Primary Hemocyte Research Articles

Molecular and functional characterization of granulin-like molecules of insects

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. Here, we report on the identification of two partial transcripts encoding granulin-like molecules from Aa23 embryonic cells of Aedes albopictus and primary haemocytes from Manduca sexta. Both these partial transcripts had the characteristic 12-cysteine motif that is a hallmark of the granulin family and they represent the first granulin mRNA transcripts identified from insects. Moreover, we demonstrate that the recombinant granulin molecule that we originally cloned in the goldfish, induced proliferation of both Aa23 embryonic cells and primary haemocytes. Interestingly, this proliferative effect was upregulated in the presence of the intracellular symbiotic bacterium Wolbachia pipientis. Thus, granulin appears to be a highly conserved growth factor not only in lower vertebrates but also invertebrates.

Eicosanoids mediate insect hemocyte migration

Hemocyte migration toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating migration in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hypotheses that (1) insect hemocytes are able to detect and migrate toward a source of N-formyl-Met-Leu-Phe (fMLP), the major chemotactic peptide from Escherichia coli and (2) that pharmaceutical modulation of eicosanoid biosynthesis inhibits hemocyte migration. We used primary hemocyte cultures prepared from fifth-instar tobacco hornworms, Manduca sexta in Boyden chambers to assess hemocyte migration toward buffer (negative control) and toward buffer amended with fMLP (positive control). Approximately 42% of negative control hemocytes migrated toward buffer and about 64% of positive control hemocytes migrated toward fMLP. Hemocyte migration was inhibited (by >40%) by treating hornworms with pharmaceutical modulators of cycloxygenase (COX), lipoxygenase and phospholipase A 2 (PLA 2) before preparing primary hemocyte cultures. The influence of the COX inhibitor, indomethacin, and the glucocorticoid, dexamethasone, which leads to inhibition of PLA 2, was expressed in a dose-dependent way. The influence of dexamethasone was reversed by injecting arachidonic acid (precursor to eicosanoid biosynthesis) into hornworms before preparing primary hemocyte cultures. The saturated fatty acid, palmitic acid, did not reverse the inhibitor effect. These findings support both our hypotheses, first that insect hemocytes can detect and respond to fMLP, and second, that insect hemocyte migration is mediated by eicosanoids.

Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways

Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO is produced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO production in these cells is poorly understood. The present study focused on the role of cell signalling pathways in NO production by primary haemocytes from the snail Lymnaea stagnalis. When haemocytes were challenged with PMA (10 microM) or the beta-1,3-glucan laminarin (10 mg/ml), an 8-fold and 4-fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase) inhibitors L-NAME (N(G)-nitro-L-arginine methyl ester) and L-NMMA (N(G)-monomethyl-L-arginine) were found to block laminarin- and PMA-induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased the phosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKC inhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular-signal-regulated kinase) pathway (PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO production were observed following exposure to laminarin or PMA. The greatest effect on NO production was seen with GF109203X and U0126, with PMA-induced NO production inhibited by 94% and 87% and laminarin-induced NO production by 50% and 91% respectively. These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOS activation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first report that shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.

Development of primary shrimp hemocyte cultures of Penaeus chinensis to study white spot syndrome virus (WSSV) infection

Hemocytes were withdrawn from hearts of Penaeus chinensis, cultured with seven kinds of culture media, the hemocytes remained viable for about 20 days using Leibovitz's 2 × L-15 medium consisting of 20% fetal bovine serum, 20% GIT, and 2% glucose, adjusting osmolarity with sodium chloride, pH 7.2–7.4, and incubated at a temperature of 25 °C. Different concentrations of virus inoculum were inoculated into the primary cultured hemocytes, and diverse cytopathic effects were observed with an inverted phase-contrast microscope. WSSV infection was confirmed by immunocytochemical tests with monoclonal antibodies against the virus. These revealed that WSSV could infect hemocytes of P. chinensis and propagate in the nuclei of infected cells.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

Modes of phagocytosis of Gram-positive and Gram-negative bacteria by Spodoptera littoralis granular haemocytes

Haemocytes are the main immunocompetent cells in insect cellular immune reactions. Here, we show that in Spodoptera littoralis, granular haemocytes are the primary phagocyte haemocytes, both in vivo and in vitro. The “trigger” and “zipper” modes of engulfment known in mammal macrophages are active, in vivo, in S. littoralis granular haemocytes, together with macropinocytosis. Lipopolysaccharide as well as lipoteichoic acid inhibit the binding of both Gram-positive ( Corynebacterium xerosis) and Gram-negative ( Escherichia coli) bacteria on granular haemocytes. In addition, different ligands can inhibit the binding of E. coli. Most of these inhibitors are known as ligands of scavenger receptors in mammal macrophages and we hypothesise that one of the receptors present on S. littoralis granular haemocytes could be a scavenger-like receptor.

Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.

We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.

Deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car 1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car 1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Total Haemocyte Counts and Haematopoiesis in Saduria Entomon (Linnaeus, 1758) (Isopoda, Valvifera) From the Baltic Sea Infected With the Yeast Cryptococcus La Urentii (Kufferath) Skinner*)

AbstractStudies were made of the haemolymph and haematopoiesis of 22 specimens of Saduria entomon, naturally infected with the yeast Cryptococcus laurentii. In 18 of these haemocytes were also counted. Their total haemocyte count was 693, which is 3 times the figure in healthy S. entomon. Their haemolymph contained granulocytes, hyaline cells, and C. laurentii cells. Granulocytes contained phagocytosed C. laurentii cells, while hyaline cells did not contain phagocytosed yeast. Haematopoiesis was clearly taking place in the gut wall of the S. entomon specimens under study. In the 4 remaining individuals the haemolymph was filled with C. laurentii cells among which only single hyaline cells with phagocytosed yeast cells were observed. In their gut wall only yeast cells accumulated. The results obtained support our earlier findings that in S. entomon granulocytes are the primary phagocytotic haemocytes, and that hyaline cells can be taken to be prohaemocytes capable of proliferation under specific conditions.

Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens

Microplitis demolitor polydnavirus (MdPDV) is associated with Microplitis demolitor, a parasitic wasp that attacks the larval stage of the lepidopteran Pseudoplusia includens. Previously, we observed that MdPDV induced several alterations in the granular cells and plasmatocytes of P. includens, the primary haemocytes involved in regulating the cellular immune response toward M. demolitor and other parasites. In examining the mechanisms underlying immunosuppression of this host, we found that MdPDV induced apoptosis of granular cells. Granular cells underwent apoptosis both when virus was injected into the haemocoel of P. includens larvae and after infection with MdPDV in vitro. Characteristics of MdPDV-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of DNA into a 200 bp ladder. Although MdPDV induced changes in the ability of plasmatocytes to adhere to foreign surfaces, apoptosis of this morphotype was not observed. Examples from the literature suggest that some viruses promote their own survival by suppressing apoptosis of host cells. However, since polydnaviruses are likely to be transmitted vertically, we suggest that MdPDV promotes its own survival by inducing apoptosis of host immune cells which would otherwise kill the developing M. demolitor egg.