Extracellular Neutrophil Elastase Research Articles

IL-27 Promotes Neutrophil Extracellular Trap Generation in Sickle Cell Disease

Introduction: Neutrophil Extracellular Traps (NETs) are neutrophil-derived damage-associated molecular patterns (DAMPs) composed of decondensed chromatin decorated with citrullinated histones and neutrophil proteases. Recently, we have shown that caspase-4-dependent activation (cleavage) of the pore-forming protein, Gasdermin-D (GSDMD) in neutrophils promotes NETs generation leading to lung injury in SCD. IL-27, an anti-inflammatory cytokine with some pro-inflammatory properties is significantly elevated in the plasma of SCD patients, however, it remains debatable whether IL-27 signaling in neutrophils serves to promote or inhibit NETs generation in SCD. Methods: Citrated blood samples from SCD or race matched control humans were incubated with saline, hemin (20 mM), or hemin (20 mM) + recombinant human IL-27 (200 ng/mL) for 15 minutes at room temperature. Platelet-poor-plasma from the three treatment groups were incubated with sytox green and fluorescent Abs against neutrophil elastase (NE) and histones for in situ staining of extracellular DNA, NE and histones, respectively. Imaging flow cytometry was used to detect circulating NETs (cNETs) as 1-2 mm fragments triple-positive for extracellular DNA, NE, and histones. Results: Incubation with hemin led to significant increase in cNETs concentration in SCD but not control human blood, which was further increased following incubation of SCD human blood with hemin in presence of IL-27. Conclusion: Our current findings suggest that IL-27 signaling promotes NETs generation by neutrophils in SCD patient blood. Currently, studies are underway to identify the molecular mechanism underlying IL-27-dependent NETs generation in SCD.

IL-8 Triggers Neutrophil Extracellular Trap Formation Through an Nicotinamide Adenine Dinucleotide Phosphate Oxidase- and Mitogen-Activated Protein Kinase Pathway-Dependent Mechanism in Uveitis.

To explore the mechanism underlying IL-8-induced neutrophil extracellular trap (NET) formation in patients with ocular-active Behçet's disease (BD) and the effect of inhibiting NET formation on the severity of inflammation in experimental autoimmune uveitis (EAU) mice. The serum extracellular DNA and neutrophil elastase (NE) and IL-8 levels in patients with ocular-active BD, the expression of myeloperoxidase, NE, and histone H3Cit in IL-8-induced neutrophils isolated from healthy controls, and the effects of NETs on HMC3 cells were detected. Female C57BL/6J mice were immunized with IRBP651-670 to induce EAU and EAU mice received intravitreal injection of the CXCR2 (IL-8 receptor) antagonist SB225002 or PBS. The serum levels of extracellular DNA, NE, and keratinocyte-derived chemokine (the mouse ortholog of human IL-8) and expression of myeloperoxidase, NE, and histone H3Cit in mouse retinas were detected. Disease severity was evaluated by clinical and histopathological scores. Serum keratinocyte-derived chemokine expression levels in EAU mice and IL-8 expression levels in patients with ocular-active BD increased. IL-8 notably increased NET formation in a dose-dependent manner through an nicotinamide adenine dinucleotide phosphate oxidase and mitogen-activated protein kinase pathway dependent mechanism. IL-8-induced NET formation damaged HMC3 cells in vitro. Pretreatment with SB225002 notably ameliorated the production of NETs in EAU mice. Our data confirm that NET formation is induced by IL-8. IL-8-induced NET formation was found to be related to mitogen-activated protein kinase and nicotinamide adenine dinucleotide phosphate pathways. Pretreatment with the CXCR2 antagonist SB225002 alleviated neutrophil infiltration and suppressed NET formation in EAU mice.

Two-dimensional molybdenum disulfide nanosheets evoke nitric oxide-dependent antibacterial effects.

