The excellent review by Gladwin and Vichinsky1 underscores the role of ischemia-reperfusion injury (IRI) in the pathobiology of sickle cell anemia. In particular, IRI seems to play a significant role in the development of acute chest syndrome, a lung injury that is the second most common cause of hospitalization and death in this hereditary condition. Several mechanisms may participate in the induction of IRI associated with the acute chest injury, such as pulmonary infection, embolization of fat from bone marrow, and vasoocclusion from sequestration of sickled erythrocytes, activated monocytes,2 and neutrophils adherent to activated endothelial cells.3 The purpose of this commentary is to provide a new paradigm to explain the exaggerated proinflammatory response of sickle cell disease to pathogens and pathogen molecular associated patterns (PAMPs). It is suggested that the heightened inflammatory response is a result of synergistic stimulation of innate immune receptors by PAMPs and danger associated molecular patterns (DAMPs) that are by-products of IRI.
It has been posited in other examples of IRI4 that innate immune mechanisms play a significant role in causing neutrophilic dominant inflammation. Cytokine interleukin (IL)-1β has been particularly implicated in the causation of IRI based on evidence of its increased secretion in animal IRI models.4 In addition, there is convincing evidence for the fundamental role of IL-1 cytokines in IRI as IL-1α/β knock-out animals exhibit marked reduction of IRI and conversely IL-1 receptor antagonist knock-out animals exhibit increased IRI. Further support for this theory is derived from experiments demonstrating improvement in acute neutrophilic inflammation with IL-1β targeted therapy (IL-1β TT) in many of these same IRI models.4 The latter observations are compatible with the suggestion by Dinarello5 that causation of cytokine mediated diseases can be established with “specific receptor blockade or cytokine neutralization.”
Increased secretion of IL-1β in IRI remains an enigma and it is known that IL-1β secretion can occur after PAMPs stimulation of innate immune receptors, such as cytoplasmic cryopyrin-inflammasome (NALP–3 Inflammasome), the cytoplasmic NALP-1 inflammasome, and cell membrane toll-like receptors. In healthy animal models of IRI, it is unlikely that PAMPs stimulate IL-1β secretion, as release of cytokines occurs temporally after blood vessel occlusion. In the absence of the involvement of PAMPs, it has been posited4 that biochemical events associated with IRI, such as hypoxia and metabolic acidosis, lead to necrosis of cells with the formation of DAMPs, that is, uric acid/urate (UA) and calcium pyrophosphate crystallization, extracellular adenosine triphosphate, and intracellular hypokalemia. DAMPs in turn are known to stimulate IL-1β secretion by the NALP–3 Inflammasome.6–8 Hence in IRI the NALP–3 Inflammasome could function as innate immune sentries that are capable of detecting by-products of stressed and dying cells.
As the NALP–3 Inflammasome may be involved in IRI pathogenesis, the following is a short review of salient investigations involving this multiprotein innate cytoplasmic structure. Hoffman et al9 identified the cryopyrin-encoding gene (C1AS1/NLRP3) on chromosome 1q44 by investigating familial cold auto-inflammatory syndrome, a rare autoinflammatory, autosomal dominant syndrome characterized by cold induction of IL-1β secretion that causes fever, neutrophilic leukocytosis, and neutrophilic leukocyte infiltrated dermatosis. The cryopyrin gene discovery led to recognition of the NALP–3 Inflammasome which is comprised of a cytoplasmic macromolecular protein complex containing cryopyrin and other adaptor proteins.6 Two other rare and seemingly unrelated autosomal dominant periodic fever syndromes (ie, Muckle-Wells and neonatal onset multisystem inflammatory disease) with dysregulated 1L-1β production were found to have mutations on the same gene. All three autoinflammatory hereditary syndromes referred to as cryopyrin-associated periodic syndromes (CAPS) are exceptionally responsive to IL-1β TT, which provided unequivocal evidence for IL-1β mediation in human disorders with dominant neutrophilic inflammation. Subsequently, investigators6–8 recognized that the NALP–3 Inflammasome can be stimulated by DAMPs to secrete IL-1β. Release of IL-1β in turn causes a cascade of proinflammatory molecular events, such as upregulation of vascular adhesion molecules (intercellular adhesion molecule), IL-6 release, increased neutrophil and monocyte chemokines, IL-17A secretion, and stimulation of phospholipase-A2 activation with formation of leukotriene B4, all of which can promote and magnify neutrophilic inflammation.
