James J. Yun

Chemokine monokine induced by IFN-γ/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production

Monokine induced by IFN-γ (MIG; CXC chemokine ligand (CXCL)9) is important in T lymphocyte recruitment in organ transplantation. However, it is not known whether this chemokine, in addition to its chemotactic properties, exerts any effect on T lymphocyte effector functions. For in vivo studies, we used a previously characterized murine model of chronic rejection. The recipient mice were treated with anti-MIG/CXCL9 Ab; graft-infiltrating cells were analyzed for IFN-γ production. For in vitro studies, exogenous CXCR3 ligands were added to CD4 lymphocytes in MLRs, and the proliferative responses were measured. Separate experiments quantitated the number of IFN-γ-producing cells in MLRs by ELISPOT. Neutralization of MIG/CXCL9, in the in vivo model, resulted in significant reduction in the percentage of IFN-γ-producing graft-infiltrating T lymphocytes. In vitro experiments demonstrated that 1) exogenous MIG/CXCL9 stimulated CD4 lymphocyte proliferation in a MHC class II-mismatched MLR, 2) MIG/CXCL9 also increased the number of IFN-γ-producing CD4 lymphocytes in ELISPOT, 3) neutralization of MIG/CXCL9 in MLR reduced T lymphocyte proliferation, 4) IFN-γ-inducible protein 10/CXCL10 and IFN-inducible T cell α chemoattractant/CXCL11 had similar effects on T lymphocyte proliferation, 5) MIG/CXCL9 stimulated T lymphocyte proliferation in MHC class I- and total MHC-mismatched MLRs, 6) neutralization of CXCR3 reduced MIG/CXCL9-induced T lymphocyte proliferation and the number of IFN-γ-positive spots on ELISPOT, and 7) the proliferative effects of MIG/CXCL9 were mediated via an IL-2-independent pathway and were controlled by IFN-γ. This study demonstrates that MIG/CXCL9 stimulates T lymphocyte proliferation and effector cytokine production, in addition to its chemotactic effects. This novel observation expands our current understanding of MIG/CXCL9 biology beyond that of mediating T cell trafficking.

Combined blockade of the chemokine receptors CCR1 and CCR5 attenuates chronic rejection.

Background—Chemokine-chemokine receptor interaction and the subsequent recruitment of T-lymphocytes to the graft are early events in the development of chronic rejection of transplanted hearts or cardiac allograft vasculopathy (CAV). In this study, we sought to determine whether blockade of chemokine receptors CCR1 and CCR5 with Met-RANTES affects the development of CAV in a murine model. Methods and Results—B6.CH-2bm12 strain donor hearts were transplanted heterotopically into wild-type C57BL/6 mice (myosin heavy chain II mismatch). Recipients were treated daily with either Met-RANTES or vehicle starting on postoperative day 4 and were euthanized on postoperative days 24 and 56. We found that Met-RANTES significantly reduced intimal thickening in this model of chronic rejection and that Met-RANTES markedly decreased the infiltration of CD4 and CD8 T lymphocytes and MOMA-2+ monocytes/macrophages into transplanted hearts. Met-RANTES also suppressed the ex vivo and in vitro proliferative responses of recipient splenocytes to donor antigens. Finally, Met-RANTES treatment was associated with a marked reduction in RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels in the donor hearts. Conclusions—Antagonism of the chemokine receptors CCR1 and CCR5 with Met-RANTES attenuates CAV development in vivo by reducing mononuclear cell recruitment to the transplanted heart, proliferative responses to donor antigens, and intragraft RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels. These findings suggest that chemokine receptors CCR1 and CCR5 play significant roles in the development of chronic rejection and may serve as potential therapeutic targets.

The role of MIG/CXCL9 in cardiac allograft vasculopathy.

