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PubMedDate not supplied

Monoclonal antibodies against Manduca sexta hemocytes bind Aedes aegypti hemocytes: characterization of six monoclonal antibodies that bind hemocytes from both species.

The mechanisms by which hemocytes mediate a mosquito's defense response to parasites or pathogens are not well understood. This is due in part to difficulty in collecting intact mosquito hemocytes for experiments and to a lack of reagents, such as antibodies. Our objectives were to collect adult Aedes aegypti hemocytes under conditions suitable for immunofluorescence microscopy, and to test whether monoclonal antibodies, generated against larval Manduca sexta hemocytes, bind adult Ae. aegypti hemocytes. We present immunofluorescence micrographs of M. sexta and Ae. aegypti hemocytes stained by six monoclonal antibodies. Two antibodies, MS11 and MS32, immunolocalized to hemocyte nuclei in both species. On Western blots, these antibodies generate one signal at approximately 40 kDa and four others between 10 and 25 kDa. Immunofluorescence staining patterns of the other four antibodies were more complex. That these antibodies bind hemocytes from both species suggests significant molecular similarities exist between hemocytes from evolutionarily divergent species.

Aedes↗
PubMed2005

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta.

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.

Animals↗
PubMed1989

Differential adherence of M line Biomphalaria glabrata hemocytes to Schistosoma mansoni and Echinostoma paraensei larvae, and experimental manipulation of hemocyte binding.

The ability of M line strain Biomphalaria glabrata hemocytes to adhere to mother sporocysts (MS) of PR1 Schistosoma mansoni or to MS or daughter rediae (DR) of Echinostoma paraensei was studied using an in vitro hemocyte adherence assay. Hemocytes were significantly more likely to bind to S. mansoni MS than to E. paraensei MS or DR. Hemocyte adherence to E. paraensei MS or DR was significantly increased if glutaraldehyde-fixed larvae were used as targets. Also, E. paraensei MS pretreated with the lectin concanavalin A (Con A) were more likely to be bound by hemocytes than MS pretreated with Con A in the presence of the competing sugar, alpha-methyl mannoside. Pretreatment of hemocytes with Con A increased their ability to bind E. paraensei sporocysts, but the effect was small compared to that achieved by pretreatment of MS with Con A. The lectin probably did not function as a bridging molecule between hemocytes and MS but, rather, altered the MS surface in a way that facilitated adherence. Similarly, adherence to E. paraensei MS was significantly increased if the MS were pretreated with cell-free M line plasma prior to use in adherence assays. Our results indicate that the two parasites provoke fundamentally different responses from M line hemocytes in vitro and that the living tegument can be modified by host humoral factors and by lectins such that hemocyte binding is significantly increased.

Animals↗
PubMed2006

Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide.

We determined the biochemical characteristics of nitric oxide synthase (NOS) in hemocytes of the crayfish Procambarus clarkii and investigated the roles of hemocyte-derived NO in host defense. Biochemical analysis indicated the presence of a Ca2+ -independent NOS activity, which was elevated by lipopolysaccharide (LPS) treatment. When bacteria (Staphylococcus aureus) and hemocytes were co-incubated, adhesion of bacteria to hemocytes was observed. NO donor sodium nitroprusside (SNP) significantly increased the numbers of hemocytes to which bacteria adhered. Similarly, LPS elicited bacterial adhesion and the LPS-induced adhesion was prevented by NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Finally, plate count assay demonstrated that addition of LPS to the hemocytes/bacteria co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU), and that L-NMMA reversed the decreasing effect of LPS on CFU. The combined results demonstrate the presence of a Ca2+ -independent LPS-inducible NOS activity in crayfish hemocytes and suggest that hemocyte-derived NO is involved in promoting bacterial adhesion to hemocytes and enhancing bactericidal activity of hemocytes.

Animals↗
PubMed2005

A hemocyte-specific integrin required for hemocytic encapsulation in the tobacco hornworm, Manduca sexta.

Upon encountering an object recognized as foreign, insect hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding hemocyte antigen, which was revealed to be the same integrin beta subunit. A cDNA for this M. sexta integrin beta1 was cloned and characterized. Integrin-beta1 mRNA was detected by Northern analysis in hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin beta1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-beta1 RNA into larvae resulted in decreased integrin beta1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation.

Amino Acid Sequence↗
PubMed1999

Cadmium turnover in the hemocytes of Mercenaria mercenaria (L.) in relation to hemocyte turnover.

