The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.     

An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway

The protease-activated receptor 2, expressed on keratinocytes but not on melanocytes, has been ascribed functional importance in the regulation of pigmentation by phagocytosis of melanosomes. Inhibition of protease-activated receptor 2 activation by synthetic serine protease inhibitors requires keratinocyte-melanocyte contact and results in depigmentation of the dark skinned Yucatan swine, suggesting a new class of depigmenting mechanism and agents. We therefore examined natural agents that could exert their effect via the protease-activated receptor 2 pathway. Here we show that soymilk and the soybean-derived serine protease inhibitors soybean trypsin inhibitor and Bowman-Birk inhibitor inhibit protease-activated receptor 2 cleavage, affect cytoskeletal and cell surface organization, and reduce keratinocyte phagocytosis. The depigmenting activity of these agents and their capability to prevent ultraviolet-induced pigmentation are demonstrated both in vitro and in vivo. These results imply that inhibition of the protease-activated receptor 2 pathway by soymilk may be used as a natural alternative to skin lightening.     

A role for collagen phagocytosis by fibroblasts in scar remodeling: an ultrastructural stereologic study

A role for collagen phagocytosis and intracellular degradation by fibroblasts during remodeling activity has been suggested by studies on several connective tissues characterized by high rates of collagen turnover and remodeling. The possible importance of such activity in the normal remodeling of scar tissue has been studied by a quantitative ultrastructural stereologic measure of collagen phagocytosis by fibroblasts at various post-wounding intervals in mouse skin scars. The results demonstrate a correlation between the peak periods of such phagocytic activity and the interval during which collagen fiber reorientation across the scar appears to take place.     

Monitoring of phagocytic activity in histiocytic cytophagic panniculitis

Histiocytic cytophagic panniculitis presents with subcutaneous panniculitis. Histologically, it is characterized by phagocytosis of blood cells in the subcutaneous tissue and bone marrow. One patient with histiocytic cytophagic panniculitis is described in whom hemophagocytosis macrophages and histiocytes was observed histologically and was confirmed in vitro measuring phagocytosis by peripheral blood monocytes by means of chemiluminescence. In vitro measurements of phagocytosis corresponded well with the clinical course. Chemiluminescence for measuring phagocytosis in vitro may be suitable for analyzing disease activity and for testing therapeutic compounds in vitro.     

AIDS患者巨噬细胞对白念珠菌的吞噬和灭活功能研究

目的:明确AIDS患者巨噬细胞对白念珠菌的吞噬和杀灭活性功能。方法:提取AIDS患者和健康者的单核细胞,诱导分化为巨噬细胞。荧光显微镜和流式细胞仪测定巨噬细胞对白念珠菌的吞噬活性;CFU计数法分析巨噬细胞对白念珠菌的杀灭活性;测定巨噬细胞内亚硝酸盐含量,分析呼吸爆炸相关产物一氧化氮的生成;Western blot法测定巨噬细胞磷酸化酪氨酸激酶的表达。结果:AIDS患者的巨噬细胞对白念珠菌的吞噬、杀灭能力、一氧化氮及磷酸化Syk的蛋白均低于健康者。结论:AIDS患者巨噬细胞吞噬和杀灭白念珠菌活性降低。     

Effects of cepharanthin on neutrophil chemotaxis, phagocytosis, and reactive oxygen species generation.

The effects of cepharanthin on inflammatory parameters such as neutrophil chemotaxis, phagocytosis and reactive oxygen species (ROS) generation, were examined. Cepharanthin significantly decreased the levels of O2-, H2O2, and OH. generated by neutrophils. H2O2 and OH. generated in a cell-free, xanthine-xanthine oxidase system were also reduced in the presence of cepharanthin. However, the drug did not affect neutrophil chemotaxis or phagocytosis. The present study indicates that cepharanthin is an effective ROS scavenger, exerting its anti-inflammatory action by reducing the potent ROS species excessively generated in tissues and organs, especially at the sites of inflammation.     

The phagocytosis of Candida albicans blastospores and germ tubes by polymorphonuclear leukocytes.