T lymphocytes play a critical role in chronic rejection of transplanted hearts, or cardiac allograft vasculopathy (CAV). However, the molecular mediators of T lymphocyte recruitment in CAV are incompletely defined. We hypothesized that the chemokine, monokine induced by interferon-gamma (MIG/CXCL9), which induces T lymphocyte migration in vitro, participates in T lymphocyte recruitment in CAV. In a previously characterized MHC II-mismatched murine model of CAV, intragraft MIG/CXCL9 gene transcript and protein levels increased on days 7, 14, and 24 days after transplantation, paralleling T lymphocyte recruitment and preceding intimal thickening. Antibody neutralization of MIG/CXCL9 significantly reduced CD4(+) T lymphocyte infiltration and intimal thickening in this model. MIG/CXCL9 was produced by graft-infiltrating MOMA-2+ macrophages in early and late stages of CAV. And, although T lymphocytes did not produce MIG/CXCL9, recipient CD4(+) T lymphocytes were required for sustained intragraft MIG/CXCL9 production and CAV development. These findings demonstrate that 1) MIG/CXCL9 plays an important role in CD4(+) T lymphocyte recruitment and development of CAV, 2) MOMA-2+ macrophages are the predominant recipient-derived source of MIG/CXCL9, and 3) recipient CD4 lymphocytes are necessary for sustained MIG/CXCL9 production and CAV development in this model. Neutralization of the chemokine MIG/CXCL9 may have therapeutic potential for the treatment of chronic rejection after heart transplantation.

Early and late chemokine production correlates with cellular recruitment in cardiac allograft vasculopathy.

BACKGROUND Cardiac allograft vasculopathy (CAV) remains the leading cause of late mortality in heart transplant recipients. Activated T lymphocytes and macrophages infiltrate the donor heart before vascular intimal thickening develops, but the specific mediators of mononuclear cell recruitment leading to CAV are unknown. Therefore, we sought to define the relationship between chemokine gene expression and production, T lymphocyte and macrophage recruitment, and intimal thickening in a murine model of CAV. METHODS B10.A or B10.BR strain hearts were transplanted heterotopically into B10.BR mice. Recipients were killed at 1, 4, 7, 14, and 30 days. Donor hearts were assayed for chemokine gene expression with ribonuclease protection and for protein with ELISA. Intragraft cellular infiltration was defined immunohistochemically. Intimal thickening was quantitated morphometrically. RESULTS Early and late patterns of intragraft chemokine expression associated with distinct cellular infiltration were identified. First, transient MIP-2 and MCP-1/JE production in isografts and allografts correlated with neutrophil and macrophage infiltration. MCP-1/JE production and macrophage infiltration was greater in allografts than isografts. Second, allografts demonstrated sustained lymphotactin, RANTES, and IP-10 expression, beginning at day 4, correlating with persistent macrophage and T lymphocyte infiltration. Intimal thickening became evident at 14 days. Isografts did not display the late pattern of sustained chemokine gene expression, cellular infiltration, or intimal thickening. CONCLUSIONS Transient, early MIP-2, and MCP-1/JE production in isografts and allografts correlated with neutrophil and macrophage recruitment, and is likely related to ischemia-reperfusion. In allografts, the delayed induction of chemokines specific for macrophages and T lymphocytes correlated with mononuclear cell infiltration and preceded intimal thickening. This study thus demonstrates a dual pattern of chemokine induction correlating with intragraft mononuclear cell recruitment, associated with ischemia-reperfusion and CAV development. Chemokine-directed interventions may interfere with leukocyte trafficking and inhibit CAV development.

Bridge to lung transplantation using short-term ambulatory extracorporeal membrane oxygenation

Severe respiratory failure refractory to mechanical ventilation can occur in patients awaiting lung transplantation (LTx). Use of extracorporeal membrane oxygen (ECMO) to bridge these patients to LTx is associated with considerable morbidity and mortality. Most techniques rely on femoral cannulation, thereby immobilizing patients. This in turn can lead to rapid deconditioning and predisposes patients to nosocomial pneumonia, deep venous thrombosis, and muscle wasting. This case highlights the use of upper-extremity ECMO, which was established without intubation in a critically ill transplant candidate, allowed ambulation, and ultimately served as a successful bridge to LTx.

Rantes production during development of cardiac allograft vasculopathy.

