Integrin β1 subunit from Ostrinia furnacalis hemocytes: Molecular characterization, expression, and effects on the spreading of plasmatocytes

When lepidopteran larvae are infected by a large quantity of pathogens or parasitized by nonadaptive parasitoids, hemocytes in the hemocoel will encapsulate these foreign invaders. Cellular encapsulation requires hemocytes, particularly plasmatocytes, to change their states from nonadhesive, spherical cells into adhesive, spreading cells. However, it is unclear how the changes of plasmatocytes are regulated. Here we report that the integrin β1 subunit from hemocytes of Ostrinia furnacalis (Ofint β1) plays an important role in regulating the spreading of plasmatocytes. The full length cDNA sequence (4477 bp) of Ofint β1 was cloned from hemocytes. Phylogenetic analysis showed that Ofint β1 belonged to the integrin βPS family of Drosophila melanogaster with highest sequence identity (78.7%) to the β-integrin of Pseudoplusia includens. Structural analysis of the deduced amino acid sequence indicated that Ofint β1 had similar functional domains to known β-integrins in other lepidopteran insects. RT-PCR, Northern blotting, Western blotting and immunohistochemical analyses showed that OfINT β1 was expressed mainly in hemocytes, especially in plasmatocytes, and weakly in fat body, Malpighian tubes and epidermis. After hemocytes had spread onto slides, fewer antibodies to OfINT β1 bound to the surface of plasmatocytes. Furthermore, anti-OfINT β1 serum clearly inhibited the spreading of plasmatocytes. Together these results indicate that OfINT β1 may play an important role in regulating the spreading of plasmatocytes.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

Differential adhesion of the haemocytes of Leucophaea maderae (Blattaria) to a glass surface

A system for the study of insect haemocytes in vitro is described. The system was used to analyse the adhesive properties of the haemocytes of the cockroach, Leucophaea maderae. The two main types of haemocytes, plasmatocytes and granulocytes, showed considerable differences in adhesive properties, which allowed the production of nearly homogeneous monolayers consisting of either plasmatocytes or granulocytes. The much stronger adhesion of the plasmatocytes is discussed in relation to their role in phagocytosis and encapsulation.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

Plasmatocytes from the moth Pseudoplusia includens induce apoptosis of granular cells

The primary immune response toward internal parasites and other large foreign objects that enter the insect hemocoel is encapsulation. Prior studies indicated that granular cells and plasmatocytes are the two hemocyte types required for capsule formation by the moth Pseudoplusia includens (Lepidoptera: Noctuidae). Capsules formed by P. includens also have a defined architecture with primarily granular cells attaching directly to the target, multiple layers of plasmatocytes adhering to this inner layer of granular cells, and a monolayer of granular cells attaching to the capsule periphery. Dye-exclusion assays indicated that granular cells die shortly after attaching to the capsule periphery, leaving a basal lamina-like layer around the capsule. In examining the mechanisms underlying granular cell death, we found that culture medium preconditioned by plasmatocytes induced apoptosis of granular cells. Characteristics of plasmatocyte-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of nuclear DNA. Plasmatocyte-conditioned medium did not induce apoptosis of other hemocyte types, and medium conditioned by other hemocyte types did not induce apoptosis of granular cells. The adhesive state of granular cells and plasmatocytes also affected levels of apoptosis. Conditioned medium from spread plasmatocytes induced higher levels of granular cell apoptosis than medium conditioned by unspread plasmatocytes. Reciprocally, spread granular cells underwent significantly higher rates of apoptosis than unspread granular cells in medium conditioned by spread plasmatocytes. In situ analysis indicated that granular cells on the periphery of capsules also undergo apoptosis. Collectively, our results suggest that spread plasmatocytes release one or more factors that induce apoptosis of granular cells, and that this response is important in the final phases of capsule formation.     

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.     

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.     

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.     

Interference with function of plasmatocytes of Heliothis virescens in vivo by calyx fluid of the parasitoid Campoletis sonorensis

Summary Immature stages of the ichneumonid parasitoid, Campoletis sonorensis, develop within the haemocoel of its noctuid host, Heliothis virescens. The host cannot encapsulate the parasitoid egg owing to the suppressive effect of the polydnavirus-laden calyx fluid injected by the female parasitoid during oviposition. We have examined the effects of injection of calyx fluid on the following haemocytic manifestations of the immune system of 5th-instar larvae of H. virescens: encapsulation, nodulation, phagocytosis, erythrocyte rosetting and coagulation. Of these phenomena, only those requiring the formation of a multicellular sheath of plasmatocytes were affected. In general, encapsulation was fully suppressed; all of the C. sonorensis eggs and most of the glass rods implanted as targets were devoid of attached haemocytes 3 days after implantation although a few of the latter were coated by a sparsely distributed layer of granulocytes. Plasmatocytes also appeared to be present in thicker depositions of haemocytes. In nodulation, only the second, encapsulation-like phase was inhibited. The resistant first stage, involving the entrapment of particles by haemocytes, only resulted in the formation of amorphous, disorganized nodules. Granulocyte-dependent aspects of the immune system (phagocytosis, rosetting and possibly coagulation and the first stage of encapsulation and nodulation) occurred normally. The data suggest that in 5th-instar hosts injection of calyx fluid acts specifically on plasmatocyte function.     

Hemocytic encapsulation of implants in the tickDermacentor variabilis

Implants of Epon, inserted inDermacentor variabilis (Say) through incisions in the cuticle, were encapsulated by hemocytes. We followed this process at intervals of 1, 3, 6, 12 and 24 h, and every 24 h thereafter up to 120 h. Degranulation of Type 1 granulocytes and coagulation of hemolymph were first seen at 1 h after implantation and were the earliest evidence of encapsulation. By 3 h after implantation, the degranulation and disintegration of granulocytes had formed a matrix at the Epon surface. From 6 h until encapsulation was completed, plasmatocytes and granulocytes continued to respond to degranulation and formed multiple cell layers around the Epon implant. The capsule was complete at 72 h after implantation. Completion was marked by decreasing degranulation, migration of hemocytes from the outermost layers of the capsule, and by the appearance of loosely attached hemocytes on the outer surface of the capsule. The most common junctional complex observed was gap junctions.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera,Carabidae)

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.     

Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4 h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8 h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR.     

Plasmatocyte spreading peptide induces spreading of plasmatocytes but represses spreading of granulocytes.

In most Lepidoptera, plasmatocytes and granulocytes are the two hemocyte classes capable of adhering to foreign targets. Previously, we identified plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it induced plasmatocytes to rapidly spread on foreign surfaces. Here we examine whether the response of plasmatocytes to PSP1 was influenced by cell density or culture conditions, and whether PSP1 affected the adhesive state of granulocytes. Plasmatocyte spreading rates were clearly affected by cell density in the absence of PSP1 but spreading was density independent in the presence of PSP1. PSP1 also induced plasmatocytes in agarose-coated culture wells to form homotypic aggregations rather than spread on the surface of culture wells. In contrast, granulocytes rapidly spread in a density independent manner in the absence of PSP1, but were dose-dependently inhibited from spreading by the addition of peptide. An anti-PSP1 polyclonal antibody neutralized the spreading activity of synthetic PSP1. This antibody also neutralized the plasmatocyte spreading activity of granulocyte-conditioned medium, and significantly delayed plasmatocyte spreading when cells were cultured at a high density in unconditioned medium. These results suggested that the spreading activity derived from granulocytes is due in part to PSP1. Pretreatment of plasmatocytes with trypsin had no effect on PSP1-induced aggregation but PSP1-induced aggregations were readily dissociated by trypsin. This suggested that PSP1 is not an adhesion factor but induces adhesion by stimulating a change in the cell surface of plasmatocytes. Synthetic PSP1 also induced aggregation of plasmatocytes from other Lepidoptera indicating that regulation of hemocyte activity by PSP1-related peptides may be widespread. Arch.     

