Living and dying for inflammation: neutrophils, eosinophils, basophils.

Neutrophils, eosinophils, and basophils play essential roles during microbe-induced and sterile inflammation. The severity of such inflammatory processes is controlled, at least in part, by factors that regulate cell death and survival of granulocytes. In recent years, major progress has been made in understanding the molecular mechanisms of granulocyte cell death and in identifying novel damage- and pathogen-associated molecular patterns as well as regulatory cytokines impacting granulocyte viability. Furthermore, an increased interest in innate immunity has boosted our overall understanding of granulocyte biology. In this review, we describe and compare factors and mechanisms regulating neutrophil, eosinophil, and basophil lifespan. Because dysregulation of death pathways in granulocytes can contribute to inflammation-associated immunopathology, targeting granulocyte lifespan could be therapeutically promising.

Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis.

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.

Inflammation-associated autophagy-related programmed necrotic death of human neutrophils characterized by organelle fusion events.

The most common form of neutrophil death, under both physiological and inflammatory conditions, is apoptosis. In this study, we report a novel form of programmed necrotic cell death, associated with cytoplasmic organelle fusion events, that occurs in neutrophils exposed to GM-CSF and other inflammatory cytokines upon ligation of CD44. Strikingly, this type of neutrophil death requires PI3K activation, a signaling event usually involved in cellular survival pathways. In the death pathway reported in this study, PI3K is required for the generation of reactive oxygen species, which somehow trigger the generation of large cytoplasmic vacuoles, generated by the fusion of CD44-containing endosomes with autophagosomes and secondary, but not primary, granules. Neutrophils demonstrating vacuolization undergo rapid cell death that depends on receptor-interacting protein 1 kinase activity and papain family protease(s), but not caspases, that are most likely activated and released, respectively, during or as a consequence of organelle fusion. Vacuolized neutrophils are present in infectious and autoimmune diseases under in vivo conditions. Moreover, isolated neutrophils from such patients are highly sensitive toward CD44-mediated PI3K activation, reactive oxygen species production, and cell death, suggesting that the newly described autophagy-related form of programmed neutrophil necrosis plays an important role in inflammatory responses.

Budesonide is effective in adolescent and adult patients with active eosinophilic esophagitis.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus characterized by dense tissue eosinophilia; it is refractory to proton pump inhibitor therapy. EoE affects all age groups but most frequently individuals between 20 and 50 years of age. Topical corticosteroids are effective in pediatric patients with EoE, but no controlled studies of corticosteroids have been reported in adult patients. METHODS: We performed a randomized, double-blind, placebo-controlled trial to evaluate the effect of oral budesonide (1 mg twice daily for 15 days) in adolescent and adult patients with active EoE. Pretreatment and posttreatment disease activity was assessed clinically, endoscopically, and histologically. The primary end point was reduced mean numbers of eosinophils in the esophageal epithelium (number per high-power field [hpf] = esophageal eosinophil load). Esophageal biopsy and blood samples were analyzed using immunofluorescence and immunoassays, respectively, for biomarkers of inflammation and treatment response. RESULTS: A 15-day course of therapy significantly decreased the number of eosinophils in the esophageal epithelium in patients given budesonide (from 68.2 to 5.5 eosinophils/hpf; P < .0001) but not in the placebo group (from 62.3 to 56.5 eosinophils/hpf; P = .48). Dysphagia scores significantly improved among patients given budesonide compared with those given placebo (5.61 vs 2.22; P < .0001). White exudates and red furrows were reversed in patients given budesonide, based on endoscopy examination. Budesonide, but not placebo, also reduced apoptosis of epithelial cells and molecular remodeling events in the esophagus; no serious adverse events were observed. CONCLUSIONS: A 15-day course of treatment with budesonide is well tolerated and highly effective in inducing a histologic and clinical remission in adolescent and adult patients with active EoE.

Long-term budesonide maintenance treatment is partially effective for patients with eosinophilic esophagitis.

BACKGROUND & AIMS: Topical corticosteroids are effective in inducing clinical and histologic remission in patients with eosinophilic esophagitis (EoE). However, the best long-term management strategy for this chronic inflammatory disease has not been determined. METHODS: In a randomized, double-blind, placebo-controlled, 50-week trial, we evaluated in 28 patients the efficacy of twice-daily swallowed budesonide (0.25 mg each) to maintain quiescent EoE in remission. Pretreatment and posttreatment activity was assessed clinically, endoscopically, histologically, immunohistologically, and by endosonography. The primary end point was the therapy's ability to maintain EoE in histologic remission. Secondary end points were efficacy in symptom control, prevention of tissue remodeling, and safety. RESULTS: In patients given low-dose budesonide, the load of esophageal eosinophils increased from 0.4 to 31.8 eosinophils/high-power field (P = .017). In patients given placebo, the load increased from 0.7 to 65.0 eosinophils/high-power field (P = .0001); this increase was significantly greater than in patients given budesonide (P = .024). The symptom scores developed in a similar manner in the 2 groups. Budesonide, but not placebo, reduced noneosinophilic markers of inflammation, epithelial cell apoptosis, and remodeling events. Compared with control individuals, patients had significantly thickened esophageal walls, based on endosonography (3.05 vs 2.18 mm; P < .0001). Budesonide therapy was associated with a significant reduction in mucosal thickness (0.75-0.45 mm; P = .025), but epithelial thickness remained stable (261.22 vs 277.23 µm; P = .576). No serious adverse events occurred. CONCLUSIONS: Low-dose budesonide is more effective than placebo in maintaining EoE in histologic and clinical remission. Signs of esophageal remodeling showed a trend toward normalization. Long-term administration of topical corticosteroids was well tolerated without induction of epithelial atrophy.