Nanomaterials are currently being explored as novel antimicrobial agents. In this study, we first investigated the ability of two-dimensional (2D) molybdenum disulfide (MoS2) nanosheets to trigger neutrophil extracellular traps (NETs) using neutrophil-differentiated HL-60 cells as well as primary human peripheral blood neutrophils. We then addressed whether the MoS2 nanosheets themselves function as antibacterial agents. We found that MoS2 and Na2MoO4 both triggered NETs, as evidenced by the quantification of neutrophil elastase (NE) activity and immunofluorescence staining of extracellular NE, as well as scanning electron microscopy. The release of NETs was found to be nitric oxide (NO)-dependent. We also found that the MoS2 nanosheets but not the soluble salt prompted acellular NO production in the presence of NaNO2. The acellular generation of NO, suggestive of nanozyme properties of the MoS2 nanosheets, was demonstrated by electron paramagnetic resonance analysis. Electrochemical analysis using cyclic voltammetry confirmed the redox transition of the MoS2 nanosheets. Finally, MoS2 nanosheets inhibited the growth of Escherichia coli in the presence of sodium nitrate. Taken together, MoS2 nanosheets triggered cellular effects as well as acellular antibacterial effects, and we provided evidence for nitrite reductase-like properties of MoS2.

In vitro investigation on extracellular traps formation of cat polymorphonuclear leucocytes against Toxoplasma gondii

Neutrophil granulocytes are one of the most important defenders of the innate immune system in the host. Moreover, neutrophils are able to reach the inflammation area and kill the pathogens using various defense strategies including neutrophil extracellular traps (NETs). However, there is still not enough information available regarding the innate immunity against Toxoplasma gondii in cats that are both definitive and intermediate hosts of this parasite. Therefore, we investigated the in vitro NETs formation which is induced by cat polymorphonuclear leucocytes (PMNs) against T. gondii tachyzoites. Firstly, PMNs were isolated from cat venous blood samples by using discontinues Percoll dilutions (72%, 63%, 54%, and 45%). Afterward, MN-tachyzoites cocultures were stained against histone (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) by using monoclonal antibodies and were examined under a fluorescence microscope. The effect of different parasite doses (1:1, 1:3, and 1:5) and incubation times (30, 60, 90, and 120 min) on NETs formation was also evaluated. The presence of the extracellular DNA content was measured using a fluorometer. Confluent Vero cell cultures were used to assess the effect of NETs on the tachyzoites viability. The classical structures of NETs, such as extracellular DNA, NE, H3, and MPO were microscopically observed in the NETs formation released from cat PMNs. The amount of extracellular DNA increased in parallel with the incubation time (p ? 0.001). The influence of the tachyzoites dose on the NETs formation was not statistically significant (p > 0.05). Zymosan was used as a positive control in the experiments and it was shown to be an important inducer for the NETs formation. In conclusion, as mentioned previous studies and considering our results, the NETs may be a conserved strategy to control T. gondii infection in hosts because of the immobilization and lethal effects.

Faecal neutrophil elastase-antiprotease balance reflects colitis severity

Given the global burden of diarrheal diseases on healthcare it is surprising how little is known about the drivers of disease severity. Colitis caused by infection and inflammatory bowel disease (IBD) is characterised by neutrophil infiltration into the intestinal mucosa and yet our understanding of neutrophil responses during colitis is incomplete. Using infectious (Citrobacter rodentium) and chemical (dextran sulphate sodium; DSS) murine colitis models, as well as human IBD samples, we find that faecal neutrophil elastase (NE) activity reflects disease severity. During C. rodentium infection intestinal epithelial cells secrete the serine protease inhibitor SerpinA3N to inhibit and mitigate tissue damage caused by extracellular NE. Mice suffering from severe infection produce insufficient SerpinA3N to control excessive NE activity. This activity contributes to colitis severity as infection of these mice with a recombinant C. rodentium strain producing and secreting SerpinA3N reduces tissue damage. Thus, uncontrolled luminal NE activity is involved in severe colitis. Taken together, our findings suggest that NE activity could be a useful faecal biomarker for assessing disease severity as well as therapeutic target for both infectious and chronic inflammatory colitis.

Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA.

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism. NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

Ischaemic stroke and the recanalization drug tissue plasminogen activator interfere with antibacterial phagocyte function

BackgroundStroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE.Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here.MethodsEx vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy.ResultsMPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release.r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA.ConclusionsIntracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies.r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.

