Bronchiolitis Obliterans Syndrome Complicating Lung or Heart-Lung Transplantation

John A. Belperio, MD; Kathleen Lake, PharmD; Henry Tazelaar, MD; Michael P. Keane, MD; Robert M. Strieter, MD; Joseph P. Lynch, III, MD


Semin Respir Crit Care Med. 2003;24(5) 

In This Article

Pathogenesis of Bronchiolitis Obliterans Syndrome

The mechanisms responsible for BOS are undoubtedly complex and may involve both alloimmune-dependent and -independent mechanisms, acting alone or in combination.[5,126] Acute allograft rejection is undoubtedly the most important mechanism,[5,16] but additional mechanisms (discussed following here) contribute. Injury to airway ECs is likely a pivotal early event leading to BO. Local recruitment of inflammatory cells and release of cytokines likely drive this inflammatory/fibrotic process. Clinical and animal models suggest that lymphocytes and lymphocyte products play critical roles in acute allograft rejection; their role in BO is less well defined.[16,127,128,129] It is likely that both T-helper (Th)-1 and Th-2 responses contribute to the pathogenesis of BO.[118,130] Activated alveolar macrophages, by release of proinflammatory and profibrotic cytokines, appear to play important roles in early inflammatory events as well as chronic fibroproliferation.[131,132] Neutrophilic recruitment and activation, with release of neutrophil products, may amplify the inflammatory process in BO.[61,62,133,134] Interactions between immune cells and airway ECs,[16,135,136,137] smooth muscle cells,[16,62] mesenchymal cells (including FBs)[138,139,140] appear to be integral to the fibrotic process. Bronchial ECs (BECs) produce proinflammatory cytokines (e.g., IL-6, IL-8), which may recruit lymphocytes and neutrophils to the allograft.[135,141] In addition, BECs express several growth factors that stimulate smooth muscle cell migration and proliferation.[137] FB migration and proliferation are critical to the pathogenesis of BO, both in animal models[139] and in humans.[138,140] A complex interplay between diverse cells (both immune and nonimmune) and their products is pivotal. In the following sections, we discuss specific mechanisms that are likely of importance in BO.

The Role of T Lymphocytes in the Pathogenesis of BOS

Activated T lymphocytes play cardinal roles in AR[16] and likely play important contributory roles in the pathogenesis of LBB[129] and BO.[130] T lymphocytes are the critical cells driving allogeneic immune responses. T cells recognize genetically encoded polymorphisms between members of the same species (termed allorecognition).[142,143] MHC antigens, also known as HLAs, are present on all donor cells and are the main targets of the recipient's immune response to donor antigens.[142,143] MHC class I antigens (HLA A, B, C in humans), present on the surface of all nucleated cells, are the target of cytotoxic CD8+ T cells, whereas class II antigens (HLADP, DQ, DR in humans) are the target of CD4+ T cells.[142,143] MHC class II antigens are found on antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, B cells), and can be induced on endothelial and epithelial cells.[142,143]

MHC molecules contribute to allograft rejection by both direct- and indirect- allorecognition pathways.[144,145,146,147,148,149,150] The direct pathway involves donor APCs that are carried as "passenger leukocytes" into the recipient. These donor APCs migrate to recipient lymphoid tissue where they activate recipient T cells creating alloreactive T cells (Fig. 5, Panel I). Recipient T cells directly recognize donor MHC molecules ("cross reactivity" or "molecular mimicry").[151,152] Alloreactive T cells infiltrate the allograft and release cytokines/chemokines, which activate other cells (e.g., macrophages, B cells, and plasma cells) and elicit the release of growth factors, cytotoxic mediators, and alloantibodies, which may cause allograft injury (see Fig. 5, Panel I).[153] Passenger leukocytes are no longer present in the allograft within weeks of posttransplantation, ending the direct pathway of allorecognition.

