulate c-Kit signaling via a novel proteolytic mechanism. These data suggest that targeting alloimmune responses and restoring quiescence of mast cells may attenuate the development of fibroproliferative and obstructive distortions of bronchiolar architecture in lung allografts.
Abbreviations: c-Kit receptor tyrosine kinase, c-Kit • CC chemokine ligand, CCL • cyclosporin A, CSA • c-Kit ligand, KL • macrophage/monocyte chemoattractant protein-1, MCP-1 • macrophage inhibitory protein-1
Alloimmune responses in lung transplants induce complex signaling interactions among resident and recruited inflammatory cells, which orchestrate the progressive fibrotic constriction and obliteration of bronchiolar airways. Loss of epithelial integrity due to imbalances in regenerative and apoptotic pathways results in epithelial cell denudation and airway lumenal occlusion (1–4). Responses of the epithelial compartment also involve goblet cells and dendritic cells (5–7), with additional cellular heterogeneity contributed by infiltrating lymphocytes, recruited neutrophils, and intermingling mesenchymal cells (8). Mast cells reside in quiescence on both sides of the airway basement membrane barrier in the epithelial and mesenchymal compartments until activated by inflammatory signaling (9). Dynamic changes in mast cell populations of lung allografts correlate with episodic rejection and bronchiolar fibrosis (10), yet their role in mechanisms regulating the development of obliterative bronchiolitis (OB) remain unclear. Tissue microenvironments program the differentiation of progenitor cells into subpopulations of mature mast cells with distinct phenotypes and organ-specific functions (11). In the lungs, mast cells contribute to homeostatic and pathologic mechanisms regulating airway caliber and responsiveness via diverse pathways involving acute and chronic release of stored or newly synthesized mediators. Expression of granule-associated serine proteases permits immunohistochemical localization of tissue mast cells, with identification of connective tissue or mucosal subtypes in rodents based on expression of chymases, rat mast cell protease (RMCP) I or RMCP II, respectively (12). Mature rat tracheas exhibit a characteristic distribution of RMCP I+ cells in the submucosa and RMCP II+ cells in the epithelium and basal lamina (12). Mast cell responses to changes in their local milieu include proliferation, migration, degranulation, and apoptosis, which are critical cellular events all linked by signaling upon ligation of c-Kit receptor tyrosine kinase (13–15). Quiescent mast cells demonstrate limited c-Kit expression, which may be upregulated on activated subpopulations in remodeling tissues (16). Pathways regulating surface c-Kit expression include internalization of dimerized receptors initiated by binding of c-Kit ligand (KL, stem cell factor) or proteolytic release of the receptor's extracellular domain (17–20). Therefore, understanding mechanisms controlling c-Kit expression on mast cells in remodeling airways may clarify their unique responses to alloimmune-mediated injury.