This in vitro study investigates the phagocytosis and killing rate of Candida albicans blastospores and germ tubes by polymorphonuclear leukocytes. Suspensions of C. albicans blastospores and germ tubes were incubated for 60 and 150 min with a neutrophil suspension. The rate of phagocytosis was found to be 92 +/- 3% for blastospores, but only 9.5 +/- 2.5% for germ tubes. The candidacidal activity rate was 29 +/- 6% for blastospores, but only 5.5 +/- 1.5% for germ tubes. Electronmicroscopically, cytoplasmic and plasma membrane alterations of phagocytized yeast cells lying in phagosomes were observed. Short germ tubes surrounded by a phagosomal membrane were found in neutrophils. Older, longer germ tubes were seen in an extracellular position. Usually, several neutrophils were adjacent to these long tubes. Obviously phagocytosis could not take place owing to the size of the germ tubes. The findings indicate that the transition of the yeast phase to the mycelium phase in a case of Candida infection may be a mechanism enabling the parasite to escape phagocytosis by the host.     

Suppressive effects of linoleic acid on neutrophil oxygen metabolism and phagocytosis

On the basis of recent reports that the proportion of linoleic acid (C18:2Cis 9,12), a free fatty acid, is markedly decreased in acne comedones and that tetracycline is effective against acne comedones by acting directly as an antioxidant on infiltrating neutrophils, we investigated the effect of linoleic acid on several inflammatory parameters of neutrophils, including neutrophil chemotaxis, phagocytosis, and generation of reactive oxygen species (ROS). Linoleic acid significantly decreased phagocytosis and the generation of O2-, H2O2, and OH.by neutrophils, whereas it did not significantly inhibit neutrophil chemotaxis or decrease the ROS levels generated in a cell-free, xanthine-xanthine oxidase system. The present study seems to suggest that decreased levels of linoleic acid in acne comedones contribute, in part, to the worsening of acne inflammation by the failure of low levels of linoleic acid to suppress neutrophil phagocytosis and ROS generation.     

Effects of UVB treatment on neutrophil function in psoriatic patients and healthy subjects

To evaluate if whole body UVB irradiation has effects on the neutrophil function, eleven patients with mild to moderate psoriasis and 14 healthy subjects were treated with whole body UVB irradiation 3 times weekly for 4 weeks. Eight healthy untreated subjects served as controls. After 2 weeks of treatment the individual change of phagocytosis measured by the ingestion of IgG-coated particles was related to the pre-treatment value both in the psoriatic patients and healthy subjects (p less than 0.05 and p less than 0.001, respectively). Similar results were obtained for ingestion of IgG-C3b-coated particles. Thus, the change in phagocytic rate seemed to be dependent on the functional activity before treatment. UV irradiation of PMNs in vitro did not influence the phagocytic rate. After 4 weeks of UV treatment the healthy subjects showed a significant decrease in the rate of phagocytosis of IgG-C3b particles (p less than 0.02) and of the serum chemokinetic activity (p less than 0.01). In the psoriatic patients the mean chemokinetic activity in heated sera was decreased after 2 weeks (p less than 0.02). There appeared to be no relation between improvement of psoriasis and changes in PMN function. In an untreated group of healthy subjects no significant changes in neutrophil function were found. The results indicate that there is a change in PMN function during UVB treatment. The degree of change seems to vary, not only between individuals but possibly also between groups, e.g. healthy subjects compared with psoriatic patients.     

Effect of palmitic acid on neutrophil functions in vitro

BACKGROUND: It has been shown that in acne comedones the proportion of linoleic acid is markedly decreased, while palmitic acid is significantly increased. We previously reported that the decreased proportion of linoleic acid, which markedly suppresses neutrophil reactive oxygen species (ROS) generation and phagocytosis, contribute to the worsening of acne inflammation. The aim of this study was to examine the effect of palmitic acid on neutrophil functions in vitro. METHODS: We investigated the effect of palmitic acid on inflammatory parameters such as neutrophil chemotaxis, phagocytosis, and ROS generation. Reactive oxygen species generation in a cell-free, xanthine-xanthine oxidase system was also assessed. The species examined were superoxide radical anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH.). RESULTS: Palmitic acid significantly decreased H2O2 generation both by neutrophils and in the xanthine-xanthine oxidase system, while neutrophil chemotaxis and phagocytosis as well as O2- and OH. generation by both systems were not markedly affected in the presence of palmitic acid. CONCLUSIONS: The present study suggests that palmitic acid may be involved in the pathogenesis of acne inflammation from a standpoint of oxidative tissue injury.     

