Crayfish Hemocytes Research Articles

The cytotoxic reaction of hemocytes from the freshwater crayfish, Astacus astacus

Crayfish hemocytes displayed cytotoxic capacity towards all tested mammalian tumor and nontumor cell lines. The ratio required for the cytotoxic action of effector cells to target cells was at least 1:1. The lysis of the target cells required a minimum of 1 hr to become detected. After separation and isolation of the hemocyte populations of crayfish, the semigranular and granular cells retained their cytotoxic capacity. These cells contain the prophenoloxidase activating (proPO) system, a complement-like pathway, which in an activated form lyses semigranular cells in vitro, but failed to kill the tested target cells.

Exocytosis of the prophenoloxidase activating system from crayfish haemocytes

Lipopolysaccharides (LPS) and the β-1,3-glucan laminarin G, both of which specifically activate the prophenoloxidase (proPO) activating system of crayfish haemocyte lysate, were found to induce degranulation (exocytosis) and subsequent lysis in vitro of monolayers of semigranular haemocytes from the crayfish,Pacifastacus leniusculus, (Table 1, Fig. 1 b), whereas the granular cells were unaffected (Fig. 1 c). Exocytosis of isolated semigranular or granular cells in vitro could also be evoked by the Ca2α ionophore A23187 (Table 2, Fig. 1 d). In this case, the whole proPO system was released from the cellular vesicles in its inactive form, since the secreted material contained protease and prophenoloxidase as inactive proenzymes, which could be activated if LPS or β-1,3-glucans were added (Table 3). The anion channel blocker SITS, which inhibits exocytosis in several systems, prevented degranulation triggered by β-1,3-glucan, LPS, or ionophore. It is concluded that, in arthropods, LPS serve as an indicator of Gram negative bacteria and β-1,3-glucan as an indicator of fungi. These non-self molecules elicit both the exocytotic release of the proPO system from the semigranular cells and the subsequent biochemical activation of this system.

Hemocyte lysate enhancement of fungal spore encapsulation by crayfish hemocytes

Crayfish hemocytes exhibited a stronger encapsulation reaction to fungal blastospores of Beauveria bassiana coated with hemocyte lysate, than to blastospores treated with plasma or buffer, indicating an opsonic function of hemocyte lysate proteins. Five proteins of the prophenoloxidase activating system in the hemocytes were attached to foreign surfaces (including the blastospores) after activation and it is suggested that these attaching proteins (one being phenoloxidase) are responsible for the opsonic function of the hemocyte lysate on crayfish blood cells.

Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte lysate

Lipopolysaccharides were found to activate a protease, which in turn triggered activation of prophenoloxidase, both enzymes present in crayfish haemocytes. Minute amounts of lipopolysaccharides (10 −10 g/ml) enhanced protease activity, whereas high concentrations (10 −4 g/ml) prevented activation. A lipopolysaccharide-inhibited protease preparation could be activated by β1,3-glucans, showing that these non-self molecules triggered activation of the prophenoloxidase activating system, in which both the protease and prophenoloxidase are constituents, in two different pathways. It is therefore suggested that the prophenoloxidase activating system is an important recognition system in invertebrates and has interesting similarities with the complement system of vertebrates.

The prophenoloxidase activating system in crayfish

1. 1. A preparation (designated 0–40 fraction) containing stable prophenoloxidase (proPO) and other dormant components of the prophenoloxidase activating system was obtained from crayfish hemocytes. 2. 2. Activation of proPO in the 0–40 fraction was elicited by β1,3-glucans, SDS, trypsin or heat, while a protease inhibitor, p-NPGB, inhibited activation of proPO by β1,3-glucans but, not activation by SDS or heat. 3. 3. Ca 2+ was always necessary for the activation of proPO and treatment of the 0–40 fraction with EDTA caused irreversible inactivation of proPO activating system, seemingly leaving proPO intact. 4. 4. The enzyme responsible for activating proPO could be separated from proPO and this enzyme was found to be inhibited by p-NPGB. This enzyme could activate proPO in the 0–40 fraction treated with EDTA. 5. 5. Protease activity increased more than 10-fold in the 0–40 fraction after the incubation with β1,3-glucans and Ca 2+. 6. 6. The proPO activating system may operate as a recognition system in crayfish, and it is suggested that this system functions as a complement-like system in arthropods.

Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro.

To study the role of the prophenoloxidase activating system, an enzyme cascade located in the haemocytes of crustaceans, in the cellular defences of the freshwater crayfish, Astacus astacus in vitro, monolayer cultures of mixed or separated haemocyte populations, isolated by density gradient centrifugation, were challenged with the bacterium, Moraxella sp. pre-coated with phenoloxidase and the other attaching proteins in crayfish haemocyte lysate (HLS), or in the case of controls, with saline or Moraxella sp. pre-incubated in saline alone. Examination of the coverslips 1 h after inoculation revealed that, in the mixed haemocyte cultures, most of the cells had undergone profound degranulation and lysis following exposure to the HLS-coated bacteria. Cell lysis was also evident in the experimental semigranular cell monolayers, but not in the controls, although in those controls treated with the saline-incubated bacteria, the semigranular haemocytes had undergone degranulation without lysis. In contrast, the granular cells appeared to be unaffected by the saline-incubated Moraxella sp., and with the HLS-coated bacteria displayed only marked degranulation. Greater numbers of bacteria were always associated with the cells or cell remnants in the experimental cultures compared to the controls. We suggest that the attaching proteins of the prophenoloxidase cascade are strong nonself signals for the haemocytes, causing them to degranulate and release previously cell-bound recognition factors into the haemolymph, where they are free to trigger activation of adjacent haemocytes.

β-l, 3 GLUCAN ACTIVATION OF CRUSTACEAN HEMOCYTESIN VITROANDIN VIVO

The effects of 13-1 ,3 glucans on the hemocytes of the freshwater crayfish, Astacus astacus, and the shore crab, Carcinus maenas, were studied in vitro and in vivo to determine the role ofthe prophenoloxidase activating system, in the cellular defense reactions of crustaceans. In vitro, phagocytosis of the bacterium, Moraxella sp. was significantly raised by addition of laminarin, a $-l ,3 glucan, simultaneously with the test particles to the hemocytes in monolayer cultures. Both the proportion of cells ingesting one or more bacterial particles and the number of bacteria taken up by individual cells were increased, and the responses were found to be time dependent and dose related. Glucose, dextran, cellulose, and chitin had no stimulatory influence on the cells, and the agglutination of erythrocytes by crab hemocytes or serum was unchanged by glucan incubation. Examination of the monolayers under phase contrast mi croscopy, revealed that the glucans induced degranulation and occasionally lysis in the crayfish hemocytes, and vacuolation in the crab hemocytes. In vivo, injection of f3-1,3 glucans (0.2 mg laminaran/ml hemolymph) into the hemocoel of A. astacus or C. maenas caused a rapid, marked reduction in the number of circulating hemocytes, indicating that a cellular defense reaction was initiated. Since prophenoloxidase in the hemocytes is specifically activated by /3-1,3 glucans, and in the activated form phenoloxidase is “?�sticky,” it is suggested that certain proteins of the prophenoloxidase activating system may serve as opsonins, and possibly constitute an important recognition mechanism in crustaceans.

Isolation and properties of a protease inhibitor in crayfish ( Astacus astacus) cuticle

1. 1. A protease inhibitor isolated from the cuticle of the crayfish, Astacus astacus, was found to be active against subtilisin and protease(s) of the fungus, Aphanomyces astaci, but not trypsin, chymotrypsin and A. laevis protease(s), when using casein as substrate. 2. 2. The inhibitor had similar tolerance towards physicochemical treatments, the same specificity and identical antigenic properties as the protease inhibitors present in the crayfish haemocytes. This suggests that the inhibitor in the cutilce could originate from the haemocytes. 3. 3. The possible biological function of the protease inhibitor in the cuticle is discussed.