Sickle cell patients are atypical in that they experience exaggerated inflammatory responses to pathogens that normally cause mild respiratory infections.1 Compared with wild mice, observations indicate transgenic sickle mice require lower concentrations of lipopolysaccharide (LPS) to cause lung injury and increased secretion of IL-1β and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid.10 A similar exaggerated inflammatory response was noted in hypoxia murine studies, as neutrophilia and activated adherent leukocytes were significantly greater in transgenic sickle mice compared with wild-type mice.11 Hence compared with appropriate controls there seems to be heightened inflammatory responses to PAMPs and hypoxia in sickle cell disease and in transgenic sickle mice.
It is posited that sickle cell lung injury may involve costimulation of the NALP–3 Inflammasome by PAMPS and DAMPs, leading to exaggerated proinflammatory responses marked by IL-1β secretion and subsequent induction of neutrophilic inflammation. The evidence for this hypothesis is as follows:
1. Therapeutic control of CAPS with IL-1β TT provides the scientific support that IL-1β is a profoundly significant cytokine capable by itself of setting off an inflammatory cascade with dominant neutrophilic inflammation.
2. Neutrophilic inflammation is prominent in sickle cell disease and in transgenic sickle mice subjected to hypoxia and reperfusion.11 Compared with wild mice, transgenic sickle mice exhibit a subclinical proinflammatory state marked by elevated neutrophil counts and soluble vascular adhesion molecules.10 In the same experiments, after LPS challenge there is heightened inflammatory response in the transgenic sickle mice as compared with wild mice in terms of increased cytokine expression, airway tone, and death.
3. Monocytes from sickle cell anemia patients incubated with endothelial cells caused an increase expression of endothelial adhesion molecules as compared with the normal monocytes.2 Antibodies to IL-1β and TNF-α blocked activation of the endothelium by monocytes.2 These studies indicate that IL-1β and TNF-α activate inflammatory endothelial responses and that there are mechanisms intrinsic to sickle cell monocytes that enhance endothelial cell activation. This provides further support for the concept that sickle cell disease has heightened proinflammatory responses.
4. UA elevations are frequently observed in sickle cell disease, probably because of a combination of hemolysis and nucleotide breakdown in tissues affected by IRI, leading to hypoxia and metabolic acidosis. UA and urates are particularly insoluble in acidosis as the solubility of urates is 1 to 4 mg/dL in a pH range of 3 to 6 versus 15 mg/dL or higher in pH >7.0. The presence of high UA in combination with the focal ischemic induced metabolic acidosis provides optimal milieu for crystallization of urates, which can stimulate the NALP–3 Inflammasome.
5. Giamarellos-Bourboulis et al12 demonstrated synergistic enhancement of IL-1β secretion from normal human monocytes costimulated by LPS and urate crystals. The authors state that “the synergy between urate crystals and LPS was directed at the level of the NALP–3 Inflammasome.”12
These observations provide the basis for the hypothesis that DAMPs and PAMPs are capable of costimulating the NALP–3 Inflammasome and possibly other innate receptors such as toll-like receptors by different PAMPs and thereby explain sickle cell amplified inflammatory responses to microbes in combination with hypoxic effects from IRI.
It is further posited that sickle cell chest injury and other IRI complications could benefit with blockade of IL-1β using any of several commercially available biologics, such as an IL-1β receptor blocker (anakinra; Kineret), IL-1β TRAP (rilanocept; Arcalyst), and monoclonal anti-IL-1β antibodies (canakinumab by Novartis).13 The use of these biologics over a 2 year or more time span in CAPS has been efficacious with excellent safety profiles. Moreover rilanocept and canakinumab13 have half lives of a minimum of 2 weeks. Hence a single injection of these biologics would theoretically be adequate in reducing the proinflammatory effects of IL-1β in acute chest injury and could be used as an adjunct to reduce severity of this and other IRI induced complications in sickle disease.
The author is hopeful that this paradigm will stimulate clinical trials with IL-1β TT in transgenic sickle mice and eventually in subjects with sickle cell disease.
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