Tubular sprouting as a mode of vascular formation in a colonial ascidian (Tunicata).

Although phylogenetically related to vertebrates, invertebrate chordate tunicates possess an open circulatory system, with blood flowing in lacunae among organs. However, the colonial circulatory system (CCS) of the ascidian Botryllus schlosseri runs in the common tunic and forms an anastomized network of vessels, defined by simple epithelium, connected to the open circulatory system of the zooids. The CCS originates from epidermal evagination, grows, and increases its network accompanying colony propagation. New vessels are formed by means of mechanisms of tubular sprouting which, in their morphogenesis and molecular regulation, are very similar to those occurring in other metazoans, particularly during vertebrate angiogenesis. From the apex of new vessels, epithelial cells detach and migrate into the tunic, while exploring filopodia extend toward the tunic and possibly guide vessel growth. Immunohistology showed that growth factors fibroblast growth factor-2 and vascular endothelial growth factor and the receptor vascular endothelial growth factor receptor-1 participate in sprouting, associated with cell proliferation. As in vertebrates, these factors may regulate cell migration, proliferation, sprouting, and tube formation. Our data indicate that similar, conserved signals were co-opted in the sprouting processes of two nonhomologous circulatory systems, that of ascidian CCS, and vertebrate circulatory systems, by recruitment of the same signaling pathway.     

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.     

Phagocytosis associated chemiluminescence of hemocytes in Mytilus edulis (Bivalvia)

The respiratory burst associated with phagocytosis by Mytilus edulis hemocytes was investigated by measurement of luminol-dependent chemiluminescence (LDCL). After experimental parameters (number of cells, quantity of stimulus) were determined, the biochemical mechanisms involved in the chemiluminescent process were investigated using inhibitors of oxygen radicals and enzymes. In particular, catechol-like phenols suggested the involvement of NADPH-oxidase and peroxidase in oxidative metabolism of mussel hemocytes. The variability of LDCL response observed among individuals and separated hemocyte subpopulations strongly suggests a variable immunocapacity depending on hemogram composition. Using a specific monoclonal antibody to discriminate different hemocyte types, the eosinophilic granulocytes appeared to exhibit the highest LDCL activity.     

Chimeras and histocompatibility in the colonial ascidian Botryllus schlosseri

Chimeras of B. schlosseri were prepared by pairwise combination of colonies sharing one allele at the fusibility gene locus (AD = AC chimeras). A frequent resorption of one of the partners was observed and the resorption time was shown to be significantly correlated with the relative size, the resorbing partner being usually the larger. Both in the whole chimeras, AD = AC, and in the separated partners, (AD)AC, the fusibility of AC was frequently altered. In AD = AC the fusion did not prevent entirely AC fusion with BC. The fusion frequency with BC was significantly higher than with BD and significantly higher in (AD)AC than in AD = AC. Repeated rejections with BC or both BC and BD, repeated fusions with BD, simultaneous or successive fusions and rejections especially with BD, over a period of several months, indicated a long lasting competitive interaction of AD and AC in (AD)AC chimeras. The persistence in these chimeras of the AD cell population was also confirmed by the chimeric electrophoretic pattern of PGI in most of them.     

Continuous development precludes radioprotection in a colonial ascidian

Colonial organisms provide a unique experimental system for stem cell biology. The colonial Urochordate Botryllus schlosseri reproduces sexually as well as by continuous asexual budding. Adjacent colonies with a shared histocompatibility allele undergo vascular fusion and establish a common blood circulation, performing natural transplantation. Fused colonies become chimeras, often with complete somatic replacement of the host cell genotype by the fused parabiont. We attempted to establish a radioprotection assay for the somatic stem cells that induce long-term chimerism in Botryllus. We demonstrate over a range of radiation doses that neither autologous nor allogeneic cell transplantation enhances survival of host colonies. This suggests that high mitotic index associated with continuous asexual development leads to radiosensitivity of organs and structures essential to survival during engraftment. We observe that radiation induces uncontrolled epithelial cell proliferation in abnormally terminated buds, suggesting that stem cells are not required for the initial stages of bud development.     

Phagocytosis mediates specificity in the immune defence of an invertebrate, the woodlouse Porcellio scaber (Crustacea: Isopoda)

Specificity and memory are the hallmarks of the adaptive immune system of vertebrates. However, phenomena of specificity upon priming of immunity have recently been demonstrated also in invertebrates, which rely exclusively on innate immune defence. It has been suggested that phagocytosis might represent a core candidate for such specificity in invertebrates. We here developed in vitro phagocytosis measurements for different bacteria in the woodlouse Porcellio scaber (Crustacea: Isopoda). After immune priming with heat-killed bacteria, hemocytes showed increased phagocytosis of a previously encountered bacterial strain compared to other bacteria. These data support the role of phagocytosis in invertebrate immunological specificity and suggest a high degree of specificity that even enables to differentiate between strains of the same bacterial species.     

Balancing selection on allorecognition genes in the colonial ascidian Botryllus schlosseri

Allorecognition is the capability of an organism to recognize its own or related tissues. The colonial ascidian Botryllus schlosseri, which comprises five genetically distinct and divergent species (Clades A-E), contains two adjacent genes that control allorecognition: fuhc^sec and fuhc^tm. These genes have been characterized extensively in Clade A and are highly polymorphic. Using alleles from 10 populations across the range of Clade A, we investigated the type and strength of selection maintaining this variation. Both fuhc genes exhibit higher within-population variation and lower population differentiation measures (F_ST) than neutral loci. The fuhc genes contain a substantial number of codons with >95% posterior probability of d_N/d_S > 1. fuhc^sec and fuhc^tm also have polymorphisms shared between Clade A and Clade E that were present prior to speciation (trans-species polymorphisms). These results provide robust evidence that the fuhc genes are evolving under balancing selection.     

Phagocytosis of mast cell granules by fibroblasts of the human gingiva

Summary Mast cell granules have been detected ultrastructurally within the cytoplasm of fibroblasts in fibrous hyperplastic lesion of the human gingiva. This finding is interpreted as phagocytosis of mast cell granules by fibroblasts. It is estimated that phagocytosis of mast cell granules occurred in four to six per cent of the fibroblasts. The result of present study suggests that mast cells play some role in fibroblast activity not only in animals as reported previously, but also in human connective tissue.     

Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri

The present paper describes the immune role played by a recently identified (Gasparini et al. 2008) member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri.B. schlosseri RBL (BsRBL) can activate phagocytes through: (i) induction of their directional movement towards the source of the molecule; (ii) modification of cytoskeleton, required for shape changes; (iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and (iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognised by anti-IL1α and anti-TNFα antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three-dimensional model for BsRBL is presented.     

Light Microscopic and Ultrastructural Evidence of in Vivo Phagocytosis of Helicobacter pylori by Neutrophils

Helicobacter pylori is believed to cause chronic active gastritis. Infection/colonization of the gastric mucosal surface induces a mucosal inflammatory reaction in the form of lymphocytic aggregates, plasma cells and, particularly, neutrophils, which may, in turn, damage the mucosal epithelium. In vitro studies demonstrate that, in culture, the bacilli are readily phagocytosed by neutrophils, this evoking a neutrophilic oxidative burst. However, it has been claimed that neutrophils do not phagocytose H. pylori in vivo. In this study of 19 endoscopic biopsies of gastric mucosa with H. pylori -associated gastritis, Cresyl violet staining for light microscopy and electron microscopy areusedto demonstrate that, in vivo, neutrophils actively phagocytose and destroy the bacilli in the epithelial intercellular space and in the mucin on the surface of the mucosa. Direct contact of neutrophils with H. pylori was observed in 17 of 17 cases by light microscopy and in 4 of 15 cases by electron microscopy. Phagocytosis by neutrophils was seen in 14 of 17 cases by light microscopy and in 3 of 15 cases by electron microscopy. It was most evident in the surface mucus coat where ``wolf packs'' of neutrophils were seen attacking the microbes. Ultrastructurally, neutrophil phagolysosomes contained both intact and partially digested bacteria,convincing evidence that the primary function of neutrophils in chronic active gastritis is to destroy H. pylori organisms. This study leaves open the question of whether, or how, neutrophils damage the gastric mucosa.     

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes

Phagocytosis of monosaccharide-binding latex particles by guinea-pig polymorphonuclear leucocytes (PMNs) was investigated. When neuraminic acid or glucuronic acid was chemically bound to the surface of latex particles the degree of phagocytosis was somewhat lower than that for bare, glucose-, mannose- or rhamnose-binding latex particles. These experimental findings were interpreted in terms of the kind of monosaccharide and the surface potential and surface hydrophobicity of latex particles. As a result, it was concluded that the inhibitory action of neuraminic acid or glucuronic acid on phagocytosis arises from an increase in the surface hydrophilicity of latex particles brought about by the hydroxyl groups and negative charges on the chemically bound neuraminic acid or glucuronic acid molecules. Physically adsorbed glucuronic acid molecules were also found to decrease the degree of phagocytosis of latex particles by PMNs, though not so remarkable as the chemically bound monosaccharide molecules.     

Phagocytosis of Leishmania donovani amastigotes is Rac1 dependent and occurs in the absence of NADPH oxidase activation

Macrophages produce little superoxide during phagocytosis of Leishmania donovani amastigotes. In this study, we characterized molecular events associated with L. donovani amastigotes uptake by mouse macrophages, to further define the mechanisms by which they are internalized without triggering superoxide production. Using transient transfections, we first showed that internalization of L. donovani amastigotes is mediated by the GTPases Rac1 and Arf6, of which Rac1 is recruited and retained on parasite-containing phagosomes. Next, we showed that, whereas internalization of amastigotes induced no superoxide release, co-internalization of serum-opsonized zymozan and amastigotes resulted in superoxide production. Furthermore, in co-internalization experiments, we detected superoxide production in over 95% of phagosomes containing IgG-opsonized SRBC compared to 5% of amastigote-harboring phagosomes. These results suggest that amastigotes evade the ability of macrophages to produce superoxide during phagocytosis. Consistently, we observed that amastigotes induced barely detectable phosphorylation of the NADPH oxidase component p47phox, leading to a defective phagosomal recruitment of p67phox and p47phox. Finally, we showed that amastigotes disrupt phagosomal lipid raft integrity, potentially interfering with NADPH oxidase assembly. Collectively, our results indicate that internalization of L. donovani amastigotes is a Rac1- and Arf6-dependent process that occurs in the absence of significant NADPH oxidase activation.     

Phagocytosis of Mycoplasma pneumoniae and Acholeplasma laidlawii measured as inhibition of [3H]uridine uptake by macrophages

Many studies of the interaction between phagocytes and mycoplasmas have given controversial results. This is probably due both to the small size of the microorganisms and their ability to attach to the cell membrane, making it difficult to distinguish between adsorption and ingestion. To overcome these difficulties we took advantage of a phenomenon we noted occuring concomitantly with phase-contrast microscope-monitored phagocytosis of heat-killed C. albicans, i.e., a reduction of [³H]uridine uptake by macrophages from culture medium. This approach allowed us to measure the ability of mouse peritoneal macrophages and the macrophage-like P 388 D 1 continuouscell line to phagocytose Mycoplasma pneumoniae and Acholeplasma laidlawii. Live, UV-killed and specific antiserum-opsonized mycoplasmas were tested. A. laidlawii was ingested under all the conditions mentioned above, while live M. pneumoniae was not phagocytosed unless UV-killed. Phagocytosis of UV-killed M. pneumoniae was directly verified by transmission electron microscopy studies. Data obtained with opsonized M. pneumoniae indicated no ingestion by mouse peritoneal macrophages and incomplete phagocytosis with P388 D 1 macrophages, suggesting that different responses by different types of phagocytes can be observed. In spite of a lack of information concerning the biological meaning of the inhibition of macrophage RNA metabolism during phagocytosis, our data suggest that this phenomenon may be used to study the phagocytosis of microorganisms which are difficult to visualize.     

用血红蛋白酶释放法检测巨噬细胞的吞噬能力

以鸡红细胞作为靶细胞,小鼠腹腔巨噬细胞为效应细胞研究了巨噬细胞的吞噬能力。经低渗处理后,血红蛋白酶释放的多少以它氧化邻苯二胺所产生的颜色反应表示吞噬百分比。对红细胞数与OD值关系、不同粘附时间和不同吞噬时间与吞噬能力的关系进行了研究。方法灵敏、可靠、客观,有推广应用的价值。     

Phagocytosis of opsonised fluorescent platelets by neutrophils to detect antiplatelet antibody

A platelet immunofluorophagocytosis method was developed to detect the presence of antiplatelet antibody. The method combines the advantages of immunofluorescence of antibody-coated platelets, the ability of neutrophils to phagocytose opsonised platelets, and the property of methylene blue to quench FITC-generated fluorescence of extracellular particles. Paraformaldehyde-fixed equine platelets in suspension were treated with rabbit antiequine platelet serum and FITC-conjugated goat antirabbit IgG. Viable equine neutrophils were incubated with these platelets at 37°C for 15–30 minutes. Phagocytosis was evaluated by fluorescence microscopy immediately after addition of 1% methylene blue to neutrophil platelet suspensions. Extracellular and free platelets were rendered nonfluorescent by methylene blue, whereas intracellular platelets remained unaffected and appeared highly fluorescent depending on the antiserum dilution. Phagocytosis was significantly (P <0.05) increased in the presence of complement. Addition of divalent cations (Ca²⁺ and Mg²⁺) did not increase phagocytosis, whereas EDTA significantly (P <0.05) reduced phagocytosis irrespective of the presence of complement. Sensitivity of the method can be increased by quantifying phagocytosis using flow cytometry.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

自制复方中药汤剂灌胃对小鼠淋巴细胞转化率和巨噬细胞吞噬功能的影响

目的:观察自制复方中药汤剂对实验小鼠淋巴细胞转化率和巨噬细胞吞噬功能的影响。方法:取昆明小鼠40只,分为对照组、复方中药低剂量组(2.5mg/kg/天)、中剂量组(2.5mg/kg/天)和高剂量组(50mg/kg/天),连续灌胃给药15天后,采尾静脉血瑞氏染色观测淋巴细胞转化率,取腹腔液瑞氏染色观测巨噬细胞吞噬功能。结果:三种剂量复方中药均可明显提高淋巴细胞的转化率(与对照组比较,P<0.01)。各组巨噬细胞吞噬功能与对照组比较显著增强(P<0.01),且中剂量组明显高于低、高剂量组(P<0.05)。结论:复方中药汤剂灌胃对小鼠免疫功能有明显的调节和增强作用。     

Modulation of phagolysosome biogenesis by the lipophosphoglycan of Leishmania

Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.     

Morphological Evidence of Neutrophil-Tumor Cell Phagocytosis (Cannibalism) in Human Gastric Adenocarcinomas

The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis has been documented by light microscopy in various human carcinomas, but little is known about the cellular pathological processes and the morphological changes involved. In an attempt to clarify the nature of this phenomenon, the authors' ultrastructural studies on the relationships among neutrophils and tumor cells in human gastric carcinomas are reviewed and analyzed. At the electron microscopy level, apoptotic neutrophils were found within vacuoles of adenocarcinoma cells in 2 cases. They showed either early apoptotic morphology with perinuclear chromatin aggregation but cytoplasm integrity or late apoptotic morphology with uniform, collapsed nucleus and tightly packed cytoplasmic granules. A light microscopy review of 200 cases of resected gastric carcinomas identified 22 cases (11%) that were characterized by neutrophil-tumor cell phagocytosis (cannibalism). TUNEL staining confirmed the presence of apoptotic neutrophils within the cytoplasm of the tumor cells. This study provides light and electron microscopic evidence of apoptotic neutrophils phagocytosed by gastric adenocarcinoma cells. The morphological features of neutrophil-tumor cell phagocytosis (cannibalism) would suggest a particular mechanism of tumor-immune escape in human gastric carcinoma.