The turnover of cadmium (Cd) in the hemocytes of the quahog Mercenaria mercenaria L. was investigated using 109Cd radiolabeling, and compared to the rate of hemocyte turnover as estimated following [3H]thymidine incorporation. Quahogs were injected with 5 microl of 0.8 microM 109Cd, and the hemocytes sampled over a 60-day depuration period. Additional organisms were injected with 15 microl of 4.7 microM [3H]thymidine and radioactivity monitored over a 60-day period. Assuming that the loss of 3H from the hemocytes during the first 27 days of the experiment is due to hemocyte turnover, results indicate that Cd turnover rate (t 0.5 = 25.3 +/- 6.3 days) was similar to hemocyte turnover rate (t 0.5 = 28.5 +/- 14.3 days). This suggests that hemocytes retain Cd internally throughout their circulatory lifespan.

Animals↗
PubMed1994

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

Animals↗
PubMed1984

Hemocyte lysate enhancement of fungal spore encapsulation by crayfish hemocytes.

Crayfish hemocytes exhibited a stronger encapsulation reaction to fungal blastospores of Beauveria bassiana coated with hemocyte lysate, than to blastospores treated with plasma or buffer, indicating an opsonic function of hemocyte lysate proteins. Five proteins of the prophenoloxidase activating system in the hemocytes were attached to foreign surfaces (including the blastospores) after activation and it is suggested that these attaching proteins (one being phenoloxidase) are responsible for the opsonic function of the hemocyte lysate on crayfish blood cells.

Animals↗
PubMed2004

Syndecan-dependent binding of Drosophila hemocytes to laminin alpha3/5 chain LG4-5 modules: potential role in sessile hemocyte islets formation.

Heparin-column chromatography and elastase-digestion of medium from hemocyte Kc167 gave Drosophila laminin alpha3/5betagamma trimer, alpha3/5LG2-3 and alpha3/5LG4-5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with alpha3/5betagamma trimer or alpha3/5LG4-5, but not with alpha3/5LG2-3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of syndecan in Kc167 cells impaired the binding, but that of dally or dally-like did not. Green fluorescent protein-expressing hemocytes also bound surface with alpha3/5betagamma trimer or alpha3/5LG4-5 module. Thus, syndecan-dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2-A8 segments of Drosophila.

Amino Acid Sequence↗
PubMed2003

Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia).

Hemocytes of the American lobster (Homarus americanus H. Milne Edwards) were classified after examination of Wright-Giemsa stained cytocentrifuge preparations by brightfield light microscopy. Eleven hemocyte types were identified using morphologic criteria. The classification system was then used to monitor changes in the differential hemocyte count (DHC) of lobsters infected with the Gram positive coccus Aerococcus viridans var. homari, etiologic agent of gaffkemia. The appearance of less mature hemocytes in the DHCs of lobsters in the late stages of infection was similar to the 'left shift' of vertebrate inflammation. Results from this study suggest that DHCs can be used to assess and characterize inflammation in H. americanus and possibly other crustaceans.

Animal Diseases↗
PubMed2005

[Agglutination and phagocytosis of foreign abiotic particles by hemocytes of the blowely, Calliphora vicina in vivo. I. Dynamics of hemocyte activity during larval development].

Three types of Calliphora larval hemocytes have been revealed to be involved in phagocytosis of abiotic foreign particles: thrombocytoids, larval plasmatocytes and plasmatocytes I. Thrombocytoids are the quickest to respond to the appearance of invaders. The onset of test particle entrapment by thrombocytoid cytoplasmic fragments was observed, depending on the larval age within 0.5-5.0 min after injection. Separated fragments were fused, forming strands or roundish agglutinates. Phagocytosis of carbon, carmine or Indian ink particles by larval plasmatocytes occurs far more lately, and no earlier than 20-30 min after injection. Plasmatocytes I are capable of foreign particles adhesion on their surface, with a subsequent morule formation, and of engulfing these particles. These two events start in different time periods: adhesion occurs in 5-10 min, while phagocytosis is observed in 1--3 h. The rate of test particle entrapment and stability of agglutinales clearly depends on the larval age. The most pronounced reaction of hemocytes to foreign particles may be observed by the end of feeding and crop emptying. The second, somewhat less expressed rise of activity occurs in mature larvae not long before the onset of pupariation. Diapause induction is accompanied by reducing activities of both plasmatocytes and thrompocytoids. The importance of different hemocyte types in cellular immune reaction of Calliphora vicina larvae, and co-ordination between plasmatocytes and thrombocytoids are discussed.