Heterotopic or orthotopic implantation of rodent tracheal allografts replicates the airway pathology of OB observed in lung transplant recipients, yielding critical insights into complex cell signaling interactions at the airway epithelial–mesenchymal interface (21, 22). Peribronchiolar recruitment of inflammatory leukocytes mediated by CC and CXC chemokines contributes to the fibrotic distortion of airway architecture in tracheal allografts (23, 24). Immunosuppression with cyclosporin A (CSA) modifies the airway remodeling in tracheal allograft models of OB by blocking the activation and proliferation of T lymphocytes, inhibiting cytokine synthesis, and attenuating activity of other inflammatory cells in the alloimmune response (21). In this article, we investigate the responses of epithelial and mesenchymal mast cells to changes in tracheal tissue microenvironments induced by allogeneic transplantation. Alloimmune-dependent mechanisms provoke mast cell activation and degranulation, altering protease phenotypes and c-Kit expression of subpopulations in remodeling microenvironments. Allotransplantation also induces airway mast cell expression of CC chemokine ligand (CCL) 2 (macrophage/monocyte chemoattractant protein-1 [MCP-1]) and CCL3 (macrophage inhibitory protein-1
Anastomotic Tracheal Allograft Model Tracheas of adult male Brown Norway or Lewis rats were anastomosed to those in recipient Lewis rats in allogeneic or syngeneic transplantation, respectively, as previously described (25). Briefly, tracheas were excised from killed donors and placed in sterile saline solution at 4°C. After induction of anesthesia in recipients, endotracheal intubation was performed with administration of 50% supplemental oxygen by a small animal ventilator. After exposure of the trachea by midline neck incision, the cephalad end of the donor trachea was sutured to the native trachea and the distal end tied off. Animals were extubated after recovery from anesthesia with resumption of spontaneous respiration and moved to a warm oxygen-rich holding cage. Rats in the immunosuppression group were treated with CSA (15 mg/kg subcutaneously; Novartis, East Hanover, NJ). Transplanted and recipient native tracheas were harvested from killed animals and lavaged with a 1-ml aliquot of sterile phosphate-buffered saline (PBS), which was stored at –70°C before analysis. Six animals were included in each experimental group with evaluation at 3, 7, or 14 d after transplantation. All experimental procedures were approved by the Committee on Animal Research of the University of California, San Francisco (San Francisco, CA). Tracheal tissues were washed in PBS, fixed in 4% paraformaldehyde (PFA), and embedded directly in paraffin or incubated in PBS containing 30% sucrose for 18 h at 4°C before freezing at –70°C in Tissue-Tek OCT compound (Miles, Elkhart, IN), before sectioning to 5 µm thickness. Tissues were stained with hematoxylin and eosin, with histologic features examined and granulation tissue severity scored (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe) by a pulmonary pathologist blinded to the experimental conditions.
Immunohistochemistry
Immunoblotting
CCL2 Enzyme-Linked Immunosorbent Assay
Cell Culture
FACS Analysis
Statistical Analysis
Allograft Airway Remodeling To investigate early airway remodeling, the temporal progression and severity of tissue granulation in anastomosed tracheal grafts were analyzed. Allogeneic transplantation of tracheal grafts induced fibroproliferative changes evident in the posterior membranous (or intercartilaginous) region at 3 d (Figure 1A). By Day 7, allograft tissues demonstrated diffuse airway thickening due to peritracheal inflammation, with progressive lumenal involvement at Day 14 that included epithelial denudation and partial occlusion. By contrast, syngeneically transplanted tracheal grafts showed preserved airway epithelium and remained morphologically similar to native tracheas throughout the time points analyzed. Allograft tracheas harvested at 3 or 7 d from experimental animals treated with the immunosuppressive agent, CSA, exhibited normal airway architecture and intact airway epithelial layers (Figure 1A). Tissues harvested at 14 d demonstrated airway morphology similar to that observed in 7 d untreated allografts. Granulation tissue severity scores of CSA-treated tracheal allografts were lower than those obtained for untreated airway tissues at each time point (Figure 1B). Allotransplantation induced inflammation and fibroproliferative remodeling in anastomosed tracheal grafts, which were both attenuated and delayed in tracheas harvested from animals treated with CSA immunosuppression.
Distribution and Protease Phenotypes of Tracheal Mast Cells To identify the normal distribution of subpopulations of mast cells expressing c-Kit receptor and granule-associated RMCP I or II, fluorescence immunohistochemistry was performed on native tracheas. Immunohistochemical signals for RMCP I localized predominantly in the subepithelial compartment, with the greatest abundance and concentration located in the intercartilaginous region (Figure 2A). By contrast, cells immunoreactive for RMCP II were distributed primarily in the epithelial layer (Figure 2B). Cells expressing c-Kit were distributed throughout the trachea, but were also concentrated in the posterior membranous region (Figure 2A). Co-localization of fluorescent signals demonstrated cells expressing both c-Kit and either RMCP I or II, with some immunoreactive cells expressing either RMCP I or II, exclusively. These data show that mast cells reside in abundance in both the epithelial and mesenchymal compartments of the intercartilaginous region, an area involved in early transplant-induced airway remodeling.
