The role of hemolymph in the initial cellular attachment to foreign cells by the hemocytes of the silkworm, Bombyx mori

The effects of silkworm hemolymph, $\text{Bombyx mori}$, on the frequency of hemocyte-binding were studied using the scanning electron microscopy ( SEM ) and a rosette assay with goose erythrocytes ( GRBC ). Hemocytes from the last instar larvae were able to bind to GRBC $\text{in vitro}$ int he absence of hemolymph. Hemocytes type involved in sponteneous cytoadherence was mainly the granular cell, while both plasmatocyte and prohemocyte were also observed to adhere to GRBC. However, the frequency of these two hemocytes binding to GRBC was low compared with that of granular cells. Observations with the SEM showed that the presence of hemolymph increased the number of GRBC-binding hemocytes, especially the granular cells and plasmatocytes. Therefore, hemolymph probably had a significant role in the attachment of granular cells and plasmatocytes to foreign erythrocytes.     

Lectin-dependent recognition of foreign cells by hemocytes of the mussel, Mytilus edulis

Phagocytosis of human erythrocytes (rbc) by hemocytes of the mussel Mytilus edulis was found to be influenced by four heterologous lectins. The effects were examined in the absence of Ca++ ions under three experimental conditions: when the lectins were bound to 1) both hemocytes and rbc, 2) only hemocytes, but not to rbc, and 3) only rbc, but not to hemocytes. The lectins used included: albumen gland agglutinin from Helix pomatia (HPA), wheat germ agglutinin (WGA), Ricinus-120 (Ric-120) and Concanavalin-A (Con A). HPA, WGA and Ric-120, for which both hemocytes and A-rbc possess receptors, strongly enhanced uptake of A-rbc. This lectin-mediated phagocytosis was abolished by addition of specific sugars either to lectin-pretreated rbc (HPA, WGA) or to a pretreated hemocyte monolayer (Ric-120); this indicated the stimulation of phagocytosis by the binding of lectin to carbohydrate determinants at the surface of hemocytes and target cells. On the other hand, HPA which binds to hemocytes, but not to O-rbc, did not influence phagocytosis of these rbc; and Con A which binds to A-rbc, but not to hemocytes, also failed to stimulate phagocytosis. These findings reveal the importance of carbohydrate determinants on the surface of hemocytes as well as on target cells in recognition and in lectin-mediated phagocytosis of foreign cells by Mytilus hemocytes.     

Purification and characterization of silkworm hemocytes by flow cytometry

Hemocyte functions are well-investigated in the silkworm, Bombyx mori, however, detailed analysis of each hemocyte subset has been hampered by the lack of appropriate separation method. Here we use an array of flow cytometric analyses to characterize silkworm hemocytes with various molecular probes, such as propidium iodide, green fluorescence protein, monoclonal antibodies, and fluorescent lectins. Of these, separation using propidium iodide was the simplest and provided most reliable results for the isolation of the hemocyte subsets. cDNAs were then synthesized from these sorted populations and subset-specific gene expression was examined by RT-PCR. Granulocytes, plasmatocytes, and oenocytoids expressed different classes of immune genes, suggesting that they have multiple roles in silkworm immunity. In contrast, a contribution of spherulocytes to immunity was not documented in that they failed to express most of the genes. The functions of spherulocytes are thus likely to be distinct from those of the other three hemocyte subsets.     

The role of protein A in the attachment of staphylococci to influenza-infected cells

The adherence of staphylococcal protein A-containing Cowan I bacteria to influenza-infected cells was enhanced up to 5 times following incubation of the monolayers with antiinfluenzal serum, but not following treatment with nomimmune serum. Significantly increased binding of Cowan I bacteria was detected at antiinfluenzal serum dilutions as high as 1:40,960. None of the several antibody concentrations tested enhanced the binding of staphylococcal protein A-negative staphylococci. In addition, extracellular staphylococcal protein A was found to inhibit complement-mediated cytolysis of antibody-coated, virus-infected cells. The possible significance of staphylococcal protein A in the synergistic interaction between staphylococcal and influenza virus infections is discussed.     