Background. RANTES (regulated on activation, normal T cell expressed and secreted) production has been shown to correlate with mononuclear cell recruitment and precede intimal thickening in cardiac allograft vasculopathy (CAV). However, the cells that produce RANTES in CAV are undefined. Therefore, in an MHC II–mismatched murine model of CAV, we sought to (1) define the cellular sources of RANTES and (2) determine the role of CD4+ lymphocytes in RANTES production during CAV development. Methods. B6.CH-2bm12 strain donor hearts were transplanted heterotopically into wild-type (WT) or CD4 knockout (CD4KO) C57BL/6 mice (MHC II mismatch). No immunosuppression was used. Recipients were sacrificed at 7, 14, and 24 days. Intragraft RANTES gene expression and protein levels were determined with ribonuclease protection assay and ELISA, respectively. At days 7 and 24, RANTES production by graft-infiltrating cells was defined with intracellular RANTES staining and multicolor FACS analysis. Intimal thickening was quantitated morphometrically. In murine hearts and in six explanted human hearts with advanced CAV, RANTES was also localized immunohistochemically. Results. NK, NKT, and &ggr;&dgr;+ cells, in addition to CD4+, CD8+ lymphocytes, and CD11b+ macrophages, produced RANTES in early and late stages of CAV. RANTES-producing NK, NKT, and &ggr;&dgr;+ cells tripled in number during CAV development; by day 24, NK and &ggr;&dgr;+ cells each outnumbered CD4+ lymphocytes and CD11b+ macrophages. The presence of CD4+ lymphocytes was required for sustained RANTES production in allografts, which correlated with mononuclear cell recruitment and preceded intimal thickening. In murine and explanted human hearts with advanced CAV, RANTES immunolocalized with graft-infiltrating mononuclear cells and vessel wall cells. Conclusions. We present evidence that other cell types in addition to CD4+, CD8+ T lymphocytes, and CD11b+ macrophages contribute significantly to RANTES production in CAV. In this MHC II–mismatched murine model of CAV, sustained RANTES production requires CD4+ lymphocytes, correlates with mononuclear cell recruitment, and precedes intimal thickening. In experimental and human CAV, vessel wall cells may also produce RANTES. Interventions aimed at inhibiting RANTES production in CAV may need to target several types of cells, and neutralization of RANTES bioactivity may reduce mononuclear cell recruitment and CAV development.

CD40 Signaling Replaces CD4+ Lymphocytes and Its Blocking Prevents Chronic Rejection of Heart Transplants

Chronic rejection remains the major obstacle to long term survival in heart transplant recipients. The cellular and molecular mechanisms that underlie chronic rejection are not known, and their discovery can form the basis of clinical intervention. Several investigators have suggested that the development of chronic rejection in solid organ transplants is dependent on help mediated by CD4+ lymphocytes. Importantly, the mechanism through which help is provided has not been fully delineated in transplant rejection. Using a murine heterotopic heart transplant model without immunosuppression, this study defines the functional role of CD4+ lymphocytes in chronic rejection. In an MHC class II-mismatched model, we demonstrate that chronic rejection was absolutely contingent on the presence of CD4+ lymphocytes. Importantly, here we report that signaling through CD40 can replace the requirement of CD4+ lymphocytes, demonstrated by the development of chronic rejection in CD4 knockout recipients treated with a CD40-activating mAb (FGK45). The return of rejection appears to be a CD8+ lymphocyte-dependent process, noted by the absence of rejection in FGK45-treated recombinase-activated gene knockout (CD4+ and CD8+ lymphocyte-deficient) recipients. The CD40 signaling pathway works independently of B7-CD28 costimulation, as indicated by the development of severe chronic rejection in CD28 knockout recipients. Importantly, this study provides evidence that CD40 ligand-targeted therapies may prevent chronic rejection only in strain combinations where CD4+ lymphocyte help is absolutely required.

CD8+ lymphocytes augment chronic rejection in a MHC class II mismatched model.