β-1,3-Glucan enhancement of protease activity in crayfish hemocyte lysate

1. 1. A serine protease is present in crayfish haemocyte lysate, which is involved in activation of prophenoloxidase and a coagulation process. Since prophenoloxidase can be activated by β-1,3-glucans, experiments were carried out to determine if these carbohydrates exert some effect on the serine protease. 2. 2. The results showed that the serine protease can be specifically activated by the glucans and that a similar response is produced by a linear pentaose with (1 → 3)-β- d-glycosyl residues. 3. 3. The serine protease was highly specific and hydrolyzed chromogenic substrates such as Bz-Ile-Glu-( γ- O-Piperidyl)-Gly-Arg-PNA-HCl. 4. 4. The activity of the glucan-treated protease was found to be short lived, and it is suggested that this could be an important mechanism in the regulation of a coagulation process and prophenol-oxidase activation.

Light and time correlated migration of invasive hemocytes in the crayfish compound eye

AbstractThe number and radial distribution of hemocytes discovered periodically invading the retinas of crayfish compound eyes (Figs. 1–3) were measured under a variety of light and dark adaptation programs (Fig. 4, Table 1). Overall the density of these cells in the eye was significantly less in light than in darkness (Figs. 5–8, Table 2). The corresponding hemocyte ebb and flow occurred in intercellular spaces between retinulas, not in blood vessels which are absent from decapod crustacean eyes everywhere distal to the basement membrane.Hemocytes under all conditions were most dense just below the basement membrane. Within the retina their occurrence ranged from maximum at the basement membrane to minimum or zero near the distal tip of the rhabdom, beyond which they rarely occurred (Figs. 5, 6, Table 3). The effectiveness of light in reducing hemocyte concentrations was maximal distally and minimal proximally and hence had a gradient inverse that for cell density (Fig. 8).Bilateral experiments with one eye covered and the other exposed showed that light could independently and unilaterally induce hemocyte emigration from the retina (Fig. 5). But in time‐of‐day experiments hemocyte movements occurring before the programmed cyclic diurnal illumination changes proved that endogenous control mechanisms must also participate (Fig. 7). Such experiments also showed the fewest retinal hemocytes (≈︁0) at 18:00 hours, when the rate of rhabdom synthesis should be near maximum.So far no direct phagocytic uptake of identifiable photoreceptor membrane has been found in crayfish retinal hemocytes. Nevertheless these cells were seen to make junctionlike contacts with retinular cells and appeared to be actively ingesting interstitial detritus in the retina at levels coincident with the radial extent of the rhabdoms (Figs. 1–3). Previously phagocytic wandering cells were known to participate in photoreceptor membrane turnover in teleost eyes and in reptile pineal organs although fixed retinal pigment epithelial cells are normally involved in this function in birds and mammals.

Light and electron microscopic studies of crayfish hemocytes.

Using light and electron microscopy, three hemocyte types are described in the hemolymph of the crayfish. The coagulocyte comprises 65% of the total hemocyte number and contains medium-sized cytoplasmic granules, abundant dilated rough endoplasmic reticulum, and a highly developed Golgi complex. It rapidly undergoes cytolysis in vitro and participates in coagulation by releasing the contents of its granules to the hemolymph. The granulocyte comprises 31% of the total hemocyte number and is capable of phagocytosis. It contains large, irregularly shaped cytoplasmic granules, a moderately developed Golgi complex, and moderate amounts of non-dilated rough endoplasmic reticulum. During coagulation in vitro, the cell attaches and spreads onto the substratum; this is followed by a slow intracellular granule breakdown and cytolysis. The amebocyte comprises 4% of the total hemocyte number and it is also capable of phagocytosis. It possesses small cytoplasmic granules, many vacuoles, a moderately developed Golgi complex, and large amounts of smooth endoplasmic reticulum. It is distinguished from the other two cell types by being stable and motile in vitro.

Immunity in the invertebrates. The role of serum factors in phagocytosis of erythrocytes by haemocytes of the freshwater crayfish (Parachaeraps bicarinatus).

SummaryAn in vitro cell culture system is described which allows a quantitative study of the phagocytosis of erythrocytes by haemocytes from the freshwater crayfish (Parachaeraps bicarinatus). The studies showed that specific senmi opsonins were essential for efficient phagocytosis. These results are discussed in relationship to similar findings in other invertebrates and to the evolution of vertebrate antibodies.