T. Sminia

Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry

SummaryThe morphology and the ultrastructure of the blood and connective tissue cells of Lymnaea stagnalis were studied. Special attention was paid to the role of these cells in the cellular defense mechanism (phagocytosis). This problem was investigated in injection experiments with enzyme histochemistry and electron microscopy.The blood contains one type of cell, the amoebocyte. The amoebocytes are very active in phagocytosis. They phagocytoze various particulate materials like India ink, trypan blue, colloidal silver, ferritin, bacteria and zymosan granules. Digestion of the phagocytozed biotic material was observed. The cells show a very strong peroxidase activity. The acid phosphatase activity is weak. It increased after phagocytosis. Numerous amoebocytes loaded with phagocytozed inert material were still found three months after injection. It is concluded that migration of phagocytes to the exterior via various epithelia, as found in other molluscs, is of minor importance.In the connective tissue 8 different cell types were distinguished: 1. pore cells, 2. granular cells, 3. vesicular connective tissue cells, 4. amoebocytes, 5. fibroblasts, 6. undifferentiated cells, 7. pigment cells, 8. muscle cells.Pore cells are characterized by numerous invaginations of the cell membrane bridged by cytoplasmic tongues. Probably these cells produce haemocyanin. Granular cells contain numerous cysteine rich glycoprotein granules. The contents of these granules are released by exocytosis. It is suggested that these cells are involved in the production of blood proteins. The empty looking vesicular connective tissue cells appeared to contain large amounts of glycogen. Obviously these cells have nutritive functions.True fixed macrophages were not observed in the connective tissue. Only the amoebocytes phagocytoze in large amounts various materials. Furthermore, the pore cells show a limited phagocytosis capacity.

Macrophages in the central nervous system of the rat

In an immunohistochemical study using monoclonal antibodies, which exclusively recognize cells of the monocyte-macrophage lineage, and monoclonal antibodies against the Ia-antigen, we describe the occurrence of macrophages in the developing and adult central nervous system (CNS). In normal adult brain, no macrophages could be detected in the CNS parenchyma; only in the meninges and the choroid plexes were a few macrophages found. During ontogeny, numerous phagocytic cells infiltrated the CNS parenchyma; these cells which did not express Ia are blood-borne. About three weeks after birth, all macrophages had disappeared from the CNS. As microglia in adult and developing brain do not stain with the anti-macrophage antibodies, we suggest that microglial cells are not related to the mononuclear phagocyte system and do not have a hematogenous origin.

The origin of osteoclasts: An immunohistochemical study on macrophages and osteoclasts in embryonic rat bone

SummaryThe origin of osteoclasts was studied in embryonic rat bone primordia using a set of monoclonal antibodies (ED1, ED2, and ED3) that exclusively recognize monocytes and macrophages. ED1 recognizes monocytes and macrophages. Mononuclear phagocytes which were ED1 positive were found in the perichondrium/periosteum of developing bone. These cells started to infiltrate the primordia when the cartilage became hypertrophic. During bone formation, multinucleated ED1-positive cells with the morphological characteristics of osteoclasts were found in the developing bone marrow cavity and against the bone collar. The present findings support the notion that osteoclasts arise by fusion of mononuclear phagocytes derived from blood monocytes.

Low-dose cyclosporin A induces relapsing remitting experimental allergic encephalomyelitis in the Lewis rat

Experimental allergic encephalomyelitis (EAE) in Lewis rats is an acute monophasic autoimmune disease. It can be treated prophylactically and therapeutically with high doses of cyclosporin A (CsA). Here we demonstrate that low-dose CsA does not prevent a first attack of EAE, but, on the contrary, induces a chronic relapsing form of the disease in 100% of Lewis rats examined. Possible explanations for the high relapse rate after low-dose CsA treatment are discussed. Further studies will be needed to evaluate the immunological mechanisms responsible for these results.

Encapsulation of foreign materials experimentally introduced into the freshwater snail Lymnaea stagnalis

SummaryThe encapsulation of experimentally introduced material was studied in Lymnaea stagnalis. Abiotic (yvelon sponge) and biotic materials (autografts, allografts and xenografts) were implanted into the cephalopedal blood sinus.Within 24 hrs the yvelon sponge is completely invaded and encapsulated by round amoebocytes. Amoebocytes are the only type of blood cell in L. stagnalis. The number of capsule amoebocytes increases until 3 days after implantation. These capsule amoebocytes are normal blood amoebocytes. This inclusion is based on morphological observations as well as on the results of labelling experiments (blood amoebocytes were labelled with India ink or 3H-thymidine). These data are in accordance with evidence that amoebocytes may have more than one function.From 24 hrs onwards a progressive flattening of the capsule amoebocytes was observed. In 7-day-old capsules 3 strata were distinguished: inner and outer strata consisting of tightly packed, flattened amoebocytes, and a middle stratum still composed of round amoebocytes. The cells of the outer stratum possess an extensive granular endoplasmic reticulum. During the further process of encapsulation the cells of the inner stratum become loaded with lysosomes. The middle stratum disappears: the cells flatten and become part of the other two strata. In the outer stratum, collagenous connective tissue fibrils and ground substance are deposited between the cells. This situation is reached about 1 month after implantation. Capsule formation is completed by that time: during the next 5 months, no major structural changes occur in the capsules.The reactions to biotic materials appeared to be quite different, and show that L. stagnalis is able to discriminate between different types of graft. Autografts and allografts are not encapsulated—only a transient amoebocyte reaction occurs at the cut surfaces of the transplants—whereas xenografts evoke a severe host reaction: they are infiltrated and encapsulated by amoebocytes. It is suggested that the discrimination mechanism is located in the plasma membrane of the amoebocytes.The encapsulation of xenografts ends in a capsule consisting only of the collagenous connective tissue stratum. The cells of the inner stratum infiltrate the graft and phagocytose degenerated graft tissue.Autoradiographic experiments using 3H-proline as a marker for collagen synthesis showed that the flattened cells of the outer stratum produce collagenous connective tissue fibrils, indicating that these cells are fibroblasts. In experiments using ink or 3H-thymidine labelled amoebocytes it was shown that these fibroblasts are transformed amoebocytes. Finally it is suggested that amoebocytes also have the capacity to transform into myofibroblasts.

Haematopoiesis in the freshwater snail Lymnaea stagnalis studied by electron microscopy and autoradiography.

SummaryIn order to investigate haematopoiesis in the freshwater pulmonate Lymnaea stagnalis, the blood cells and the connective tissue of this snail were studied by light and electron microscopy as well as by autoradiography.In the circulating blood only one type of cell, the amoebocyte, is present. Amoebocytes also occur in the connective tissue (tissue amoebocytes) as single cells, in small groups or in large accumulations. Study of the morphology and ultrastructure of blood and tissue amoebocytes shows that no differences exist between these cells, indicating that L. stagnalis does not possess a well-defined haematopoietic organ. This assumption is supported by the following observations: 1. both blood and tissue amoebocytes can act as phagocytes, 2. blood and tissue amoebocytes both have the capacity to divide (i.e. incorporate tritiated thymidine) and 3. the percentage of dividing cells in the blood and in the connective tissue is the same. These quantitative data indicate furthermore that there is no difference in the relative importance of the blood and the connective tissue in the process of haematopoiesis.Comparison of tritiated thymidine labelled cells with unlabelled amoebocytes showed that these cells do not differ with respect to their morphology and ultrastructure. Moreover, amoebocytes involved in phagocytosis and encapsulation of foreign materials or in wound healing still have the capacity to divide.The percentages of tritiated thymidine labelled amoebocytes in different snails varied considerably. It is suggested that this variation reflects differences in the physiological state of the individual snails.

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

SummaryBlood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per μl haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of 3H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.

Sieve structure of slit diaphragms of podocytes and pore cells of gastropod molluscs

SummaryThe ultrastructure of the slit diaphragms between the pedicels of the podocytes of the prosobranch Viviparus viviparus and between the cytoplasmic tongues of the haemocyanin producing pore cells of the pulmonate Lymnaea stagnalis was investigated. In both cell types 2 diaphragms are present in the slits. They form a 3-dimensional sieve structure with holes of respectively 90 × 110 Å (podocyte) and 200 × 220 Å (pore cell). Injection experiments showed that the size of the holes of the pore cell sieve matches that of particles which can be ingested by this cell type. The substructure of the sieves of the molluscs is compared to that of the 2-dimensional sieve of the podocytes of the mouse and the rat.

Histological and ultrastructural observations on wound healing in the freshwater pulmonate Lymnaea stagnalis

SummaryThe process of wound healing in Lymnaea stagnalis was studied by light and electron microscopy. Snails were wounded by making incisions in the skin.The observations showed that the wounds are closed by muscular contraction and by formation of thrombi of blood amoebocytes. These thrombi form a large amoebocyte plug. During the first 72 hrs after incision thin tubules (diameter 175–225 Å) were observed between the amoebocytes in the plug. Possibly these tubules represent a blood clotting protein. The round amoebocytes constituting the plug can be regarded as normal blood amoebocytes. First, ultrastructurally they closely resemble the amoebocytes of the circulating blood. Second, not only blood amoebocytes but also plug amoebocytes of snails injected with India ink before incision contained ink particles, indicating that the cells are of one type. Apparently due to phagocytosis of cell debris the number of lysosomes in plug amoebocytes increased during the first days after incision.Eighteen to twenty four hrs after incision the first signs of differentiation of round plug amoebocytes into flattened cells were observed. Between these cells collagen was seen from 3–5 days after incision and onwards. It is suggested that these flattened amoebocytes produce collagen fibrils. These cells are structurally different from collagen producing fibroblasts and from muscle cells of the surrounding connective tissue. Transformations of amoebocytes into these two latter cell types were not found.Ninety days after incision the connective tissue in the wound area is still different from that on non-injured sites.

Alterations in the internal defence system of the pond snail Lymnaea stagnalis induced by infection with the schistosome Trichobilharzia ocellata

In order to investigate whether the schistosome Trichobilharzia ocellata interferes with defence activities in its snail intermediate host Lymnaea stagnalis, aspects of the immune system of infected snails and of non-infected controls were compared. The elimination of injected live Staphylococcus saprophyticus bacteria starts at a lower rate in infected snails 1 and 5 weeks after exposure to the parasite, but then proceeds faster than in control snails. During the first 3 weeks of infection, when only mother sporocysts are present, the haemocytes of the infected snails have an increased capacity to phagocytose rabbit red blood cells in vitro. From 5 weeks onwards, when mother and daughter sporocysts are present but cercariae are not yet mature, the phagocytic activity decreases to below control level. The number of circulating haemocytes is also higher in infected snails than in controls at this time. Moreover, the cells are larger, have more inclusions and an increased surface area with many long, branched, spiked pseudopods. The development of the parasite is retarded in a subpopulation of snails in which the haemolymph plasma agglutinates erythrocytes with high titres, compared to a subpopulation with low haemagglutinating activity. The haemagglutinating activity in infected snails of the first decreases significantly from 6 weeks onwards.