Allotransplantation Alters Tracheal Mast Cell Phenotypes To determine whether alloimmune mechanisms induced responses in airway mast cells, phenotypes of mast cells in the intercartilaginous region were examined in allografted tracheas in the absence or presence of CSA immunosuppression. Allograft tissues harvested at 3 d from untreated animals demonstrated immunoreactive signals for RMCP I in both the epithelial and mesenchymal layers (Figure 3A). Untreated tracheas harvested at 7 or 14 d demonstrated decreases in RMCP I expression compared with those harvested at 3 d or to native tissues (Figure 3B). By contrast, expression of RMCP I by mast cells in tracheal allografts harvested from CSA-treated animals remained similar to that in native tissues, although signals were also present in the epithelial layer. Untreated tracheal allografts demonstrated similar decreases in immunoreactive signals for RMCP II at 3, 7, or 14 d compared with native tracheas (Figures 3C and 3D). Whereas RMCP II expression in 3-d allograft tracheas from CSA-treated animals was similar to that in native tissues, levels in 7- or 14-d tissues were decreased compared with those in native tracheas (Figure 3D). Although expression of RMCP I or II decreased in tracheal allografts, immunoreactive signals for c-Kit increased compared with those in native tissues (Figures 3E and 3F). c-Kit expression decreased by 7 d and remained low at 14 d, at levels that were decreased compared with those in native tissues (Figure 3F). c-Kit expression in 3-d allografts from CSA-treated animals was higher than that in native tracheas, but lower than that observed in tissues from untreated allografts. Synchronous changes in allograft c-Kit levels, which were attenuated by CSA treatment, suggest that mast cells respond early to alloimmune-mediated injury via dynamic adjustments of c-Kit expression.
Mast Cell Degranulation A rapid decrease in expression of RMCP I and RMCP II in the posterior membranous region of allografted tracheas, which was both delayed and attenuated by CSA immunosuppression, suggested degranulation as a cause of the loss of RMCP I and II signals. To determine whether mast cell degranulation occurs in remodeling allografts, we examined levels of mast cell tryptase, a secretory granule–associated tryptic serine protease, in fluid lavaged from harvested tracheas. Immunoblot analysis of tracheal lavage fluid (TLF) from tissues harvested from the untreated experimental group demonstrated tryptase in all six allografts at 3 d (Figure 4A). Levels increased in TLF from four animals at 7 d, and decreased or remained unchanged in two others. Immunoreactive bands for tryptase decreased or disappeared in TLF at 14 d. By contrast, tryptase was detectable in only one TLF aliquot from animals harvested from the CSA treatment group at 3 d. Tryptase was absent from TLF at 7 d in these animals, and appeared in four animals by 14 d. Further analysis of the intercartilaginous region of untreated or CSA-treated tracheal allograft tissues revealed differences in localization of RMCP I immunoreactive signals. Signals for RMCP I in native or syngeneically transplanted tracheas remained focused and discreet, whereas those in allograft tissues appeared diffuse (Figure 4B). By contrast, RMCP I signals in CSA-treated allografts were similar to those observed in native tracheas or syngeneic grafts. These data suggest that allotransplantation induced mast cell activation and degranulation with release of tryptic and chymotryptic serine proteases in the airways.
