The inhibition of serum opsonins by a carbohydrate and the opsonizing effect of purified agglutinin on the clearance of nonself particles from the circulation of Helix pomatia

The serum of Helix pomatia agglutinates enzyme-treated erythrocytes and also possesses opsonizing properties. The agglutinating as well as the opsonizing activity could be inhibited by N-acetylglucosamine, indicating an identicalness of these serum components. As this observation supports the hypothesis that agglutinins may function as opsonins, purified agglutinins from the albumin gland of Helix pomatia, from the sponge Axinella polypoides, and Con A were utilized to sensitize foreign cells prior to their injection into the hemocoel of H. pomatia. Helix agglutinin revealed a strong opsonic effect on the elimination of the nonself particles from the circulation of the snail. It is assumed that serum opsonins of H. pomatia may couple certain nonself materials to the surface of cells in different clearance organs and that hemocytes possess membrane-associated agglutinins which mediate their attachment to trapped foreign particles.     

Identification of agglutinin receptors on hemocytes of Helix pomatia

The attachment of opsonized foreign particles to phagocytic cells indicates the occurrence of opsonin receptors on the surfaces of the phagocytes. There is good evidence that naturally occurring hemagglutinins may serve as opsonins in invertebrates. To prove the occurrence of agglutinin receptors on the hemocytes of an invertebrate, the interaction of various agglutinins with Helix pomatia hemocytes was investigated. A positive agglutination reaction was obtained with Ricinus, Axinella, anti-H_eel, Ulex, concanavalin A, and Limulus agglutinins. The known specificities of these agglutinins and the influence of carbohydrases on the agglutinability of Helix cells have led to the conclusion that the carbohydrate components of the binding sites include galactose, fucose, mannose or glucose or both, and N-acetylneuraminic acid or polygalactose.     

Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

The removal of human erythrocytes of the A₁ and B types from the circulation of the gastropod Helix pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A₁ and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A₁ erythrocytes injected 12–19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A₁ erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A₁ erythrocytes in the agglutinating extract of the sponge Aaptos papillata.     

A galactose specific agglutinin, a blood-group H active polysaccharide, and a trypsin inhibitor in albumin glands and eggs of Arianta arbustorum (Helicidae).

Eggs and albumin glands of the land snail Arianta arbustorum contain a powerful agglutinin which reacts specially with rabbit erythrocytes. The agglutination can be inhibited completely by di-, tri-, and oligosaccharides with α-glycosidically (1 → 6) bound galactose residues. β-Linked sugars do not inhibit the agglutinin. The agglutinin activity is not dependent on Ca²⁺ ions. Eggs and albumin glands also contain a blood-group active polysaccharide which, unlike the polysaccharide from the albumin gland of Helix pomatia (Baldo, B. A., and Uhlenbruck, G. 1973. Cross-reactive human blood group H-active polysaccharide from Helix pomatia. I. Detection with catfish anti-H and eel sera. Immunology, 25, 1–13) does not react with anti-H_eel, but does react with the agglutinins of Evonymus europaeus and Laburnum alpinum. The Arianta polysaccharide has been purified and shown to be galactogen. Finally, the occurrrence of a strong trypsin inhibitor has been demonstrated in the extracts of eggs and albumin glands. The inhibitor has been separated by column chromatography. The precipitation lines of both substances have been identified in the immunoelectrophoretogram of the extracts of albumin glands and eggs.     

Phagocytosis as a cellular immune response mechanism in the American lobster, Homarus americanus.

Procedures to quantitate accurately the in vitro phagocytosis of sheep erythrocytes and Aerococcus viridans var. homari (formerly Gaffkya homari) bacteria by hemocytes of the American lobster were utilized to assess the effect of various vaccines on the humoral and cellular defense mechanisms of the lobster. Both the percent of hemocytes showing phagocytosis and the number of particles phagocytosed increased markedly in many of the animals injected with Pseudomonas perolens cells or endotoxin. A lesser response was elicited by A. viridans cellular vaccines. In vivo phagocytosis was observed in the circulatory system of lobsters infected with A. viridans var. homari.Natural hemolymph agglutinins for sheep or rabbit erythrocytes were not affectd by any vaccines. Hemocyte numbers dropped initially after administration of P. perolens endotoxin or cells. The bactericidin level was enhanced in samples taken from vaccinated animals.     

