Apoptotic Hemocytes Research Articles

Quantitative assessment of apoptotic hemocytes in white spot syndrome virus (WSSV)-infected penaeid shrimp, Penaeus monodon and Penaeus indicus, by flow cytometric analysis

A study was carried out to determine the percentage of apoptotic hemocytes in WSSV-infected Penaeus monodon and Penaeus indicus at different time intervals by DNA fragmentation assay using flow cytometry and agarose gel electrophoresis. Shrimp were injected with WSSV, and hemolymph at different time intervals was collected to determine apoptotic hemocytes. Apoptotic hemocytes were increased in course of infection and were found to be higher in P. indicus in comparison with P. monodon. Furthermore, DNA fragmentation of WSSV-injected hemocytes was also evaluated by the agarose gel electrophoresis and characteristic intranucleosomal DNA ladder patterns were detected in the DNA extracted from hemocytes of WSSV-infected shrimp, but not in the DNA of hemocytes from uninfected shrimp.

Apoptosis and Adhesion of Hemocytes During Molting Stage of Silkworm, Bombyx mori

To clarify the regulatory mechanism of the rapid changes in the hemocyte density in the silkworm, Bombyx mori, during ecdysis, we evaluated the relationship between the hemocyte density and the incidence of apoptosis during this stage. We also evaluated the role of the sugar chains on the adhesion of hemocytes by analyzing the effects on the hemocyte density of the injection of enzymes that cut sugar chains and monosaccharides into the body cavity. The hemocyte density was increased in the molting stage and spinning, and then decreased after the ecdysis. During spinning, the diameter of the granulocytes markedly increased, in which fatty granules in the cytoplasm increased, becoming foamy. They were identified to be apoptotic hemocytes using the Hoechst staining and the Comet assay. The decrease in the hemocyte density during spinning was mainly caused by the apoptosis of granulocytes. Next, we focused on the fluctuation of hemocyte density during the molting stage. Examination of the changes in the hemocyte density induced by injecting glycoside hydrolases, neuraminidase, sialic acid, or monosaccharides into the body cavity during the fourth molt stage and the third day in fifth instar larva demonstrated that the alteration of hemocyte density was regulated by the attachment and detachment of hemocytes via a selectin ligand, sugar chains. As with the injection of glycoside hydrolase, neuraminidase, sialic acid and fucose raised the hemocyte detachment, and it was assumed that the selectin ligands include the sialyl Lewis x like sugar chains, the same as mammalian lymphocytes.

Branchial lesions associated with abundant apoptotic cells in oysters Ostrea edulis of Galicia (NW Spain)

An experiment to evaluate differences in growth, mortality and disease susceptibility among Ostrea edulis stocks was performed. Five families were produced from each of 4 oyster populations (Irish, Greek and 2 Galician). The spat were transferred to a raft in the Ria de Arousa (Galicia, Spain) for grow-out. Monthly samples of each family were histologically processed from 2001 to 2003. One of the pathological conditions discovered by this study was the occurrence of extensive branchial lesions characterized by haemocytic infiltration and loss of branchial architecture. Furthermore, abundant atypical cells occurred among the haemocytes in the lesions in the branchial connective and epithelial tissues, but rarely in the mantle. These cells were contracted in size with nuclei showing chromatin condensation and fragmentation. Some nuclear chromatin aggregated under the nuclear membranes into crescent shapes, whereas others were uniformly dense. Those characteristics suggested that the cells were apoptotic haemocytes, which was confirmed by transmission electron microscopy (TEM) and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay using the Apoptag Kit on paraffin sections. A low prevalence of gill lesions was detected in some, but not all, families of every origin peaking in July 2002 and April 2003. No etiologic agent was identified by either histology or TEM; thus, the cause of the abundance of apoptotic cells remains unclear.