Mast Cell CC Chemokine Expression in Tracheal Allografts Our results suggest that tracheal mast cells both responded and contributed to alloimmune-mediated airway inflammation. To determine whether mast cells regulate signaling interactions among inflammatory cells, we identified expression of CC chemokines in remodeling tracheal allografts. Immunohistochemical analysis demonstrated increased expression of CCL2 or CCL3 in epithelial, subepithelial, intercartilaginous, and peritracheal tissues of allografts (Figures 5A and 5B). By contrast, CCL2 and CCL3 were minimally detectable in native recipient tracheas or syngeneic grafts (data not shown). Immunoreactive signals for CCL2 or CCL3 at 3 or 7 d, respectively, also co-localized with those for RMCP I in both the epithelium and mesenchyme, thus identifying mast cells as a source of these CC chemokines in allogeneically transplanted tracheas (Figures 5A and 5B). Analysis of TLF revealed an early increase in the levels of CCL2 in allograft airways which diminished by 7 or 14 d, approaching levels similar to those seen in native or syngeneic grafts (Figure 6A). By contrast, levels of CCL2 in TLF from CSA-treated animals were lower than those in untreated allografts. Whereas immunohistochemical analysis also demonstrated increased expression of CCL2 in 3-d untreated allografts (Figure 6B), tissues harvested from animals in the CSA immunosuppression group showed diminished CCL2 expression relative to that in 3 d untreated tissues. However, in CSA-treated tissues CCL2 levels at 7 d exceeded those at 3 d (Figure 6C). CCL3 expression was increased at 7 d in untreated tracheal allografts and exceeded the highest levels detected in CSA-treated tissues at 3 d (Figure 6E). These data suggest that epithelial and mesenchymal mast cells express CCL2 and CCL3 in response to allogeneic tracheal transplantation.
CC Chemokines Regulate Mast Cell c-Kit Expression Our data also demonstrated that dynamic changes in mast cell c-Kit expression occurred as tissue expression of CCL2 and CCL3 varied. To determine whether expression of CC chemokines regulate mast cell responses, we examined the effects of CCL2 and CCL3 on cell surface c-Kit expression. Incubation of RBL 2H3 cells (a well-characterized model of rodent mast cells [27]) with either CCL2 or CCL3 decreased c-Kit receptor expression as detected by FACS analysis (Figure 7A). The magnitude of c-Kit receptor downregulation induced by CCL2 or CCL3 was similar to that induced by KL. Co-incubation of cells with CSA attenuated the decrease in surface c-Kit expression induced by either CC chemokines or KL. Pretreatment of cells with PP2, an Src family kinase inhibitor, had no effect on c-Kit downregulation induced by CCL2 or CCL3 (Figure 7B). By contrast, PP2 blocked the decrease in surface c-Kit receptor induced by KL. Treatment of cells with protease inhibitors blocked CCL2- or CCL3-induced c-Kit downregulation, suggesting that CC chemokines induce loss of surface c-Kit expression via a mechanism involving surface proteolysis of portions of the receptor's extracellular domain (28). These data identify a novel mechanism whereby CC chemokines downregulate c-Kit expression by inducing receptor cleavage which can be attenuated by CSA.
