m biophys's List: neutrophil UCSD Proj II

• The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes
• M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF
• Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo
• The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection
• We hypothesized that degradation of the DNA framework of NETs could represent the underlying mechanism to explain the contribution of Sda1 to GAS resistance to neutrophil killing and virulence potential. To examine this interaction, we developed assays for real-time visualization of NET formation by freshly isolated human neutrophils via the Sytox family (Molecular Probes) of fluorescent DNA stains. Because these stains do not penetrate cell membranes effectively, fluorescent DNA staining is specific to secreted extracellular DNA (e.g., NETs) or to nuclear DNA of dead cells. By using this live-cell imaging technique, we were able to observe distinct NET formation by isolated neutrophils within 10 min of isolation. The rapid appearance of NETs supported the conclusions of Brinkman et al. who concluded that NET phenomenon is an active process independent of necrosis or apoptosis by using differential staining of formalin-fixed cells 1 V. Brinkmann, U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D.S. Weiss, Y. Weinrauch and A. Zychlinsky, Neutrophil extracellular traps kill bacteria, Science 303 (2004), pp. 1532–1535. View Record in Scopus | Cited By in Scopus (232)[1]. Over the course of 15 min, the wt M1 GAS strain was able to effect a dose-dependent elimination of NETs, while significant quantities of NETs persisted in companion assays by means of the M1ΔSda1 mutant. For gain-of-function analysis, we tested L. lactis, which lacked the ability to eliminate NETs upon coincubation with human neutrophils. Heterologous expression of GAS Sda1 in L. lactis allowed the transformed bacteria to degrade NETs (Figure 3A). These studies revealed DNase Sda1 to be necessary and sufficient for effective bacterial degradation of NETs and correlated directly with the respective ability of the various bacterial strains to survive extracellular killing when coincubated with neutrophils (Figure 1D).
• Additional studies were performed to clarify whether Sda1-mediated degradation of NETs per se represented a key pathogenic phenotype in the early stages of GAS infection in vivo. Biopsies from mice infected with the wt M1 GAS or the M1ΔSda1 mutant at 4 hr post subcutaneous injection revealed similar levels of neutrophil infiltration and early abscess formation (Figure S3A). The number of recruited leukocytes stimulated by the wt strain and M1ΔSda1 mutant was also similar when the bacteria were implanted into subcutaneous tissue cages for accurate serial sampling over 72 hr (Figure S3B). Purulent exudates from the early stages of abscess formation in the subcutaneous challenge model (4 hr) were collected for DNA staining to visualize NET formation in vivo. In each of four mice infected with wt M1 GAS in which clear exudates of intact neutrophils were collected, no NETs could be visualized (Figure 3B). In contrast, NETs were common in the purulent exudates obtained from a group of mice challenged with the identical inoculum of the M1ΔSda1 mutant (Figure 3B). These findings support the mechanistic linkage of GAS Sda1 DNase-mediated enhancement of bacterial survival and NET degradation ex vivo (Figures 1B–1E and 3A) with enhanced bacterial virulence potential in vivo (Figure 2).

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• Many bacterial pathogens produce extracellular DNases, and whether these proteins have a role in bacterial pathogenesis has been a long-standing question. Now, two papers in Current Biology prove a role for streptococcal DNases in evading the innate immune response

  • escaping from the NET that is formed by neutrophils as an immune respose
• James Musser and colleagues began to elucidate the role of DNases when they reported that the extracellular DNase activity of a group A Streptococcus (GAS) strain contributes to disease progression and protects from killing by polymorphonuclear leukocytes
• They speculated that the protection resulted from bacterial DNases degrading neutrophil extracellular traps (NETs), which are released by activated neutrophils as part of the innate immune response
• NETs comprise a DNA scaffold 'decorated' with antimicrobial agents such as histones and components from neutrophil granules
• In the first Current Biology paper, Buchanan et al. looked at the role of the DNase Sda1 from an M1 serotype GAS strain. Initial in vitro work revealed that a sda1 mutant showed reduced DNase activity and increased killing by neutrophils. A fluorescence-microscopy protocol that allowed live imaging of NET formation by neutrophils indicated that the DNase activity of Sda1 was necessary and sufficient to degrade NETs. Finally, a correlation was made between NET degradation and GAS pathogenicity in vivo — in a mouse model of necrotizing fasciitis, the sda1 mutant was significantly less virulent than the wild-type strain, and when exudates from abcesses in mice in the early stages of infection were examined for the presence of NETs, it was shown that significantly more NETs were present in mice infected with the sda1 mutant than in mice infected with wild-type GAS
• The second study looked at the role of the DNase EndA from the Streptococcus pneumoniae serotype 4 strain TIGR4. The DNase activity of EndA and a role for this activity in degrading the DNA scaffold of NETs were both confirmed in vitro. Beiter et al. then established that NETs are present in a C57BL/6 mouse model of TIGR4 pneumococcal pneumonia, and that the removal of endA was associated with a decrease in TIGR4 virulence in vivo. Further detailed characterization of the competitive index after simultaneous infection with TIGR4 and either TIGR4 endA or an endA⁺ revertant showed that the endA strain was outcompeted by both the wild-type and the revertant strain in the lungs and bloodstream but not in the upper respiratory tract. As Beiter et al. also established that pneumococci are trapped but not killed by NETs and that EndA activity allowed the pneumococci to escape from NETs, these observations suggest that the S. pneumoniae DNase EndA has a crucial role in virulence, allowing the bacteria to escape from the innate immune response in the upper respiratory tract and establish an invasive infection

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• Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase
• Neutrophils are one of the first lines of defense against invading microbes (Kanthack and Hardy, 1895; Nathan, 2006). These cells are terminally differentiated, and they have a short life span and low levels of gene expression. When they reach the circulation, they are already equipped with the proteins required to kill microorganisms (Borregaard and Cowland, 1997). Neutrophils in circulation are directed by cytokines into infected tissues, where they encounter invading microbes. This encounter leads to the activation of neutrophils and the engulfment of the pathogen into a phagosome. In the phagosome, two events are required for antimicrobial activity. First, the presynthesized subunits of the NADPH oxidase assemble at the phagosomal membrane and transfer electrons to oxygen to form superoxide anions. These dismutate spontaneously or catalytically to dioxygen and hydrogen peroxide. Collectively, superoxide anions, dioxygen, and hydrogen peroxide are called reactive oxygen species (ROS; Hampton et al., 1998). Second, the granules fuse with the phagosome, discharging antimicrobial peptides and enzymes. In the phagosome, microorganisms are exposed to high concentrations of ROS and antimicrobial peptides. Together, they are responsible for microbial killing (Klebanoff, 1999)
• Recently, we described a novel antimicrobial mechanism of neutrophils. Upon activation, neutrophils release extracellular traps (neutrophil extracellular traps [NETs]; Brinkmann et al., 2004). NETs are composed of chromatin decorated with granular proteins. These structures bind Gram-positive and -negative bacteria, as well as fungi (Urban et al., 2006). NETs provide a high local concentration of antimicrobial molecules that kill microbes effectively. NETs are abundant at inflammatory sites, as shown for human appendicitis and an experimental model of shigellosis
• Recently, NETs were shown to be relevant in vivo in
• streptococcal infections (Molloy, 2006)
• The release of intact chromatin decorated with cytoplasmic proteins into the extracellular space is unprecedented. We describe that activated neutrophils initiate a process where first the classical lobulated nuclear morphology and the distinction between eu- and heterochromatin are lost. Later, all the internal membranes disappear, allowing NET components to mix. Finally, NETs emerge from the cell as the cytoplasmic membrane is ruptured by a process that is distinct from necrosis or apoptosis. This active process is dependent on the generation of ROS by NADPH oxidase. In an infection, ROS formation may contribute to the following two antimicrobial pathways: intraphagosomal killing in live neutrophils and NET-mediated killing post mortem
• To analyze NET formation, we monitored individual neutrophils with live-cell imaging through four different channels. First, we recorded the phase-contrast image to determine the morphology. Second, to assess cell viability, neutrophils were loaded with calcein blue, a dye that is retained in the cytoplasm of living cells and rapidly lost upon cell death. Third, the neutrophils were incubated in the presence of Annexin V, which binds to phosphatidylserine (PS). PS is localized to the inner leaflet of the cell membrane. Annexin V can only bind to PS of cells undergoing apoptosis, when PS is transferred to the outer leaflet, or after membrane rupture, when Annexin V can enter into the cell. Thus, if the plasma membrane breaks, the cells lose the vital dye and are stained with Annexin V simultaneously. If a cell undergoes apoptosis, it will first become Annexin V–positive and later lose the vital dye. Fourth, to detect the appearance of NETs, we used fluorescently labeled Fab fragments of monoclonal antibodies against the complex composed of histone 2A, histone 2B, and DNA (Fig. 1 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200606027/DC1; Losman et al., 1992) or neutrophil elastase (Fig. S3 and Video 2). In viable neutrophils, neither Fabs nor Annexin V have access to their targets. When NETs emerge or cells die, Fabs and Annexin V can bind; because of the increase in the local concentration, they become detectable
• Purified peripheral blood neutrophils were activated with PMA and monitored by live-cell imaging for 240 min. Initially, neutrophils flattened and formed numerous intracellular vacuoles (Fig. 1 a and Video 1).   After 80 min, nuclei lost their lobular shape, and they expanded and filled most of the intracellular space (Fig. 1 b). At this time, the cells were viable because they retained calcein blue (Fig. 1, b and h, blue) and excluded Annexin V (green). After 220 min of activation, progressively more cells lost the calcein blue marker and concurrently stained with Annexin V (Fig. 1, d, j, and p), indicating rupture of the plasma membrane
• NETs only became detectable when the cell membrane was ruptured. As shown in Fig. 1, NETs (Fig. 1 p, red) appeared exactly when cells lost the vital dye and simultaneously became positive for Annexin V (Fig. 1 j). This clearly demonstrates that NETs emerge from dying neutrophils. This also shows that NETs are not released by apoptotic neutrophils because PS was not exposed before the rupture of the plasma membrane, which is indicated by the loss of calcein blue. In contrast, apoptotic neutrophils became Annexin V positive before the integrity of the plasma membrane was compromised (unpublished data)
• 60 min after stimulation, the nuclei started to lose their lobules, and the chromatin began to decondense while the nuclear membrane remained intact (Fig. 2, b and f).   At this time point, the space between the inner and outer nuclear membrane dilated (0 min, 18.8 ± 3.9 nm; 60 min, 27.9 ± 5.8 nm). 120 min after stimulation, the nuclear membranes formed distinct vesicles (Fig. 2, c and g), and by 180 min the nuclear envelope disintegrated into numerous small vesicles and the chromatin decondensed (Fig. 2, d and h). These vesicles originated from the nuclear envelope, as demonstrated by immune detection of the lamin B receptor, which is a component of the inner nuclear membrane (Fig. 3, a and d; Worman et al., 1988), and by immunofluorescence staining of nuclear membrane proteins (Fig. 3, c and f).   By 180 min, most granules disappeared (Fig. 2, d and h)
• Disintegration of the nucleus and granules allows the formation of NETs. Transmission electron microscopy (a–h) and confocal immunomicroscopy (i–l) of neutrophils. Cells were incubated for 180 min in medium without stimulation (a, e, and i) or activated with 20 nM PMA for 60 (b, f, and j), 120 (c, g, and k), or 180 min (d, h, and l; all incubated for a total of 180 min). e–h are higher magnifications of a–d. (a and e) Naive neutrophils show a lobulated nucleus with clearly defined eu- and heterochromatin and numerous cytoplasmic vacuoles, even after 180 min of incubation. (i) Immunofluorescence staining for nuclear (red, histone–DNA complex antibodies) and granular components (green, neutrophil elastase antibodies) is clearly separated in naive neutrophils. Granules are distributed through the globular cell and exhibit a patchy cytoplasmic pattern in this projection of a confocal z stack. (b) After 60 min of stimulation, nuclei are less lobulated. (f) The space between inner and outer nuclear membrane is dilated. (j) Nuclei and granules are now individually visible because the cell has flattened out. (c) After 120 min, most nuclei no longer show separation of eu- and heterochromatin, and in some cells the nuclear envelope starts to disintegrate into a chain of vesicles surrounding the DNA (arrows). (g) DNA and cytoplasm are no longer separated by a membrane (arrows). (k) Cells lose their distinct granule pattern and show colocalization (yellow) of neutrophil elastase (green) and chromatin (red), especially at the nuclear periphery (arrows). (d) Most neutrophils stimulated for 180 min are nearly entirely filled with decondensed chromatin. (h) Small areas are occupied with residual granules and numerous vesicles. (l) Some cells already released NETs
• Figure 2
• The nuclear membrane vesiculates after activation. Ultrathin cryosections of neutrophils stimulated with 20 nM PMA for 60 (a) and 180 (d) min and labeled with an antibody against the nuclear envelope–specific lamin B receptor. Bound antibody was detected with a gold-conjugated secondary antibody. In 60-min–stimulated neutrophils, the lamin B receptor was found on the inner nuclear membrane, as expected (a, arrows). 180 min after stimulation, the lamin B receptor was found in vesicles (d, arrows) in the cytoplasm. Nuclear material (d, arrowheads) is decondensed and not enclosed by a membrane. (b) 60 min after activation, neutrophils have flattened out and exhibit a lobulated nucleus and numerous granules
• Figure 3
• With the loss of nuclear and granular membranes, the decondensed chromatin came into direct contact with cytoplasmic and granular components. We showed by immunofluorescence that 120 min after activation the granular marker neutrophil elastase colocalized with chromatin, although some granular punctuate staining remained (Fig. 2 k). Later in the activation, the granular staining was lost and all the neutrophil elastase was associated with chromatin (Fig. 2 l). The intracellular mixing of granular components and chromatin was supported by the observation that most of the neutrophil elastase released by the cell remained bound to NETs (Fig. 2 m). Interestingly, the plasma membrane integrity of activated neutrophils was not affected by disruption of internal membranes, allowing the intracellular mixing of NET components (Fig. 2 d). Similar morphological changes were observed when neutrophils were infected with bacteria (Fig. 5)
• We compared the cell death that leads to NET formation to apoptosis and necrosis. We induced apoptosis (Fig. 4 a) by incubating neutrophils with anti-Fas antibodies for 18 h (Fadeel et al., 1998).   In these conditions, 70% of the cells were dead. Apoptotic neutrophils showed the classical morphology, including condensation of chromatin and fragmentation of nuclei without rupture of the nuclear envelope, as well as cytoplasmic vacuolization. Organelles in the cytoplasm remained intact
• Neutrophils incubated with high concentrations of secreted pore-forming toxins from Staphylococcus aureus for 15 min when >70% of the cells were dead showed typical necrotic morphology (Fig. 4 b; Genestier et al., 2005). The nuclei lost their structure, and lobules fused into a homogenous mass without segregation into eu- and heterochromatin and did not make NETs, even in long incubation periods
• In contrast to cells forming NETs (Fig. 4 c), the nuclear membranes remained intact and clearly separated nucleoplasm from cytoplasm. The structure of organelles was not affected. Fig. 4 c shows a neutrophil activated with PMA for 4 h to promote NET formation. The most salient morphological differences in comparison to the apoptotic and necrotic cells are disintegration of the nuclear envelope and mixing of nuclear and cytoplasmic material, loss of internal membranes, and disappearance of cytoplasmic organelles. Neutrophils activated with PMA did not proceed to have a necrotic morphology, even after longer incubation
• Neither apoptosis nor necrosis induce NETs. Neutrophils were stimulated with 20 ng/ml anti-Fas antibodies for 18 h to induce apoptosis, incubated with 25 µg/ml of pore-forming secreted toxins from S. aureus for 15 min to induce necrosis, or activated with 10 nM PMA for 4 h to induce NETs. (a–c) Transmission electron micrographs. (a) Neutrophil treated with anti-Fas antibodies show characteristic apoptotic morphology, including nuclear condensation and fragmentation as well as cytoplasmic vacuolization. (b) Features of necrosis in neutrophils treated with toxins are the loss of segregation into eu- and heterochromatin and of the nuclear lobules. The nuclear envelope, as well as the granules, remains intact. (c) Neutrophils undergoing NET-forming active cell death exhibit a morphology clearly different from both apoptosis and necrosis. The nuclear membranes are entirely fragmented while most of the granules are dissolved, allowing direct contact and mixing of nuclear, cytoplasmic, and granular components. Bars, 2 µm. (d) TUNEL analysis of different forms of cell death reveals that 60% of the anti-Fas–treated neutrophils have fragmented DNA, whereas toxin- and PMA-treated neutrophils do not. n = 200 cells/condition. (e) Neither apoptosis nor necrosis lead to NET formation, as revealed by quantifying extracellular DNA. The experiment was repeated at least three times with neutrophils from independent donors with similar results. The data shown is a representative triplicate experiment and presented as a mean value ± the SD
• Figure 4
• DNA fragmentation, as detected by TUNEL, is a hallmark of apoptosis and was only apparent after stimulation with anti-Fas antibodies (Fig. 4 d), but not when the cells died by necrosis after incubation with S. aureus toxins. Neutrophils activated to make NETs were also TUNEL negative. We quantified NET formation under these different conditions by digesting the DNA scaffold of NETs with an endonuclease and measuring the content of released DNA in the supernatant (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200606027/DC1). NETs were detected after PMA activation (Fig. 4 e), but not after incubation with Fas antibody or treatment with S. aureus toxins. Together, these data indicate that neither apoptosis nor necrosis lead to NET formation, and that NET-inducing cell death is different from both apoptosis and necrosis by morphological and molecular criteria
• Figure 6
• e–h) Transmission electron micrographs. Neutrophils isolated from CGD patients 3 h after PMA activation (e and f) showed no morphological signs of NET formation. The nuclei are still lobulated and granules are clearly visible. (g and h) GO-stimulated neutrophils isolated from CGD patients at the moment of NET formation. The plasma membrane ruptured, allowing the release of NETs. The nuclear membrane disintegrated, allowing the mixing of nuclear and granular components

• Isolation and quantification of NETs
    NETs generated by activated neutrophils were digested with 500 mU/ml micrococcal nuclease (MNase; Worthington Biochemical Corp.). The nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at 4°C until further use. Total DNA was isolated from naive neutrophils with DNazol supplemented with 1% polyacryl carrier (Molecular Research Center) according to the manufacturer's instructions and solubilized in TE buffer. NETs or genomic DNA was quantified using Picogreen dsDNA kit (Invitrogen) according to manufacturer's instructions. Total DNA of neutrophils and PBMCs was isolated from four different donors and quantified. 10⁶ neutrophils had 6.2 µg DNA (±0.27 SD) and 10⁶ PBMCs contained 6.3 µg DNA (±0.81 SD). Percentage of released NET-DNA was calculated by dividing the amount of isolated NET-DNA through the average genomic DNA content.

    <!-- null -->Isolation and quantification of neutrophil elastase
    Neutrophils were activated for indicated times with PMA. Neutrophil elastase in suspension was collected in culture supernatants. NET-associated neutrophil elastase and NET-DNA was isolated by incubating the cells in medium containing 500 mU/ml MNase for 10 min. Samples were resuspended in 1 M NaCl 0.02% Triton X-100. Total neutrophil elastase was measured from unstimulated neutrophils lysed with 0.02% Triton X-100 in 1 M NaCl. Neutrophil elastase activity was quantified with 100 µM of the peptide substrate N-(Methoxysuccinyl)-Ala-Ala-Pro-Val 4-nitroanilide for 15 min at room temperature. Optical density was measured at 405 nm (Microplate reader EL800; BIO-TEK Instruments).

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• Figure 9
• Resistance of viscous material-producing strain 17 against the phagocytic activity of human neutrophils. Strain 17 cells were not internalized by neutrophils though many of these cells were bound to the cell surface of neutrophils (A, arrows). In contrast, viscous material non-producing strain 17-2 cells were internalized and the ingested bacteria appear to be enclosed within cytoplasmic vacuoles (B, asterisks)

• Figure 2
• Figure 2
• Neisseria gonorrhoeae N313 (Opa₅₇) triggers different ultrastructural modifications of the cell surface in HeLa lines, depending on the expression of either CEACAM3 or CEACAM1. Subconfluent monolayers were infected with an m.o.i. of 100 for 30 min at 37°C. (A−D) SEMs of infected cells showing large cellular protrusions extending from the cell surface of HeLa-CEACAM3 (A and C) and tightly fitting pseudopods enclosing bacteria that adhere to HeLa-CEACAM1 (B and D). (D) shows a detail of (B). Interactions between N313 and a CEACAM6-expressing HeLa cell line were indistinguishable at the ultrastructural level from those with HeLa-CEACAM1 (not shown). (E and F) TEMs of infected HeLa-CEACAM3 (E) and HeLa-CEACAM1 (F). Images are representative of three independent experiments

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• The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation
• Figure 3
• Ultrastructural (TEM) analysis of different stages of phagocytosis of healthy RgpB-treated PMNs by macrophages (hMDMs). Individual panels show (a) freshly isolated, RgpB-treated PMN with characteristic segmented nucleus (n) and variety of cytoplasmic granules; (b) two PMNs engulfed within the phagocytic cup, arrows pointing at the PMN intact cell membrane; (c) fully engulfed PMN, black arrowheads indicate the intact nuclear membrane; (d) digested PMN within a macrophage, arrowheads point at remnants of PMNs. M, the macrophage nuclei

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• eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles
• A candidate mechanism suggested by these early reports was that eosinophil granule proteins might be mobilized from within intracellular granules into vesicles that traffic to and release extracellularly at the cell surface, a process termed piecemeal degranulation (PMD).^{<!--bib12-->12, <!--bib13-->13, <!--bib14-->14}
• (d) Immunogold electron microscopy demonstrates CD63 labeling primarily on the cytoplasmic surface of eosinophil granule limiting membranes
• Figure 2
• Major basic protein (MBP) is localized to specific granules and cytoplasmic vesicles in human eotaxin-stimulated eosinophils. Immunonanogold electron microscopy revealed MBP-positive vesicles surrounding (a and b) and arising (c) from granules undergoing depletion of their contents. (aii) is the boxed area of (ai) and shows a tubular vesicle (arrowheads) at the surface of a specific granule. Note that MBP is present in the granule and in the attached vesicle. In (b), a pool of vesicles around a secretory granule is labeled for MBP. Observe that (aii) and (b) show specific granules in progressive stages of content emptying, characterized by reduced electron density. In (c), a linear array of small, MBP-positive vesicles bud from a granule surface into the cell surface. The inset shows a vesicle fused with the plasma membrane at higher magnification. In (d), a granule, which demonstrates its disarranged core and matrix, is densely labeled for MBP. A similar image is seen at low magnification in ai (arrows). Bar, 800 nm (ai)
• Figure 3
• Large tubular carriers actively transport MBP. (a–c) Eosinophil sombrero vesicles (EoSVs; arrows) are observed in the cytoplasm of activated eosinophils by transmission electron microscopy (TEM) after immunonanogold labeling for MBP. Vesicles are seen beneath the plasma membrane in the cytoplasm (a), fused with the plasma membrane (b) and attached to an enlarged partially empty granule (c), typically indicative of PMD. In (d), EoSVs, isolated by subcellular fractionation from unstimulated eosinophils, are densely labeled for MBP. Note that MBP is preferentially localized within the vesicle lumen. Eosinophils were stimulated by eotaxin as described in Materials and methods section
• Figure 4
• Eosinophil sombrero vesicles (EoSVs) are significantly increased within HES eosinophils. (a) Significant increases in EoSV numbers are observed within eosinophils from a patient with HES compared to normal donors (^*P<0.05). (bi and bii) The boxed area shows EoSVs (arrows), with typical morphology, close or associated with an emptying specific granule. A total of 958 EoSVs were counted in 35 cell sections showing the entire cell profile ad nucleus. Bar, 600 nm (bi); 250 nm (bii). Gr, specific granules; N, nucleus; HES, hypereosinophilic syndrome.
• Figure 5
• Ultrastructure of an eosinophil from a patient with hypereosinophilic syndrome. (ai) Specific granules (Gr) with typical crystalloid cores are observed together with enlarged granules with disassembled cores (boxed area in higher magnification in aii). Arrows indicate eosinophil sombrero vesicles (EoSVs)
• Figure 6
• A major pool of MBP-loaded vesicles is tightly associated with secretory granules in unstimulated eosinophils. (a) Immunonanogold electron microscopy reveals MBP labeling within and around specific granules in an unstimulated eosinophil. (b) In higher magnification, labeling around granules is imaged as a consistent pool of MBP-positive vesicles. Labeling is observed in both round small and large vesiculotubular (arrowheads) carriers. Note that the vesicles are closely associated with the granule
• Figure 7
• Ultrastructure of isolated eosinophil granules. (ai and bi) Transmission electron microscopy of a granule-enriched fraction isolated by subcellular fractionation shows typical EoSVs, the lumens of which are highlighted in pink in (aii and bii). EoSVs are imaged as open, curved, tubular-shaped structures. EoSVs are seen around and in close association with isolated granules. Granules were isolated and fixed for optimal membrane preservation as described in Materials and methods section. Bar, 250 nm (a and b). Gr, specific granules

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• Transmission electron micrographs of lung tissue from mice with OVA-induced eosinophilia. Control mice treated with the isotype control Ab showed no sign of eosinophil apoptosis at 8 h (A) and 24 h (not shown). In mice treated with anti-Fas mAb there were numerous apoptotic eosinophils in the lung tissues at both 8 and 24 hours after treatment (B and C, respectively). The apoptotic eosinophils were rarely engulfed although macrophages (labeled M) commonly occurred in the tissue (B). By 24 h a majority of the apoptotic eosinophils exhibited signs of secondary necrosis and severe inflammation was recorded including neutrophil infiltration (arrow) and derangement of the epithelial lining (labeled E)
• Figure 4
• Figure 4
• Figure 5
• These micrographs illustrate characteristic eosinophil phenotypes present in mouse airways in this study: (A) viable non-degranulating eosinophil, the only phenotype found in lung tissues of allergen challenged animals; (B) apoptotic eosinophil exhibiting nuclear condensation, cell shrinkage, and an intact cell membrane; (C) an apoptotic eosinophil exhibiting secondary necrosis involving cell membrane rupture and piecemeal degranulation; (D) a cytolytic eosinophil exhibiting chromatolysis, cell membrane rupture, and spilling of electron-dense specific granules into the tissue (arrow)
• Observations in allergen challenged animals demonstrating anti-Fas-induced mucus-exudate plugs (A,C,D,E) compared to control isotype antibody (IgG) treatment (A,B)
• Histochemical staining with periodic acid-Schiff reagent (PAS) illustrated mucus-containing cells (B,C) and so did the transmission electron micrograph (D)

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• Effects of cigarette smoke (CS) on eosinophils (EOS), important cells involved in the pathogenesis of chronic lung diseases such as asthma, were studied in vitro. EOS were isolated from healthy and mildly atopic donors and exposed to soluble components of cigarette smoke (CSE). Viability and apoptosis were assessed by flow cytometry after staining with propidium iodide and Annexin-V. Activation was determined by release of newly-synthesized (IL-8, IL-6) mediators and by phosphorylation of MAPKs. CSE effects on ultrastructural morphology and production of neutrophil chemotactic factors in CSE-activated EOS were also evaluated. CSE concentrations from 0-2.5% were non-toxic for up to 18-24 hours of exposure. However, CSE at 2.5% activated EOS as evidenced by ultrastructural degranulation: release of IL-8 and IL-6, and increased expression of the MAPK, c-Jun. Supernatants from CSE-activated EOS were found to be significantly chemotactic for neutrophils. These results suggest that CS may aggravate lung inflammation by activating EOS which, in turn, release inflammatory mediators promoting inflammatory cell recruitment and lung remodeling

  • cse activating eos by promoting them to degranulate and release pro-inflametory mediators and adherent molecules which in turn promotes inflammatory cell recruitment and affects the ultrastructural morphology and production of neutrophil chemotactic factors
• A representative electron micrograph of untreated eosinophils cultured for 18h is shown in Fig. 3A
• In this cell some granules are electron-transparent suggesting partial degranulation (“piecemeal degranulation”) while the majority of granules appear normal, containing core protein, visible as a large crystal structure within the granule. The CSE-exposed eosinophil shown in Fig. 3B appears enlarged in comparison to the untreated cell (Fig. 3A) and most of its granules are electron transparent, lacking the core crystal. Its cytoplasm contains numerous vesicles and an area of “cloudy swelling” suggestive of hydropic degeneration due to cellular stress. Electron micrographs of cells that were incubated with GM-CSF [10ng/ml] and TNF-α [5000U] are shown in Figs. 3C and 3D. In the eosinophil incubated with GM-CSF and TNF-α, but not exposed to CSE (Fig. 3C), the granule content and the crystal core appear more electron-dense than the untreated control eosinophil (Fig. 3A) suggesting that GM CSF and TNF-α support cell viability and stability despite the physiologic stress imposed by culture conditions. Its cytoplasm has a vesicular appearance suggesting “piecemeal degranulation” as a result of TNF α activation of the cell. While the eosinophil shown in Fig. 3D was also exposed to 2.5% CSE for 18h the majority of its granules contain their crystal core, suggesting that in the presence of GM CSF and TNF-α eosinophil structure is more preserved. However, in some granules the crystals appear partially dissolved and many vesicles are scattered within the cytoplasm suggesting that CSE is enhancing “piecemeal degranulation” in response to the culture conditions. Thus, while the culture conditions activate eosinophils to some degree, exposure to CSE appears to augment eosinophil activation as evidenced by loss of granule contents
• In this cell some granules are electron-transparent suggesting partial degranulation (“piecemeal degranulation”) while the majority of granules appear normal, containing core protein, visible as a large crystal structure within the granule. The CSE-exposed eosinophil shown in Fig. 3B appears enlarged in comparison to the untreated cell (Fig. 3A) and most of its granules are electron transparent, lacking the core crystal. Its cytoplasm contains numerous vesicles and an area of “cloudy swelling” suggestive of hydropic degeneration due to cellular stress. Electron micrographs of cells that were incubated with GM-CSF [10ng/ml] and TNF-α [5000U] are shown in Figs. 3C and 3D. In the eosinophil incubated with GM-CSF and TNF-α, but not exposed to CSE (Fig. 3C), the granule content and the crystal core appear more electron-dense than the untreated control eosinophil (Fig. 3A) suggesting that GM CSF and TNF-α support cell viability and stability despite the physiologic stress imposed by culture conditions. Its cytoplasm has a vesicular appearance suggesting “piecemeal degranulation” as a result of TNF α activation of the cell. While the eosinophil shown in Fig. 3D was also exposed to 2.5% CSE for 18h the majority of its granules contain their crystal core, suggesting that in the presence of GM CSF and TNF-α eosinophil structure is more preserved. However, in some granules the crystals appear partially dissolved and many vesicles are scattered within the cytoplasm suggesting that CSE is enhancing “piecemeal degranulation” in response to the culture conditions. Thus, while the culture conditions activate eosinophils to some degree, exposure to CSE appears to augment eosinophil activation as evidenced by loss of granule contents.

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• The neutrophil is a major effector cell of innate immunity. Exocytosis of granules and secretory vesicles plays a pivotal role in most neutrophil functions from early activation to the destruction of phagocytosed microorganisms. Neutrophil granules contain a multitude of antimicrobial and potentially cytotoxic substances that are delivered to the phagosome or to the exterior of the cell following degranulation. This review summarises current knowledge of granule biology and highlights the effects of neutrophil degranulation in the acute inflammatory response