Increased in vivo secretory activity of neutrophil granulocytes in patients with psoriasis and palmoplantar pustulosis

Summary The relationship between psoriasis and palmoplantar pustulosis (PPP) is uncertain, as is the role of the neutrophil granulocyte in these conditions. In a previous comparative study of the rate of polymorphonuclear leucocyte (PMN) phagocytosis of IGG- and IgG-C3b-coated particles, an increased uptake rate was found in both diseases. Further information on the in vivo activity of PMNs in these conditions may be obtainable by determining the level of lactoferrin (LF) in serum from such patients, since LF serves as a specific marker of the turnover and activity of the circulating pool of neutrophils. In this study on 19 patients with psoriasis and 20 patients with PPP, elevated levels of LF were found in both conditions. In contrast, the levels of lysozyme and ₂-microglobulin, which are markers of monocyte-macrophage and lymphocyte activity, respectively, were normal. This suggests the selective activation of neutrophils in these disorders. LF was significantly correlated (P<0.05 and 0.001, respectively) to the rates of phagocytosis of IgG- and IgG-C3b-coated particles, but not to the chemotaxis of isolated PMNs. There was no correlation between the severity of the disease and the levels of serum LF. The data suggest the increased in vivo activity of neutrophils in psoriasis and PPP.     

Membrane Activity in Histiocytic Diseases of the Skin

Study of the histiocytes from a large variety of skin diseases demonstrates a close relationship between the cell processes peculiar to those cells and their degree of phagocytic activity. The membrane structures, as well as the dense bodies localized in the peripheral cytoplasm, suggest a type of endocytosis distinct from pinocytosis and phagocytosis.     

Granulocyte function in the aged

22 patients (10 female, 12 male) aged between 60 and 76 were tested in vitro with regard to granulocyte function (i.e., leucotaxis, chemotaxis, capability of phagocytosis, intracellular killing of bacteria, and oxidase reaction). Most of the patients showed reduced chemotactic activity of the granulocytes, although the motility of these cells was normal. With regular phagocytosis of candida albicans cells--either active or heat-inactivated the intracellular killing rate of bacteria was clearly decreased in all cases. The intracellular oxidase activity of granulocytes always showed normal function.     

Deficient phagocytic function in Papillon-Lefèvre syndrome.

The function of microphages has been studied in vitro in a 5 year-old girl with Papillon-Lefèvre syndrome. The results showed a weakness in chemotatic activity, a defect of intracellular killing of Staphylococcus aureus, and an almost normal phagocytosis but decreased intracellular killing of Candida albicans by the microphages.     

Annular elastolytic giant cell granuloma: an unusual case with lesions arising in non-sun-exposed areas

A 70-year-old man with a 2-year history of annular elastolytic giant cell granuloma associated with diabetes mellitus was reported. The lesions mainly developed in non-sun-exposed areas. Histologic examination revealed phagocytosis of elastic fibers by histiocytic cells. Immunoperoxidase staining for lysozyme disclosed positive reactivity within the cytoplasm of these histiocytic cells. Electron microscopic study also showed elastic fibers and numerous lipid-like substances in the cytoplasm of these cells. These findings indicate high phagocytolytic activity by these infiltrating cells. In our case, actinic damage was not considered to be a primary causative factor, and a possible pathogenesis was also discussed.     

Inhibitory effect of azelaic acid on neutrophil functions: a possible cause for its efficacy in treating pathogenetically unrelated diseases

Summary It has been shown that acne, hyperpigmentation and lentigo malignant are more or less related pathogenetically to reactive oxygen species (ROS). It has recently been reported that azelaic acid is effective in treating these conditions and that it possesses anti-enzymatic and anti-mitochondrial activity, including cytochrome-P₄₅₀ reductase and 5-reductase in microsomal preparations with nicotinamide adenine dinucleotide phosphate (NADPH). We therefore investigated the effects of azelaic acid on human neutrophil functions, such as chemotaxis, phagocytosis and ROS generation. ROS generation in a cell-free system was also assessed. The results revealed that neutrophil chemotaxis and phagocytosis as well as ROS generated in a xanthine — xanthine-oxidase system were not significantly changed in the presence of azelaic acid. However, azelaic acid markedly decreased O ₂ ^– and OH generated by neutrophils. It may be concluded that the reported clinical effectiveness of azelaic acid is partly due to its inhibitory action on neutrophil-generated ROS, leading to a reduction both in oxidative tissue injury at sites of inflammation and in melanin formation.     

Toll样受体与抗白念珠菌感染

Toll样受体蛋白家族属于动物模式识别受体家族,对天然免疫和获得性免疫都有调控作用.与抗白念珠菌感染免疫有关的主要是Toll样受体2和4.Toll样受体2和4可以分别识别病原体,也可以与其他的Toll样受体以二聚体的形式参与识别,或者与其他模式识别受体,如Dectin-1、甘露糖受体等共同识别.免疫细胞表面Toll样受体识别白念珠菌后主要通过MyD88依赖的信号传递途径.激活白细胞的趋化作用,增强吞噬细胞的吞噬、杀伤功能,促进相关细胞因子的释放,从而起到抗白念珠菌的作用.     

Disorders of phagocytic and fungicide function of granulocytes in chronic muco-cutaneous candidiasis]

The granulocytes which are distributed by blood circulation close to the extern and intern body surfaces as well as in all organs protect the organism from microbial perils by phagocytosis and intracellular killing of bacteria and fungi so constituting a very important component of the granulocytic functions seriously impairs the host resistance and may entail a state of persisting infectious disease. As chronic mucocutaneous candidosis (CMCC) represents such a persistent generalized infection, we studied in vitro several functional activities of the granulocytes of 5 patients suffering from CMCC. 2 of them presented a non-familiar type of CMCC, the remaining 3 patients (father and 2 daughters) were subject to the hereditary type of CMCC. For comparison, by the same way we investigated the granulocytic functions of 51 clinically and immunologically healthy adult persons. Each of our 5 patients exhibited a reduced ability of the granulocytes to phagocytize and kill Candida albicans. In the CMCC family, the father had a marked deficiency of the oxidase activity of the granulocytes whereas in his daughters, the oxidase deficiency proved to be of a minor grade. In the sera of both daughters a phagocytosis inhibiting factor could be assumed to exist in addition to the granulocytic abnormalities. When heat-inactivated Candida albicans cells, however, were used for experiments, the granulocytes of each patient were able to phagocytize the germs at the same rate as did the granulocytes taken from the controls. With regard to alterations of the T cell function previously reported in CMCC, in all patients we also could demonstrate various symptoms of a T cell-dependent immunodeficiency. The results of the present in vitro-experiments furnish good evidence of a state of fundamental deficiency of the microphages in patients with CMCC, which may be the dominating cellular factor in the aetiopathogenesis of CMCC.     

Release of enzymes from human leucocytes during incubation with Neisseria gonorrhoeae

The effect of Neisseria gonorrhoeae on release of enzymes from human leucocytes was determined. Supernatants from incubation mixtures containing leucocytes and gonococci were assayed for activity of the cytoplasmic enzyme, lactic acid dehydrogenase, as well as for activity of the hydrolytic enzymes, β-glucuronidase and lysozyme, which are found primarily in leucocyte granules. Thirty-minute incubation of leucocytes with pilated T1 gonococci resulted in a negligible release of lactic acid dehydrogenase and little release of β-glucuronidase even at bacteria to leucocyte ratios as high as 50 to 1. Lysozyme release, however, was significant at this ratio and at 20 to 1 but not at 5 to 1. Incubation with non-pilated T4 bacteria yielded no significant release of lactic acid dehydrogenase or β-glucuronidase, but it caused a significant release of lysozyme at bacteria to leucocyte ratios as low as 2 to 1. These results suggested that the lysozyme release might be related to the degree of phagocytic activity since, at low ratios, T4 was readily ingested but T1 was not. Consistent with this hypothesis, serum which promoted the phagocytosis of the pilated gonococci also stimulated lysozyme release at low ratios of T1 to leucocyte. Absorption of the serum with T1 abolished the opsonic effect and markedly diminished the amount of lysozyme released.     

Activity of the phagocytic process in patients with atopic dermatitis

The phagocytic activity was determined in 18 patients with atopic dermatitis who were treated in the Dermatology Department, Medical Academy in Wroc?aw. Reduced phagocytic activity was found in 12 patients. In 3 cases this was caused by a defect in phagocytic cells, in the remaining 9 cases this was due to disturbances of the opsonizing properties of the serum. The serum of these patients caused mainly a fall of the bactericidal activity of the leucocytes, and this was caused more frequently by a deficit of the opsonizing factors than by the presence of inhibitor. In the sera attenuating the engulfing and intracellular killing of bacteria presence of immune complexes was demonstrated. The disappearance of immune complexes during treatment restored normal function of the serum during the process of phagocytosis.