Granular Hemocytes Research Articles

Analysis of immune defences of the wax moth, Galleria mellonella, with anti-haemocytic monoclonal antibodies

The effects of anti-haemocyte monoclonal antibodies on the cellular defence reactions of the wax moth, Galleria mellonella, were investigated. One of these monoclonal antibodies, termed GM, reacted with a ca 100 kDa protein only found associated with the granular haemocytes. Blocking of this protein with the monoclonal antibody caused a specific reduction in nodule formation, in the attachment of bacteria to granular haemocytes and in the adhesion of these cells to glass substrates. Other monoclonal antibodies with differing specificity had no effect on these processes. In contrast to the granule haemocytes, the adhesive and phagocytic properties of the plasmatocytes were apparently unaffected by the presence of the GM monoclonal antibody. The mechanism by which these monoclonal antibodies inhibit the reactions of the granular haemocytes is still nuclear, although it is suggested that they act by blocking the activity of the 100 kDa protein during degranulation events of these cells following contact with foreign material. The similarity of this protein with other adhesion-promoting molecules is discussed.

Morphological and biochemical characterization of Procambarus clarki blood cells

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40–0.78 μm). Granular cell contained large eosinophilic granules (1.79–3.05 μm). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Antioxidant enzymes associated with the blood cells and haemolymph of the mussel Mytilus edulis

Immunocytochemical localisation of the antioxidant enzymes catalase, superoxide dismutase (SOD, both the Cu/Zn and the Mn form) and glutathione peroxidase (GPX) was carried out on blood cells from the mussel Mytilus edulis at both the light and electron microscope level. The results showed the SOD (Cu/Zn) antibody localised in association with the plasma membrane of the hyalinocytes. The granular haemocytes gave a positive reaction for all the antibodies investigated. Catalase was localised in small electron dense granules indicating that these granules are peroxisomes. Both forms of the SOD enzyme were associated with granules, with the labelling density generally lower for the Mn form. Biochemical measurements of antioxidant enzyme activities supported the immunocytochemical results. Catalase and SOD activities were present in blood cells but not detectable in haemolymph. The SOD activity was indicated to be due to both Cu/Zn and Mn forms of the enzyme (respectively, 75·9 and 24·1% of total activity). GPX activity was present in the cells but predominantly in the haemolymph (87·5% of total).

In vitro Interactions between Bivalve Hemocytes and the Oyster Pathogen Haplosporidium nelsoni (MSX)

Resistance to disease caused by the oyster pathogen Haplosporidium nelsoni (MSX) has developed in nature and through selective breeding; however, examination of tissue slides shows no evidence that phagocytic cells are involved. To investigate this phenomenon further, we quantified in vitro phagocytosis of plasmodial stages of H. nelsoni by hemocytes from 2 oyster and 1 mussel species. Our results show that hemocytes from oysters Crassostrea virginica, whether selected or unselected for resistance to MSX disease, did not ingest plasmodia unless the parasites were damaged or killed. Time-lapse photography suggested that granular hemocytes retreated rapidly after initial contact with living plasmodia and thatnearby agranular hemocytes showed little movement

Cellular Responses of Oysters Infected with Haplosporidium nelsoni: Changes in Circulating and Tissue-Infiltrating Hemocytes