Parasitism and venom of ectoparasitoid Scleroderma guani impairs host cellular immunity

Venom is a prominently maternal virulent factor utilized by parasitoids to overcome hosts immune defense. With respect to roles of this toxic mixture involved in manipulating hosts immunity, great interest has been mostly restricted to Ichneumonoidea parasitoids associated with polydnavirus (PDV), of which venom is usually considered as a helper component to enhance the role of PDV, and limited Chalcidoidea species. In contrast, little information is available in other parasitoids, especially ectoparasitic species not carrying PDV. The ectoparasitoid Scleroderma guani injects venom into its host, Tenebrio molitor, implying its venom was involved in suppression of hosts immune response for successful parasitism. Thus, we investigated the effects of parasitism and venom of this parasitoid on counteracting the cellular immunity of its host by examining changes of hemocyte counts, and hemocyte spreading and encapsulation ability. Total hemocyte counts were elevated in parasitized and venom‐injected pupae. The spreading behavior of both granulocytes and plasmatocytes was impaired by parasitization and venom. High concentration of venom led to more severely increased hemocyte counts and suppression of hemocyte spreading. The ability of hemocyte encapsulation was inhibited by venom in vitro. In addition to immediate effects observed, venom showed persistent interference in hosts cellular immunity. These results indicate that venom alone from S. guani plays a pivotal role in blocking hosts cellular immune response, serving as a regulator that guarantees the successful development of its progenies. The findings provide a foundation for further investigation of the underlying mechanisms in immune inhibitory action of S. guani venom.     

Cloning,expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

Abstract Chitinase catalyzes β‐1,4‐glycosidic linkages in chitin and has attracted research interest due to it being a potential pesticide target and an enzymatic tool for preparation of N‐acetyl‐β‐D‐glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, OfCht5, the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1 665‐bp nucleotides. Phylogenic analysis suggested OfCht5 belongs to the Group I insect chitinases. Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate precipitation and metal chelating chromatography, about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supernatant. OfCht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward α‐chitin. When chitooligosaccharides [(GlcNAc)_n, n= 3–6] were used as substrates, OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)₄ and predominantely hydrolyzed the second glycosidic bond from the non‐reducing end. Together with β‐N‐acetyl‐D‐hexosaminidase OfHex1, OfCht5 achieved its highest efficiency in chitin degradation that yielded N‐acetyl‐β‐D‐glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of OfCht5 versus OfHex1 in the range of 9 : 1–15 : 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

Arthropod immune system. V. Activated immunocytes (granulocytes) of the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) show increased number of microtubules and nuclear pores during immune reaction to foreign tissue

The German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) has two major immunocytes (blood cells) (granulocytes (GRs) and plasmatocytes). The GRs participate both in encapsulation and phagocytosis of nonself tissue. Structurally, the GRs are flattened and discoid, and contain, among other organelles, microtubules that are arranged in the form of a bundle in their peripheral region in the plane of flattening. If one implants a foreign tissue in the cockroach's abdomen, the GRs become activated and begin to encapsulate the implant by flattening and wrapping around it. The activated GRs show considerable increase in the number of both the microtubules and the nuclear pores of the nuclear envelope. Such structural changes in an activated arthropod immunocyte and their functional significance in its immune reaction against a foreign tissue have not been previously reported. We believe that the large number of microtubules is necessary not only to maintain the flattened nature of the GRs, but also to ensure the formation of an effective capsule against the deforming and shearing forces of the foreign tissue. And to keep up with the rapid assembly of new microtubules during encapsulation, the nucleus apparently triggers the synthesis of tubulin via ribosomes, its nuclear pores serving as channels for molecular transport to and from the nucleus. A structural and functional analogy between GRs and human platelet has also been suggested.