Modified Hemagglutination-Inhibition Test for Rubella Employing Human Group O Erythrocytes

A hemagglutination-inhibition (HI) test for rubella is described which utilizes human group O, rather than 1-day-old chick, erythrocytes. The test was found to be as sensitive and reproducible for detection of rubella antibody as HI tests employing chick erythrocytes. Advantages to the use of human erythrocytes are (i) they are more available, (ii) it is unnecessary to absorb natural agglutinins from human test sera, and (iii) heparin-MnCl₂-treated sera do not agglutinate human erythrocytes, as is sometimes the case with chick erythrocytes. Factors influencing the reliability of the test are discussed.     

Erythrocyte Sensitization by Blood Group-Specific Bacterial Antigens

Human and chicken erythrocytes are readily coated in vitro by blood group active protein-lipopolysaccharides and lipopolysaccharides from E. coli O₈₆ and E. coli O₁₂₈. Serum albumin, α₂- and β-lipoproteins inhibit this sensitization. Blood group B specific agglutination of erythrocytes with B or B-like antigens was obtained with antibodies purified by adsorption on and elution from B erythrocytes. Anti-blood group B and E. coli O₈₆-specific antibodies could be eluted from E. coli O₈₆-coated O erythrocytes. Eel anti-H(O) serum agglutinated O erythrocytes and only those A₁B red cells which were coated with blood group H(O) active E. coli products. Blood group active substances specifically inhibited agglutination of lipopolysaccharide-coated erythrocytes by anti-B and anti-H(O) agglutinins. Demonstrable amounts of lipopolysaccharide could only be removed from coated erythrocytes by washing them at elevated temperatures (58°C) in physiological solutions. Red cell sensitization with B active E. coli O₈₆ substances was achieved in vivo in a minority of severely diseased infants and in germ-free and ordinary chicks which were in tourniquet shock after treatment with cathartics. Therefore, a possible mode by which erythrocytes of patients with severe intestinal disorders acquire antigens is the fixation of bacterial substances to their surfaces, if there are not enough of the normally interfering plasma factors present.     

Activation of heart contractility of the edible snail <Emphasis Type="Italic">H. pomatia</Emphasis> by thrombin. Study of the role of cAMP

Thrombin acts on mammalian cells through the specific, so-called protease-activated receptors (PARs). The thrombin action is mediated via three out of four known types of these receptors—PAR_1,3,4. Mammalian thrombin receptors, apart from performance of other functions, control cardiac and vascular contractility. It is not known whether receptors of such kind exist in invertebrate animals. In the present work we have showed for the first time that thrombin in the concentration range of 0.01–1 units/ml increases amplitude of contractions of the isolated heart ventricle of the edible snail Helix pomatia. Its effect is reproduced by peptide ligands of receptors PAR₁ and PAR₄ that have sequences Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and Glu-Tyr-Pro-Gly-Lys-Phe (QYPGKF), respectively. A potent activator of cardiac contractility of H. pomatia is serotonin. A comparative study of the mechanisms of action of serotonin and thrombin on the edible snail heart was carried out. cAMP participates in transduction of signal from serotonin receptors. On the membrane preparation from the H. pomatia heart, it was shown that thrombin and peptide ligands PAR₁ and PAR₄, unlike serotonin, did not increase adenylyl cyclase activity. Thus, mechanism of activation of cardiac contractility of H. pomatia by thrombin differs from that of serotonin. It is suggested that molluscs have receptors homologous to protease-activated mammalian receptors.     

Streptococcus pneumoniae Invades Erythrocytes and Utilizes Them to Evade Human Innate Immunity

Streptococcus pneumoniae, a Gram-positive bacterium, is a major cause of invasive infection-related diseases such as pneumonia and sepsis. In blood, erythrocytes are considered to be an important factor for bacterial growth, as they contain abundant nutrients. However, the relationship between S. pneumoniae and erythrocytes remains unclear. We analyzed interactions between S. pneumoniae and erythrocytes, and found that iron ion present in human erythrocytes supported the growth of Staphylococcus aureus, another major Gram-positive sepsis pathogen, while it partially inhibited pneumococcal growth by generating free radicals. S. pneumoniae cells incubated with human erythrocytes or blood were subjected to scanning electron and confocal fluorescence microscopic analyses, which showed that the bacterial cells adhered to and invaded human erythrocytes. In addition, S. pneumoniae cells were found associated with human erythrocytes in cultures of blood from patients with an invasive pneumococcal infection. Erythrocyte invasion assays indicated that LPXTG motif-containing pneumococcal proteins, erythrocyte lipid rafts, and erythrocyte actin remodeling are all involved in the invasion mechanism. In a neutrophil killing assay, the viability of S. pneumoniae co-incubated with erythrocytes was higher than that without erythrocytes. Also, H₂O₂ killing of S. pneumoniae was nearly completely ineffective in the presence of erythrocytes. These results indicate that even when S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they can still invade erythrocytes. Furthermore, in the presence of erythrocytes, S. pneumoniae can more effectively evade antibiotics, neutrophil phagocytosis, and H₂O₂ killing.     

A cell membrane-associated lectin of the oyster hemocyte

The presence of a lectin in association with hemocytes of the American oyster, Crassostrea virginica, has been demonstrated by utilizing a microhemagglutination assay. The plasma membrane association of this lectin is shown by its copurification with the plasma membrane fraction of disrupted hemocytes, using sucrose density gradient centrifugation, and also by the binding of ¹²⁵I-labeled glycoproteins to intact hemocytes at 4°C. Based upon agglutinating spcificity for a range of vertebrate erythrocytes, both untreated and enzyme-treated, along with hemagglutination-inhibition assays and crossed-absorption tests, it is apparent that there are also two serum (soluble) lectins, each having a distinct serological agglutination specificity, and that the hemocyte membrane-associated lectin has a specificity that is identical with one of these two serum lectins. It is proposed that the hemocyte membrane-associated lectin may be a true integral membrane protein, and therefore may function as a membrane receptor in nonself recognition by molluscan hemocytes.     

Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer,Chilo suppressalis

Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cells, with an EC₅₀ value of 18.7 ± 5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca²⁺ release from the hemocytes of C. suppressalis.     

Immune response of the prawn, Macrobrachium rosenbergii, to bacterial infection

Serum agglutinins of Macrobrachium rosenbergii found in normal serum were reactive toward eight bacterial species and type A human red blood cells. Absorption studies indicated the ability of the agglutinins to distinguish between different bacterial species as well as between certain bacteria and red blood cells. Agglutinin titers were approximately the same for the bacteria, whereas those for red blood cells were significantly higher. A virulent strain of Vibrio anguillarum was used in infectivity experiments. An LD₅₀ value was determined between 5 × 10⁶ and 10⁷ cells/animal, and an attempt was made to immunize the animals using formalin-killed cells. The animals did not respond to the vaccination, as there was neither an increase in the level of circulating agglutinins nor the LD₅₀ level 6 days after injection. Structural and functional traits of serum agglutinins are markedly different from vertebrate antibodies.     

Quantification of cytotoxic hemocytes of Mytilus edulis using a cytotoxicity assay in agar

When a thin layer of agar containing a mixture of erythrocytes and Mytilus hemocytes is prepared on slides, the occurrence of plaques of lysed target cells can be observed around a limited number of hemocytes. These hemocytes remain completely intact cells and are viable as evidenced by their ability to phagocytose target cells and/or to form pseudopods. The number of hemocytes releasing cytotoxic molecules has been shown to vary greatly between different animals. The same holds true for the total number of circulating hemocytes, although no correlation exists between the number of hemocytes in the circulation and the percentage of cytotoxic blood cells.     

