J. Lai

In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.

A Mouse Model of Orthotopic Vascularized Aerated Lung Transplantation

Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient‐type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.

Cutting edge: acute lung allograft rejection is independent of secondary lymphoid organs

It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

CCR2 Regulates Monocyte Recruitment As Well As CD4+ Th1 Allorecognition After Lung Transplantation

Graft rejection remains a formidable problem contributing to poor outcomes after lung transplantation. Blocking chemokine pathways have yielded promising results in some organ transplant systems. Previous clinical studies have demonstrated upregulation of CCR2 ligands following lung transplantation. Moreover, lung injury is attenuated in CCR2‐deficient mice in several inflammatory models. In this study, we examined the role of CCR2 in monocyte recruitment and alloimmune responses in a mouse model of vascularized orthotopic lung transplantation. The CCR2 ligand MCP‐1 is upregulated in serum and allografts following lung transplantation. CCR2 is critical for the mobilization of monocytes from the bone marrow into the bloodstream and for the accumulation of CD11c+ cells within lung allografts. A portion of graft‐infiltrating recipient CD11c+ cells expresses both recipient and donor MHC molecules. Two‐photon imaging demonstrates that recipient CD11c+ cells are associated with recipient T cells within the graft. While recipient CCR2 deficiency does not prevent acute lung rejection and is associated with increased graft infiltration by T cells, it significantly reduces CD4+ Th1 indirect and direct allorecognition. Thus, CCR2 may be a potential target to attenuate alloimmune responses after lung transplantation.

CD4+ T Lymphocytes Are Not Necessary for the Acute Rejection of Vascularized Mouse Lung Transplants

Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4+ T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4+ T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4+ T cell-mediated allorecognition pathways. CD4+ T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4+ T cells, but does not delay acute rejection when CD4+ T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.

Five-year update on the mouse model of orthotopic lung transplantation: Scientific uses, tricks of the trade, and tips for success

It has been 5 years since our team reported the first successful model of orthotopic single lung transplantation in the mouse. There has been great demand for this technique due to the obvious experimental advantages the mouse offers over other large and small animal models of lung transplantation. These include the availability of mouse-specific reagents as well as knockout and transgenic technology. Our laboratory has utilized this mouse model to study both immunological and non-immunological mechanisms of lung transplant physiology while others have focused on models of chronic rejection. It is surprising that despite our initial publication in 2007 only few other laboratories have published data using this model. This is likely due to the technical complexity of the surgical technique and perioperative complications, which can limit recipient survival. As two of the authors (XL and WL) have a combined experience of over 2500 left and right single lung transplants, this review will summarize their experience and delineate tips and tricks necessary for successful transplantation. We will also describe technical advances made since the original description of the model.

Apyrase treatment prevents ischemia-reperfusion injury in rat lung isografts

OBJECTIVE Endothelial cells express the ectoenzyme ectonucleoside adenosine triphosphate diphosphohydrolase, an apyrase that inhibits vascular inflammation by catalyzing the hydrolysis of adenosine triphosphate and adenosine diphosphate. However, ectonucleoside adenosine triphosphate diphosphohydrolase expression is rapidly lost following oxidative stress, leading to the potential for adenosine triphosphate and related purigenic nucleotides to exacerbate acute solid organ inflammation and injury. We asked if administration of a soluble recombinant apyrase APT102 attenuates lung graft injury in a cold ischemia reperfusion model of rat syngeneic orthotopic lung transplantation. METHODS Male Fisher 344 donor lungs were cold preserved in a low-potassium dextrose solution in the presence or absence of APT102 for 18 hours prior to transplantation into syngeneic male Fisher 344 recipients. Seven minutes after reperfusion, lung transplant recipients received either a bolus of APT102 or vehicle (saline solution). Four hours after reperfusion, APT102- and saline solution-treated groups were evaluated for lung graft function and inflammation. RESULTS APT102 significantly reduced lung graft extracellular pools of adenosine triphosphate and adenosine diphosphate, improved oxygenation, and protected against pulmonary edema. Apyrase treatment was associated with attenuated neutrophil graft sequestration and less evidence of tissue inflammation as assessed by myeloperoxidase activity, expression of proinflammatory mediators, and numbers of apoptotic endothelial cells. CONCLUSIONS Administration of a soluble recombinant apyrase promotes lung function and limits the tissue damage induced by prolonged cold storage, indicating that extracellular purigenic nucleotides play a key role in promoting ischemia-reperfusion injury following lung transplantation.