Chronic rejection, or cardiac allograft vasculopathy (CAV), remains the leading cause of late death in heart transplant recipients. The precise role and contributions of T lymphocyte subsets to CAV development remains unknown. Methods. Donor hearts from B6.C-H2bm12 mice were transplanted into T lymphocyte subset knockout recipients and T lymphocyte-reconstituted nude recipients. No immunosuppression was used. Intimal proliferation was measured morphometrically. In vitro studies were performed to analyze the donor-specific activation status of recipient CD8+ lymphocytes by examining cellular proliferation, interleukin-2 secretion, and interleukin-2R&agr; expression. Intracellular cytokine staining assay was performed to determine both the profile and source of intragraft cytokines. Results. Hearts transplanted into wild-type recipients developed severe CAV by 24 days. Intimal lesions were absent in the hearts that were transplanted into nude and CD4−/− knockout mice (containing CD8+ lymphocytes). In contrast, the donor hearts in CD8−/− knockout recipients (containing CD4+ lymphocytes) developed CAV, but significantly less than in wild-type. Adoptive transfer of T lymphocyte subset populations into nude recipients confirmed that CAV was absolutely contingent on CD4+ lymphocytes, and that CD8+ lymphocytes played an additive role in intimal lesion progression in the presence of CD4+ lymphocytes. Although CD8+ lymphocytes alone did not cause CAV in vivo, we demonstrated that MHC class II disparate alloantigens activated CD8+ lymphocytes both in vivo and in vitro. Finally, both CD4+ and CD8+ lymphocytes contributed to the intragraft IL-2 and IFN-&ggr; production. Conclusions. In this MHC class II mismatched murine model, CAV is a T lymphocyte dependent event, and absolutely contingent on the presence of CD4+ lymphocytes. Furthermore, CD8+ lymphocytes (1) are activated by MHC class II disparate antigens and (2) play a significant role in the progression of lesion development. Finally, both CD4+ and CD8+ lymphocytes contribute to CAV development via secretion of IFN-&ggr;, a known mediator of CAV in this model.

Staged surgical approach to neonates with aortic obstruction and single-ventricle physiology

BACKGROUND The surgical management of neonatal systemic outflow obstruction and complex single ventricle pathology is variable. METHODS In 15 neonates (12 boys and 3 girls) with complex forms of single-ventricle pathology and aortic coarctation or interruption, an initial strategy of banding the pulmonary artery and repair of the obstruction from a left thoracotomy was undertaken. RESULTS The median age at operation was 6 days (range 2 to 33 days) and the median weight was 3.3 kg (range 2 to 4.6 kg). There were no early deaths and one late death after the initial surgical palliation. Of the 14 survivors, 8 have undergone a bidirectional cavopulmonary anastomosis. The median age for bidirectional Glenn was 9.75 months (range 3.5 to 26 months). Seven infants have required Damus-Kaye-Stansel reconstruction for subaortic obstruction (one early death). The median age of the Damus-Kaye-Stansel procedure was 4 months (range 3 weeks to 9 months). Thirteen of 15 patients (87%) are alive and 6 have proceeded to a Fontan operation (median follow-up 68 months). A single failing Fontan required takedown to bidirectional Glenn and central shunt. CONCLUSIONS Our experience suggests that this high-risk subgroup of neonates with aortic obstruction and single-ventricle pathophysiology is safely managed by initial pulmonary artery banding palliation and repair of aortic obstruction. This strategy, careful surveillance, and early relief of subaortic stenosis can maintain acceptable anatomy and hemodynamics for later bidirectional Glenn and Fontan procedures.

CD8 lymphocytes are sufficient for the development of chronic rejection

Background. The role of CD8 lymphocytes, in chronic rejection or cardiac allograft vasculopathy (CAV), is incompletely understood. The purposes of this study were to determine whether CD8 lymphocytes, in the absence of CD4 lymphocytes, are capable of causing the intimal lesions of CAV; and if so, to define the effector mechanism(s) of CD8 lymphocytes. Methods. We modified a previously characterized major histocompatibility complex class II mismatched murine model of CAV. Wild-type CD8 lymphocytes were transferred to nude mice followed by heterotopic heart transplantation. Recipient mice were then treated with a CD40 activating antibody, which is known to provide help for CD8 lymphocyte activation, in the absence of CD4 lymphocytes. Donor hearts were harvested on day 40 posttransplantation and analyzed for cellular infiltrates and intimal thickening. In separate experiments, isolated perforin −/−, Fas ligand (FasL) −/−, and interferon (IFN)-&ggr; −/− CD8 lymphocytes were transferred to nude mice followed by identical experimented protocol. Results. With adaptive transfer of wild-type CD8 lymphocytes, the donor hearts were infiltrated with activated CD8 lymphocytes and displayed significant intimal lesions. Adoptive transfer of perforin −/− and FasL −/− CD8 lymphocytes to nude mice resulted in similar patterns of CD8 lymphocyte infiltration and similar severity of intimal lesions. The donor hearts from IFN-&ggr; −/− CD8 lymphocyte reconstituted recipients displayed minimal intimal lesions, although CD8 lymphocytes were present in the allografts. Conclusions. Unprimed CD8 lymphocytes in the absence of CD4 lymphocytes can cause intimal lesions of CAV. CD8 lymphocytes production of IFN-&ggr;, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.