• Normally, neutrophils are silently removed by apoptosis, followed by phagocytosis. However, if phagocytosis fails, apoptotic cells undergo secondary necrosis
• Detailed electron microscopic assessment of neutrophil activation and death modes revealed that up to 14% of the neutrophils were undergoing secondary necrosis, whereas apoptotic or primary necrotic structural cells were rarely found
• Under normal physiological conditions, neutrophils are silently and swiftly eliminated through apoptosis, followed by phagocytosis by alveolar macrophages 2. However, if the phagocytosis system fails, apoptotic cells die through secondary necrosis, a pro-inflammatory event associated with cell membrane disruption and extracellular spreading of cell contents 3
• Special focus was given to neutrophil infiltration and clearance, due to the possibility of finding direct evidence for secondary necrosis of neutrophils to be a significant feature of the inflammation
• Taking the pathogenic potential of necrosis into consideration 5, 6, this study suggests neutrophil secondary necrosis to be a potential pathogenic mechanism during an intense neutrophil-rich lung inflammation
• Histological observations, such as extent of inflammation, infiltrating cells and occurrence of cell debris, were examined on 6-µm haemotoxylin and eosin-stained paraffin sections, co-stained with the DNA-marker Hoechst 33342 (H33342<!-- HIGHWIRE EXLINK_ID="28:2:268:1" VALUE="H33342" TYPEGUESS="GEN, PIRDB, SPROT" --> [GenBank] <!-- /HIGHWIRE -->; Sigma, St Louis, MO, USA). Neutrophils were identified as cells with polymorph nucleus, apoptotic cells by intensely DNA-stained condensed nuclei, and necrotic cells and cell debris as vague and blurry positive DNA staining
• The terminal deoxynucleotide transferase-meditated dUTP nick-end labelling (TUNEL) technique, performed according to the manufacturer’s protocol (Intergen Company, New York, NY, USA) as previously described 8, in combination with H33342<!-- HIGHWIRE EXLINK_ID="28:2:268:2" VALUE="H33342" TYPEGUESS="GEN, PIRDB, SPROT" --> [GenBank] <!-- /HIGHWIRE -->, was used to identify apoptotic cells in lung sections, as discussed in another study 9. When using the TUNEL technique it is important to consider that not only apoptotic cells are labelled, but also necrotic cells and free apoptotic cell nuclei stain positive. Therefore, no separation of these stages was performed using this analysis; the differentiation was performed with electron microscopy
• For quantification, high-resolution digital images of the lung parenchyma were randomly obtained (4–6 images per slide) and the total number of TUNEL-positive cells/nuclei in each image was counted (blinded) using the ImageJ® software (1.30v; National Institutes of Health, Bethesda, MD, USA) and expressed as cells·mm^-2
• For quantification of positivity within areas of intense neutrophil infiltration and inflammation (IF), selected areas within sections displaying numerous TUNEL-positive staining were photographed and analysed as previously described
• At least 90 neutrophils per time-point were counted, with the exception being controls for which a very limited number of neutrophils were found, and the results were expressed as percentage of the total number of cells. The areas studied via TEM were consciously chosen to include IF, as the aim of the present study was to investigate the inflammatory process in these areas
• Transmission electron microscopic images exemplifying the different ultrastructural criteria used to determine the fate of lung neutrophils. a) Resting neutrophil with normal nucleus and intact primary and secondary granules. b) Activated neutrophil, exemplified here by occurrence of cytoplasmatic protrusions and phagosomes. c) Apoptotic neutrophil with characteristic chromatin condensation and separation of euchromatin and heterochromatin. d) Former apoptotic neutrophil, in the process of secondary necrosis. The electron-dense nuclei, ruptured cell membrane and largely dissolved/released cytoplasm should be noted. e) Cluster of neutrophil-derived cell debris
• Table 2
• the vast majority of the neutrophils were activated with granulae alterations and/or intracellular phagosomes (fig. 3a). A marked increase in apoptosis, and primary and secondary necrosis as well as cell debris (i.e. extracellular neutrophil granules, free condensed nuclei and other cell remnants) was regularly seen in IF 24 h after LPS administration (fig. 3a and b)
• Neutrophils undergoing primary necrosis were found, though only a few scattered cells, indicating that the increase in neutrophil-derived cell debris was mainly due to secondary necrosis
• a) Neutrophils (N) were primarily activated with granulae alterations or phagosomes (indicated by arrows) in lumen amidst cell debris (arrowheads), such as free apoptotic nuclei and neutrophil-derived granulae. b) Secondary necrosis of neutrophils (cells with condensed and electron-dense nuclei and ruptured cell membrane) were extensively seen in IF. c) Only scattered dying structural cells were found, illustrated by a type-II pneumocyte undergoing primary necrosis, with early signs of chromatolysis and rupture of the cell membrane (arrowhead)

  • chromatolysis: the solution and disintegration of the chromatin of cell nuclei
• Fig. 4
• Several fundamental questions therefore remain regarding the nature of secondary necrosis in the lung. For example, it is still unclear which cell types actually undergo secondary necrosis, and are therefore the source of the LDH found in BAL, and if/how this release relates to modes of cell death, such as primary necrosis 13. Furthermore, until now, virtually nothing has been known about when and where in the inflammatory process secondary necrosis takes place
• This study shows that in IF, only scattered pneumocytes displayed ultrastructural signs of apoptosis, whereas signs of primary necrosis (electron-lucid cytoplasm and damaged cell membrane, for example) were more commonly found. Although this finding supports the notion that necrosis rather than apoptosis is the usual death mode of structural cells 15, it must not be forgotten that apoptosis of structural cells occurs more frequently in more chronic situations. The study also found numerous apoptotic neutrophils and neutrophils in the process of secondary necrosis, suggesting that the apoptotic process is not too swift to be detected. This implies that apoptotic neutrophils undergoing secondary necrosis are the primary source of LDH, and the fact that secondary necrotic neutrophils occurred amidst apoptotic neutrophils and extracellular neutrophil-derived cell debris suggests a rapid turnover of neutrophils
• In the regions of mild and moderate inflammation, apoptosis and subsequent macrophage phagocytosis carried out swift and silent neutrophil elimination, resulting in sufficient clearance of infiltrating neutrophils. A similar silent removal process may also have occurred in IF, to an as yet unknown extent. However, as demonstrated through TEM analysis, in IF a significant proportion of the neutrophils underwent secondary necrosis, suggesting the clearance to be insufficient. Taken together, the present data suggest secondary necrosis to be a rare phenomenon in large areas of the lung parenchyma (with mild-to-moderate neutrophil infiltration) but a common fate of lung neutrophils in IF