Panel I direct allorecognition. (A) Donor antigen presenting cells (APCs) carried in the donor lung allograft parenchyma ("passenger leukocytes") migrate to the recipient lymphoid tissue. (B) Here the APCs directly activate recipient CD4+ and CD8+ T cells. (C) Alloreactive CD4+ "T helper" cells migrate to the lung allograft along a chemokine gradient. These T helper cells release cytokines/chemokines, which activate CD8+ T cells to become cytotoxic CD8+ T cells (which release cytokines and perforin/ granzymes); B cells to release alloantibodies; and mononuclear phagocytes to release cytokines/chemokines/growth factors) recruiting more leukocytes, ultimately leading to allograft injury. (D) Alloreactive CD8+ "T cytotoxic" cells migrate to the lung allograft and release cytokines/perforin/granzymes. Panel II Indirect allorecognition. (A) Immature/precursor APCs are released from the bone marrow and travel to the lung allograft as surveillance cells or secondary to signals occurring within the allograft (i.e., ischemia reperfusion injury). (B) Immature APCs process donor antigens from the allograft. (C) Maturing APCs loaded with donor antigen migrate through lymphatic to lymphoid tissue. (D) Here recipient APCs present and activate recipient T cells. (E) Alloreactive T cells through cytokine/chemokine release activate mononuclear phagocytes, B cells, and T cells to cause persistent lung allograft injury and eventually BOS.

The indirect pathway of allorecognition involves recipient APCs that have processed donor cells and present donor MHC molecules, eliciting a T cell response[144,145,146,147,154,155,156,157,158] (see Fig. 5, Panel II). However, persistent T cell alloresponses to antigens on recipient APCs and to donor APC MHC proteins may be a major driving force for chronic allograft rejection.[159,160]

Co-Stimulation Is Required for T Cell Activation and the Pathogenesis of BOS

Full activation of T cells requires two distinct yet synergistic signals. "Signal I" is the recognition of the antigen by the T cell receptor (TCR) in the context of self-MHC molecules. A second co-stimulatory signal is not antigen specific. Without a co-stimulatory signal, T cells undergo abortive activation leading to anergy or apoptosis.[161,162] Many T cell surface molecules are receptors for co-stimulatory signals, of which the CD28 molecule is best characterized. CD28 has two ligands B7-1 (CD80) and B7-2 (CD86); both are expressed primarily on activated APCs (Fig. 6).[163] T cells that receive this co-stimulatory signal produce activating cytokines, chemokines, and anti-apoptotic signals.[152] T cells also express cytotoxic T lymphocyte antigen 4 (CTLA-4), which also binds B7-1 and B7-2. In contrast to CD28, CTLA-4 interaction with B7 transmits an inhibitory signal that terminates the immune response (see Fig. 6).[164] Another important pathway in T cell activation is the CD40:CD40 ligand (CD40L) pathway. CD40 is expressed on APCs; its ligand, CD40L (CD154), is expressed on activated T cells. Stimulation of CD40 elicits APC expression of B7; antibody production from B cells; and upregulation of cytokine production and adhesion molecules.[165] The importance of CD40/CD40L interaction was demonstrated in a murine model of BOS. Fibro-obliteration failed to develop even in fully mismatched tracheas transplanted heterotopically when recipient mice lacked CD40L (CD154)-/-.[166] In a rodent model of BOS, heterotopic tracheal allografts displayed increased expression of B7-2 without increased expression of B7-1 compared with syngeneic controls.[167] Selective inhibition of CD28/B7-1 interactions did not attenuate rodent BOS. However, inhibition of both CD28/B7-1 and CD28/B7-2 interactions decreased intragraft cytokines (e.g., TNF-α, IL-2, and INF-γ) and inhibited fibro-obliteration.[167]

"Signal II" T cell co-stimulatory molecule activation. (A) Antigen-loaded dendritic cells (DCs) migrate through the lymphatics to lymphoid tissue. In the lymphoid tissue resting T cells express CD28 molecules. (B) Loaded DCs express B7-2 and interact with T cell CD28 molecules, signaling the DC to express B7-1 along with B7-2 molecules. (C) Fully activated DCs expressing B7-1 and B7-2 can interact with either "activating" co-stimulatory CD28 molecules or "inhibitory" co-stimulatory molecules CTLA-4.