The protozoan parasite Haplosporidium nelsoni (MSX) elicits an inflammatory-type response in oysters, Crassostrea virginica. We assayed circulating hemocytes of oysters exposed to H. nelsoni to quantify the effects of parasitism, selection for resistance, and season on total and differential counts. All factors had a significant (P < 0.02) effect on total counts, but explained relatively little of the overall variance [season (12%) > selection (4%) > infection (2%)]. Circulating hemocyte densities increased with infection intensity in resistant animals, but were depressed in susceptible oysters with light infections. Counts in both groups were lowest in August and highest in May, at which times densities in susceptible oysters were reduced (P < 0.02) compared to those in resistant animals. No differences existed in November. Susceptible oysters may be more debilitated by infections than resistant animals, resulting in impaired circulation and depressed cell counts. The number of tissue-infiltrating hemocytes increased with infection intensity (P < 0.0001), but showed no statistically significant (P > 0.05) association with numbers of circulating hemocytes in individual oysters. Size-frequency analysis with a Coulter counter indicated that the proportion of large cells (presumed to be granular hemocytes) was lower (P < 0.0001) in susceptible oysters, which were also heavily infected, compared to resistant oysters, which had very few infections. The loss of granular hemocytes may stem from degranulation associated with tissue damage and inflammation. Present evidence suggests that the principal role of the hemocyte response in MSX disease is to plug lesions, remove debris, and repair tissues and that these functions may help oysters survive infection.

Crayfish α-macroglobulin and 76 kDa protein; Their biosynthesis and subcellular localization of the 76 kDa protein

A 76 kDa protein from the freshwater crayfish, Pacifastacus leniusculus, which previously has been demonstrated to mediate the attachment and degranulation of crayfish haemocytes, was shown to be synthesized in the haemocytes. This was demonstrated by immunoprecipitation of [ 35S]methionine-labelled haemocyte preparations. With the use of immunogold staining and electron microscopy it was shown that this protein is localized to the granules of both the semigranular and granular haemocytes, suggesting that after synthesis, the 76 kDa protein is deposited into these organelles. One protein present in the plasma of freshwater crayfish, the α-macroglobulin-like proteinase inhibitor, was also found to be synthesized in the haemocytes.

Plumbagin-induced ultrastructural haemocytic response of Dysdercus koenigii F.

The phytochemical ptumbagin, isolated from Plumbago zeylanica L., was applied in 2 and 5 μl doses of 0.1 % concentration on the surface of Dysdercus koenigii F. adults and its effect on the haemocytes of the bug, studied by transmission electron microscopy. All the five haemocyte types viz. the prohaemocyte, plasmatocy te, granular haemocyte, oenocytoid, and the adipohaemocy te are affected within a period of 24–48 hr.The effect begins with an acute state of vacuolization of the cells and a gradual destruction of the organelles, followed by dissolution of the plasma membrane and passing out of the internal organelles, like the mitochondria and the endoplasmic reticulum. This phenomenon is responsible for a consistent elimination of the haemocytes from the blood, leading to lowering of the resistance and mortality of the insects.

Antibacterial activity in the haemocytes of the shore crab,Carcinus maenas

The presence of antibacterial activity in the haemocytes of the shore crab,Carcinus maenas(L.) (Crustacea: Decapoda), was investigated using a selection of Gram-positive and Gram-negative bacteria from geographically diverse waters. Preliminary investigations into the relationship between this activity and the prophenoloxidase activating system (proPO) were also carried out. Antibacterial activity against both Gram-positive and Gram-negative organisms were found to reside exclusively in the granular haemocytes and eight of the twelve bacteria tested were susceptible to this effect. Additional studies, usingPsychrobacter immobilis(=Moraxellasp.), revealed that the factor (or factors) responsible was 90% effective within 60 min and was also heat stable, independent of divalent cations, and non-lytic in character. Although antibacterial activity resides in the same cell population that carries the proPO system, there appears to be no relationship between antibacterial activity and phenoloxidase itself. Other components of the proPO system, however, may be involved.

Phagocytosis by hemocytes of the hard clam, Mercenaria mercenaria

Large granular hemocytes of Mercenaria mercenaria avidly phagocytose a variety of biological particles (red blood cells of six species, yeast, and gram-positive and gramnegative bacteria) as well as polystyrene spheres. Clam hemolymph is not necessary for phagocytosis but may have some opsonic effect in certain circumstances (e.g., low temperature and low particle density). Formaldehyde treatment of red blood cells enhances susceptibility to phagocytosis. Phagocytosis by Mercenaria hemocytes in vitro appears to be a nonspecific process.

Classification of Hemocytes in the Common Cutworm, Spodoptera litura(Lepidoptera:Noctuidae) : II.Possible Roles of Granular Plasmatocytes and Oenocytoids in the Cellular Defense Reactions

Granular plasmatocytes in Spodoptera litura (FABRICIUS), which until now have been called granular hemocytes, were specifically stained pink by May-Grunwald. They had a character of plasmatocytes: they spread on a glass slide, participated in encapsulation against large latex particles, and did not function as phagocytes against India ink. Oenocytoids, which were characterized by their rapid lysis and ovoid forms, contained a prophenoloxidase-activating-system: only oenocytoids, which were fixed with glutaraldehyde, formed melanin in the presence of DOPA. This reaction was inhibited by pNPGB, a serine-type protease inhibitor.

Classification of Hemocytes in the Common Cutworm, Spodoptera litura(Lepidoptera:Noctuidae) : I.Phase Microscopic Study

The phase microscopic observation of the hemocytes of the common cutworm, Spodoptera litura (FABRICIUS) yielded categorization into 7 classes: prohemocytes, plasmatocytes, granulocytes, spherulocytes, oenocytoids, podocytes, and granular plasmatocytes. Podocytes and vermiform cells (JONES, 1959) were classified in the same class as the "podocytes", because they morphologically transformed to the same spherical cells with fine extensions during incubation. They did not have a character of plasmatocytes. Granular plasmatocytes, which have been so far called granular hemocytes, had the character of plasmatocytes, spreading on a glass slide. Oenocytoids lucid immediately after bleeding. Total hemocyte counts were 1.4-2.1×104/μl, and the proportion of granulocytes and plasmatocytes was 37-47% and 16-35% from the 5th molting stage through 6th instar, respectively.

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus ( Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.

The ?-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus, when reacted with a ?-1,3-glucan, induces spreading and degranulation of crayfish granular cells

A β-1,3-glucan-binding protein (βGBP) was purified from crayfish plasma, and incubated with laminarin (L), a β-1,3-glucan. The βGBP reacted with laminarin (βGBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the βGBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 μg of βGBP-L, more than 82% of the haemocytes were affected. The activity of βGBP-L on granular cells was dose-dependent and a plateau was reached at 10 μg of βGBP-L. The degranulation of crayfish haemocytes induced by βGBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-βGBP antibodies also totally blocked the effect of βGBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the βGBP-L could bind to the surface of granular cells, whereas βGBP did not bind or bound very weakly to the haemocyte surface.

Cheliped regeneration in the megalopa of the mud crab,Rhithropanopeus harrisii(Gould)

Summary The process of autotomy-induced cheliped regeneration in the megalopa of the mud crab, Rhithropanopeus harrisii has been studied by light microscopy and transmission electron microscopy. Following blastema eruption, four time intervals were selected arbitrarily to cover the entire period of limb regeneration. Epidermal proliferation preceded muscle and nerve regeneration. On the basis of morphologic observations, nerve tissue regenerated from existing pedal nerve. It is proposed that blood vessels arose from epidermal differentiation, while muscle differentiated from immigrant cells. Three types of haemocyte, resembling those described in adult crustaceans, were identified in the haemolymph of the larval limb bud: granular haemocytes, hyaline cells and lipoprotein cells. Two other immigrant cell types, which appear to be primitive in nature and may be variants of a single cell type, are also described. Morphological evidence is provided to suggest that the more electron dense variant differentiate...

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 μm in diameter) and less electron-dense than the D-granules (less than 0.6 μm in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.