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• SEM micrographs of TNF-induced necrotic cell internalization. (a) Macrophage (M) demonstrating broad ruffling towards the necrotic cell (arrow). (b) Clearance of necrotic material (arrow). Macrophage (M) displays flat membrane ruffle directed towards the necrotic debris. (b') Detail of the rectangle in (b). Necrotic material (arrow) seems to be grasped by tiny protrusions emanating from the membrane ruffle of the macrophage. Note that the necrotic cell (N) has an irregular surface
• Transmission electron micrographs. (a) Macrophages cocultured with unstimulated L929sAhFas cells in the presence of HRP (control). Note HRP positive vacuoles in the macrophages as a signature of constitutive endocytosis. (b) Coculture of macrophages and apoptotic cells in the presence of HRP. The phagosome of the macrophage (M) is devoid of HRP and contains an engulfed apoptotic body (asterisk). (c) Detail of rectangle in (b). An apoptotic body internalized by a macrophage demonstrating the absence of HRP and formation of a tightly fitting phagosome. Note a double membrane structure (arrows) formed by the phagosomal and apoptotic body membranes. (d, e) Macrophages cocultured with TNF-induced necrotic cells in the presence of HRP. (d) Note absence of the HRP reaction products at the surface of the macrophage (M) and remnants of the necrotic cell (arrow). (e) Detail of rectangle in (d). Colocalization of HRP and necrotic material in the macropinosomes (arrows). Note that necrotic material is located in the spacious macropinosomes. (f, g) Macrophages cocultured with heat-induced necrotic cells in the presence of HRP. (f) Nearly complete encircling by the macrophage of a necrotic cell corpse. (g) Detail of rectangle in (f). Note formation of ruffles at the surface of the macrophage (asterisks) with underneath the creation of spacious macropinosomes with coingestion of HRP and necrotic material (arrows)

  • refer to full figure

• Note chromatolysis of nucleus and the dense cluster of fine particulates with a surrounding zone of lysis. The lysis extends entirely to cell membrane, which is still apparently intact morphologically

  • chromatolysis: the solution and disintegration of the chromatin of cell nuclei

• Neutrophil cannibalism – a back up when the macrophage clearance system is insufficient
• a massive accumulation of neutrophils occurs, which is normally cleared by macrophage phagocytosis following neutrophil apoptosis
• However, in cases of extensive apoptosis the normal clearance system may fail, resulting in extensive neutrophil secondary necrosis. The aim of this study was to explore the hypothesis that neutrophils, in areas of the lung with extensive cellular infiltration, contribute to clearance by phagocytosing apoptotic cells and/or cell debris derived from secondary necrosis
• Neutrophils are previously known to phagocytose invading pathogens and harmful particles. However, this study demonstrates that neutrophils are also able to engulf apoptotic neutrophils or cell debris resulting from secondary necrosis of neutrophils. Neutrophils may thereby contribute to clearance and resolution of inflammation, thus acting as a back up system in situations when the macrophage clearance system is insufficient and/or overwhelmed
• Neutrophils have a high turnover and are normally rapidly cleared by apoptosis, followed by macrophage phagocytosis
• During infection a large number of neutrophils are present in order to efficiently clear the infection, and studies have shown that ingestion of bacteria may delay neutrophil apoptosis
• thereby causing very large number of cells accumulating in the same area
• If the apoptotic neutrophils are not cleared away efficiently or fast enough they undergo secondary necrosis, which is a pro-inflammatory event
• Interestingly, in IF we found apparently viable neutrophils with phagosomes enclosing what appeared to be whole apoptotic neutrophils, apoptotic nuclei and other neutrophil cell remnants
• The aim of the present study was to prove the existence of this phenomenon and quantify its occurrence through detailed ultrastructural studies, and test the hypothesis that neutrophils contribute to clearance in localized areas where the macrophage system is insufficient.
• We frequently found phagocytosing neutrophils in IF and BALF, with phagosomes of varying size containing what appeared to be whole apoptotic neutrophils, apoptotic nuclei and neutrophil-derived cell debris. Phagocytosing macrophages were present in both IF and in BALF but in IF, the macrophage clearance system seemed to be insufficient (indicated by the large number of neutrophils undergoing secondary necrosis) and in addition, several macrophages in IF displayed signs of necrosis
• Due to the pro-inflammatory effect of secondary necrosis [13,14], we suggest that neutrophils in IF may contribute to resolution of inflammation by phagocytosing apoptotic neutrophils and/or neutrophil-derived cell debris
• alveolar luminal neutrophils were generally activated; both apoptosis and secondary necrosis (Figure 2A), as well as extracellular neutrophil granules, free condensed nuclei and other types of cell debris was regularly seen (Figure 2B), primarily at 24 h and onwards. Phagocytosing neutrophils were frequently found (Figure 2C–E), the numbers significantly increasing already 4 h after LPS administration, peaking at 24 h (Figure 3). When assessing the granulae content in neutrophils with large phagosomes 36 h after LPS administration, a significant decrease in the number of granulae was detected, (0.21 ± 0.07 granulae/μm²) compared to neutrophils without phagosomes (1.5 ± 0.37 granulae/μm²). We also noted that the phagosome content varied over time, from 24 h and onwards phagosomes generally contained cell remnants (e.g. apoptotic nuclei and neutrophilic granulae), and at the earlier time points mainly surfactant
• Representative transmission electron micrographs displaying neutrophils during an LPS-induced lung inflammation. In areas of intense inflammation and neutrophil infiltration highly activated neutrophils (N), characterized by e.g. phagosomes and/or cytoplasmatic protrusions, were lying amongst apoptotic neutrophils (black arrow) and cell debris (black arrowhead) (A). Also secondary necrosis (characterized by membrane rupture of cells with an otherwise apoptotic morphology) of neutrophils was regularly observed (B). Furthermore, neutrophils containing large phagosomes (asterisks) enclosing neutrophilic cell remnants such as apoptotic nuclei and neutrophil granulae (C-E) were frequently found
• Also in BALF, neutrophils containing phagosomes were found (Figure 4), detected as DNA-positive phagosomes in neutrophils (Figure 5A). The number of phagocytosing neutrophils increased significantly following LPS administration and peaked at 48 h after LPS administration when 10.8 ± 2% of the BALF neutrophils contained DNA-positive phagosomes. In control animals, none of the exceedingly rare neutrophils contained any DNA-positive phagosomes
• In areas of intense inflammation and neutrophil infiltration, a majority of the macrophages (M) were abundantly packed with multiple large phagosomes (C)
• Neutrophils are classified as professional phagocytes, and are important in resolution and clearance of pathogens [1,2]. They are known to phagocytose pathogens (including yeast and bacteria) as well as potentially hazardous substances, being a fist line defence [17]. Normally neutrophils die through apoptosis, followed by subsequent macrophage phagocytosis. However, if macrophages fail to clear the apoptotic neutrophils, apoptotic neutrophils are left in the tissue and undergoes secondary necrosis [6]
• In IF, neutrophils with phagosomes containing both what appeared to be whole apoptotic neutrophils as well as apoptotic nuclei and neutrophil derived cell debris, were frequently found. Besides neutrophils, phagocytosing macrophages were present in both IF and BALF, but in IF, the macrophage clearance system seemed to be insufficient to meet the needs (indicated by the large number of apoptotic cells, mainly neutrophils, in the process of secondary necrosis) and in addition, several macrophages in IF displayed signs of necrosis. Phagocytosing neutrophils were also found in BALF, but at lower numbers. However, the results obtained in BALF only include cells with DNA-positive phagosomes, whereas the electron microscopic study of IF includes all cells with phagosomes, suggesting the BALF-values to be falsely low
• Unfortunately, we could not determine whether neutrophils (either from BALF or IF) had phagocytosed intact apoptotic cells, or only cell remnants of secondary necrosis, i.e. free condensed nuclei, neutrophil granulae and other cell components. However, in several cases (see for example figure 2E), it did look as if whole apoptotic neutrophils were ingested
• we found that neutrophils containing large phagosomes contained less granulae. We cannot rule this out as an artefact; however, it suggests that neutrophils lose at least a part of their granulae before or during phagocytosis. This implies that attempts to identify neutrophils via labelling of their granulae proteins may prove unsuccessful in situations where neutrophils are engaged in phagocytosis

