Hemocyte Adhesion Research Articles

Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. M. demolitor eggs are never encapsulated by host haemocytes when coinfected with its associated polyndnavirus (MdPDV) whereas eggs are encapsulated within 36 h when injected into hosts without virus. In this study, infection of specific classes of P. includens haemocytes by MdPDV was examined. Electron microscopic studies indicated that MdPDV entered all haemocyte morphotypes. Northern blot analysis revealed that similar size classes of viral mRNAs were produced in granular cells, plasmatocytes and spherule cells. Expression of a 1.6 kb MdPDV mRNA in haemocytes from parasitized hosts was detectable by in situ hybridization at 2 h post-parasitism (p.p.) and continued through until day 6 p.p. By 12 h p.p., viral expression was detected in greater than 80% of the haemocytes in circulation but thereafter the percentage of haemocytes exhibiting a hybridization signal declined. Similar patterns were observed in haemocytes from larvae injected with calyx fluid or MdPDV plus venom. Granular cells and plasmatocytes from unparasitized larvae were purified on Percoll cushions and maintained in vitro. Both morphotypes were successfully infected with MdPDV and exhibited changes in morphology and adhesiveness very similar to cells from parasitized hosts. Cell-free plasma from parasitized larvae had a variable effect on haemocyte adhesion. Haemocytes cultured in plasma from 1 or 4 day p.p. larvae rapidly spread whereas cells cultured in 7 day p.p. plasma did not. Reciprocally, adhesion of haemocytes from parasitized larvae could not be rescued by cell-free plasma from unparasitized larvae. Together, these data suggest that disruption of the host encapsulation response is medicated primarily by direct infection of granular cells and plasmatocytes by MdPDV.

The roles of carbohydrates in aggregation and adhesion of hemocytes from the California mussel ( Mytilus calif ornianus)

Forty-seven carbohydrates failed to inhibit the cell aggregation and substrate adhesion behaviors of Mytilus californianus hemocytes. Treatment of hemocytes with sodium periodate and with YV-acetyl glucosaminidase interfered with these behaviors. Trypsin slightly reduced adhesion, and had no effect on aggregation. Hyaluronidase and neuraminidase failed to inhibit either behavior, and slightly enhanced them. An inhibitory effect of caffeine was not altered by hyaluronidase of N-acetyl-glucosaminidase, but neuraminidase partially reversed this inhibition. Inhibition by the calcium channel blocker verapamil was reversed by hyaluronidase. Cell-cell and cell-substrate adhesion behaviors probably involve complex carbohydrates in molluscan hemocytes.

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae.

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M(r) = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 +/- 20 micrograms/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH2-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses.

Isolation of a 90kDa protein from haemocytes of Blaberus craniifer which has similar functional and immunological properties to the 76 kDa protein from crayfish haemocytes

A 90 kDa protein has been partially purified from Blaberus craniifer haemocytes and shown to cross-react with a monospecific antiserum against a 76 kDa protein from the blood cells of the crayfish, Pacifastacus leniusculus. The Blaberus protein functioned both to enhance adhesion of the cockroach haemocytes to the substratum and to induce the degranulation of these cells by a regulated exocytosis. Cross reactivity studies showed that the Blaberus protein could also enhance the adhesion and degranulation of the Pacifastacus blood cells whilst the crayfish protein induced the same reactions with the cockroach haemocytes. The Blaberus 90 kDa protein is probably involved in controlling cell to cell cooperation during immunoreactivity by the cockroach haemocytes.

Bacteria‐induced protein P4 (hemolin) from Manduca sexta: A member of the immunoglobulin superfamily which can inhibit hemocyte aggregation

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.

A peptide containing the cell adhesion sequence RGD can mediate degranulation and cell adhesion of crayfish granular haemocytes in vitro

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (which corresponds to a fragment of fibronectin and contains its cell adhesion sequence RGD) caused degranulation and spreading of monolayers of isolated granular haemocytes of the crayfish Pacifastacus leniusculus in vitro. When coated on glass coverslips, this RGD-containing peptide could mediate cell attachment of granular cells in vitro. A control peptide, Gly-Arg-Gly-Glu-Ser (GRGES), did not have these activities. Thus, GRGDS imitates the biological activities in vitro of the cell adhesion factor recently purified from crayfish haemocytes. This suggests that the sequence Arg-Gly-Asp (RGD), which is responsible for the cell adhesion activities of a number of vertebrate proteins, may also be involved in degranulation and cell adhesion of arthropod haemocytes. This is the first report describing direct activities by an RGD-containing peptide towards invertebrate cells in vitro, and the first indication of the presence of an RGD-recognizing receptor on invertebrate haemocytes.

