TLR- and NOD2-dependent regulation of human phagocyte-specific chitotriosidase

Human chitotriosidase is specifically expressed by phagocytes, has anti-fungal activity towards chitin-containing fungi in vitro and in vivo, and is part of innate immunity. We studied the effect of toll-like receptor (TLR)- and nucleotide-binding oligomerization domain (NOD)-2 triggering on chitotriosidase expression and release by phagocytes. We find that TLR, but not NOD2 activation, regulates chitotriosidase release by neutrophils. Furthermore, both TLR and NOD2 activation resulted in diminished induction by monocytes. Lastly, NOD2 activation, but not TLR stimulation, induces chitinase expression in macrophages. We conclude that phagocyte-specific regulation is important for efficient eradication of chitin-containing pathogens.     

Marked differences in tissue-specific expression of chitinases in mouse and man.

Two distinct chitinases have been identified in mammals: a phagocyte-specific enzyme named chitotriosidase and an acidic mammalian chitinase (AMCase) expressed in the lungs and gastrointestinal tract. Increased expression of both chitinases has been observed in different pathological conditions: chitotriosidase in lysosomal lipid storage disorders like Gaucher disease and AMCase in asthmatic lung disease. Recently, it was reported that AMCase activity is involved in the pathogenesis of asthma in an induced mouse model. Inhibition of chitinase activity was found to alleviate the inflammation-driven pathology. We studied the tissue-specific expression of both chitinases in mice and compared it to the situation in man. In both species AMCase is expressed in alveolar macrophages and in the gastrointestinal tract. In mice, chitotriosidase is expressed only in the gastrointestinal tract, the tongue, fore-stomach, and Paneth cells in the small intestine, whereas in man the enzyme is expressed exclusively by professional phagocytes. This species difference seems to be mediated by distinct promoter usage. In conclusion, the pattern of expression of chitinases in the lung differs between mouse and man. The implications for the development of anti-asthma drugs with chitinases as targets are discussed.     

A novel serum chitinase that is expressed in bovine liver

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.     

Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β (pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 μg/mL), and/or nigericin (Nig, 30 μmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies. RESULTS: We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1β, but not the processing of pro-IL-1β to IL-1β, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1β, but not the processing of IL-1β, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1β processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.     

Comparative functional analysis of the human macrophage chitotriosidase

This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the β-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.     

Human chitotriosidase is expressed in the eye and lacrimal gland and has an antimicrobial spectrum different from lysozyme

Chitotriosidase is a chitinolytic enzyme expressed by maturing macrophages and preformed in neutrophil granules, suggesting a role in antimicrobial defence. Although available evidence supports a role in anti-fungal immunity, there is a lack of an obvious phenotype in humans homozygous for a mutation which renders chitotriosidase inactive. This may be explained by compensatory effects of enzymes co-expressed with chitotriosidase, such as lysozyme. We have found that chitinase is highly expressed in mouse and human eye, particularly in lacrimal glands. Chitotriosidase is the only member of the chitinase/chilectin gene cluster expressed in the murine eye. As lacrimal glands also produce lysozyme, we have asked whether chitotriosidase, in addition to its documented anti-fungal effects, has synergistic anti-bacterial properties with lysozyme. The effect of recombinant chitotriosidase on the growth of five Gram-positive (Bacillus cereus, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Staphylococcus aureus OatA(+/-)) and two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa), were tested in a luminometric assay. Recombinant chitotriosidase did not inhibit bacterial growth and did not synergize with lysozyme. Though the expression of chitotriosidase in the eye supports a role in innate immunity, the antimicrobial spectrum appears to be complementary to lysozyme and may indeed be limited to fungi.     

Kinin generation from exogenous kininogens at the surface of retinoic acid-differentiated human neuroblastoma IMR-32 cells after stimulation with interferon-γ

Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.     

Human chitotriosidase CHIT1 cross reacts with mammalian-like substrates

Humans do not synthesize chitin, yet they produce a number of active and inactive chitinases. One of the active enzymes is chitotriosidase whose serum levels are elevated in a number of diseases such as Gaucher’s disease and upon fungal infection. Since the biological role of chitotriosidase in disease pathogenesis is not understood we screened a panel of mammalian GlcNAc-containing glycoconjugates as alternate substrates. LacNAc and LacdiNAc-terminating substrates are hydrolyzed, the latter with a turnover comparable to that of pNP-chitotriose. Glycolipids or glycoproteins with LacNAc and LacdiNAc represent potential chitinase substrates and the subsequent alteration of glycosylation pattern could be a factor in disease pathogenesis.     

Cellular expression of gut chitinase mRNA in the gastrointestinal tract of mice and chickens.

Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.     

Regulation of steroid hormone biosynthesis enzymes and organic anion transporters by forskolin and DHEA-S treatment in adrenocortical cells

Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (StAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3.     

Cell-free activation of phagocyte NADPH-oxidase: tissue and differentiation-specific expression of cytosolic cofactor activity

We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Nitric oxide activates the expression of IRAK-M via the release of TNF-α in human monocytes

The activation of interleukin receptor associated kinases (IRAK) is an important event in several inflammatory processes. However, exposing monocytes to a nitric oxide (NO) donor inhibits the activity of IRAK-1 and its molecular interaction with TNF receptor associated factor-6 (TRAF6). Despite the fact that NO is known to regulate many events in the immune and vascular system, the mechanism that underlies this inhibition remains unknown. We have recently demonstrated that IRAK-M inhibits the TLR/IRAK pathway during endotoxin tolerance and thus, we hypothesized that IRAK-M may be involved in the inhibition of IRAK-1 activity in the presence of NO. Hence, we have analyzed the expression of IRAK-M in human monocytes following exposure to a NO donor (GSNO) and we have observed that GSNO was capable of inducing IRAK-M mRNA and protein expression 8 and 20 h after stimulation, respectively. It is known that NO induces the expression of TNF-alpha in monocytes and we found that exposure to TNF-alpha induced IRAK-M mRNA expression in human monocytes within 2 h of stimulation. Furthermore, the expression of IRAK-M induced by GSNO was inhibited by the presence of a blocking antibody raised against TNF-alpha. Thus, our data indicate that stimulation of human monocytes with a NO donor results in a clear induction of IRAK-M and this is dependent on the release of TNF-alpha by this kind of cells.