Hepatocyte growth factor stimulates neutrophil degranulation but not respiratory burst

Neutrophil function is regulated in part by cytokines with growth factor activities for different cell types. Hepatocyte growth factor (HGF) is a cytokine produced during injury to the liver and other organs. Neutrophils are numerous in such tissue injury sites and may be influenced by HGF. In the present study the effect of HGF on neutrophils was investigated. The data show that HGF at 1-10 ng/ml increased lysosomal enzyme release from both specific and azurophilic granules of cytochalasin-B treated neutrophils. The release of specific granule contents in response to N-formyl-methionyl-leucylphenylalanine was also increased by HGF. In contrast there were no significant effects of HGF on neutrophil respiratory burst, adherence or locomotion. It is concluded that HGF modulates neutrophil granule exocytosis.     

Priming of the neutrophil respiratory burst involves p38 mitogen-activated protein kinase-dependent exocytosis of flavocytochrome b558-containing granules

The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b(558), a component of the NADPH oxidase. This study shows that TNFalpha also increases membrane expression of flavocytochrome b(558). Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b(558) are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNFalpha and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b(558), with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNFalpha failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNFalpha and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b(558) through exocytosis of intracellular granules in a process regulated by p38 MAPK.     

Respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza A virus

Oxidants and neutrophils contribute to lung injury during influenza A virus (IAV) infection. Surfactant protein (SP)-D plays a pivotal role in restricting IAV replication and inflammation in the first several days after infection. Despite its potent anti-inflammatory effects in vivo, preincubation of IAV with SP-D in vitro strongly increases neutrophil respiratory burst responses to the virus. Several factors are shown to modify this apparent proinflammatory effect of SP-D. Although multimeric forms of SP-D show dose-dependent augmentation of respiratory burst responses, trimeric, single-arm forms either show no effect or inhibit these responses. Furthermore, if neutrophils are preincubated with multimeric SP-D before IAV is added, oxidant responses to the virus are significantly reduced. The ability of SP-D to increase neutrophil uptake of IAV can be dissociated from enhancement of oxidant responses. Finally, several other innate immune proteins that bind to SP-D and/or IAV (i.e., SP-A, lung glycoprotein-340 or mucin) significantly reduce the ability of SP-D to promote neutrophil oxidant response. As a result, the net effect of bronchoalveolar lavage fluids is to increase neutrophil uptake of IAV while reducing the respiratory burst response to virus.     

Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.     

The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity

Ribosome inactivating proteins (RIPs) inhibit protein synthesis depurinating a conserved residue in the sarcin/ricin loop of ribosomes. Some RIPs are only active against eukaryotic ribosomes, but other RIPs inactivate with similar efficiency prokaryotic and eukaryotic ribosomes, suggesting that different RIPs would interact with different proteins. The SRL in Trypanosoma cruzi ribosomes is located on a 178b RNA molecule named 28Sδ. In addition, T. cruzi ribosomes are remarkably resistant to TCS. In spite of these peculiarities, we show that TCS specifically depurinate the predicted A⁵¹ residue on 28Sδ. We also demonstrated that the C-terminal end of ribosomal P proteins is needed for full activity of the toxin. In contrast to TCS, PAP inactivated efficiently T.cruzi ribosomes, and most importantly, does not require from the C-terminal end of P proteins. These results could explain, at least partially, the different selectivity of these toxins against prokaryotic and eukaryotic ribosomes.     

Activation of the human neutrophil respiratory burst with zymosan-activated serum

Zymosan-activated serum (ZAS) stimulated a time- and concentration-dependent generation of superoxide anion (O₂^?) by human neutrophils. O₂^? production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS. O₂^? generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS. The amount of O₂^? produced by ZAS stimulated neutrophils was enhanced in the presence of extracellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS-elicited O₂^? production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O₂^? in response to 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB₄), demonstrates the stimulus specific nature of ZAS-induced desensitization. Thus, ZAS, which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represénts a relevent probe for investigating neutrophil function.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I_e. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 μM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.     

Stroma cell-derived factor 1alpha mediates desensitization of human neutrophil respiratory burst in synovial fluid from rheumatoid arthritic patients

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1alpha (SDF1alpha) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1alpha in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1alpha failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1alpha-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1alpha actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1alpha beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1alpha promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.     

Drosophila big brain does not act as a water channel, but mediates cell adhesion

The neurogenic gene Drosophilabig brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.     