A cell adhesion factor from crayfish haemocytes has degranulating activity towards crayfish granular cells

A factor able to mediate cell adhesion of semigranular and granular haemocytes of the crayfish Pacifastacus leniusculus was recently purified from crayfish haemocyte lysate (Johansson and Söderhäll, J. Cell Biol. 106, 1795–1803, 1988). It is a protein with a mass of 76 kDa, and its activity seems to be generated concomitantly with the activation of the prophenoloxidase (proPO) activating system. In this paper, we present evidence that this same protein is also responsible for the previously reported degranulating activity of a crayfish haemocyte lysate, in which the proPO system has been activated. First, the 76 kDa band in SDS-polyacrylamide electrophoresis seems to be a single protein, since in isoelectric focusing the purified cell adhesion factor fraction migrated as one band with an isoelectric point of 7.2. Second, this fraction was also able to degranulate crayfish granular cells in vitro, and third, antibodies to this 76 kDa protein, which are known to block cell adhesion, could also inhibit degranulation in vitro.

Preliminary characterization and purification of [formula omitted][formula omitted] encapsulation promoting factor: A peptide that mediates insect haemocyte adhesion

The granular cells and plasmatocytes (PLs) of Heliothis virescens form multicellular aggregations in vitro . This allows capsules to form around suitable targets. Prior trypsinization of the haemocytes abolishes their ability to encapsulate, and this function can be restored by adding plasma to the trypsinized cells. Trypsinized PLs were also unable to spread on a planar glass surface unless normal plasma was present. Plasma was subjected to a variety of treatments to determine the nature of the encapsulation promoting factor (EPF) using the in vitro encapsulation system as a bioassay. The data suggest EPF is a peptide; it is trypsin sensitive and moderately heat stable. Similar results were obtained when using spreading by trypsinized PLs as the bioassay. Dialysis using a 3,500 MW cut-off membrane also abolished encapsulation promoting activity. Protein-free extracts of plasma (crude EPF) has strong activity in both bioassays but does not agglutinate human erythrocytes. A single peak with strong activity in both bioassays was resolved after subjecting crude EPF to reversed-phase HPLC. This active material was purified after additional HPLC with a different solvent system.

Reactions of hemocytes of immune and non-immune [formula omitted][formula omitted] larvae to [formula omitted][formula omitted

Immune larval Galleria mellonella removed live Proteus mirabilis from the hemolymph less effectively than did non-immune larvae. This was attributed to a decline in total hemocyte counts, levels of plasmatocytes and granulocytes and hemocyte adhesion capacity. Immune serum possessed factors which reduced bacterial adhesion to the hemocytes. This was not due to altering bacterial surfaces but rather to irreversible suppression of hemocyte activity.

Chemokinetic behaviour of insect haemocytes in vitro.

Time-lapse microphotography was used to film the locomotory behaviour of cockroach haemocytes in vitro, and the cell tracks were analysed for speed and persistence; the percentage mobilization and the diffusion rate of the population were calculated. Haemocytes are either fast locomotor or spread moving cells, or non-motile spread or rounded cells; the first three types are plasmatocytes and their behaviour is interchangeable. Approximately 20% of the cells are motile under control conditions and there is no correlation between orthokinesis and klinokinesis. If activated haemocyte lysate supernatant (HLS), a source of components of the prophenoloxidase enzyme sequence, is added to the cell monolayer, up to 80% of the cells switch to fast locomotor behaviour, rounding up and moving faster and for longer in straight lines. Neither heat-inactivated HLS nor zymosan supernatant, used to activate HLS, had any effect. If the chemokinins present in activated HLS are also released in vivo on haemocyte activation or during cuticular wounding, then they and the induced changes in haemocyte adhesion could contribute to haemocyte recruitment to sites of infection.

(A2) Cell adhesion of crustacean haemocytes : Mats W. Johansson and Kenneth Söderhäll, Institute of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden

(A2) Cell adhesion of crustacean haemocytes : Mats W. Johansson and Kenneth Söderhäll, Institute of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden

Surface charge of insect haemocytes, examined using cell electrophoresis and cationized ferritin-binding.

Differences in the negative surface charge of haemocytes from Periplaneta americana and Schistocerca gregaria have been revealed using cell electrophoresis and cationized ferritin-binding. Although haemocyte populations from both insect species exhibit ranges of negative surface charge, both techniques show that Schistocerca haemocytes are significantly more negative than Periplaneta haemocytes. The results may help to explain why Schistocerca haemocytes adhere poorly to negative substrata, both in vitro and in vivo, and suggest that an electrostatic mechanism may be involved, at least in part, in adhesion of insect haemocytes to substrata.

Haemocytic encapsulation and the prophenoloxidase-activation pathway in the locust Schistocerca gregaria forsk

Negatively-charged Sepharose beads are not encapsulated in vivo by haemocytes of the locust Schistocerca gregaria. It has been suggested by other workers that components of the prophenoloxidase activation pathway of haemolymph might adhere to foreign surfaces and stimulate haemocyte adhesion, so one possible reason for the lack of encapsulation of beads might be due to failure of these components to adhere to the bead. Beads were thus incubated in locust haemocyte lysate supernatant, in which the prophenoloxidase pathway had been activated by Ca 2+ or Zymosan supernatant, and were then injected into the haemocoeles of locusts. Although at least 5 proteins, including phenoloxidase, could be shown to be attached to the beads, these coated beads were not encapsulated suggesting either that the putative opsonin did not attach or that none of the components is opsonic in this system. In addition, it has been shown that the prophenoloxidase pathway in locust haemocyte lysate supernatant can be partially activated in the presence of Ca 2+, strongly activated by β1,3-glucans and that production of phenoloxidase is not enhanced by the presence of bacterial LPS and is inhibited by a serine protease inhibitor. The changes in protein composition of unactivated and activated haemocyte lysate supernatant are discussed.