Increased survival and exocytosis of toxic primary granules by neutrophils in an in vitro model of Cystic Fibrosis airway disease (INC6P.310)

Abstract Cystic Fibrosis (CF) lung disease is characterized by the massive recruitment of neutrophils (PMNs) into the bronchiolar lumen. Extracellular neutrophil elastase (NE) that is released from PMN primary granules, is a strong predictor of lung function and survival in CF patients. However, the process by which CF airway PMNs exocytose HNE is not well understood and not reproduced in animal models of CF airway disease. Methods: We used a surface collagen-coated 3D porous polystyrene scaffold (Alvetex) to mimic the airway lamina propria and grew human bronchiolar H441 cells at air-liquid interface on top. In the transepithelial migration assay, blood PMNs were placed on the basal side and allowed to migrate towards to CF airway fluid placed apically, after which PMNs were analyzed by flow cytometry for rate of migration, survival, and degranulation. Results: Using this model we show that (i) CF airway fluid induces rapid transepithelial migration and survival of neutrophils, (ii) live PMNs actively release of primary granules upon exposure to CF airway fluid, and (iii) migration towards CF airway fluid induces PMN reprogramming allowing for the activation of the inflammasome and pinocytosis. Conclusions: These results suggest a primary role for the CF microenviroment in inducing increased survival and phenotypic reprogramming of PMNs and establish our model as a robust platform for deconstructing CF airway PMN dysfunction and developing new therapies for CF inflammation.

Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.

Abstract 801: Uptake of exogenous neutrophil elastase by breast cancer cells: A novel link between innate immunity, inflammation and breast cancer immunity

Abstract Introduction: High level of neutrophil elastase (NE) in tumor tissue from breast cancer (BrCa) patients is associated with poor metastasis-free survival. Modest endogenous expression of NE in some BrCa cell lines has also been observed. However, because the major source of NE is from azurophil granules of activated neutrophils, we hypothesized that breast cancer cells may take up soluble NE released by abundant inflammatory cells in the tumor microenvironment. Methods: Endogenous NE expression was evaluated in MCF7, HER18, MDA-MB-231 (231) and MDA-MB-453 BrCa cell lines by western blot analysis and RT-PCR. Uptake of soluble NE was quantified by flow cytometry by intracellular staining of NE-pulsed BrCa and compared to non-specific uptake of OVA. Dose and time course experiments were performed. Confocal immunoflourescence microscopy was used to confirm uptake and demonstrate subcellular localization of NE. BrCa cells were also co-incubated with activated neutrophils to determine if they were able to take up cell-associated NE. Results: Western blot analysis of BrCa cell lysates showed no NE protein expression in all 4 cell lines. Furthermore, RT-PCR with NE-specific primers demonstrated no NE mRNA expression in the 4 BrCa cell lines. However, BrCa cells pulsed with 5 to 25 μg/ml of soluble NE took up NE in a time and dose dependent manner, consistent with a receptor-mediated mechanism. Maximal intracellular uptake of NE by 231 cells occurred by 24 hours when NE accumulated at comparable levels to that of HL60 leukemia cells that express high levels of NE (987 vs 1300 MFI, p=ns). Uptake was enzyme-independent because it was not affected by alpha-1-antitrypsin, a protease inhibitor. Confocal microscopy demonstrated that NE accumulates in membrane bound compartments by 20 minutes after uptake, whereas there was no significant uptake of OVA by BrCa cells. Similarly, after co-incubation of 231 cells with activated neutrophils, significant uptake of NE was detected (p<.001). Conclusions: These data show that BrCa cells do not express NE but the cells take up extracellular NE by a receptor-mediated enzyme-independent mechanism. This establishes a novel link between NE, a naturally secreted mediator of inflammation and cell growth, and BrCa. Because neutrophils are the major source of NE and chronic inflammation by tumor-infiltrating neutrophils is associated with positive tumor growth, this suggests NE could mediate growth control of BrCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2011-801

Alpha-1-antitrypsin aerosolised augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro

Background:Neutrophil elastase (NE) activity is increased in lung diseases such as α1-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease...

Serine Protease Inhibitor 6-Deficient Mice Have Increased Neutrophil Immunity to Pseudomonas aeruginosa

Inflammation is a localized, protective response to trauma or microbial invasion that destroys the injurious agent and the injured tissue. Neutrophil elastase (NE), a serine protease stored in the azurophil granules of polymorphonuclear neutrophils, digests microbes after phagocytosis. NE can also digest microbes extracellularly but is associated with tissue damage and inflammatory disease. In this study, we show that polymorphonuclear neutrophils from mice deficient in serine protease inhibitor 6, a weak intracellular NE inhibitor, had increased susceptibility to self-inflicted lysis because of increased NE activity. The resulting transient increase in local extracellular NE activity was within a narrow range that resulted in the clearance of Pseudomonas aeruginosa but did not damage the lung. Therefore, deficiency in a weak intracellular inhibitor of NE results in an acute inflammatory response that protects from P. aeruginosa but does not cause lung disease.

Elevated Serum Neutrophil Elastase but Not Proteinase 3 Is Present in Patients with Myeloid Leukemia.

Proteinase 3 (P3) and neutrophil elastase (NE) are abnormally expressed in the cytoplasm of myeloid leukemia, and we have shown that the HLA-A2-restricted peptide PR1 is processed and cross-presented from both proteins by antigen-presenting cells (APC) to cytotoxic T lymphocytes (CTL). PR1-specific CTL kills leukemia cells and contributes to cytogenetic remission in patients with chronic myelogenous leukemia. Both exogenous P3 and NE can bind to monocytes and P3 promotes DC maturation in vitro. Since high levels of circulating P3 were found in patients with the ANCA-associated systemic vasculitis Wegener's granulomatosis, modulation of DC by P3 has been implicated in reversal of tolerance. The role of extracellular P3 and NE in reversing tolerance in patients with myeloid leukemia is unknown. We used an ELISA assay to assess P3 and NE level in the sera of patients with AML and CML. Serum NE level was highly increased in all 15 AML patients studied (1045±149 ng/ml, n = 15) and in all 15 CML patients (1013±128, n = 15), compared to a control group of healthy donors (54±13 ng/ml, n = 4; p = 0.0038 and p = 0.0014, respectively). In contrast, serum P3 was increased in only 5 out of 18 (28%) AML patients (47 ± 18, n = 18 ng/ml) and in 8 out of 28 (29%) CML patients (95 ± 44 ng/ml, n = 28) compared to a group of 14 healthy donors (10 ± 2.2 ng/ml; p = 0.09 and p = 0.06, respectively). There was no relationship between elevated serum NE and P3 levels (r=0.16, n=35). P3 level in untreated patients and healthy donors did not change within at least 13 months, although P3 decreased after allogeneic BMT in 5 out of 5 patients (p < 0.05) with high serum levels of P3 pre-BMT. Because P3 is expressed by myeloid progenitor cells in the bone marrow, we studied the concentration of P3 in bone marrow-derived serum (BMS). To our surprise, BMS P3 concentration in patients with myeloid leukemia (23 ± 9.5 ng/ml, n = 9) was similar to that of a control group of patients with non-myeloid hematological malignancies (27 ± 11.8, n = 5) (p = 0.08), and BMS P3 concentration was similar to sera P3 concentration in the same patient (p = 0.52). Interestingly, BMS P3 level correlated with the level of BMS GM-CSF (quantified by ELISA) in a small group of patients with AML and CML (r = 0.94, n = 9), suggesting that P3 secretion by leukemia cells may be downstream of GM-CSF. We also analyzed membrane expression of P3 and NE in 5 AML patients using flow cytometry and found that P3, but not NE, was expressed on circulating cells in 3 of 5 patients. This unique expression of P3 and NE suggests that circulating NE might be the predominant source of cross-presented PR1 antigen by APC or myeloid leukemia cells.

Neutrophil and eosinophil participation in atopic and vernal keratoconjunctivitis

Purpose. A retrospective study was conducted at three centers to examine the participation of neutrophils and eosinophils in the inflammatory processes associated with atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). Methods. Conjunctival specimens were obtained from four patients with AKC, six with VKC, and five normal subjects. Indirect immunofluorescent staining was used to localize neutrophil elastase (NE) and eosinophil granule major basic protein (MBP) in serial sections of all specimens. Results. Specimens from both AKC and VKC patients revealed extracellular deposition of NE and MBP. Control specimens showed no or minimal extracellular NE, and no MBP. Statistical analysis demonstrated significantly greater extracellular MBP deposition in AKC specimens compared to controls (p = 0.009), and VKC specimens showed significantly greater staining for both extracellular MBP (p = 0.005) and NE (p = 0.006). Conclusions. These results suggest that neutrophils, in addition to eosinophils, play an active role in the pathogenesis of AKC and VKC as evidenced by the extracellular deposition of their specific granule proteins.

Evidence for pathogenic involvementof eosinophils and neutrophilsin Churg-Strauss syndrome

<h2>Abstract</h2> <b>Background:</b> Churg-Strauss syndrome (CSS) is a multi-organ disease with tissue and blood eosinophilia. <b>Objective:</b> Our aim was to study eosinophil and neutrophil involvement in CSS. <b>Methods:</b> Eight lesional skin biopsy specimens from 6 patients with CSS and serum and blister fluid from one patient were tested for eosinophil and neutrophil activity. Indirect immunofluorescence on skin specimens used antibodies to eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase (NE). Serum and blister fluid specimens were analyzed for granule protein levels and for eosinophil-activating cytokines. <b>Results:</b> Indirect immunofluorescence showed prominent cellular and extracellular staining for EDN in skin biopsy specimens; MBP staining was less extensive. Five biopsy specimens showed marked cellular NE staining; 4 showed prominent extracellular NE. Serum and blister fluid specimens contained elevated MBP, EDN, and interleukin 5 levels and enhanced eosinophil survival in culture. Granulocyte-macrophage colony-stimulating factor and interleukin 5 were detected in blister fluid. Blister fluid contained more NE than normal serum. <b>Conclusions:</b> Both eosinophils and neutrophils likely participate in skin lesion development in CSS. (J Am Acad Dermatol 2002;47:209-16.)

Evidence for pathogenic involvementof eosinophils and neutrophilsin Churg-Strauss syndrome

Background: Churg-Strauss syndrome (CSS) is a multi-organ disease with tissue and blood eosinophilia. Objective: Our aim was to study eosinophil and neutrophil involvement in CSS. Methods: Eight lesional skin biopsy specimens from 6 patients with CSS and serum and blister fluid from one patient were tested for eosinophil and neutrophil activity. Indirect immunofluorescence on skin specimens used antibodies to eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase (NE). Serum and blister fluid specimens were analyzed for granule protein levels and for eosinophil-activating cytokines. Results: Indirect immunofluorescence showed prominent cellular and extracellular staining for EDN in skin biopsy specimens; MBP staining was less extensive. Five biopsy specimens showed marked cellular NE staining; 4 showed prominent extracellular NE. Serum and blister fluid specimens contained elevated MBP, EDN, and interleukin 5 levels and enhanced eosinophil survival in culture. Granulocyte-macrophage colony-stimulating factor and interleukin 5 were detected in blister fluid. Blister fluid contained more NE than normal serum. Conclusions: Both eosinophils and neutrophils likely participate in skin lesion development in CSS. (J Am Acad Dermatol 2002;47:209-16.)