The Role of Cytokines during the Pathogenesis of BOS

Airway wound repair requires a delicate balance between proinflammatory and antiinflammatory cytokines. Changes in this balance influence airway tissue remodeling or fibrosis. The specific mechanisms leading to BO in lung allografts have not been fully elucidated. We demonstrated elevated levels of IL-1 receptor antagonist (IL-1Ra), in BAL fluid from human LTRs with BOS compared with non-BOS.[66] Further, IL-1Ra were elevated even prior to the onset of BOS (future-BOS). This was not accompanied by elevations in IL-1β. The elevated ratio of (IL-1Ra to IL-1β) promotes a profibrotic environment.[168]

Additionally, IL-10, which down-regulates several cytokines and chemokines,[169,170] may attenuate allograft rejection. Administration of IL-10 attenuates acute and chronic allograft rejection in animal models (cardiac and lung allografts).[171,172] Further, IL-10 deficient (-/-) mice exhibit accelerated cardiac allograft rejection.[173] However, in human LTRs, we found similar levels of IL-10 in BALF in patients with BOS and non-BOS controls, casting doubt on a substantial role for endogenous IL-10 in BOS.[66] In a rodent model of BOS (i.e., heterotopic tracheal transplant), administration of IL-10 in recombinant form or by gene transfer inhibited fibro-obliteration.[172] This was only found when IL-10 was administered starting at day 5 posttransplantation and not if it was started at the time of transplantation.[172] Further investigations of timing and dose of IL-10 in animal models may clarify the role of IL-10 in chronic allograft rejection.

Transforming growth factor-β (TGF-β) is a potent inducer of collagen synthesis, FB proliferation, and FB chemotaxis[174,175] and favors the perpetuation and stabilization of the ECM.[174,175] Some studies noted increased expression of TGF-β from transbronchial biopsies and in BALF from LTRs with BOS compared with non-BOS LTRs,[26,176] whereas other studies failed to find increased expression of TGF-β.[66,176] In a rodent model of BOS, TGF-β was localized to infiltrating mononuclear cells and fibrotic tissue in the allografts.[177] In this model, administration of a functional TGF-β antagonist (i.e., soluble TGF-β IIIR) topically on day 5 posttransplantation attenuated BOS.[177] No effect was demonstrable if soluble TGF-β IIIR was given on day 0 or 10 or intramuscularly. Thus, timing and compartmentalization of TGF-β augmentation may be important in influencing fibroproliferation in BOS.

TNF-α is a proximal proinflammatory cytokine with myriad effects on inflammatory and immunologic responses, including enhanced cytolytic activity of natural killer (NK) cells, upregulation of MHC class II Ag and IL-2 receptors, and induction of T cell proliferation.[178,179] All of these biological functions are relevant to both acute and chronic allograft rejection. In animal models, TNF-α plays a key role in fibroproliferation and deposition of ECM in bleomycin-induced pulmonary fibrosis and hepatic fibrosis.[180,181] However, we found similar levels of TNF-α in BALF from LTRs with BOS as compared with non-BOS, casting doubt on a role for endogenous TNF-α in the pathogenesis of BOS.[66] Similarly, other studies found no significant differences in TNF-α in human chronic liver and renal allograft rejection.[182,183] However in an animal model of BOS, TNF-α expression was increased in allografts as compared with syngeneic controls.[184] In this model, administration of TNFR:RC, an inhibitor of TNF-α, attenuated fibroplasia.[184] Similarly, neutralizing antibodies to TNF-α, prevented murine BOS in an HLA-A2 transgenic tracheal (single-mismatched) allograft model, suggesting a role for TNF-α in the pathogenesis of BOS.[185]