Heightened inflammatory cell responses in allograft airways disrupt signaling interactions in the extracellular matrix, which cause progressive and irreversible distortions of bronchiolar architecture. Allogeneic transplantation induces denudation of apoptotic epithelial cells and recruitment of lymphocytes, and also elicits responses from mesenchymal cell populations. Synchronous loss of basement membrane integrity permits cell migration and reciprocal signaling between the normally sequestered compartments. Quiescent mast cells reside in both the epithelium and mesenchyme, and may exert unique influences on airway remodeling upon activation during tissue injury or inflammation. In this work, we show that mast cells alter their phenotypes and degranulate in response to changes in tracheal allograft microenvironments, and identify a novel mechanism whereby CC chemokines induced during the alloimmune response may regulate expression of mast cell c-Kit receptor. Transplantation exposes engrafted airways to numerous insults in the perioperative period, which may initiate acute tissue responses or propagate chronic remodeling. Several lines of evidence suggest that the activated mast cell phenotypes represent alloimmune responses, rather than changes related to surgical implantation. Tracheal mast cells in syngeneic grafts at 3, 7, or 14 d after transplantation maintained receptor phenotypes that resembled those of subpopulations characterized in native airways. By contrast, mast cell c-Kit expression increased within 3 d in allogeneic grafts. By 7 d, allograft airways demonstrated an almost complete loss of immunohistochemically detectable expression of c-Kit or RMCP I and RMCP II, which coincided with elevated TLF levels of tryptase, a stored serine protease released during degranulation by activated mast cells. CSA immunosuppression attenuated the changes in c-Kit and RMCP I or II expression by allograft mast cells, but had no effect on subpopulations in syngeneic grafts. These data suggest that changes in phenotype and activation of mast cells in tracheal allografts result from alloimmune mechanisms, rather than potential insults associated with graft harvest, implantation, or ischemia. Allotransplantation-induced changes in mast cell phenotypes may exert diverse influences on bronchiolar airway patency and remodeling. Upon their release from degranulating cells, serine proteases stored in active form in secretory granules participate in a variety of pathways, possibly regulating cell signaling and matrix protein deposition. Tryptase may control airway patency via its effects on bronchodilator and bronchoconstrictor peptides, or its regulation of airway hyperresponsiveness. Its mitogenic effects on smooth muscle cells and fibroblasts suggest that tryptase may also regulate airway caliber by influencing chronic remodeling. Chymases, such as RMCP I or II, may regulate vascular permeability or cleave extracellular matrix proteins. Both tryptase and chymase also participate in activation of matrix metalloproteinases, and thus may regulate collagen remodeling and a multiplicity of other pathways mediated by these proteases (26, 29). Changes in c-Kit expression may control mast cell numbers in allograft airways, because ligation of the receptor regulates proliferation, migration, and survival, which are potential mechanisms contributing to tissue mast cell hyperplasia. Signaling transduced via c-Kit may also control degranulation and release of stored mediators, such as tryptase and chymase. The loss of immunohistochemical signals for RMCP I or II and c-Kit suggests a variety of possible fates for allograft mast cells. Dependence of tissue mast cell identification on intact granule contents suggests that the absence of RMCP I or II represents widespread allograft mast cell degranulation, which is supported by the finding of tryptase exclusively in lavage fluids of allogeneically transplanted tracheas. The dramatic decrease in c-Kit suggests alloimmune-mediated downregulation of its expression, which may occur via receptor internalization triggered by binding of KL, a growth factor whose expression increases in other organ allografts (30). Alternative explanations for the absence of identifiable mast cells at 7 or 14 d include extensive apoptosis of allograft mast cells, their migration out of the mesenchyme or epithelium into adjacent connective tissues or the airway lumen, or their persistence in tissues as "phantom mast cells," which remain undetectable (except by electron microscopy) due to the absence of granule contents or unique surface proteins (31). Inducible expression of CC chemokines suggests that mast cells may also contribute to complex signaling pathways implicated in the pathogenesis of OB. Immunohistochemical signals for CCL2 and CCL3 localized to RMCP I-expressing mast cells in tracheal allografts. By contrast, native tracheal tissues or syngeneic grafts expressed minimal levels of CC chemokines. These data identified mast cells as a source of CC chemokines in remodeling allografts, and suggest that their expression occurred as part of the alloimmune response to transplantation. Subsequent decreases in CCL2 and