Kenneth Söderhäll

The prophenoloxidase-activating system in invertebrates.

Summary:  A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO‐activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as β‐1,3‐glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti‐microbial, cytotoxic, opsonic, or encapsulation‐promoting activities.

Role of the prophenoloxidase-activating system in invertebrate immunity

The melanization reaction, which is a common response to parasite entry in invertebrate animals, especially arthropods, is due to the activity of an oxidoreductase, phenoloxidase. This enzyme is part of a complex system of proteinases, pattern recognition proteins and proteinase inhibitors constituting the so-called prophenoloxidase-activating system. It is proposed to be a non-self recognition system because conversion of prophenoloxidase to active enzyme can be brought about by minuscule amounts of molecules such as lipopolysaccharide, peptidoglycan and beta-1, 3-glucans from micro-organisms. Several components of this system recently have been isolated and their structure determined.

The proPO-system: pros and cons for its role in invertebrate immunity

Melanisation is an important immune response in many invertebrates. Recent evidence also strongly implies that the melanisation (prophenoloxidase activating) cascade is intimately associated with the appearance of factors stimulating cellular defence by aiding phagocytosis and encapsulation reactions. However, some controversy exists in the field, and at least in flies and mosquitoes, the successful combat of some pathogens does not seem to be dependent on phenoloxidase activity. This may be because of redundancy among separate immune mechanisms, inappropriate testing, species differences or a combination thereof. Recently, by using RNA interference against phenoloxidase or in specific host-pathogen interactions where the pathogen prevents melanin production by the host, convincing data have confirmed the importance of this cascade in invertebrate innate immunity.

Crustacean haemocytes and haematopoiesis

Crustacean haemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and cell-cell communication. Classification of the haemocyte t ...

The proPO and clotting system in crustaceans

Aquaculture production has made impressive progress during the past two decades especially with regards to crustaceans, such as shrimp, prawn and lobster. Research in terms of immunity has received a high priority to control disease and to ensure long-term survival of shrimp culture. Invertebrates, including crustaceans, do not have acquired immunity, instead they have an innate immune system, which includes Ž . melanization by activation of the prophenoloxidase activating system proPO system , a clotting process, phagocytosis, encapsulation of foreign material, antimicrobial action Ž . and cell agglutination Soderhall, 1999 . Research in the area of innate immunity in arthropods is rapidly progressing, whereas shrimp immunity research has been a subject of minor interest compared to similar research performed on other crustaceans and insects. The penaeid shrimp can be a good model to use mainly because they are short lived compared to many other crustaceans. In this paper, we review what is known so far about crustacean defence mechanisms, particularly of penaeid shrimp, with special emphasis on the proPO and the clotting system.

Cellular immunity in crustaceans and the proPO system

The molecular mechanism of cellular immunity in arthropods has until recently been largely unknown, but with the development of a technique to isolate and handle the different blood cell types of crustaceans and with the purification of several proteins associated with the so-called proPO system of freshwater crayfish the processes have now begun to be better understood. In this article Mats Johansson and Kenneth Söderhäll discuss the function of the proPO system in cellular immune reactions in crustaceans and in particular the role of a protein with a molecular mass of 76 kDa, which has been shown to be involved in the communication between the different blood cell types of crayfish.

Cell adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity

The question as to how the immune defence of an invertebrate animal is initiated and coordinated has largely been unanswered. This short review focuses on recent discoveries about crayfish hemolymph proteins, which may play roles in cell adhesion events leading to initiation of phagocytosis and encapsulation. Focus will also be made on anti-oxidative enzymes that may participate in the production of reactive oxygen compounds used in the destruction of engulfed or encapsulated parasites. Peroxinectin is stored in semi-granular and granular hemocytes and released concomitant with activation of prophenoloxidase (proPO). It is a cell adhesion protein, enhancing phagocytosis and encapsulation and triggers degranulation. It is also a peroxidase, belonging to the same protein family as mammalian myeloperoxidase. Peroxinectin binds a 90-kDa peripheral cell surface superoxide dismutase (SOD) of crayfish blood cells. Integrins are transmembrane proteins present on crayfish hemocytes and commonly known to be acting as cell adhesion receptors in many events. After its release and activation, peroxinectin may opsonize foreign surfaces where it is recognized by integrins on the hemocyte. This can be a starting point for phagocytosis or encapsulation. Peroxinectin and extracellular SOD (EC-SOD) may then cooperate during a respiratory burst to destroy an ingested or encapsulated parasite.

A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning.

A lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to β-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and β-1,3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the β-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.

Hemocyte production and maturation in an invertebrate animal; proliferation and gene expression in hematopoietic stem cells of Pacifastacus leniusculus☆

Regulation of hematopoiesis in invertebrates is largely unknown, although the hemocytes are essential in immunity, performing functions such as phagocytosis, encapsulation and lysis of foreign cells. We have developed a method to isolate hematopoietic stem cells from the freshwater crayfish, Pacifastacus leniusculus, and therefore, this animal provides a powerful tool for studies on invertebrate hematopoiesis. The hematopoietic tissue of crayfish was found to be actively proliferating. Injection of a beta1,3-glucan caused a severe loss of hemocytes, followed by a rapid recovery, due to release from the hematopoietic organ. Transcripts for peroxinectin, a hemocyte cell adhesion protein, were present in the hematopoietic cells, whereas mRNA for proPO was not detected. A gene coding for a Runt-domain protein known to be involved in hematopoiesis in Drosophila and mammals, was upregulated prior to hemocyte release.We conclude that hemocytes are synthesised and partly differentiated in the hematopoietic tissue, but the final differentiation into functional hemocytes expressing proPO is not completed until the hemocytes are released into the circulation.

Proteolytic cascades and their involvement in invertebrate immunity

Bacteria and other potential pathogens are cleared rapidly from the body fluids of invertebrates by the immediate response of the innate immune system. Proteolytic cascades, following their initiation by pattern recognition proteins, control several such reactions, notably coagulation, melanisation, activation of the Toll receptor and complement-like reactions. However, there is considerable variation among invertebrates and these cascades, although widespread, are not present in all phyla. In recent years, significant progress has been made in identifying and characterizing these cascades in insects. Notably, recent work has identified several connections and shared principles among the different pathways, suggesting that cross-talk between them may be common.