Ischemia-Reperfusion Injury: Key to New Therapies for the ICU
By Doreen M. Anardi, RN
The quest to understand and manage inflammation in human disease is one that unites seemingly disparate fields of clinical practice and scientific investigation. The elucidation of the inflammatory response and laboratory investigation of disordered inflammation or inflammation "run amuck" has proceeded with much enthusiasm in the last few decades. This essay discusses one aspect of disordered inflammation that has great potential relevance to critical care: neutrophil-mediated ischemia-reperfusion injury and the promising role of anti-adhesion therapy. Many investigators feel that the time is ripe for more randomized clinical trials that use these agents.
What Clinical Conditions Can Lead to Ischemia-Reperfusion Injury?
Damage at the cellular, tissue, and organ level can be seen in the patients with myocardial infarction, stroke, mesenteric ischemia, peripheral vascular disease, limb reimplantation, organ transplantation, burns, cardiopulmonary bypass, and circulatory shock.2-6,10 The phrases we use to describe these patients are "they are third spacing" or "they have capillary leak syndrome." Patients who prompt these expressions may be unrecognizable to their loved ones because of massive accumulation of tissue fluid. The undesirable physiologic outcome of this process is the clinical syndrome of multiple organ failure, which is characterized by cardiovascular, pulmonary, central nervous system, hepatic, gastrointestinal, and renal dysfunction, accompanied by signs of generalized inflammation, which increases morbidity and mortality.4,5,7
What is Ischemia-Reperfusion Injury?
While many advances have been made in restoring perfusion following ischemic events, it has become clear that much of the tissue damage associated with ischemia is actually related to reperfusion of the ischemic tissue.2,8 Oxygen-derived free radicals are generated at the time of reperfusion and are important mediators of this injury. An important source of these free radicals is the neutrophil, which can also cause damage by releasing proteases and phospholipase products.2 Neutrophils play a key role in preventing disease: their ability to adhere to vascular endothelium and to enter the tissues is critical in order to participate in host defense and repair damaged tissue. It is this same ability to leave the intravascular space and move through the tissues that implicates the neutrophil as a central player to the pathogenesis of disorders produced by ischemia-reperfusion injury as well as acute inflammatory diseases.2 The inflammatory system evolved long before we were able to reperfuse organs, so reperfusion injury most likely represents "accidental" triggering of the inflammatory response and not a homeostatic mechanism.5
Neutrophils become activated by a variety of inflammatory stimuli, including cytokines, activated complement components, platelet activating factor, bacterial peptides or endotoxin, and the release of abundant inflammatory mediators.2 Unregulated adherence of activated neutrophils can cause direct endothelial injury, interrupting vascular integrity producing edema, hemorrhage, and thrombosis, and leading to a vicious cycle of more ischemia and further stimulation of the mediators of inflammation.2,5 This process can overwhelm the protective naturally occurring tissue- and plasma-based anti-inflammatory mechanisms and interfere with the homeostatic role of the endothelial cell.2,6
The Adhesion Cascade
Neutrophil adhesion and subendothelial migration in both the setting of host defense and pathologic endothelial injury can be simplified into five sequential steps (See Figure). First, activation of the vascular endothelial cells create a pro-adhesive condition in which the neutrophils leave the laminar flow stream. Second, random contact between circulating neutrophils and the vessel wall leads to low-affinity, adhesion-dependent leukocyte rolling in post-capillary venules at the site of inflammation. Third, these neutrophils move along the endothelial surface until they meet an interendothelial cell junction, where local activation results in firm adhesion or sticking, creating a protected microenvironment in which the neutrophils release proteases, oxidants, and other toxic products that are damaging to the endothelium and are protected from circulating anti-inflammatory agents. Fourth, this microenvironment allows the neutrophils to crawl between the endothelial cells (diapedesis) into the extravascular tissue. Finally, subendothelial migration of neutrophils is further directed by chemotactic mechanisms where further release of tissue-damaging toxic neutrophil products can continue and may directly injure tissue or cause further organ damage. 2,8,9
Amazing progress has been made in identifying and characterizing the specific proteins that direct neutrophil-endothelial interaction, endothelial activation, and neutrophil adhesion.9 These adherence molecules are currently classified into two major categories: 1) the leukocyte integrins that interact with their ligands on endothelial cells that belong to the immunoglobulin superfamily; and 2) the selectin receptors, which recognize specific carbohydrate counterstructures.2,8 The selectin receptors appear to be responsible for the initial transient adhesion that becomes rolling. Once the neutrophil is slowed by selectin-carbohydrate interactions, local stimuli activate the neutrophil to create firm integrin-immunoglobulin adhesion.2
There are three different selectins: L-selectin, found only on leukocytes; E-selectin, found only on endothelial cells; and P-selectin, which occurs on both endothelial cells and platelets. There are three subfamilies of integrins, that are of ancient origin and have been highly conserved during evolution.9 These subfamilies are differentiated by b subunits. The leukocyte b2 group mediates adhesion to endothelium. The b2 integrins are found only on leukocytes and are composed of a common b2 unit (clusters of differentiation, CD 18) linked to one of 3 a chains (CD11 a, b, or c) and are required to adhere to endothelium.9,10 CD11a/CD18 is expressed on all leukocytes, and CD11b/CD18 is expressed only on neutrophils, monocytes, and natural killer cells. CD11c/CD18 is expressed only on neutrophils, monocytes, and some lymphocytes.10
The immunoglobulin superfamily consists of cell surface proteins involved in antigen recognition, complement binding, and cell adhesion. Endothelial members of this superfamily involved in leukocyte-endothelial cell adhesion include the intercellular adhesion molecules ICAM-1 and ICAM-2, and platelet-endothelial cell adhesion molecule-1 (PECAM-1).10 In addition to endothelial cells, ICAM-1 is expressed on leukocytes, fibroblasts, and epithelial cells and binds both CD11a/CD18 and CD11b/CD18 to mediate firm adherence of leukocytes to endothelial cells.10 ICAM-2 is expressed on endothelial cells and is a ligand for CD11a/CD18. PECAM-1 is expressed on endothelial cells, localized to the intercellular junctions. It has been shown to play a role in the process of neutrophil and monocyte diapedesis between endothelial cells.10
Anti-Adhesion Therapy in Experimental Models of Human Disease
Since the protected microenvironment that is created once the neutrophil adheres to the endothelial cell is likely to render anti-oxidant and anti-protease interventions ineffective, using monoclonal antibodies to interfere with adhesion has been an exciting direction in regulating this injury.8 Monoclonal antibodies (MAb) that block neutrophil-endothelial adhesion molecules have attenuated injury in the following experimental models: intestinal ischemia; tissue reperfusion injury; shock/resuscitation; myocardial ischemia; skeletal muscle ischemia; central nervous system ischemia.2 This is a general summary and partial listing of the experimental models and anti-adhesion intervention. More detailed descriptions can be found in the cited references.
A model of tissue reperfusion using a rabbit ear isolated on its vascular pedicle demonstrated that blocking neutrophil adherence with anti-CD18 MAb greatly reduced edema and tissue necrosis. Similar results were obtained with an anti-P-selectin antibody and with an anti-L-selectin antibody.8,10 Administration of oligosaccharide to compete with selectin-endothelial binding has reduced injury during reperfusion.10
Attenuation of myocardial injury following ischemia was demonstrated by inhibiting either CD11/CD18 or P- or L-selectin-mediated adhesion.8,10 CD18 blockade preserved endothelial responsiveness to vasodilators.4 Administration of oligosaccharide to compete with selectin-endothelial binding has reduced injury during reperfusion.10 In a model of embolic stroke, both CD18 and ICAM-1 reduced experimental brain injury.8
Reperfusion injury following skeletal muscle ischemia was decreased by blocking both CD18 and P-selectin.8,10 Oligosaccharide administration to compete with selectin-endothelial binding has reduced injury during reperfusion.10 In a hemorrhagic shock and resuscitation model, representing a global ischemia-reperfusion injury, decreased microvascular injury, and improved survival with a single dose of CD18 Mab was demonstrated.8 P-selectin has also reduced injury in this model.10
P-selectin blockade reduced local ischemia-reperfusion injury in the intestine and liver.10 ICAM-1 or CD18 blockade has increased circulation in the zone of stasis in models of thermal injury. In another model of dermal vascular injury, vascular permeability and hemorrhage in the dermis were reduced by treatment with MAb to CD 18, CD11b, ICAM-1, E-selectin, and L-selectin. In addition, damage to a remote organ represented by lung vascular permeability and hemorrhage was seen following blockade with these same agents.10
Potential Approaches for Anti-Adhesion Therapy and Clinical Trials
As the experimental models demonstrate, the potential success of anti-adhesion therapy in a variety of clinical conditions is exciting to contemplate. A major concern has been the potential risk of overwhelming infection outweighing the benefits of reducing damage from ischemia-reperfusion injury. Experimental models of infection have been done in conjunction with the anti-adhesion experiments to characterize the risks. Studies have shown that blocking CD18-mediated neutrophil function does not increase infectious complications or mortality in models of bacterial peritonitis and can improve outcome in certain septic models, such as bacterial meningitis and gram-negative sepsis.8 It has been shown that, while extremely high innoculations of subcutaneous bacteria can increase infectious complications and mortality after a single dose of CD18 MAb, this was not seen with smaller, more clinically relevant bacterial quantities.8 P-selectin inhibition has not demonstrated increased susceptibility to soft-tissue infections.8 A handful of studies are in the progress of examining the effects of anti-adhesion therapy on conditions affected by ischemia-reperfusion injury. One study is an ongoing phase II clinical trial of the effects of aICAM-1 Mab on the outcome of adult patients with thermal injury.10
A phase I clinical trial has been completed in human renal allograft recipients. Patients received a cadaveric donor transplant and received conventional immunosuppression. In addition, the treatment group received aICAM-1 MAb prior to transplantation followed by a daily dose for two weeks. There were no instances of primary nonfunction in the treatment group and 78% of the patients had good-to-excellent graft function at 16-30 month follow-up. These results compared to a 20% incidence of primary nonfunction in the control group, with 56% adequate function at long-term follow-up. Data from phase II and III clinical trials testing the efficacy of aICAM-1 MAb are currently being analyzed.10
An open-label, pilot phase II clinical trial of humanized CD11/CD18 MAb in hemorrhagic shock has recently been completed.12 A single dose of CD11/CD18 MAb was administered to patients with hypotension due to hemorrhage within four hours of receiving medical attention (reperfusion). The trial demonstrated that adequate blood levels of study drug could be attained despite ongoing blood loss and replacement. Prophylactic antibiotics were given and serious soft tissue infections did not appear to be increased.12 Plans for a randomized, controlled trial are underway. Clinical trials testing the safety and efficacy of this Mab in the setting of stroke and myocardial infarction have begun.
The Future of Anti-Adhesion Therapy
Ischemia followed by reperfusion leads to significant injury in a variety of organs. Recent investigations have markedly increased our knowledge of the molecular pathways of neutrophil adhesion and the development of organ injury.9 The identification of the sequential steps of neutrophil adhesion presents a variety of potential therapeutic targets.9 Research in this area will benefit the knowledge base of many pathologic conditions related to neutrophil adhesion.
References
1. Lehr HA, et al. Circulation 1994;90(3):1580.
2. Vedder NB, et al. In: Ayres S, et al (eds). Textbook of Critical Care, 3rd ed. Philadelphia: WB Saunders; 1995:192-199.
3. Bevilacqua MP, et al. Annu Rev Med 1994;45:361-378.
4. Thiagarajan RR, et al. Thromb Haemost 1997;78: 310-314.
5. Harlan JM, et al. West J Med 1991;155:365-369.
6. Verrier E. J Cardiovasc Pharmacol 1996;27(Supp 1):S26-S30.
7. Stechmiller JK, et al. Am J Crit Care 1997;6:204-209.
8. Vedder NB, et al. Topics Mol Med 1995;1:127-140.
9. Sharar SR, et al. Semin Immunopathol 1995;16:359-378.
10. Cornejo CJ, et al. Adv Pharmacol 1997;39:99-142.
11. Cronstein BN, et al. Arthritis Rheum 1993;36:147-155.
12. Vedder N, et al. in 4th Internat Congress on Immune Consequences of Trauma Shock and Sepsis. Munich, Germany, March 4-8, 1997.
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