Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.     

Acanthamoeba castellanii: Morphological analysis of the interaction with human cornea

The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman´s membrane in 3 h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman´s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested.     

Heat shock protects cardiac cells from doxorubicin-induced toxicity by activating p38 MAPK and phosphorylation of small heat shock protein 27

Doxorubicin (DOX) and its derivatives are used as chemotherapeutic drugs to treat cancer patients. However, production of DOX-mediated reactive oxygen species (ROS) by prolonged use of these drugs has been found to cause dilative cardiomyopathy and congestive heart failure. Thus various preventive modalities have been developed to avoid this side effect. We have found that the DOX-mediated oxidant-induced toxicity in cardiac cells could be minimized by hyperthermia-induced small heat shock protein 27 (HSP27); that is, this protein acts as an endogenous antioxidant against DOX-derived oxidants such as H(2)O(2). Heat shock-induced HSP27 was found to act as an antiapoptotic protein (reducing ROS and Bax-to-Bcl2 ratio) against DOX, and its phosphorylated isoforms stabilized F-actin remodeling in DOX-treated cardiac cells and, hence, attenuated the toxicity. Protein kinase assays and proteomic analyses suggested that higher expression of HSP27 and its phosphorylation are responsible for the protection in heat-shocked cells. Two-dimensional gel electrophoresis showed six isoforms (nonphosphorylated and phosphorylated) of HSP27. Matrix-assisted laser desorption/ionization time of flight analyses showed alpha- and beta-isoforms of HSP27, which are phosphorylated by various protein kinases. Ser(15) and Ser(85) phosphorylation of HSP27 by MAPK-assisted protein kinase 2 was found to be the key mechanism in reduction of apoptosis and facilitation of F-actin remodeling. The present study illustrates that hyperthermia protects cells from DOX-induced death through induction and phosphorylation of HSP27 and its antiapoptotic and actin-remodeling activities.     

Glutamate Activates Protein Kinase B (PKB/Akt) through AMPA Receptors in Cultured Bergmann Glia Cells

Glutamate is involved in gene expression regulation in neurons and glial cells through the activation of a diverse array of signaling cascades. In Bergmann glia, Ca²⁺-permeable α-hydroxy-5-methyl-4-isoazole-propionic acid (AMPA) receptors become tyrosine phosphorylated after ligand binding and by these means form multiprotein signaling complexes. Of the various proteins that associate to these receptors, the phosphatidylinositol 3-kinase (PI-3K) deserves special attention since D3-phosphorylated phosphoinositides are docking molecules for signaling proteins with a pleckstrin homology domain. In order to characterize the role of PI-3K in AMPA receptors signaling, in the present report we analyze the involvement of the serine/threonine protein kinase B in this process. Our results demonstrate an augmentation in protein kinase B phosphorylation and activity after glutamate exposure. Interestingly, the effect is independent of Ca²⁺ influx, but sensitive to Src blockers. Our present findings broaden our current knowledge of glial glutamate receptors signaling and their involvement glutamatergic neurotransmission.Special issue dedicated to Miklós Palkovits.     

Purification of soluble alpha1,2-mannosidase from Candida albicans CAI-4

A soluble alpha-mannosidase from Candida albicans CAI-4 was purified by conventional methods of protein isolation. Analytical electrophoresis of the purified preparation revealed two polypeptides of 52 and 27 kDa, the former being responsible for enzyme activity. The purified, 52 kDa enzyme trimmed Man9GlcNAc2, producing Man8GlcNAc2 isomer B and mannose, and was inhibited preferentially by 1-deoxymannojirimycin. These properties are consistent with an endoplasmic reticulum-resident alpha1,2-mannosidase of the glycosyl hydrolase family 47. Moreover, a proteolytic activity responsible for converting the 52 kDa alpha-mannosidase into a polypeptide of 43 kDa retaining full enzyme activity, was demonstrated in membranes of ATCC 26555, but not in CAI-4 strain.     

Microbial immune suppression mediated by direct engagement of inhibitory Fc receptor

A microbial polysaccharide (glucuronoxylomannan (GXM)) exerts potent immunosuppression by direct engagement to immunoinhibitory receptor FcgammaRIIB. Activation of FcgammaRIIB by GXM leads to the recruitment and phosphorylation of SHIP that prevents IkappaBalpha activation. The FcgammaRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-alpha secretion. GXM quenches LPS-induced TNF-alpha release via FcgammaRIIB. The addition of mAb to GXM reverses GXM-induced immunosuppression by shifting recognition from FcgammaRIIB to FcgammaRIIA. These findings indicate a novel mechanism by which microbial products can impair immune function through direct stimulation of an inhibitory receptor. Furthermore, our observations provide a new mechanism for the ability of specific Ab to reverse the immune inhibitory effects of certain microbial products.     

Increased frequencies of cochlin-specific T cells in patients with autoimmune sensorineural hearing loss

Autoimmune sensorineural hearing loss (ASNHL) is the most common cause of sudden hearing loss in adults. Although autoimmune etiopathogenic events have long been suspected in ASNHL, inner ear-specific Ags capable of targeting T cell autoreactivity have not been identified in ASNHL. In this study, we show by ELISPOT analysis that compared with normal hearing age- and sex-matched control subjects, ASNHL patients have significantly higher frequencies of circulating T cells producing either IFN-gamma (p = 0.0001) or IL-5 (p = 0.03) in response to recombinant human cochlin, the most abundant inner ear protein. In some patients, cochlin responsiveness involved both CD4+ and CD8+ T cells whereas other patients showed cochlin responsiveness confined to CD8+ T cells. ASNHL patients also showed significantly elevated cochlin-specific serum Ab titers compared with both normal hearing age- and sex-matched control subjects and patients with noise- and/or age-related hearing loss (p < 0.05 at all dilutions tested through 1/2048). Our study is the first to show T cell responsiveness to an inner ear-specific protein in ASNHL patients, and implicates cochlin as a prominent target Ag for mediating autoimmune inner ear inflammation and hearing loss.     

Interleukin-6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture

We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED₅₀ of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-α; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.This work was supported in part by CONACyT grant nos. 1314P-N9507 and G28272-N.