Animals↗
PubMed2005

Contribution of circulating hemocytes to the regeneration of heavy ion beams (12C5+) irradiated hematopoietic organs in the silkworm, Bombyx mori, through the way of phagocytosis of injured cells after invasion.

Heavy ion beam irradiation has promising effects on tumor therapy. Our previous study using the domesticated silkworm, Bombyx mori, showed that this irradiation could seriously damage larval hematopoietic organs but they would regenerate later. In the in vitro irradiation, most hemocytes died when hematopoietic organs and wing discs connected with epidermis were directionally irradiated from epidermis to hematopoietic organ and then cultured so as to exclude circulating hemocytes. A few hemocytes had escaped irradiation according to extremely low hematopoiesis in vitro. Almost no hemocytes could incorporate BrdU at 60 h after irradiation, with which living and proliferating hemocytes are also labeled. In the absence of circulating hemocytes, the irradiation-escaped hemocytes in the organs were not enough for cleaning all dead cells because lots of small dead bodies remained in situ post-irradiation. After irradiating hematopoietic organs in larvae (in vivo irradiation), only a few apoptotic cells were found when given the same length of recovery time, and most hemocytes maintained normal morphology. Many hemocytes incorporated BrdU when tested at the same time as the in vitro irradiation but this number was lower than that measured for control organs. Circulating hemocytes, labeled by fluorescent microbeads through phagocytosis before irradiation, were found to have invaded the in vivo irradiated hematopoietic organs where they help the irradiation-escaped hemocytes to clear dead cells in the process of regeneration. Hematopoiesis of the regenerated hematopoietic organs did not fully recover to the level of the control organs according to the number of hemocytes produced in tissue culture. Some of the released hemocytes obviously underwent apoptosis, suggesting a far-reaching bystander effect of carbon ion beams irradiation on hemocytes inside. From these results, it is suggested that, together with irradiation-escaped hemocytes, the invaded circulating hemocytes took part in the regeneration of heavy ion beams irradiated hematopoietic organs through the way of phagocytosis of injured hemocytes in vivo.

Animals↗
PubMed2000

Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea.

During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom. This complex fluid has toxic properties and recently several venom components were characterized. In addition, it was suggested that venom might be involved in host immune suppression. For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system. Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration. Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro. Injection of 16 microg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined. Hemocyte concentration in sixth instar larvae 32 h after injection with 16 microg of venom was reduced by 56% compared to that in controls. Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P. hypochondriaca venom has cytotoxic properties. In vitro incubation of washed hemocytes for 20 h with 500 ng/microl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact. Treatment with low concentrations of venom (1.6 ng/microl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers. High-resolution microscopy of hemocyte cultures exposed to 320 ng/microl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles. Incubation of hemocytes with 320, 64, or 3.2 ng/microl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes. Treatment with 320 ng/microl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85%. Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.

Animals↗
PubMed2006

Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: cell types and their role after in vivo artificial non-self challenge.

The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an important aquacultured decapod species as well as an invasive species in some European countries. In the current investigation we characterized the different classes of circulating blood cells in A. leptodactylus by means of light and electron microscopy analysis and we explored their reaction to different latex beads particles in vivo by total and differential cell counts at 0.5, 1, 2 and 4h after injections. We identified hemocytes by granule size morphometry as hyaline hemocytes with no or rare tiny granules, small granule hemocytes, unimodal medium diameter granule hemocytes and both small and large granule containing hemocytes. The latter granular hemocytes showed the strongest phenoloxidase l-DOPA reactivity both in granules and cytoplasm. A. leptodactylus respond to foreign particles with strong cellular immune responses. All treatments elicited a total hemocyte increase with a conspicuous recruitment of large granule containing hemocytes. All hemocyte types mounted some phagocytic response but the small granule hemocytes were the only ones involved in phagocytic response to all foreign particles with the highest percentages. These results (1) depict the variability in decapod hemocyte functional morphology; (2) identify the small granule hemocyte as the major phagocytic cell; (3) suggest that the rather rapid recruitment of large granule hemocyte in all treatments plays a relevant role by this hemocyte type in defense against foreign particles, probably in nodule formation.

Animals↗