The Role of Growth Factors during the Pathogenesis of BOS

Insulin-like growth factor-1 (IGF-1) is a potent profibrotic mediator that stimulates collagen synthesis by FBs.[186,187,188,189] IGF-1 can be secreted by alveolar macrophages, FBs, ECs, and endothelial cells.[186,187,188,189] The local bioactivity of IGF-1 in the lung is regulated by a system of multiple high-affinity IGF binding proteins (IGFBP).[190] The effects of these IGFBP in vivo are complex; some IGFBP inhibit whereas other IGFBP potentiate IGF-1 receptor binding.[190] In one recent study, expression of IGF-1, IGFBP-2, and IGFBP-3 were assessed on serial BALs in a cohort of LTRs.[191] Among LTRs who developed BOS, cellular IGF-1 expression was increased in LTRs prior to BOS but not in LTRs without BOS.[191] Interestingly, IGF-1 expression was not affected by AR episodes or CMV infection. IGFBP-3 was markedly increased in LTRs who later developed BOS as compared with those without BOS; IGFBP-2 was highly expressed from all patients.[191] These data are intriguing and suggest that interaction between IGF-1 and IGFBP-3 could play a role in the pathogenesis of BOS by potentiating fibrosis. Further, IGF-1 and IGFBP-3 in BALF may serve as early biomarkers for BOS, although this needs to be confirmed in larger trials with adequate control groups.

Platelet derived growth factor (PDGF) is another mitogen for mesenchymal cells, including FBs and smooth muscle cells.[192] PDGF ligands exist as different isoforms (e.g., PDGF-AA, PDGF-BB, PDGF-AB)[192] that display differential affinity to their receptors, PDGF-Rα and PDGF-Rβ.[193,194] In human LTRs, elevated levels of biologically active PDGF in BALF were associated with BOS.[138] In a rodent heterotopic tracheal transplantation model of BOS, the expression of PDGF-AA and PDGF-Rα was increased in allografts as compared with syngeneic controls.[195] Levels of PDGF-Rβ expression were less impressive. Treatment with an inhibitor specific for the PDGF receptor reduced the myofibroproliferation associated with BOS without affecting the extent of inflammatory cells, IL-2R, or MHC class II cellular expression.[195] These studies suggest a regulatory role for PDGF on fibroplasia during the development of BOS.

The Role of Chemokines/Receptors during Chronic Lung Allograft Rejection

The hallmark of rejection is infiltration of leukocytes within the lung allograft. The ability to maintain leukocyte recruitment despite immunosuppression is pivotal in the transition from inflammation/immune response to fibro-obliteration of the airways. Chemokines can selectively mediate the local recruitment/activation of leukocytes along established chemotactic gradients. Chemokines are pivotal for the trafficking of both immune and nonimmune cells to the sites of allospecific tissue injury and may promote inflammation, fibroproliferation, and airway luminal narrowing in BOS.

The chemokine superfamily is divided into four subfamilies (C, CC, CXC, and CX3C) based on the presence of a conserved cysteine residue at the NH2-terminus.[196,197,198] Multiple studies have found increased levels of IL-8/CXCL8 in BALF from patients with BOS that correlate with airway wall neutrophilia.[61,62,133,199,200,201] We confirmed that IL-8/CXCL8 localized to actin-positive smooth muscle cells of BOS lesions.[62] Increased levels of IL-8/CXCL8 were associated with human ischemia-reperfusion injury.[202] High levels of IL-8 in human donor lungs were associated with poor graft function and early mortality.[103] These data suggest an important role for IL-8/CXCL8 and neutrophil migration in the pathogenesis of BOS.