Maintenance of IKKβ activity is necessary to protect lung grafts from acute injury

Background. Signaling pathways that target I-&kgr;B kinase &bgr; (IKK&bgr;) activation stimulate the expression of nuclear factor (NF)-&kgr;B-dependent genes and are thus believed to primarily promote inflammation and injury in solid organ grafts. Methods. We examined the role of IKK&bgr; in a mouse model of lung transplantation-mediated ischemia-reperfusion injury using NF-&kgr;B essential modulator (NEMO)-binding domain (NBD) peptide to pharmacologically inhibit IKK activation. As myeloid cells are primarily responsible for the production of acute inflammatory mediators after lung transplantation, we also investigated the effects of myeloid cell-specific IKK&bgr; gene deletion on acute lung graft injury by transplanting mutant mice. Results. When NBD was administered at a dose that partially inhibits IKK&bgr; activation, we observed attenuated lung graft injury and blunted expression of intragraft proinflammatory mediators. Surprisingly, when the dose of NBD was increased to a level that ablates intragraft IKK&bgr; activation, graft inflammation, and injury were significantly worse compared with recipients treated with control peptide. Similar to lung recipients with pharmacologically ablated IKK&bgr; activity, donor-recipient transplant combinations with a myeloid cell-specific IKK&bgr; gene deletion had marked intragraft inflammation and poor lung function. Conclusions. Our data show maintenance of IKK&bgr; activity is critical for promoting graft homeostasis with important implications for targeting NF-&kgr;B-dependent signaling pathways for treating acute lung injury.

Polarized alloantigen presentation by airway epithelial cells contributes to direct CD8+ T cell activation in the airway.

Activated T lymphocytes are abundant in the airway during lung allograft rejection. Based on respiratory viral studies, it is the current paradigm that T cells cannot divide in the airway, and that their accumulation in the lumen of the respiratory tract is the exclusive result of recruitment from other sites, such as mediastinal lymph nodes. Here, we show that CD8(+) T cell activation and proliferation can occur in the airway after orthotopic lung transplantation. We also demonstrate that airway epithelium expresses major histocompatibility class I predominantly on the apical surface, both in vitro and in vivo, and initiates CD8(+) T cell responses in a polarized fashion, favoring luminal activation. Our data identify a unique site for CD8(+) T cell activation after lung transplantation, and suggest that attenuating these responses may provide a clinically relevant target.

Orthotopic vascularized right lung transplantation in the mouse

OBJECTIVES Orthotopic left lung transplantation in the mouse, as recently developed by our laboratory, represents a physiologic model for studies in transplantation biology. However, because of the limited overall respiratory contribution of the murine left lung, left lung transplant recipients remain healthy despite immune-mediated graft necrosis. We sought to develop a lung transplantation model in which animal survival depends on graft function. METHODS Orthotopic vascularized right lung transplantations using cuff techniques were performed in syngeneic and allogeneic strain combinations. Grafts were assessed histologically or functionally by measuring arterial blood gases from 7 to 28 days after transplantation. In a parallel set of experiments, syngeneic and immunosuppressed allogeneic hosts underwent a left pneumonectomy 2 weeks after right lung transplantation, with assessment of graft function 1 week later. RESULTS We performed 40 right lung transplantations, with a survival rate of 87.5%. Syngeneic grafts remain free of inflammation as far as 28 days after transplantation. On day 7, arterial oxygen levels in syngeneic recipients (481 +/- 90 mm Hg) are equivalent to those in naive mice (503 +/- 59 mm Hg) after left hilar occlusion. Alternatively, allogeneic grafts develop histologic evidence of acute rejection, and arterial oxygen levels are significantly decreased after left hilar clamping (53.3 +/- 10.3 mm Hg). Both syngeneic and immunosuppressed allogeneic right lung recipients tolerate a left pneumonectomy. CONCLUSIONS Right lung transplantation followed by left pneumonectomy represents the first survival model of vascularized lung transplantation in the mouse and will therefore allow for the design of novel studies in experimental lung transplantation.