Esophageal dilation in eosinophilic esophagitis: effectiveness, safety, and impact on the underlying inflammation.

OBJECTIVES: Esophageal dilation often leads to long-lasting relief of dysphagia in eosinophilic esophagitis (EoE). The aim of this study was to define the effectiveness, safety, and patient acceptance of esophageal dilation in EoE. In addition, we examined the influence of dilation on the underlying esophageal inflammation. METHODS: Two databases including 681 EoE patients were reviewed. Cohort 1 consisted of patients treated with dilation alone, whereas cohort 2 included patients treated with a combination of dilation and antieosinophilic medication. Patients from cohort 1 underwent a prospective histological reexamination and an evaluation using a questionnaire. RESULTS: In total, 207 EoE patients were treated with esophageal dilation, 63 in cohort 1 and 144 in cohort 2. Dilation led to a significant increase in esophageal diameter and to an improvement in dysphagia in both the cohorts (P<0.001). After dilation, dysphagia recurred after 23+/-22 months in cohort 1 and 20+/-14 months in cohort 2. No esophageal perforation or major bleeding occurred. Among the patients surveyed, 74% reported retrosternal pain after dilation; however, all were agreeable to repeated dilation if required. Eosinophil peak infiltration, eosinophil load, and EoE-associated histological signs were not significantly affected by esophageal dilation. CONCLUSIONS: Esophageal dilation is highly effective in providing long-lasting symptom relief and can be performed safely with a high degree of patient acceptance. However, dilation is associated with postprocedural pain in most patients and does not influence the underlying inflammatory process. Symptom improvement despite persistence of inflammation suggests that tissue remodeling contributes substantially to symptom generation in EoE.

General laboratory diagnostics of eosinophilic GI diseases.

Eosinophils and gastrointestinal tract interact in an intimate and enigmatic relationship. Under inflammatory conditions, eosinophil infiltration in the gastrointestinal tract is a common feature of numerous eosinophilic gastrointestinal disorders (EGIDs). EGIDs are disorders, for which the diagnosis is relatively difficult. Nevertheless, some common laboratory techniques are currently used for their diagnosis and disease monitoring. Besides eosinophils, mast cells and T cells have also been suggested to play a role in the pathogenesis of these disorders. Here, we review the pathogenesis and common laboratory approaches applied for their diagnosis, in particular eosinophil and mast cell markers.

Cytokine expression in healthy and inflamed mucosa: probing the role of eosinophils in the digestive tract.

BACKGROUND: In eosinophilic esophagitis (EE), the esophagus is infiltrated with activated eosinophils that often evoke tissue damage, but the intestines of these patients remain unaffected. We thus hypothesized that different tissue-dwelling eosinophil populations may coexist: activated eosinophils that infiltrate the esophagus and resting eosinophils that reside in unaffected intestines. We sought to characterize different eosinophil subpopulations by comparing the expression of certain proinflammatory proteins in tissue-dwelling eosinophils at different parts of the gastrointestinal tract. METHODS: The 8 patients participating included 6 men and 2 women with a previously confirmed diagnosis of EE, whose average age was 39.4 years (range, 20-55 yr) and average disease duration was 13.6 years (range, 2-26 yr). Controls were 3 men and 1 woman, with a mean age of 43.3 years (range, 29-56 yr) with untreated functional dyspepsia who underwent diagnostic esophagogastroduodenoscopy. Six additional individuals having normal blood eosinophils were recruited for cytokine measurements in blood eosinophils. Immunofluorescence and immunoassays charted expression of CD25 and the TH2 cytokines, interleukin (IL)-4, IL-5, IL-10, and IL-13, in esophageal, intestinal, and blood eosinophils from controls and patients. RESULTS: Controls showed a small but significant proportion of intestinal, but no blood, eosinophils expressing CD25 and IL-13, suggesting physiologic activation occurring in the digestive tract. On the other hand, eosinophils infiltrating the inflamed esophageal mucosa of patients with EE showed strong evidence of activation, with most expressing CD25, IL-4, and IL-13. Moreover, IL-13-positive intestinal eosinophils were increased in patients compared with controls. CONCLUSIONS: We thus conclude that tissue-dwelling eosinophils show different and distinct cytokine expression patterns under noninflammatory and inflammatory conditions.

Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to activate macrophages and T cells. In this study, we demonstrate that recombinant MIF delays apoptosis of neutrophils in vitro. MIF action is dose and time dependent as well as specific since it was abolished with a neutralizing anti-MIF antibody. MIF, like G-CSF, delayed cleavage of the proapoptotic members of the Bcl-2 family Bid and Bax in neutrophils, suggesting that MIF inhibits apoptosis pathways proximal to mitochondria activation. Indeed, MIF also prevented release of cytochrome c and Smac from the mitochondria and subsequent activation of the critical effector caspase-3 in these cells. Moreover, we observed increased MIF plasma levels in patients with cystic fibrosis, a heterogeneous recessive genetic disorder associated with bacterial infections and delayed neutrophil apoptosis. In conclusion, MIF is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in infectious diseases.

Cathepsins and their involvement in immune responses.

The immune system is composed of an enormous variety of cells and molecules that generate a collective and coordinated response on exposure to foreign antigens, called the immune response. Within the immune response, endo-lysosomal proteases play a key role. In this review we cover specific roles of cathepsins in innate and adaptive immunity, as well as their implication in the pathogenesis of several diseases.

Cathepsins: key modulators of cell death and inflammatory responses.

Apoptosis is a key mechanism in the build up and maintenance of both innate and adaptive immunity as well as in the regulation of cellular homeostasis in almost every organ and tissue. Central to the apoptotic process is a family of intracellular cysteine proteases with aspartate-specificity, called caspases. Nevertheless, there is growing evidence that other non-caspase proteases, in particular lysosomal cathepsins, can play an important role in the regulation of apoptosis. In this review, the players and the molecular mechanisms involved in the lysosomal apoptotic pathways will be discussed as well as the importance of these pathways in the immune system and the pathogenesis of diseases.

Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation.

In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species-dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.

cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

The exact molecular mechanisms leading to delayed apoptosis, a phenomenon frequently observed in eosinophil inflammatory responses, remain largely unknown. Here, we show that cultured eosinophils purified from blood of hypereosinophilic syndrome (HES) patients exhibit delayed spontaneous death and relative resistance towards ceramide- but not CD95-mediated death. The subsequent investigation of members of the inhibitor of apoptosis (IAP) family revealed that HES but not normal eosinophils expressed high levels of cellular IAP-2 (cIAP-2) and survivin. The eosinophil hematopoietins IL-3, IL-5, and GM-CSF increased the expression of cIAP-2 and survivin in normal eosinophils in vitro. In the blood of HES patients, we observed increased concentrations of IL-3 and/or IL-5, suggesting that these cytokines are, at least partially, responsible for the elevated levels of cIAP-2 and survivin in the eosinophils of these patients. Utilizing a cell-free system in which caspase-3 was activated in eosinophil cytosolic extracts by addition of cytochrome c and immunodepletion of cIAP-2 or survivin resulted in accelerated caspase activation. These data suggest that some members of the IAP family including survivin are regulated by survival cytokines and inhibit the caspase cascade in HES eosinophils. The cytokine-dependent mechanism of delayed eosinophil apoptosis described here may also apply to other eosinophilic diseases.

Leptin is an eosinophil survival factor.

BACKGROUND: Leptin regulates food intake, as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. OBJECTIVE: We sought to examine the expression of leptin receptors on eosinophils and the effect of recombinant leptin on proapoptotic pathways in these cells. METHODS: The presence of leptin receptor was examined by means of RT-PCR and immunofluorescence analysis in freshly isolated blood eosinophils and tissue eosinophils. The effect of recombinant leptin on apoptotic pathways in eosinophils was studied by using flow cytometric, immunoblotting, and immunofluorescence techniques. RESULTS: Human eosinophils express leptin surface receptors under in vitro and in vivo conditions, and leptin delays apoptosis of mature eosinophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block eosinophil apoptosis was similar to that of GM-CSF. Leptin delayed the cleavage of Bax, as well as the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, suggesting that it blocks proapoptotic pathways proximal to mitochondria in eosinophils. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving phosphatidylinositol-3-OH kinase and mitogen-activated protein kinase-dependent pathways in eosinophils. CONCLUSION: Leptin is a survival cytokine for human eosinophils, a finding with potential pathologic relevance in allergic and parasitic diseases.

Apoptotic pathways are inhibited by leptin receptor activation in neutrophils.

Leptin regulates food intake as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. In this study, we demonstrate that human neutrophils express leptin surface receptors under in vitro and in vivo conditions, and that leptin delays apoptosis of mature neutrophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block neutrophil apoptosis was similar to G-CSF. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving PI3K- and MAPK-dependent pathways in neutrophils. Moreover, leptin delayed the cleavage of Bid and Bax, the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, as well as the activation of both caspase-8 and caspase-3 in these cells. Taken together, leptin is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in inflammatory diseases.

Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis.

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.