In both human and animal models of BOS, inflammation antedates fibro-obliteration.[61,62,133,199,200,201,203,204,205,206,207] In both human and animal models of BOS, elevated levels of RANTES/CCL5 and MCP-1/CCL2 have been noted.[65,130,208] In a rat model of BOS, neutralization of endogenous RANTES/CCL5 reduced the numbers of infiltrating graft CD4 T cells, preserved lumen patency, and attenuated early EC injury. Similarly, CCR1 deficient (-/-) mice were resistant to chronic cardiac rejection.[209] These studies suggest that CC chemokines play critical roles in the pathogenesis of chronic allograft rejection.

MCP-1/CCL2 is another CC chemokine with potent chemoattraction for mononuclear phagocytes and lymphocytes.[210,211] MCP-1/CCL2 binds and signals through CCR2.[212] We found that MCP-1/CCL2 levels in BALF from patients with AR and BOS were markedly elevated and localized to airway epithelium and mononuclear cells.[65] This suggests MCP-1/CCL2 is important in the continuum from acute to chronic allograft rejection by causing persistent accumulation of peribronchiolar leukocytes. Similarly, in a murine model of BOS, MCP-1/CCL2 localized to airway columnar epithelium and infiltrating mononuclear cells.[65] MCP-1/CCL2 levels paralleled the recruitment of mononuclear cells and cellular expression of its receptor CCR2. A murine model of BOS showed that CCR2 deficient (-/-) mice had less injury to the allograft even though lymphocyte trafficking in the allografts was unaffected.[209] These data suggest that a phenotypically distinct mononuclear phagocyte which expresses CCR2 (i.e., producing more TGF-β and PDGF) is pivotal to the pathogenesis of BOS. Similarly, elevated levels of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 in human BALF were associated with the continuum from acute to chronic rejection.[67,213] In a murine model of BOS, increased expression of ELR (glutamate leucine arginine)-negative CXC chemokines were found; this paralleled the recruitment of CXCR3-expressing mononuclear cells.[67] In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS.[67] This supports the notion that ligand/CXCR3 biology plays a pivotal role in the recruitment of mononuclear cells, a critical event in the pathogenesis of BOS (Fig. 7).

Phenotypically distinct CCR2-expressing mononuclear phagocytes and CXCR3-expressing lymphocytes are pivotal during the pathogenesis of bronchiolitis obliterans syndrome. Persistent allospecific injury of endothelial, epithelial, and mesenchymal cells leads to persistent expression of MCP-1/CCL2, MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11. These chemokines are acting in parallel (MCP-1/CCR2 recruiting phenotypically distinct CCR2 expressing mononuclear phagocytes) and (ELR negative CXC chemokines recruiting phenotypically distinct CXCR3 lymphocytes) to cause a cytokine/chemokine/growth factor cascade leading to cellular injury and mesenchymal cell matrix deposition, ultimately causing fibro-obliteration.

Chemokine/chemokine receptor biology plays an important role in renal allograft rejection.[214] CCR5Δ32 is a nonfunctional mutant allele of CCR5.[215,216,217] A multicenter study demonstrated that the homozygous expression of CCR5Δ32 was associated with prolonged renal allograft function.[214] CCR5 may be a potential target for inflammatory/fibroproliferative disorders such as allograft rejection. Both human and animal studies of BOS affirm the importance of chemokine/chemokine receptors during the pathogenesis of BOS.

In summary, the pathogenesis of BOS is a finely orchestrated process involving multiple events including allorecognition, co-stimulation, cytokines, growth factors, and chemokine/chemokine receptors. Animal models of BOS have demonstrated proof of the principle that each of these processes plays important roles in mediating the alloreactive leukocyte infiltration and ECM deposition that leads to fibro-obliteration. A greater understanding of the pathogenic mechanisms involved in BOS should pave the way for the development of pharmaceutical agents that will target each of these biological events and provide novel therapies that will ultimately enhance long-term allograft survival.


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