Steven M. Woolley

β-Chemokine function in experimental lung ischemia-reperfusion injury

Abstract Background Although chemokines are functionally important in models of ischemia-reperfusion injury, little is known about their role in lung ischemia-reperfusion injury (LIRI). This study examined the role of the β-chemokines, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T cells expressed and secreted (RANTES) in LIRI. Methods Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received anti–MIP-1α, anti–MCP-1, or anti-RANTES antibodies before reperfusion. Changes in lung vascular permeability were measured with iodine 125–labeled bovine serum albumin. Neutrophil accumulation in the lung parenchyma was determined by myeloperoxidase activity, and bronchoalveolar lavage was performed to measure leukocyte cell counts. Western blots, Northern blots, and ribonuclease protection assays assessed β-chemokine messenger RNA and protein levels. Results Animals receiving anti–MIP-1α demonstrated reduced vascular permeability compared with controls ( p p p p Conclusions MIP-1α is functionally significant in the development of LIRI. It likely exerts its effects in part by mediating the expression of proinflammatory and antiinflammatory cytokines and influencing tissue neutrophil recruitment. MCP-1 and RANTES seem to play relatively minor roles in the development of direct LIRI.

Endogenous interleukin-4 and interleukin-10 regulate experimental lung ischemia reperfusion injury

BACKGROUNDnRegulatory cytokines play functional roles in experimental heart, hindlimb, and liver ischemia reperfusion injury. However, little is known about their involvement in direct lung ischemia reperfusion injury (LIRI). These studies were undertaken to investigate the role of two regulatory cytokines, interleukin-4 (IL-4) and IL-10, in an in vivo model of LIRI.nnnMETHODSnLeft lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received either recombinant IL-4 or recombinant IL-10, or antibodies to IL-4 or IL-10 immediately before reperfusion. Lung injury was quantitated by permeability indices, lung parenchymal neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) effluent. Expression of IL-4 and IL-10 was determined by immunoblotting, and mRNA expression for early response cytokines was evaluated by ribonuclease protection assays.nnnRESULTSnIL-4 and IL-10 protein expression was significant after 2 hours of reperfusion. Animals receiving anti-IL-4 (p = 0.05) and anti-IL-10 (p = 0.01) antibodies demonstrated increased permeabilities compared with positive controls. Lung tissue neutrophil accumulation (p < 0.004) and BAL leukocyte content (p < 0.04) were also significantly increased in animals receiving anti-IL-10 antibodies. Conversely, animals receiving recombinant IL-4 and recombinant IL-10 demonstrated reduced permeabilities and lung MPO content. Both anti-IL-4 and anti-IL-10 treatment increased mRNA expression for a number of early response cytokines, including TNF-alpha and IL-1beta.nnnCONCLUSIONSnIL-4 and IL-10 are expressed in response to LIRI and function to decrease injury severity. These effects are partly due to modulated expression of early proinflammatory cytokines.

The role of the beta chemokines in experimental obliterative bronchiolitis.

Beta chemokines have been implicated in cardiac and renal allograft rejection. This study determined if antibody antagonization of beta chemokines conferred protection against the development of experimental obliterative bronchiolitis (OB) in a heterotopic rat tracheal allograft model. Rat tracheas were transplanted from Brown-Norway or Lewis donors into Lewis recipients. Rats received 200 microg/day of either anti-RANTES or anti-MCP-1 antibody for 14 days. Luminal obstruction and epithelial loss were calculated. Northern blots for MCP-1 and RANTES mRNA expression were performed, and immunohistochemistry for chemokine protein localization. There was a significant increase in airway obstruction in allografts compared to isografts (P < 0.001). Antibody-treated allografts demonstrated an amelioration of airway obstruction from 58% (vehicle allografts) to 26% (anti-RANTES) and 12% (anti-MCP-1), both of which were significant (P < 0.001). Epithelial preservation was increased in both antibody-treated groups (P < 0.001), and increased expression of MCP-1 and RANTES mRNA was present in tracheal allografts by Day 2 and maximal by Day 6. Beta chemokines are expressed during the development of experimental OB, as MCP-1 and RANTES mRNA expression increased with time from transplantation. Both MCP-1 and RANTES are functional in the formation of the fibroproliferative response that characterizes OB in this model, and their antagonization conferred protection against airway obstruction and epithelial loss.

Proinflammatory response of alveolar type II pneumocytes to in vitro hypoxia and reoxygenation.

Type II pneumocytes (T2P) are integral in preserving the integrity of the alveolar space by modulating the fluid composition surrounding the alveolar epithelium. There is also mounting evidence supporting their contribution to the development of acute inflammatory lung injury subsequent to oxidative stress. This study characterized the response of T2P to in vitro hypoxia and reoxygenation (H&R). Rat T2P from a cultured cell line (RLE‐6TN) were rendered hypoxic for 2 h, and reoxygenated for up to 6 h. Activation of signaling kinases, the nuclear translocation of proinflammatory transcription factors, and quantification of secreted cytokine and chemokine protein content were assessed. Type II pneumocytes expressed activated extracellular signal regulated kinase (ERK) 1/2 maximally at 15 min of reoxygenation. C‐jun n‐terminal kinase (JNK) and p38 activation was minimal at all time points studied. The nuclear translocation of nuclear factor kappa B (NFκB) and activator protein (AP)‐1 were dramatic after 15 min of reoxygenation. There was a significant increase in the protein secretion of CINC (p = 0.03), IL‐1β (p = 0.02), and monocyte chemoattractant protein‐1 (p < 0.001) at 6 h of reoxygenation. Type II pneumocytes respond directly to H&R. ERK 1/2 activity peaks at 15 min of reoxygenation, and correlates temporally with the nuclear translocation of NFκB and AP‐1. These signaling cascades likely promote the elaboration of proinflammatory mediators.

Alpha chemokines regulate direct lung ischemia–reperfusion injury

BACKGROUNDnAlpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia-reperfusion injury in a warm in situ hilar clamp model.nnnMETHODSnLeft lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to (125)I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting.nnnRESULTSnTreatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression.nnnCONCLUSIONSnCINC and MIP-2 are important mediators involved in direct lung ischemia-reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.