Interleukin-6 regulation of direct lung ischemia reperfusion injury.

BACKGROUND: Lung ischemia reperfusion injury continues to adversely affect patient and graft survival after transplantation. While the role of interleukin-6 has been studied in ischemia-reperfusion models of intestine, liver, and heart, its participation in lung reperfusion injury has not been characterized. METHODS: We administered recombinant interleukin-6 to rat lungs through the intratracheal route before inducing left lung ischemia and reperfusion. Multiple in-vivo indicators of left lung injury were studied, as were transactivation patterns for nuclear factor kappa B and signal transduction and activators of transcription-3. Downstream effects on the elaboration of proinflammatory chemokines and cytokines were also studied. RESULTS: Recombinant interleukin-6 reduced endothelial disruption and neutrophil sequestration in left lung and alveolar spaces, resulting in improved oxygenation after ischemia and 4 hours of reperfusion. This protection was associated with decreased nuclear factor kappa B and signal transduction and activators of transcription-3 nuclear translocation early in reperfusion, and diminished proinflammatory mediator secretion late in reperfusion. CONCLUSIONS: Further studies focusing on the effects of recombinant interleukin-6 in large animal models are warranted, as this may be a novel strategy to improve outcomes after lung transplantation. Intratracheal administration may focus its efficacy on the lung while reducing effects on other organ systems during organ procurement.

Poly (ADP) ribose polymerase inhibition improves rat cardiac allograft survival.

BACKGROUND: Heart transplantation is an accepted treatment modality for end-stage heart failure. However, acute cellular rejection (ACR) continues to be a morbid complication. Recently a novel mechanism of inflammatory allograft injury has been characterized which involves overactivation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). In the present studies, we compared the efficacy of INO-1001, a novel, potent PARP inhibitor, in limiting ACR with and without adjuvant low-dose cyclosporine (CSA). METHODS: Heterotopic heart transplantation was performed utilizing Brown-Norway strains as donors and Lewis rats as recipients. Groups received daily intraperitoneal injections of: vehicle, low-dose CSA, low-dose INO-1001, high-dose INO-1001, and low-dose CSA combined with high-dose INO-1001. Additional animals were sacrificed on postoperative Day 5 for histologic assessments of allograft inflammation, including immunohistochemistry for nitrotyrosine and poly (ADP-ribose) (the product of PARP) staining. RESULTS: PARP inhibition significantly prolonged allograft survival relative to vehicle controls. The combination of low-dose CSA and INO-1001 resulted in a marked increase in allograft survival and significant reductions in allograft rejection scores. This was associated with decreased nitrotyrosine and PAR staining in transplanted cardiac allografts. CONCLUSIONS: Pharmacologic inhibition of INO-1001 prolongs allograft survival in a dose-dependent fashion in a rodent model of heart transplantation. PARP inhibitors may permit reductions in the dose of CSA needed for adequate immunosuppression after heart transplantation.

The role of CC and CXC chemokines in cardiac allograft rejection in rats.

Acute cellular rejection is due in part to an upregulation of chemokine genes, resulting in eventual cell-mediated cytotoxicity. The role of chemokines in acute cardiac allograft rejection is not fully characterized presently. These studies compared the patterns of expression for multiple chemokines in rodent cardiac allograft rejection. Allogeneic transplants were performed from Brown-Norway donors to Lewis recipients. Survival studies utilized daily administration of neutralizing antisera to MCP-1, CINC, and MIP-1alpha. Patterns of mRNA and protein expression were determined by Northern blots and immunohistochemistry. Allogeneic controls rejected at mean of 6.5 days. Neutralization of MCP-1 (10.8 days, P<0.001) and MIP-1alpha (7.5 days, P=0.004) function, but not CINC (6.2 days, P>0.05), significantly prolonged allograft survival. Message expression for the beta chemokines studied were increased by day 2 and continued to increase until day 6 just before rejection, while CINC levels did not change as dramatically after day 2. Chemokine protein levels mirrored mRNA patterns by IHC analysis. MCP-1 and MIP-1alpha appear to play regulatory roles in cardiac allograft rejection, while CINC is expressed, but not functional, in injury development. Beta chemokine activity should be studied further in hope of developing more targeted immunosuppression, or identifying specific chemokines that may be useful for immunosurveillance purposes.

Cyclosporine modulates the response to hypoxia-reoxygenation in pulmonary artery endothelial cells.

BACKGROUND: Depletion of macrophages, neutrophils, or lymphocytes confers only partial protection against experimental lung reperfusion injury, suggesting that inflammatory responses in other cell types contribute to tissue injury. Endothelial cell activation has previously been shown to be critical to the development of ischemia-reperfusion injury in other vascular beds. Furthermore, cyclosporine (CSA) reduces in vivo lung reperfusion injury through attenuated secretion of proinflammatory mediators. These studies determined whether pulmonary artery endothelial cells (PAEC), subjected to hypoxia and reoxygenation, promote inflammation and whether CSA afforded any modulation of that response. METHODS: Isolated rat PAEC were subjected in vitro to 2 hours hypoxia followed by up to 4 hours reoxygenation. Cells were pretreated with CSA or a cremaphor vehicle. Differences in activation of signaling kinases and transcription factors were assessed, as was cytokine-chemokine protein secretion. RESULTS: There was significant signaling kinase (extracellular signal regulated kinase [ERK 1/2]) activation by 15 minutes reoxygenation, which was temporally associated with marked activation of the transcription factors nuclear factor kappa B (NFkappaB) and early growth response one (EGR-1). At 4 hours reoxygenation there were significant increases in chemokine protein secretion. The CSA decreased ERK 1/2 phosphorylation and significantly attenuated transcription factor transactivation at 15 minutes reoxygenation. The CSA was found to be selective in reducing cytokine-chemokine elaboration at 4 hours reoxygenation. CONCLUSIONS: Hypoxia-reoxygenation induces ERK 1/2 phosphorylation, as well as transactivation of the transcription factors NFkappaB and EGR-1 in PAEC. Cyclosporine selectively reduces proinflammatory mediator secretion, likely by transcriptional regulation through NFkappaB and EGR-1. This is the first demonstration of ERK 1/2 inhibition afforded by CSA.