CCL3 expression occurred with the loss of mast cells, which suggests that regulation of CC chemokine expression is also linked to mechanisms programming cell fate. Thus, mast cell–derived CC chemokines may play two distinct roles in the alloimmune response. Because CCL2 functions as a chemoattractant for graft infiltrating mononuclear phagocytes (23), its expression by epithelial and mesenchymal mast cells may contribute to early leukocyte recruitment during the development of OB. Our data also suggest that inducible expression of CC chemokines may regulate signaling transduced via c-Kit. In contrast to Src kinase–mediated receptor internalization signaled by KL, similar decreases in surface c-Kit levels induced by CCL2 or CCL3 were dependent upon proteolytic activity. These data suggest a novel CC chemokine-dependent pathway that downregulates c-Kit expression independently of tissue KL availability. Such a mechanism might serve to control both the magnitude and duration of c-Kit–dependent mast cell responses in remodeling allografts. Immunosuppression impedes the development of fibroproliferation and luminal occlusion in tracheal allografts, which serves as a useful model of intervention to deduce the contributions of effector cells and molecules to the alloimmune response. As previously shown in allograft models using implanted tracheas (21), our data demonstrate that CSA also preserves airway integrity and delays luminal remodeling in donor grafts anastomosed directly to recipient tracheas. Allotransplantation-induced phenotypic changes in tracheal mast cells provided a novel opportunity to characterize the effects of CSA on these cell- and tissue-specific responses in vivo. CSA-treated allografts demonstrated either attenuated or delayed changes in the expression of c-Kit, RMCP I, RMCP II, tryptase, CCL2, and CCL3 in allograft tissues at different time points. At 3 d, CSA maintained allograft c-Kit expression at low levels more characteristic of native tissues, suggesting that it either blocks upregulation of c-Kit or stabilizes mast cell numbers. Compared with untreated allograft tissues at 7 or 14 d, which demonstrated an almost complete absence of c-Kit signals, CSA-treated tissues showed stable c-Kit expression reminiscent of native tissues. In vitro data show that CSA blocks proliferation and enhancement of survival induced by c-Kit ligation, and also increases c-Kit mRNA and protein levels (32), suggesting possible mechanisms that might explain the observed changes at different time points in vivo. Preservation of tissue signals for RMCP I or RMCP II and the relative lack of tryptase in TLF of CSA-treated animals suggest that CSA blocked allotransplantation-induced mast cell degranulation. These in vivo results substantiate other data showing that CSA blocks degranulation of rodent or human mast cells induced by both c-Kit–dependent and –independent mechanisms (33–35). Two additional novel observations include the ability of CSA to attenuate mast cell expression of CC chemokines in allograft tissues and to block CCL2- or CCL3-induced proteolytic downregulation of surface c-Kit levels. Thus, CSA may maintain or restore mast cell quiescence by stabilizing c-Kit expression or cell numbers, which may minimize the contributions of degranulated mediators or newly-released CC chemokines to the alloimmune response. In summary, our data demonstrate that allogeneic tracheal transplantation provokes unique mast cell responses with dynamic changes in stored, secreted, and surface-expressed signaling proteins. Activation of mast cells localized to the intercartilaginous, posterior membranous region of the trachea involved in early remodeling suggests that discreet changes in tissue microenvironments program cell-specific phenotype switches. Degranulation of serine proteases, alterations in population size, and expression of CC chemokines implicate signaling via c-Kit as a unifying pathway that coordinates critical mast cell responses. Dynamic regulation of the surface proteolysis of c-Kit by CC chemokines suggests a novel regulatory pathway, which might limit mast cell contributions to the inflammatory response by restoring cellular quiescence. Further investigations of the intersection between c-Kit and CC chemokine signaling pathways involving in vivo models, such as mast cell–deficient animals, may provide critical insights into the contributions of mast cell responses to airflow obstruction and airway fibrogenesis in lung allografts.
The authors thank Paul F. Dazin for helpful discussions and Linda Vona-Davis for statistical analysis, and acknowledge the excellent technical assistance of David Dreizin, Tracy L. Evans, Sang H. Sung, Andrew Toy, and Mai Tran. This work was supported by National Institutes of Health grant HL-64897, a research grant from the Research Evaluation and Allocation Committee and an Individual Investigator Grant from the Academic Senate of the University of California, San Francisco School of Medicine, and the Diamond Family Fund. Received in original form December 8, 2003 Received in final form February 23, 2004
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Abstract
Introduction
, MIP-1