IL-7 receptor mediates tyrosine phosphorylation but does not activate the phosphatidylinositol-phospholipase C-gamma 1 pathway.

IL-7 is a glycoprotein involved in the regulation of lymphocyte precursor growth. In addition, it has a comitogenic effect on mature T cells but not on mature B cells. The exact mechanism whereby IL-7R mediates these cell growth properties remains unknown. Because many growth factor receptor systems on various cell types transduce signals by activating a tyrosine kinase, we have studied here the effect of IL-7R ligation on protein tyrosine phosphorylation. We found that human rIL-7 consistently induced tyrosine phosphorylation of five major proteins, of 175, 155, 135, 110, and 85 kDa, and five minor proteins. The effect of human rIL-7 on tyrosine phosphorylation of these substrates was concentration and time dependent. One of the known substrates that is phosphorylated on tyrosine residues after binding of growth factors to their receptors is the phosphoinositide-specific phospholipase C. Several phospholipase C isozymes have been recently recognized; one isozyme, phospholipase C-gamma 1, was demonstrated to be phosphorylated rapidly after ligand binding to the platelet-derived growth factor receptor and the T cell Ag receptor. We show here that, in contrast to Ag receptor ligation, activation of IL-7R does not induce tyrosine phosphorylation on phospholipase C-gamma 1. Consistent with these results, human rIL-7 failed to increase phosphatidylinositol turnover and did not induce a rise in cytosolic free Ca2+ in the thymocytes, mature T cells, or pre-pre-B cells. The results indicate that the IL-7R mediates the activation of the tyrosine phosphorylation pathway but does not induce the phosphatidylinositol-phospholipase C pathway.     

Orexin mediates initiation of sexual behavior in sexually naive male rats, but is not critical for sexual performance

The hypothalamic neuropeptide orexin mediates arousal, sleep, and naturally rewarding behaviors, including food intake. Male sexual behavior is altered by orexin receptor-1 agonists or antagonists, suggesting a role for orexin-A in this naturally rewarding behavior. However, the specific role of endogenous orexin-A or B in different elements of male sexual behavior is currently unclear. Therefore, the current studies utilized markers for neural activation and orexin cell-specific lesions to test the hypothesis that orexin is critical for sexual motivation and performance in male rats. First, cFos expression in orexin neurons was demonstrated following presentation of a receptive or non-receptive female without further activation by different elements of mating. Next, the functional role of orexin was tested utilizing orexin-B conjugated saporin, resulting in orexin cell body lesions in the hypothalamus. Lesions were conducted in sexually naive males and subsequent sexual behavior was recorded during four mating trials. Lesion males showed shortened latencies to mount and intromit during the first, but not subsequent mating trials, suggesting lesions facilitated initiation of sexual behavior in sexually naive, but not experienced males. Likewise, lesions did not affect sexual motivation in experienced males, determined by runway tests. Finally, elevated plus maze tests demonstrated reduced anxiety-like behaviors in lesioned males, supporting a role for orexin in anxiety associated with initial exposure to the female in naive animals. Overall, these findings show that orexin is not critical for male sexual performance or motivation, but may play a role in arousal and anxiety related to sexual behavior in naive animals.     

Helicobacter pylori does not mediate the formation of carcinogenic N-nitrosamines

Background. Both N‐nitroso compounds and colonization with Helicobacter pylori represent known risk‐factors for the development of gastric cancer. Endogenous formation of N‐nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N‐nitroso compounds. Based on experiments with a noncarcinogenic N‐nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N‐nitrosamines. Materials and Methods. Bacteria were grown in the presence of 0–1000 µM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3. Results. Incubation of Neisseria cinerea (positive control) with 500 µM morpholine and 500 µM nitrite, resulted in a significant increase in formation of N‐nitrosomorpholine, but there was no significant induction of N‐nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions. Conclusion. H. pylori does not induce formation of the carcinogenic N‐nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficultly nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.     

Regulation of the phagocyte respiratory burst by small GTP-binding proteins

Bacteria phagocytosed by leukocytes are killed and degraded by toxic oxygen metabolites produced in the phagosome via an NADPH oxidase. NADPH oxidase activity is regulated by small GTP-binding proteins in response to phagocytic stimuli. In this review, Gary Bokoch focuses on the role of Rac in regulating this important phagocytic process.