White spot syndrome virus (WSSV) interaction with crayfish haemocytes

WSSV particles were detected in separated granular cells (GCs) and semigranular cells (SGCs) by in situ hybridisation from WSSV-infected crayfish and the prevalence of WSSV-infected GCs was 5%, whereas it was 22% in SGCs. This indicates that SGCs are more susceptible to WSSV and that this virus replicated more rapidly in SGCs than in GCs and as a result the number of SGCs gradually decreased from the blood circulation. The effect of haemocyte lysate supernatant (HLS), containing the degranulation factor (peroxinectin), phorbol 12-myristate 13-acetate (PMA), the Ca 2+ ionophore A23187 on GCs from WSSV-infected and sham-injected crayfish was studied. The results showed that the percentage of degranulated GCs of WSSV-infected crayfish treated with HLS or PMA was significantly lower than that in the control, whereas no significant difference was observed when treated with the Ca 2+ ionophore. It was previously shown that peroxinectin and PMA have a degranulation effect via intracellular signalling involving protein kinase C (PKC), whereas the Ca 2+ ionophore uses an alternative pathway. HLS treatment of GCs and SGCs from WSSV-infected crayfish results in three different morphological types: non-spread, spread and degranulated cells. The non-spread cell group from both GCs and SGCs after treatment with HLS had more WSSV positive cells than degranulated cells, when detected by in situ hybridisation. Taken together, it is reasonable to speculate that the PKC pathway might be affected during WSSV infection. Another interesting phenomenon was that GCs from non-infected crayfish exhibited melanisation, when incubated in L-15 medium, while no melanisation was found in GCs of WSSV-infected crayfish. However, the phenoloxidase activities of both sham- and WSSV-injected crayfish in HLS were the same as well as proPO expression as detected by RT-PCR. This suggests that the WSSV inhibits the proPO system upstream of phenoloxidase or simply consumes the native substrate for the enzyme so that no activity is shown. The percentage of apoptotic haemocytes in WSSV-infected crayfish was very low, but it was significantly higher than that in the sham-injected crayfish on day 3 or 5 post-infection. The TEM observation in haematopoietic cells (hpt cells) suggests that WSSV infect specific cell types in haematopoietic tissue and non-granular hpt cells seem more favourable to WSSV infection.

An Entomopathogenic Bacterium, Xenorhabdus nematophila. Causes Hemocyte Apoptosis of Beet Armyworm, Spodoptera exigua

Entomopathogenic nematode, Steinernema carpocapsae, carries the symbiotic bacterium, Xenorhabdus nematophila, and enter host insect hemocoel. In the infected host, nematodes release the bacteria, which in turn cause lethal hemolymph septicemia. This study was designed to test a hypothesis that hemolymph septicemia of beet armyworm, Spodoptera exigua, is triggered by X. nematophila through a programmed cell death (= apoptosis) of the hemocytes. Morphological changes of the hemocytes after the bacterial infection were similar to cell changes undergoing a typical apoptosis. At 4-8 h post-infection, the cell membrane blebbing and apoptotic vesicles were observed and the nuclear membrane was broken apart. At 12 h postinfection, the overall cell shape was lost externally and nuclear condensation was evident internally, where genomic DNA was fragmented into small pieces. Proportion of apoptotic hemocytes increased with postinfection time and reached a maximum level when the hemocytes were in complete septicemia. Increase of FAD-glucose dehydrogenase (EC 1.1.99.10) activity preceded initial hemocyte apoptosis after the bacterial infection. The infected plasma or the culture broth of X. nematophila contained apoptosis-inducing factors. The apoptosis factors were extracted into organic fraction and turned out to be relatively high hydrophobic chemical nature after further fractionation assay. These results indicate that X. nematophila induces hemocyte apoptosis by its secretory material(s), which lead to hemolymph septicemia and immunodepression of the target insect hosts.

4-Nonylphenol induces immunomodulation and apoptotic events in the clam Tapes philippinarum

The effects of 4-nonylphenol (NP) on functional responses of haemocytes from the clam Tapes philippinarum were investigated after 7 d exposure to sublethal NP concentrations (0, 0 + ace- tone, 0.025, 0.05, 0.1 and 0.2 mg l -1 NP). Haemocytes from both controls and exposed clams were col- lected, and the effects of NP on uptake of the vital dye Neutral Red (NR), superoxide dismutase (SOD) and lysozyme activities, total haemocyte count (THC) and volume of circulating cells were evaluated. The capability of NP to induce apoptosis was also investigated. Exposure of clams to 0.2 mg l -1 NP significantly increased (p < 0.05) NR uptake when compared with controls, suggesting that NP caused alterations in cell membrane stability. Significant decreases in both SOD and lysozyme activ- ity were observed from 0.05 mg l -1 NP (p < 0.01 and p < 0.05, respectively) with respect to controls, indicating that NP causes oxidative stress and reduces the immunocompetence of the exposed clams. From 0.05 mg l -1 NP, the apoptotic index (percentage of haemocytes showing positivity to the TUNEL reaction) significantly increased (p < 0.001). Apoptotic haemocytes showed shrinkage and lost their amoeboid shape. Moreover, NP exposure significantly increased THC (p < 0.05 at 0.2 mg l -1 NP) and caused a different distribution in size frequency. On the basis of the threshold concentration values, the most sensitive endpoints for NP were SOD, lysozyme and apoptosis assays. Our results highlight a relationship between NP exposure and changes in the functional responses of haemocytes, sug- gesting that the contaminant induces immunomodulation in T. philippinarum, mainly by altering cell morphology, cell membrane stability and enzymatic activities, and by promoting oxidative stress and apoptotic events.

Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian Botryllus schlosseri.

Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating professional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution.