Mechanisms Underlying Therapeutic Attenuation of the Pro-Inflammatory State Following Cardiopulmonary Bypass

Joseph C. Cleveland, Jr., MD and Alden H. Harken, MD
Department of Surgery, Division of Cardiothoracic Surgery
University of Colorado Health Sciences Center
Denver, Colorado

“Supposing is good, but finding out is better” – Mark Twain

Before we summarize our current research, it might prove helpful to briefly review our laboratory’s past. Our interest in the early to mid 1990’s was directed toward understanding the neutrophil’s role in mediating cellular injury. We realized that the neutrophil was centrally involved in orchestrating a vigorous host mediated pro-inflammatory state following injury. It was still unclear to us, however, whether specific signals directed the neutrophil to its target, and whether the neutrophil received its “marching” orders from a distance or from a local tissue interaction. These questions were subsequently explored in a series of experiments directed by Dr. Gene Moore which employed a rodent gut ischemia-reperfusion injury model. We found that neutrophils acquired the ability to deliver several times their “killing “ power when they had previously received signals of trouble elsewhere – for example gut ischemia-reperfusion. This finding led to one of our early observations – that a cell (or neutrophil) could be “primed” or “reprogrammed” or “preconditioned”. While we still debate the definition of priming amongst ourselves, we generally believe a “primed” cell to behave differently when encountering a subsequent stimulus based on that cell’s immediate past history. This concept of priming will re-emerge later as a central concept in our laboratory’s (and others) efforts to delineate ischemic preconditioning.

Clinically, all of us have observed the seemingly out of proportion derangement of pulmonary function which occurs in a variety of ischemia-reperfusion injuries. Understanding the nature of this acute lung injury also became an important component of our laboratory’s research. Drs. David Fullerton, Robert McIntyre, and Brett Sheridan utilized an isolated pulmonary artery vascular ring model in a series of experiments to further characterize the role of the neutrophil in acute lung injury. These experiments implicated the neutrophil as a prime offender in mediating the acute pulmonary hypertension associated with sepsis. Further, the intracellular signaling in pulmonary vascular smooth muscle also seemed to be altered in the setting of various shock or ischemia – reperfusion injuries. The understanding of neutrophil mediated intracellular signaling proved clinically useful as our group (and many other laboratories) showed that nitric oxide and nitric oxide donors could attenuate much of the exaggerated pulmonary vasoreactivity during ischemia-reperfusion events. While the majority of these research efforts focused on understanding the destructive effects of a “primed” neutrophil, we began to question whether cells could be constructively “primed”.

Our laboratory became interested in the concept of ischemic preconditioning, as it appeared to be a perfect paradigm of “constructive priming”. Ischemic preconditioning describes myocardium that is rendered tolerant to an ischemia-reperfusion injury by a preceding brief episode of ischemia. Drs. Harken, Cleveland, Meldrum, Meng, and Banerjee led this component of our laboratory’s research as we sought to delineate the intracellular signals responsible for preconditioning. We felt that if preconditioning were ever to be used clinically, ischemia would not be a psychologically acceptable stimulus to surgeons, or our patients. Initially, we focused our efforts at the receptor level and found that activation of either purinergic (adenosine based) or alpha -adrenoreceptors was capable of partly reproducing the protective effects of antecedent ischemia. We subsequently became interested in protein kinase C activation, as a considerable amount of work implicates this intracellular kinase as an important mediator of ischemic preconditioning. Any good surgical laboratory constantly seeks relevance. It appeared that PKC activates the mitochondrial potassium-ATP channel (KATP channel) in order to achieve the protected or “preconditioned” status. Dr. Joe Cleveland recognized the sulfonylurea oral diabetic agents as inhibitors of the K-ATP channel. He proposed that oral hypoglycemic agents might therefore prevent protective cardiac preconditioning and thus explain the paradoxical increase in cardiac events long recognized in diabetics on oral agents versus diabetics in insulin.

While we had focused the majority of our laboratory efforts upon understanding the role of “primed” neutrophils and “preconditioned” cardiomyocytes, Dr. Dan Meldrum brought previous experience with macrophage signaling to our laboratory. Our laboratory began then to examine the role of the circulating blood monocyte and resident cardiac macrophage and their cytokine signals during ischemia-reperfusion. This series of investigations into macrophage physiology uncovered a pivotal role of TNF during myocardial surgical stress. In retrospect, the linkage between TNF and neutrophils seemed intuitive, but as Mark Twain quipped(this quote is displayed on the wall of our laboratory), “Supposing is good, but finding out is better”. This link between macrophage cytokine signaling and neutrophils has developed into our most recent area of interest in which we are examining the relationships between TNF and the interleukins (IL-1, IL-6, IL-8, IL-10, IL-12, and IL-18). With this brief overview of our laboratory’s experience in the last 15 years, the following represent some of our current research interests:

• The role of the TNF receptor in mediating neutrophil recruitment during Acute Lung Injury. This project focuses on the intracellular signaling that occurs with TNF receptor stimulation. Specifically, the role of the TNF p55 receptor in lipopolysaccaride induced lung injury is examined in both mice and alveolar macrophages.
• The role of the TNF receptor in mediating macrophage heat shock production. Heat shock protein is produced during a variety of surgical stresses and may be modified by TNF. Dr. Julie Heimbach is working to define the signaling in macrophages as it relates to post-translational modification of the Heat Shock Protein 70 family.
• Dr. Tom Robinson and Dr. Charles Cairns are tracking the inner mitochondrial membrane potential and propose this to be a proximal determinant of myocellular health and happiness.
• Dr. Ben Pomerantz is exploring IL-18 cytokine signaling in the human myocardium during ischemia-reperfusion.
• Dr. Stephanie Miller and Dr. Craig Selzman postulate that MIP-2 not only attracts cytokine spewing macrophages into blood vessel walls; but also directly stimulates vascular smooth muscle proliferation leading to atherosclerosis.
• Dr. David Ciesla, Dr. Rick Gonzales, and Dr. Gene Moore have worked out the pacifying influence of a hypoosmolar stress (hypertonic saline) on post-injury “primed” neutrophils in order to attenuate the hyperinflammatory state leading to MOF.
• Dr. Casey Calkins, Dr. Robert McIntyre, and Dr. Chris Siliman have identified lipid breakdown products from transfused, stored blood that provoke inflammation directly.
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