Regulatory effects of temperature and hemolymph on inhibition and enhancement of attachment and filopodia function of phagocytic granular cells of the silkworm, Bombyx mori

The effect of temperature on the adhesiveness and filopodia elongation of granular cells was examined in the presence or absence of hemolymph. The incubation in hemolymph remarkably increased the number of adherent cells almost irrespective of temperatures. Marked inhibition of attachment was seen in incubation media without hemolymph or with 20 % hemolymph but at much lower temperatures. Filopodia elongation was markedly inhibited at a low temperature even when the cells were incubated in hemolymph alone. However, the number of filopodia-elongated cells was increased by raising the temperature even without hemolymph. These results show that hemolymph of B. mori is suggested to contain at least two kinds of humoral factors which affect the initial phases of cellular reactions to foreign materials. One is to promote attachment and the other to activate filopodia function. Moreover, it is shown that attachment is a temperature-independent process in the presence of hemolymph unlike filopodia elongation. However, since both attachment and filopodia elongation were increased by raising the temperature, temperature is considered to be another important cofactor in enhancement of foreignness-recognition and binding ability.     

The role of basal cells in attachment of columnar cells to the basal lamina of the trachea

The mechanism by which basal cells play a role in attachment of airway epithelium to the basal lamina has not been determined. Our hypothesis is that basal cells form a structural bridge between columnar cells and the basal lamina via hemidesmosomes, the cytoskeleton, and desmosomes. To evaluate this hypothesis, we determined the percentage of the columnar cell surface area associated with attachment to the basal lamina and the basal cell in tracheal epithelia of different heights. Tracheas from mice, hamsters, rats, bonnet monkeys, cats, and sheep were prepared for electron microscopy by standard techniques. The height of the epithelia ranged from 8.6 microns in the hamster to 56.8 microns in the sheep. The number of basal cells/100 microns ranged from 3.4 in the hamster to 21.4 in the sheep. The percentage of the basal lamina covered by basal cells increased from 32.6 in the hamster to 94.7 in the sheep. In the shorter epithelia of the hamster, 32% of the columnar cell attachment to the basal lamina was indirect through basal cells, and in the taller epithelia of the sheep, 92% of the columnar cell attachment was by this means. Conversely, the percentage of columnar cell surface in contact with the basal lamina decreased from 67.4% in the hamster to 5.3% in the sheep. These data demonstrate that basal cells play a role in attachment of columnar epithelium to the basal lamina by presenting a surface area for cell-to-cell attachment, thus acting as a bridge between columnar cells and the basal lamina.     

The initial attachment of cemental fibrils to the root dentin surface in acellular and cellular cementogenesis in rat molars.

To elucidate the initial attachment mechanism of cemental fibrils to the root dentin surface in acellular and cellular cementogenesis, developing rat molars were observed by light microscopy and scanning electron microscopy combined with NaOH maceration. The NaOH maceration was used to observe details of the positional association of cemental and dentinal fibrils during cementogenesis. An initial hematoxylin stained, cementum layer began to form on the root dentin surface with the first dentin mineralization in both acellular and cellular cementogenesis. The initial attachment of cemental fibrils to the dentin surface also began at this point. At the initial attachment the intermingling of cemental and dentinal fibrils occurred only in places. With advanced cementogenesis the initial cementum layer became the fibril-poor cemento-dentinal junction. This suggests that cemental fibrils attach on the initial cementum layer, and not directly on dentinal fibrils, so that the layer results in the fibril-poor cemento-dentinal junction. The present study suggests that an intervening adhesive is necessary for the cemento-dentinal attachment at any stage of cementogenesis in rat molars.     

Cellular recognition of foreign materials by Bombyx mori phagocytes: II. Role of hemolymph and phagocyte filopodia in the cellular reactions

The plasmatocytes and granular cells are the most important hemocyte types involved in the cellular reactions of insects. These two hemocytes are obviously characterized with the adhesive morphology. Particularly, the granular cells in Bombyx mori have many long filopodia, and they are considered to play roles in catching the foreign materials invading into the hemocoel and also in obtaining some phagocytic signals during elongation process. The second part of this mini-review is a continuation of the previous summary (1) in which the immunocompetent cells in B.mori were described. Here, possible role of hemolymph factor is presented with much attention to the filopodial function of phagocytic granular cells of B.mori.     

The role of T cells and adjuvant in the immune response of mice to foreign erythrocytes

The splenic response of CBA mice, deprived of thymus-derived cells (T cells), to the injection of different doses of sheep erythrocytes, has been compared to that obtained in control mice which possess T cells. The effect of pertussis injected with the antigen was measured in both groups of mice in terms of the number of plaque-forming cells (PFC) to sheep red blood cells (SRBC) of the five main immunoglobulin classes of mice. The following conclusions have been reached:

• a Increasing the antigen concentration does not overcome the depression in the immune response resulting from an absence of T cells; consequently this evidence is incompatible with the role of T cells being solely one of concentration of antigen to B cells.
• b As the antigen dose is increased, the direct effect of adjuvant on B cells is also increased, whereas it is argued that an inverse relationship exists for the effect of adjuvant on T cells in the antigen dose range used here.
• c The lower the concentration of antigen, the greater is the dependence of the adjuvant effect of pertussis on the presence of T cells. This is far apparent for γM-PFC than for non-γM-PFC.
• d A significant proportion of γM-PFC are produced from precursor cells, which do not appear to require T cells for their differentiation into antibodyforming cells.
• e It is possible that adjuvant and T cells stimulate B cells in a similar way. Previously published evidence, which was thought to support the antigen-concentration hypothesis of T cell action, may have been an artefact due to the direct action of adjuvant (or adjuvant-like properties of the antigen preparation itself) on B cells binding antigen.
• f An effect consisting of an impaired γG_l response is described in fetal liver reconstituted and one-year old normal mice. The effect is independent of irradiation received during reconstitution and may be due to a partial deficiency of T cells in old age.

     

The role of E receptors in the attachment of thymocytes and T lymphocytes to human target cells

Human thymocytes, activated T lymphocytes, and neuraminidase-treated T cells possess the distinct capacity of forming conjugates with various human cell lines. The present study investigated whether E receptors, which endow human T cells with their capacity to bind sheep red blood cells (SRBC), are involved in this phenomenon. Monoclonal antibodies to human T cells and various simple sugars were studied for their effect on the attachment of human T cells to target cells. A-22, a monoclonal antibody to the E receptor, inhibited the formation of E rosettes by T cells and SRBC, and reacted in immunofluorescent-staining assays with the majority of human thymocytes and peripheral T cells, and with T-cell lines capable of forming E rosettes. When human thymus cells were treated with A-22 antibody they showed a reduction of up to 70% in their capacity to attach to the GM-4762 lymphoblast cell line and the K-562 myeloid line. Antibody treatment of the target cells, rather than of the thymus cells, had no effect on the formation of conjugates between thymus cells and target cells. Treatment of thymus cells with various monoclonal antibodies to T cells which do not react with the E receptor had no inhibitory effect. The exposure of human thymus cells to various simple sugars (D-mannose, D-fucose, galactose, and lactose) markedly reduced their capacity of forming conjugates with target cells. Exposure of neuraminidase-treated peripheral blood lymphocytes and of activated T cells to A-22 antibody inhibited their attachment to human target cells. The present study suggests that E receptors play a role in the attachment of human thymus cells and activated T cells to other human cells, and raises the possibility that these T-cell receptors may be involved in the process of recognition of "self" structures by human T lymphocytes.     

The initial attachment and subsequent behavior of osteoblastic cells and oral epithelial cells on titanium

The interaction between implant materials and bone cells or oral epithelial (OE) cells contributes to the clinical success of dental implants. The functional activity of cells in contact with an implant is determined by its surface properties. Before cells attach, extracellular matrix (ECM) in the serum deposits on the substrate; rounded cells then attach and spread upon it. Cells form focal adhesions and polarize, then start to migrate or proliferate to form colonies. Comparison of the attachment and behavior of osteoblastic cells on titanium (Ti) and hydroxyapatite (HA) revealed that more cells attached on HA and that these spread more rapidly than on Ti. In contrast, cells did not form good stress fibers or vinculin-positive focal adhesions on HA, whereas the cells on Ti possessed well-defined and polarized stress fibers. The initial attachment of OE cells to Ti was inferior to that on polystyrene culture dish or glass, and the OE cell migration area, indicated by the deposition of LN5, was smaller on Ti than on the other materials. This review summarizes data on the attachment and behavior of osteoblastic cells and OE cells on biomaterials, which may suggest future improvements in surface properties.     

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goal of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of na?ve spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion.     

The use of nylon pouches to prevent cellular attachment to implanted materials

A nylon pouch system has been developed to permit the in vivo exposure of implantable materials to tissue fluids without complications arising from cellular attachment or from the bacterial attack. SEM observations have indicated that the pouches were effective in reducing the ingress of bacteria as well as reducing, and in some cases eliminating, cell infiltration through their mesh structure. Over a 7 d implantation period, the exterior surfaces of the nylon pouches were heavily infested with microorganisms but there was minimal growth of bacteria within the pouches. Non-infected pouches that were implanted for up to 25 wk showed heavy deposits of cellular matter on the external surfaces of the pouches and minimal deposits within the pouches.     