Effect of substratum wettability and charge on adhesion in vitro and encapsulation in vivo by insect haemocytes

The circulating leucocytes of insects, the haemocytes, adhere to and encapsulate foreign material that enters the insect's body cavity. The thickness of the capsule depends not only on the insect species but also on the nature of the object concerned, a fact that is of great importance to invading parasites and pathogens. In this paper, some of the factors that may stimulate haemocyte adhesion and subsequent encapsulation of the object have been investigated using abiotic materials with surfaces of different charge and wettability. The negativity and wettability of surfaces of polystyrene beads and plates can be increased by pretreatment with acid, and adhesion of haemocytes to these modified surfaces has been examined in vivo and in vitro. A similar proportion of haemocytes of the locust Schistocerca gregaria adhere to the plates in vitro, irrespective of the changes in charge and wettability, but the adhesion of haemocytes of the cockroach Periplaneta americana is proportional to the increases in both parameters. These differences in cell behaviour are reflected in vivo: cockroach haemocytes form thicker capsules around more hydrophilic and more negatively charged polystyrene beads, while locust cells encapsulate both types of surface to the same, minimal, degree. Positively and negatively charged Sepharose beads are encapsulated more thickly than are neutral beads in cockroaches; negatively charged Sepharose beads are not encapsulated at all in locusts. There are thus obvious differences between the two species in the ways in which their haemocytes respond to these modified abiotic surfaces. It is suggested that capsule thickness in vivo depends on the initial cell-substratum contact; different surfaces stimulate the cell to different extents, which in turn causes variations in the recruitment of other cells to the capsule.

Cellular immune responses of spruce budworm larvae to Entomophthora egressa protoplasts and other test particles

The protoplast stage of two isolates of Entomophthora egressa developed normally and eventually produced conidiophores when injected into larvae of the spruce budworm, Choristoneura fumiferana. The spruce budworm hemocytes never made long-term contact with the protoplasts either in vivo or in vitro. The protoplasts made active, short-term contact with spruce budworm granulocytes both in vivo and in vitro. Total larval hemocyte counts (THC) initially declined when larvae were injected with protoplasts, growth medium (MGM), or Escherichia coli. The recovery rate to THC control levels was similar for MGM and protoplasts and supports the concept of nonrecognition of protoplasts by the hemocytes. The granulocytes were important in both nodulation and phagocytosis of E. coli and Bacillus cereus, whereas the plasmatocytes were important in phagocytosis. In in vitro studies, spruce budworm granulocytes did not adhere to rod-shaped hyphal bodies, spherical hyphal bodies, or germinating spherical hyphal bodies of E. egressa, whereas the granulocytes readily encapsulated the hyphae. There was no evidence for the production by the protoplasts of metabolites which might interfere with hemocyte adhesion. When protoplasts contacted Tenebrio molitor granulocytes, the protoplasts reacted by increasing the number of protoplasmic extensions and by granule discharge. The process of granule discharge may be an active protoplast defense mechanism. The sporangiospores of Absidia repens and Rhizopus nigricans adhered to spruce budworm granulocytes; however, the number of A. repens spores per granulocyte and the level of granulocytes with spores decreased in the presence of phenylthiourea. The adhesion of A. repens spores to granulocytes was enhanced by N-acetylglucosamine, whereas glucosamine, sucrose, fucose, fructose, arabinose, and galactose either had no effect on or reduced spore adhesion. Thus, the chitin (or its subunits) in the hyphal wall may initiate the granulocyte response.

Humoral factors promoting the adhesive properties of the granular cells and plasmatocytes of the silkworm, Bombyx mori, and their possible role in the initial cellular reactions to foreignness

In this study the differential adhesion of hemocytes of Bombyx mori to a glass surface was analyzed using a microincubation system under agar, and the effects of hemolymph on the adhesive properties of each hemocyte type and on the surface features were examined employing the scanning electron microscope (SEM). The two main types of hemocytes, plasmatocytes and the granular cells, showed much stronger adhesion to a glass surface, especially when hemocytes were incubated with media containing autologous hemolymph. Observations with the SEM clearly showed the presence of humoral factors which promoted the elongation of filopodia of the granular cells. In addition, the initial cellular reactions to goose erythrocytes (GRBCs) injected into the hemocoel of larvae were also observed with the SEM. Results suggest that filopodia of the granular cells play an essential role when the granular cells attach to the surface of GRBCs. The promoted elongation of filopodia of the granular cells in the presence of hemolymph is discussed with regard to the initial cellular reactions to foreignness.