Allergic fungal sinusitis: An immunohistologic analysis

Background: Allergic fungal sinusitis is a noninvasive form of fungal sinusitis that has recently been delineated as a distinct clinicopathologic entity. It is increasingly recognized as a cause of chronic sinusitis, with the primary causative agents being members of the Dematiaceae fungus family. Although its immunopathogenesis has not been elucidated, the eosinophil is a prominent inflammatory cell on histologic examination. Objective: We sought to characterize the involvement of eosinophils in sinus tissue and accompanying mucin from patients with allergic fungal sinusitis. As a comparison, neutrophil and mast cell involvement was also evaluated in the same group of patients. Methods: Tissue specimens from 8 patients with allergic fungal sinusitis, along with 8 nasal polyp specimens from patients without allergic fungal sinusitis, were stained by using indirect immunofluorescence for eosinophil granule major basic protein (MBP). Neutrophil elastase and mast cell tryptase staining was also performed on the same allergic fungal sinusitis and nasal polyp tissues. Results: MBP was diffusely localized within the mucin, showing intense staining at the periphery and variable staining of degenerated cell clusters throughout. Extracellular MBP in the mucin was strikingly greater than intact eosinophil staining. Diffuse extracellular neutrophil elastase was also present in the mucin. Mucinous areas showed no tryptase localization. Adjacent nonmucinous areas of respiratory mucosa showed predominantly cellular staining with eosinophil MBP, neutrophil elastase, and mast cell tryptase. MBP staining of nasal polyps showed a predominantly cellular pattern with focal areas of extracellular deposition. Conclusions: Given the known toxicities of eosinophil granule MBP and neutrophil elastase, their extracellular presence supports the contribution of these proteins in the pathogenesis of allergic fungal sinusitis and further indicates that eosinophil and neutrophil activation occurs in the disease. (J Allergy Clin Immunol 2000;106:1096-101.)

Assessment of eosinophil and neutrophil participation in atopic dermatitis: Comparison with the IgE-mediated late-phase reaction

We hypothesized that repeated IgE-mediated late-phase reactions are critical in the pathogenesis of atopic dermatitis (AD). Prior studies have shown that extracellular deposition of eosinophil granule major basic protein (MBP) occurs in lesional AD skin, despite a paucity of infiltrating eosinophils, and that deposition of both neutrophil and eosinophil granule proteins occurs in the IgE-mediated late-phase reaction. We evaluated the participation of both eosinophil and neutrophil granule proteins in AD. Cutaneous biopsy specimens and serum and urine samples were obtained from 22 patients with AD. Lesional tissue was examined by means of immunofluorescence for neutrophil elastase and lactoferrin and for eosinophil granule MBP, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Serum levels of elastase, MBP, EDN, and ECP and urine levels of MBP, EDN, and ECP were measured. Marked extracellular deposition of at least one of the eosinophil granule proteins was present in the dermis of 15 of the 22 AD skin specimens, but minimal or no extracellular neutrophil elastase or lactoferrin deposition was observed in any specimens. Serum and urine levels of MBP, EDN, and ECP in the patients were elevated when compared with those of normal controls, whereas serum levels of neutrophil elastase were not elevated. Serum MPB levels correlated with extent of body surface involvement. These results suggest that eosinophil degranulation occurs in AD but that neutrophil degranulation does not. Although eosinophil degranulation is prominent in both the late-phase reaction and in AD, the lack of neutrophil degranulation in AD demonstrates differences in the inflamamtory reactions. (J ALLERGY CLIN IMMUNOL 1994;94:120-8.)

Eosinophil and Neutrophil Degranulation in Ophthalmic Lesions of Wegener's Granulomatosis

Although eosinophils and neutrophils have been identified in orbital and corneoscleral tissues in some patients with Wegener's granulomatosis, their role in the pathogenesis of the disorder is not completely understood. Nine specimens from six patients with Wegener's granulomatosis and autopsy controls from patients without ophthalmic disease were evaluated with indirect immunofluorescence for eosinophil granule major basic protein and neutrophil elastase. Extracellular deposition of both major basic protein and elastase was identified in orbital tissues from all the patients with Wegener's granulomatosis. Two of the specimens were from enucleated eyes with corneoscleral disease; extracellular deposition of eosinophil major basic protein was identified in one eye, and extracellular neutrophil elastase was deposited in both eyes in lesional areas. None of the control tissues showed major basic protein or elastase deposition. These findings suggest that both eosinophils and neutrophils participate in the pathogenesis of the orbital and corneoscleral manifestations of Wegener's granulomatosis.

825 Deposition of extracellular neutrophil elastase (ELAST) and eosinophil major basic protein (MBP) in the bronchial tree epithelium in fatal asthma (FA)

825 Deposition of extracellular neutrophil elastase (ELAST) and eosinophil major basic protein (MBP) in the bronchial tree epithelium in fatal asthma (FA)