Investigation of the role of signal transduction in attachment of ocular melanoma cells to matrix proteins: inhibition of attachment by calmodulin antagonists including tamoxifen

We investigated the role of signal transduction systems in the attachment of human uveal melanoma cells to matrix proteins. Ocular melanoma cells established from primary tumours attached rapidly to all substrates examined. Preferred substrates of attachment were collagens type I, III and IV and fibronectin rather than laminin, gelatin, arginine-glycine-aspartine, vitronectin, poly-I -lysine or plastic. All cells showed rapid attachment to the preferred substrates (80% within 10 min). Manipulation of intracellular cyclic AMP or protein kinase C activity had relatively little effect on cell attachment. In contrast, attachment was significantly reduced by manipulating either intracellular calcium or calmodulin. After 15 min at 37°C, the calcium ionophore ionomycin (5 m) reduced attachment to 25%, and TMB8 (50 M), which can reduce intracellular calcium, reduced attachment to 60%. The experimental calmodulin antagonist J8 (25 m), a substituted naphthalene sulphonamide, reduced attachment to 40%. Similarly tamoxifen (25 M), which has calmodulin antagonist activity in vitro,reduced attachment to 55%. Both J8 and tamoxifen inhibited cell attachment to a wide range of matrix proteins, suggesting that this effect on attachment is not dependent on the presence of specific adhesion receptors. Reduction of ocular melanoma tumour cell/matrix interactions through manipulation of intracellular calcium or calmodulin may therefore merit further investigation as a possible approach to reducing metastatic spread.     

Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells

The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.     

The role of cytokines in the initiation,expansion, and control of cellular immunity to tuberculosis

Summary: Tuberculosis (TB) results from an interaction between a potent immune response and a chronically persistent pathogen. The ability of Mycobacterium tuberculosis (Mtb) to induce a strong immune response while being able to resist the ability of the host to clear bacteria provides an excellent tool with which to investigate the role of specific cytokine pathways on the induction, expansion, and control of the effector T-cell response. In this review, the role of interleukin-12p40 (IL-12p40), IL-12p70, IL-23, and IL-27 in the immune response to Mtb are described. We show that IL-12(p40)₂ acts to mediate the activation of dendritic cells to become responsive to homeostatic chemokines. We also show that IL-12p70 is required for the optimal interferon-γ (IFN-γ) T-cell response, which is required for control of Mtb growth. IL-23 can induce IFN-γ responses in the lung if IL-12 is not present, but its major role is in supporting the IL-17 response within the lung. Neither IL-23 nor IL-17 is required for early control of Mtb in the lung. IL-23 and IL-17, however, can be instrumental in vaccine-induced protection. Finally, IL-27 limits protective immunity in the lung, but it is also required for long-term survival. These cytokines are therefore key players in the immune response to TB.     

Contribution of circulating hemocytes to the regeneration of heavy ion beams (12C5+) irradiated hematopoietic organs in the silkworm, Bombyx mori, through the way of phagocytosis of injured cells after invasion

Heavy ion beam irradiation has promising effects on tumor therapy. Our previous study using the domesticated silkworm, Bombyx mori, showed that this irradiation could seriously damage larval hematopoietic organs but they would regenerate later. In the in vitro irradiation, most hemocytes died when hematopoietic organs and wing discs connected with epidermis were directionally irradiated from epidermis to hematopoietic organ and then cultured so as to exclude circulating hemocytes. A few hemocytes had escaped irradiation according to extremely low hematopoiesis in vitro. Almost no hemocytes could incorporate BrdU at 60 h after irradiation, with which living and proliferating hemocytes are also labeled. In the absence of circulating hemocytes, the irradiation-escaped hemocytes in the organs were not enough for cleaning all dead cells because lots of small dead bodies remained in situ post-irradiation. After irradiating hematopoietic organs in larvae (in vivo irradiation), only a few apoptotic cells were found when given the same length of recovery time, and most hemocytes maintained normal morphology. Many hemocytes incorporated BrdU when tested at the same time as the in vitro irradiation but this number was lower than that measured for control organs. Circulating hemocytes, labeled by fluorescent microbeads through phagocytosis before irradiation, were found to have invaded the in vivo irradiated hematopoietic organs where they help the irradiation-escaped hemocytes to clear dead cells in the process of regeneration. Hematopoiesis of the regenerated hematopoietic organs did not fully recover to the level of the control organs according to the number of hemocytes produced in tissue culture. Some of the released hemocytes obviously underwent apoptosis, suggesting a far-reaching bystander effect of carbon ion beams irradiation on hemocytes inside. From these results, it is suggested that, together with irradiation-escaped hemocytes, the invaded circulating hemocytes took part in the regeneration of heavy ion beams irradiated hematopoietic organs through the way of phagocytosis of injured hemocytes in vivo.