Neutrophil Adhesion to Vascular Prosthetic Surfaces Triggers Nonapoptotic Cell Death

Vascular prosthetic infections are intransigent clinical complications associated with excessive rates of death and complications. It has been estimated that the number of vascular graft procedures performed in the United States per year exceeds 500,000, including peripheral vascular reconstruction and coronary bypass grafts. Because of the limitations of prosthetic materials in small vessel configurations, they are used in less than a third of these procedures. Infections occur in 2% to 12% of implanted vascular prostheses despite the use of systemic antibiotic prophylaxis. When these events unfold, they are associated with death rates of 30% to 50% and limb loss in approximately one third to one half of survivors. 1–4 The epidemiology of vascular prosthetic infections supports the hypothesis that implant infections occur because of contamination of the prosthesis with small numbers of bacteria that find the prosthetic surface favorable for survival. 5,6 The recognition of matrix proteins that coat implant surfaces by bacterial adhesion receptors 7; the complex three-dimensional structure of these surfaces, which may provide bacteria with niches in which to hide; and the fact that material-adherent bacteria become encapsulated in a secreted glycocalyx 8,9 all contribute to enhanced bacterial survival in the prosthetic graft milieu. An alternative explanation, not exclusive of the former, is that foreign bodies weaken the ability of host immune cells to kill bacteria. Using an implanted subcutaneous Teflon cage, Zimmerli et al 10 in 1984 observed that neutrophils adherent to the Teflon surface become impaired in their ability to kill bacteria, whereas nonadherent neutrophils recovered from the cage milieu displayed normal bacterial killing ability. Nonbiologic surfaces, including polystyrene, glass, expanded polytetrafluoroethylene (ePTFE), and Dacron are potent neutrophil stimuli able to elicit activation responses independent of exogenous/soluble agonists. 11–17 Neutrophils readily adhere to and spread on these surfaces. Additional intracellular signals triggered by these adhesive events result in degranulation and a massive production and secretion of reactive oxygen species into the surrounding medium. 11–13 Henson et al 18,19 coined the term “frustrated phagocytosis” to indicate that these activation responses may represent a failed attempt of neutrophils to phagocytose substrates too large to be engulfed. Reactive oxygen species produced by neutrophils are central to their ability to kill bacteria. Patients with chronic granulomatous disease who fail to mount a respiratory burst are therefore subject to life-threatening bacterial infection. 20 Reactive oxygen species produced during phagocytosis are confined to phagosomes, whereas surface-adherent neutrophils appear to release their antibacterial substances, including reactive oxygen species, into the surrounding. 13,18 Henson and Johnston 19 contemplated whether reactive oxygen species that are released into the medium when neutrophils are challenged by stimulatory surfaces can cause damage to surrounding tissues. One can also speculate whether reactive oxygen species produced by neutrophils can cause self-destruction. Consistent with the latter, Tsan 21 showed that treatment of neutrophils with 12-myristate 13-acetate (phorbol ester) resulted in an increase in cell membrane permeability within a few hours. This correlated with hydrogen peroxide production by the activated neutrophils and was blocked by treatment of neutrophils with catalase, but not with superoxide dismutase. These data suggested that hydrogen peroxide produced by neutrophils could lead to self-killing. More recently, Takei et al 22 have shown that suspended neutrophils treated with phorbol ester die by a mechanism that they considered distinct from either apoptosis or necrosis. Nuclear changes such as swelling and fusion of nuclear lobes, followed by an increase in membrane permeability, were observed within minutes to hours of treatment. 22 The goal of this study was to test the hypothesis that neutrophil adhesion to prosthetic graft materials decreases neutrophil viability. We report that neutrophil adhesion to uncoated and plasma-coated ePTFE and Dacron results in a rapid cell death. In contrast, neutrophils adherent to polystyrene surfaces coated with fibrinogen, immunoglobin (IgG), or fetal bovine serum (FBS) for the same length of time remain viable. We also show that pretreatment of neutrophils with N-acetyl-L-cysteine (NAC), cytochalasin D, or cyclosporin A significantly improves neutrophil viability after adhesion to ePTFE and Dacron. NAC, a reactive oxygen intermediate scavenger, protects neutrophils from phorbol ester-induced cell death. 22 Cytochalasin D, an inhibitor of actin filament assembly, prevents the production of reactive oxygen intermediates by surface-adherent neutrophils. 23,24 Cyclosporin A is an inhibitor of calcineurin 25–29 and the mitochondrial megachannel. 30–32 Oxidative stress results in activation/opening of the mitochondrial megachannel. This in turn leads to a rapid membrane depolarization, uncoupling of oxidative phosphorylation, loss of metabolic intermediates, mitochondrial swelling, and ultimately cell death. 33,34 These data suggested that reactive oxygen intermediates produced by neutrophils adherent to ePTFE and Dacron affect cell viability. Further, within 3 hours of implantation into a subcutaneous pouch on the back of rats, numerous ePTFE- and Dacron-adherent neutrophils were dead. Taken together, these data demonstrate that prosthetic materials trigger a rapid cell death both in vitro and in vivo.