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• Whereas induction of macrophage apoptosis by pathogens may adversely affect the host immune response to infection, acceleration of neutrophil apoptosis following phagocytic interaction with bacteria appears essential for the resolution of infection. This idea is supported by the finding that some bacterial pathogens alter normal phagocytosis-induced neutrophil apoptosis to survive and cause disease. This review summarizes what is currently known about modulation of phagocyte apoptosis by bacteria and describes a paradigm whereby bacteria-induced neutrophil apoptosis plays a role in the resolution of infection

• Swollen mitochondria and intact mitochondria are indicated by arrowheads and arrows, respectively

  • disruption of plasma membrane and organelles, nuclei condensation (not chromatin rearrangement in the nuclei) and swollen mitochondria => necrosis

• TEM of a necrotic cell: the disruption of plasma membrane and organelles is observable
• TEM of an apoptotic (A) and a normal (N) cell. The characteristic chromatin rearrangement appears in A, strongly different from its normal organization (N)
• SEM of a necrotic cell. Numerous lesions appear on the cell surface
• SEM of an apoptotic cell. Surface blebbing is evident
• FF of normal cell, nuclear envelope. The regular distribution of nuclear pores is visible
• FF of apoptotic cell. The nuclear envelope shows a characteristic clustering (asterisc) of nuclear pores

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• Nowadays physicians prefer to speak of periodical (pollinosis) or persistent (house dust and such) allergic rhinitis or conjunctivitis
• One does not become allergic for the entire pollen grains, but for certain proteins present in or around the pollen grains. These proteinscan been biochemically classified and get then a name composed from the first three letters ofthe genus of the plant which produces the pollen,followed by a number. The allergen Lol 1 is allergene nr 1 from the genus Lolium (Rye Grass). Sometimes also the first letter of the species is added to the name: Lol p 1 is allergene nr 1 from Lolium perenne
• An allergic reaction to a certain allergen that also occurs after contact with an allergen from another source is also called cross-reaction. Who is sensitive to pollen belonging to a certain genus, for example Birch pollen, is more likely to suffer also from pollen of other groups belonging to the Birch family, like Alders and Hazels: they carry allergens that are quite similar. Cross-reactions can also occur between allergens from completely different origin, like Birch pollen, apples and nuts
• 1. Pollen allergens (antigens) are released in the air and reach the air ways and so the mucosa 
  2. Once in the blood stream of the mucosa the antigens are taken up by cells of the immune system (Antigen-Presenting-Cells = APC), broken down into bits and bound to specific proteins called AHL to be "presented" at the cell surface. 
  3 and 4. These APCs stimultate B and Th2 cells to produce the antigen-binding protein  
  5. Th2 cell stimulates the B cell  
  6. to make even more antigen-binding protein  
  7. B cell in het blood plasma secretes antigen-binding protein (IgE) 
  8. By B cell secreted antigen-binding proteins bind to the surface of mast cells (also called mastocyte) 
  9. Therefore the mast cell become more sensitive to antigens 
  10. After the mast cell has bound antigens it produces vesicles with histamin and other hormones 
  10. The histamin is released and caused an allergic reaction
• During the sensibilization phase the allergens are taken up (engested by phagocytosis) by cells of the immune system, like macrophages and dendric cells. Next these cells (also named APC = antigen presenting cells) partly break down the entered allergens and "present" the remaining bits bound to certain proteins, the HLA's or Human Leukocyte Antigens, to the cell surface: the so-called antigen presentation. The antigens bound to HLA can now be recognized by T-cells which subsequently stimulate B-cells toproduce a specific antibody. B-cells can also be directly activated
• When a sensibilized person comes in contact with the allergen, the effector phase is provoked
• The allergen binds then to the allergen-specific IgE at the surface of the mastcells. In the mastcells numerous granula are present; these are vesicles filled with proteolytic (= proteins degradating) enzymes, heparine (prevents blood coagulation) and moreover the hormone Histamine. As a consequence of the binding of the allergen to the IgE at the surface of the cell the granula releasetheir contents in the surroundings, the so-called "degranulation". The effect can be usually sensed within 20 minutes: the early reaction. Especially the Histamine is responsible for the direct symptoms, it causes a burning feeling, itching and dilatation of the periphere blood capillaries
• About six hours after the early reaction the late reaction occurs. This is a true inflammatory reaction caused by the hormones released at the beginning of the allergic reaction
• The secretion vesicles or secretion granula (= grains) that contain a.o. the hormone Histamine are shown in blue. The cells degranulate, which means that they discharge the contents of the granula into their surroundings. Along with degranulation the dark vesicles swell and become grainy.
• Prevention of hay fever
• Treatment of hay fever

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• NETs provide for a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly independent of phagocytic uptake. In addition to their antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of the pathogens. Furthermore, delivering the granule proteins into NETs may keep potentially injurious proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of inflammation
• High-resolution scanning electron microscopy has shown that NETs consist of stretches of DNA and globular protein domains with diameters of 15-17 nm and 25 nm, respectively. These aggregate into larger threads with a diameter of 50 nm ^[1]. However, under flow conditions, NETs can form much larger structures, hundreds of nanometers in length and width
• While it was originally proposed that NETs would be formed in tissues at a site of bacterial/yeast infection, NETs have also been shown to form within blood vessels during sepsis (specifically in the lung capillaries and liver sinusoids). Intra-vascular NET formation is tightly controlled and is regulated by platelets, which sense severe infection via platelet TLR4 and then bind to and activate neutrophils to form NETs. Platelet-induced NET formation occurs very rapidly (in minutes) and does not result in death of the neutrophils. NETs formed in blood vessels can catch circulating bacteria as they pass through the vessels

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• nonconventional functions of the phagocytic NADPH oxidase (NOX2). The image  depicts a neutrophil extracellular trap (NET), formation of which depends on  reactive oxygen species generated through NOX2 activity