Lisa A. Spencer

Thrombospondin‐1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin‐1 (TSP‐1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP‐1 in the skin, using a keratin 14 expression cassette. TSP‐1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full‐thickness skin wounds was greatly delayed in TSP‐1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP‐1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP‐1 preferentially interfered with wound healing‐associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially.

Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune‐polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2‐associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL‐4, IL‐13, IL‐6, IL‐10, IL‐12, IFN‐γ, and TNF‐α. We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2‐promoting IL‐4 but not Th1‐inducing IL‐12. Moreover, a large quantity of IFN‐γ was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus‐dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.

Immunoregulatory roles of eosinophils: a new look at a familiar cell

Eosinophils are usually considered as end‐stage degranulating effector cells of innate immunity. However, accumulating evidence has revealed additional roles for eosinophils that are immunoregulatory in nature in both the adaptive and innate arms of immunity. Specifically, eosinophils have key immunoregulatory roles as professional antigen‐presenting cells and as modulators of CD4+ T cell, dendritic cell, B cell, mast cell, neutrophil, and basophil functions. This review addresses the emerging immunoregulatory roles of eosinophils with a focus on recent data that support this new paradigm. Recognizing both the effector and immunoregulatory functions of eosinophils will enable a fuller understanding of the roles of eosinophils in allergic airways inflammation and may be pertinent to therapies that target eosinophils both for their acute and ongoing immunomodulatory functions.

Eosinophils in innate immunity: an evolving story

Eosinophils are innate immune leukocytes found in relatively low numbers within the blood. Terminal effector functions of eosinophils, deriving from their capacity to release their content of tissue-destructive cationic proteins, have historically been considered primary effector mechanisms against specific parasites, and are likewise implicated in tissue damage accompanying allergic responses such as asthma. However, the past decade has seen dramatic advancements in the field of eosinophil immunobiology, revealing eosinophils to also be key participants in many other facets of innate immunity, from bridging innate and adaptive immune responses to orchestrating tissue remodeling events. Here, we review the multifaceted functions of eosinophils in innate immunity that are currently known, and discuss new avenues in this evolving story.

Eosinophils and Th2 immunity: contemporary insights

Eosinophils, innate immune leukocytes elicited by Th2 cells, have long been associated with the effector arm of Th2 immune responses. However, accumulating data over the past decade reveal a much more dynamic picture of Th2 immunity, where eosinophils are present very early in response to Th2‐inducing agents and function in the initiation of Th2 immunity. Here we discuss recent data showing immune functions of eosinophils distinct from their previously appreciated tissue‐ and helminth‐destructive capacities, providing strong evidence for a new paradigm of Th2 immunity defined by a dynamic interplay between eosinophils and T cells.

Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins

Eosinophils generate and store a battery of proteins, including classical cationic proteins, cytokines, chemokines, and growth factors. Rapid secretion of these active mediators by eosinophils is central to a range of inflammatory and immunoregulatory responses. Eosinophil products are packaged within a dominant population of cytoplasmic specific granules and generally secreted by piecemeal degranulation, a process mediated by transport vesicles. Large, pleiomorphic vesiculotubular carriers were identified recently as key players for moving eosinophil proteins from granules to the plasma membrane for extracellular release. During secretion, these specialized, morphologically distinct carriers, termed eosinophil sombrero vesicles, are actively formed and direct differential and rapid release of eosinophil proteins. This review highlights recent discoveries concerning the organization of the human eosinophil secretory pathway. These discoveries are defining a broader role for large vesiculotubular carriers in the intracellular trafficking and secretion of proteins, including selective receptor‐mediated mobilization and transport of cytokines.

Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Human Eosinophils Secrete Preformed, Granule-Stored Interleukin-4 Through Distinct Vesicular Compartments

Secretion of interleukin‐4 (IL‐4) by leukocytes is important for varied immune responses including allergic inflammation. Within eosinophils, unlike lymphocytes, IL‐4 is stored in granules (termed specific granules) and can be rapidly released by brefeldin A (BFA)‐inhibitable mechanisms upon stimulation with eotaxin, a chemokine that activates eosinophils. In studying eotaxin‐elicited IL‐4 secretion, we identified at the ultrastructural level distinct vesicular IL‐4 transport mechanisms. Interleukin‐4 traffics from granules via two vesicular compartments, large vesiculotubular carriers, which we term eosinophil sombrero vesicles (EoSV), and small classical spherical vesicles. These two vesicles may represent alternative pathways for transport to the plasma membrane. Loci of both secreted IL‐4 and IL‐4‐loaded vesicles were imaged at the plasma membranes by a novel EliCell assay using a fluoronanogold probe. Three dimensional electron tomographic reconstructions revealed EoSVs to be folded, flattened and elongated tubules with substantial membrane surfaces. As documented with quantitative electron microscopy, eotaxin‐induced significant formation of EoSVs while BFA pretreatment suppressed eotaxin‐elicited EoSVs. Electron tomography showed that both EoSVs and small vesicles interact with and arise from granules in response to stimulation. Thus, this intracellular vesicular system mediates the rapid mobilization and secretion of preformed IL‐4 by activated eosinophils. These findings, highlighting the participation of large tubular carriers, provide new insights into vesicular trafficking of cytokines.

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Intragranular Vesiculotubular Compartments are Involved in Piecemeal Degranulation by Activated Human Eosinophils

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule‐stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil‐specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30–60 min with eotaxin, regulated on activation, normal, T‐cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule‐stored cytokines and cationic proteins.