Hydrolytic enzymes associated with the granular haemocytes of the marine musselMytilus edulis

The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis. Arylsulphatase activity and immunocytochemical localization of beta-glucuronidase and elastase were demonstrated within the large granules of the haemocytes. Lysozyme and cathepsin B were both localized within all sizes of granule, however, at high dilutions the primary antibody against lysozyme was also restricted to the large granules. The labelling density for cathepsin B antibody tended to be very low. Antibodies for cathepsin G showed a clear, discrete labelling which was restricted to the granules of haemocytes containing small granules. The fact that antibodies raised against human proteinases recognize invertebrate enzymes suggests that there must be a certain degree of structural similarity between the human proteinases and the enzymes present in the mussel haemocytes indicating either convergence or conservation of the enzyme molecules. The presence of a range of hydrolytic enzymes including proteinases, glycosidases and sulphatases within the large granules shows that these granules are a form of lysosome. The reduction in activity of lysosomal enzymes in haemocytes following adhesion to glass is evidence for release of the enzymes from the granules (degranulation). The possibility of a serine protease being specifically associated with the small granules and its role as a cytolysin are discussed.

Observations on the Multitubular Pod Inclusions of the Granular Hemocytes of the Cockroach Periplaneta americana

Observations on the Multitubular Pod Inclusions of the Granular Hemocytes of the Cockroach Periplaneta americana

Effect of plumbagin on haemocytes ofDysdercus koenigii F.

Effect of plumbagin, a phytochemical, on the haemocytes ofDysdercus koenigii was studied after topical application. Scanning electron microscopical studies showed deformity in surface morphology in almost all the 5 types of haemocytes categorized in the bug, especially that of granular haemocytes and plasmatocytes, which are devoid of their filopods in the treated insects. The fat droplets of adipohaemocytes shrink while oenocytoid is affected to a lesser degree. Plasma membrane of all the cells becomes fragile leading to a gradual loss of cytoplasm and ultimately only a few interconnected cytoplasmic strands are left. Ultrathin sections revealed a highly vacuolized condition and disintegrating organelles which pass out of the cells due to ruptures of very thin plasma membrane. Total and differential haemocyte counts performed after 24 and 48h of treatment showed a drastic reduction of all the types i.e. the prohaemocytes disappear from blood, the number of granular haemocytes and plasmatocytes declines while oenocytoids and adipohaemocytes comprise the major part of counts. Because of the damages caused to haemocytes and the suppression of filopodial elongations of plasmatocytes and granular haemocytes (the types that are active in defense mechanism), it can be inferred that cellular defense reactions ofDysdercus koenigii are reduced after plumbagin treatment.

Immunocytochemical localization of a calcium‐binding phosphoprotein in hemocytes of heterodont bivalves

Calcium-binding phosphoprotein particles are the most abundant extracellular proteins in the hemolymph of heterodont bivalves, and granular hemocytes are the most abundant cells in the same fluid. In this study, the hemocytes of Rangia cuneata were examined ultrastructurally and probed with anti-phosphoprotein IgG to demonstrate that the granulocytes are a probable source of the hemolymph phosphoprotein. The granulocyte cytoplasm is laden with large vesicles containing an amorphous homogenous matrix and variable numbers of electron-dense particles; the latter are ultrastructurally similar to the extracellular phosphoprotein. The vesicle particles and matrix are related forms of the hemolymph phosphoprotein as evidenced by heavy gold labeling when Lowicryl sections were sequentially treated with rabbit-anti-phosphoprotein IgG and colloidal gold-anti-rabbit IgG. The vesicles may be the loci for posttranslational phosphorylation and eventual secretion of the calcium-binding phosphoprotein, or alternatively the vesicles may be digestive structures which degrade internalized phosphoprotein.

A morphological study of wound response and telson regeneration in postlarvalLimulus polyphemus(L.)

Summary The morphology of telson regeneration in postlarval Limulus polyphemus has been studied by light and transmission electron microscopy. Three types of ‘haemocyte’ (granular haemocyte, plasmatocyte and spherical dark cell) have been identified in the circulation. The characteristics and variants of these types are discussed in relation to clotting and to regeneration of tissues pre- and post-moult. It is suggested that plasmatocytes serve as pluripotential cells for the differentiation of tissues of the telson, in particular, nerve and muscle. This study provides a framework against which effects of xenobiotics on telson regeneration can be assessed.