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.     

Response of eastern hemlock looper hemocytes to selected stages of Entomophthora egressa and other foreign particles

Indirect evidence for the natural existence of the free-protoplast stage of the fungus Entomophthora egressa in the eastern hemlock looper, Lambdina fiscellaria fiscellaria, is presented. The protoplasts were viable after 72 hr postinjection and subsequent development in the host produced conidia characteristic of E. egressa. The hemocytes studied (plasmatocytes, granular cells, and spherule cells) did not adhere to the protoplasts either in vivo or in vitro. Cells of Escherichia coli and sporangiospores of Absidia repens adhered to the granular cells in vitro. The granular cells adhered to the hyphae of Rhizopus nigricans in vitro. The spherule cells strongly adhered to the hyphae and hyphal bodies of E. egressa in vitro. The protoplasts, hyphae, and conidia of E. egressa and the hemocytes of L. fiscellaria fiscellaria adhered to positively charged DEAE-Sephadex beads and not to negatively charged CM-Sephadex beads. Aspects of active and passive strategies for protoplast evasion of host hemocytes are discussed with some emphasis on hemocyte-protoplast electrostatic repulsion and active secretion of hemocyte inhibitors by the protoplasts.     

Inhibition of human collagenase activity by a small molecular weight serum protein.

Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α₂-macroglobulin and a smaller serum component which eluted after α₁-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α₁-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α₁-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.     

Strains of protoplasts of Entomophthora egressa in spruce budworm larvae

Protoplasts of strain 458 (S458) and strain 521 (S521) of Entomophthora egressa had LD₅₀s of 620 and 8.8 cells/insect, respectively, for sixth-instar spruce budworm larvae. Both protoplast strains exhibited biphasic growth profiles with comparable growth rates in the larval hemocoel. The growth rates of the fungal strains increased as the total hemocyte counts declined. Hemocytopenia was greatest and most rapid in larvae containing S521 protoplasts. Protoplasts of S521 exposed to α-mannosidase and β-N-acetylglucosaminidase were as virulent as the control cells. β-Galactosidase reduced protoplast virulence.     

The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles

Hemocytes of Mytilus edulis were examined cytologically and cytochemically. On the basis of structure, staining reactions, and phagocytic behavior, they were divided into two main groups: basophilic hemocytes and eosinophilic granular hemocytes (granulocytes). The basophilic cells were further divided into small lymphocytes and larger phagocytic macrophages reactive for lysosomal hydrolases. Mitosis was observed in granulocytes and in small lymphoid cells, believed to be the stem cells for the basophilic cell line. A few cells appeared to be intermediate between lymphocytes and small granulocytes. Macrophages were the main cell type involved in the clearance of injected carbon particles. However, granulocytes did show some phagocytic activity. Brown cells displaying apparent amoebocytic behavior were found to contain Fe³⁺ and Pb²⁺ in cytoplasmic inclusions, some of which were also reactive for β-glucuronidase and glucosaminidase. These cells appear to have a separate origin from the hemocytes.     

Epidermal growth factor-urogastrone receptor: selective alteration in simian virus 40 transformed mouse fibroblasts

Comparative data on the properties of four thiol proteinase inhibitors, and of four serine proteinase inhibitors (two subtilisin and two trypsin inhibitors) isolated from seeds of Vigna are presented. They were similar in their molecular weights (5000–15,000) and dissociation constants (10^?8–10^?9m). The range of isoelectric points of the thiol proteinase inhibitors was 6.5 to 10.6, and of the serine proteinase inhibitors was 5.0 to 5.9. The amino acid compositions of one papain isoinhibitor, one of subtilisin, and one of trypsin are presented. Papain inhibitor A1 and subtilisin inhibitor 2a were low in cystine. All of the inhibitors were stable upon heating to 80 °C for 5 min at low pH. The subtilisin inhibitor did not bind to catalytically inactive subtilisin derivatives, whereas the papain inhibitor was stoichiometrically bound to the Hg or thioacetamide derivatives of papain. Incubation of the subtilisin inhibitor with catalytic amounts of subtilisin led to the formation of a modified form with the same inhibitor activity as the native inhibitor but with a different electrophoretic mobility. There was no indication of a similar modification of the papain inhibitor by papain. Separate sites are present on the trypsin-chymotrypsin inhibitors for trypsin and chymotrypsin. The papain inhibitors have the same binding sites for papain and ficin.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: I. Occurrence of thiol proteinase inhibitors in plants and purification from Vigna unguiculata subsp. cylindrica

Inhibitors of the thiol proteinase, papain (EC 3.4.22.2), were shown to be present in 11 species of 10 genera of plants. The inhibitor activity was nondialyzable, and precipitated by ammonium sulfate. Tissue cultures from a number of plant genera consisting of rapidly dividing cells contained latent papain inhibitor that could be activated upon heating. Four isoinhibitors of plant thiol proteinases from seeds of the legume Vigna unguiculata subsp. cyclindrica were purified to apparent homogeneity by acrylamide gel electrophoresis with or without sodium dodecyl sulfate. The inhibitors were present in very small amounts compared to the trypsin inhibitors and the degree of purification of the homogeneous isoinhibitors on the assumption that all were present initially in equal amounts was 15,000- to 60,000-fold. The isoinhibitors did not inhibit pepsin, bromelain, and the serine proteinases, trypsin, chymotrypsin, and subtilisin. They were specific for papain, chymopapain, and ficin but their inhibition of the proteinase, esterase, and amidase activities of the three enzymes differed.     

Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata)

Morphological characteristics and functions of hemocytes were used to compare the immunological effects of biological and chemical stress in the freshwater snailLymnaea palustris. Animals were either infected by a trematode parasite (Metaleptocephalus sp.), or exposed to environmental contaminants, namely atrazine and hexachlorobenzene (HCB). Three populations of circulating hemocytes, morphologically and cytochemically distinct (round cells, hyalinocytes, granulocytes), were identified in both control and parasitized or pesticide-exposed snails. After 6 h of exposure, HCB and atrazine resulted in 8-fold increases in the mean total number of hemocytes, whereas only a 2.2-fold increase was observed 6 h after cercaria emission in parasitized snails. The impact of HCB was limited to the first 24 h of exposure, whereas long-lasting effects of atrazine were observed. Hyalinocytes and, to a lesser extent, round cells contributed most to the increases in hemocyte density in pesticide-exposed snails. Parasitism and atrazine treatment resulted in significant increases of lectin-stained hemocytes, whereas exposure to HCB did not affect the percentages of stained and unstained cells. Hemocyte phagocytic activity increased in HCB-exposed snails but with no concomitant change of the oxidative burst. Opposite results were obtained in atrazine-treated snail hemocytes, with unchanged phagocytosis and decreased phorbol 12-myristate 13-acetate-stimulated production of reactive oxygen intermediates. No increase in phagocytosis, or in the production of reactive oxygen intermediates, was observed in hemocytes from parasitized snails. Infection with the immunologically compatible trematode parasiteMetaleptocephalus sp. and exposure to atrazine generated similar reactions from circulating hemocytes, whereas a different response pattern was observed in HCB-exposed snails. This revised version was published online in July 2006 with corrections to the Cover Date.     

ACTION OF PAPAIN AND FICIN ON TADPOLES AND ARBACIA EGGS

Living tadpoles and Arbacia eggs are not digested by ficin or papain although the dead organisms are. Arbacia eggs develop in papain solutions but the cells become separated. Development is normal in ficin and trypsin solutions.     

Nitric oxide production by hemocytes of the ascidian Styela plicata

Ascidian hemolymph contains various types of blood cells (hemocytes), which are believed to be involved in defense mechanisms. We have studied nitric-oxide (NO) synthase activity in hemocytes of the ascidian Styela plicata after exposure to lipopolysaccharide (LPS). To investigate which cell types are involved in NO production, we first identified, by electron microscopy, the types of hemocytes previously described, mainly by light microscopy, by others. Five types of blood cells could be recognized in the hemolymph: granulocytes, hemoblasts, lymphocyte-like cells, morula cells, and pigment cells. The lymphocyte-like cells produced the most NO. In agreement with studies of other invertebrates, nitrite generation did not change after LPS stimulation in assays in vitro, under either different concentrations of LPS or different time periods. Therefore, we performed an in vivo assay by injecting a known quantity of Escherichia coli into the tunic of the ascidians in order to investigate possible differences in NO levels. No increase of NO occurred accompanying the inflammatory reaction suggesting that another molecule in the pathway was involved. We found that nuclear factor κB (NFκB) was activated. Since NFκB is involved in the production of many substances related to immune responses, additional molecules might also be generated in response to E. coli infection. These observations may improve our understanding of the reaction of animals to eutrophic conditions.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.     

Hemocyte classification and differential counts in the dungeness crab, Cancer magister

The primary purposes of this research were to describe and classify the circulating hemocytes of Cancer magister and devise a method for making differential hemocyte counts for crustaceans. C. magister hemocytes were classified using two simple criteria: the presence or absence of cytoplasmic granules and staining characteristics of the granules, if present. Hyalinocytes (HC) were devoid of granules, intermediate granulocytes (IG) contained basophilic granules or a mixture of basophilic and acidophilic granules, and eosinophilic granulocytes (EG) contained large, acidophilic granules. Hemocyte renewal and a hypothetical maturation sequence of C. magister hemocytes are described and discussed. Differential counts revealed that granulocytes were more abundant than hyalinocytes. For 22 crabs, the mean percentage (and range) of each hemocyte class was: IG, 65.97 (57.50–73.80); EG, 17.76 (4.70–26.47); and HC, 16.25 (3.40–34.67). After additional data are collected and analyzed, the routine use of differential counts may prove to be a valuable method for monitoring the status and health of C. magister and perhaps other crustaceans as well.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.     

Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations

Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.     

Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts

A plasmalemma-bound NADH oxidation system (Lin 1982 Proc Natl Acad Sci USA 79: 3773-3776) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH stimulated O₂ consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which have been treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 versus 9.4 kilocalories per mole per degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5 to 25°C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K⁺ uptake into cell volume of the protoplast. However, the NADH-stimulated K⁺ uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed one extra peptide band with ~42 kilodalton molecular weight.     

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis.     

A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.