Effect of Temperature on the Morphometrical and Physical Parameters of Erythrocytes and Polymorphonuclear Leucocytes in <Emphasis Type="Italic">Carassius gibelio</Emphasis> (Bloch)

Dynamics of the morphometric and physical properties of hemocytes of the Prussian carp Carassius gibelio (Bloch) under the influence of a temperature factor has been studied with atomic force microscopy in experiments in vitro. It is found that, at a low incubation temperature (5°C), as opposed to room temperature (20°C), morphometric parameters change in erythrocytes; at a high temperature (40°C) they change in polymorphonuclear leucocytes. The low incubation temperature reduces the adhesion and elasticity of polymorphonuclear leucocytes and erythrocytes of C. gibelio, whereas a high incubation temperature leads to a decrease in adhesion in polymorphonuclear white blood cells.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

A Volume Regulatory Response Can Be Triggered by Nucleosides in Human Erythrocytes, a Perfect Osmometer No Longer

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells. Further, because ectonucleotidases may scavenge ATP or ADP or act as a source for extracellular adenosine and therefore modulate P receptor activation and RVD, we also determined their activity in intact erythrocytes. We found relatively low ectoATPase but significant ectoADPase and ectoAMPase activities. When erythrocytes were exposed to hypotonic medium alone, they swelled as expected for an osmometric response and showed no RVD. Activation of P2 receptors by exogenous ATP or ADP did not trigger RVD, whereas P1 agonists adenosine and adenosine-5′-N-ethylcarboxamide induced significant RVD. The effect of adenosine-5′-N-ethylcarboxamide was dose-dependent (maximal RVD of 27%; apparent K_½ of 1.6 ± 1.7 μm). The RVD induced by adenosine was blocked 80% with the non-selective P1 antagonist 8-(p-sulfophenyl theophylline) or the P1-A_2B inhibitor MRS1754, but not by inhibitors of P1 subtypes A₁, A_2A, and A₃. In addition, forskolin (an inducer of intracellular cAMP formation) could mimic the effect of adenosine, supporting the idea of P1-A_2B receptor activation. In conclusion, we report a novel P1-A_2B receptor-mediated RVD activation in mature human erythrocytes and thus indicate that these long held perfect osmometers are not so perfect after all.     

The effect of ACTH and adrenal steroids on K transport in human erythrocytes

The effect of ACTH and adrenal steroids on K transport in human erythrocytes has been studied. A new method of calculation has revealed that in normal human erythrocytes the K transport is not independent of external K concentration as had previously been thought. The equation describing the relationship is, K influx (m.eq./liter cells hour) = [K]_pi/(0.697 + 0.329 [K]_pi) in which [K]_pi refers to the plasma K concentration at the beginning of the experiment. At the physiological plasma K concentration of 4.65 m.eq./liter, K influx is 2.09 m.eq./liter cells hour; K efflux is 1.95 m.eq./liter cells hour and is independent of plasma K concentration. The effect of the infusion of ACTH and adrenal steroids on the K content of the erythrocytes was also studied. Infusions of ACTH or cortisone do not cause the expected loss in erythrocyte K content and may well cause a gain. Infusions of ACTH and cortisone decrease the rate of K influx and efflux slightly at all stages of the infusion, as measured in vitro in blood samples drawn at various times during and following the infusion. However, the erythrocytes incubated in vitro do not exhibit the same changes in K content as are found in vivo. Hydrocortisone added to normal cells in vitro also decreases both influx and efflux of K, without affecting the K content of the cells.     

Electrophoretic and immunologic studies of a hemagglutinin in the hemolymph of the decapod Macropipus puber

Serum agglutinins directed against antigens of the A-B-O blood groups of human erythrocytes have been detected in the blood of male Macropipus puber crabs. The hemagglutinating activity was constant in the specimens examined of which the intermolt stages ranged from C2 to D2. These hemagglutinins, which are not group specific, are proteinaceous and with an electrophoretic mobility similar to some human immunoglobulins. Their molecular weights approximate 300,000.     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.