Haemocytes and basement membrane formation in Rhodnius

During the period between feeding and ecdysis in the fourth instar larva of Rhodnius the basement membrane below the epidermis of the abdomen increases about threefold in thickness. This increase takes place during the time, about 5 to 7 days after feeding, when the plasmatocytes settle and flatten in great numbers on the inner surface of the membrane.By a process beginning with pinocytosis the plasmatocytes accumulate their characteristic periodic acid Schiff (PAS)-positive inclusions, often with rod-like or tubular contents. These inclusions are discharged and spread out on the surface of the basement membrane. The inclusions and the membrane have the same staining properties and contain protein, carbohydrate, and lipid. Similar material is slowly deposited in less compact form between the plasmatocytes which encapsulate foreign bodies.The oenocytoids (granular cells) discharge the electron dense contents of their inclusions into the haemolymph rather later. The subsequent fate of this material is not known.     

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells from a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.     

Fine structure of the hemocytes and nephrocytes of Argas (Persicargas) arboreus (Ixodoidea: Argasidae)

The fine structure of the hemocytes and nephrocytes in Argas (Persicargas) arboreus is described and compared with that of similar cells in other tick species and insects. The hemocytes are of three types: prohemocytes, with a relatively undifferentiated cytoplasm lacking granular inclusions and probably serving as progenitors of the other hemolymph cell types; plasmatocytes, containing abundant mitochondria, cisternae of rough endoplasmic reticulum (RER), and free ribosomes, as well as some small granular inclusions; granulocytes, the predominant cell type in the hemolymph, containing numerous granules of variable electron density and maturity, and pseudopodia-like processes on the cell surface. Plasmatocytes and granulocytes are phagocytic and possibly also have other functions in the tick body. Cells with intermediate features appear to be in a stage of transition from plasmatocyte to granulocyte. Nephrocytes contain vacuoles enclosing fibrillar material, some electrondense granules, and moderate amounts of the active organelles—mitochondria, RER, and ribosomes. The nephrocyte is surrounded by a basal lamina and its plasma membrane infolds to form many deep invaginations coated by a fine fibrillar material. Openings to these invaginations are closed by membranous diaphragms. Coated tubular elements connect the surface invaginations with large coated vesicles, which appear to be specialized for internalization of proteins from the hemolymph. The dense granules may represent an advanced stage of condensation of ingested protein and thus may be lysosomal residual bodies, or they may develop by accumulation of secretory products.     

Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.     

Laminin,fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane

Light and electron microscope immunohistochemical methods were used to study the distribution of several proteins in rat skeletal muscle. The aims were to identify components of muscle fiber basement membrane and to compare the small fraction (0.1%) of the basement membrane that extends through the synaptic cleft at the neuromuscular junction with the remaining, extrasynaptic portion. Synaptic basement membrane is functionally specialized and plays important roles in neuromuscular function and regeneration. Laminin, fibronectin, collagen IV, collagen V, and a collagenous protein (high-salt-soluble protein [HSP]) are all present in muscle fiber basement membrane. Laminin and collagen IV are concentrated in basal lamina (the feltlike, inner layer of the basement membrane) and are shared by synaptic and extrasynaptic regions. Fibronectin, also present synaptically and extrasynaptically, is present in basal lamina and in the overlying reticular lamina. Collagen V and HSP are present throughout extrasynaptic basement membrane but are absent from synaptic sites; HSP is concentrated in the reticular lamina and on the outer surface of the basal lamina. These results, together with experiments reported previously (Sanes and Hall, 1979. J. Cell Biol: 83:357--370), provide examples of three classes of components in muscle fiber basement membrane--synaptic, extrasynaptic, and shared.     

Demonstration of microfibrils in Bruch's membrane of the eye

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.     

Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

Molecular and biochemical aspects of nematode collagens.

Collagens are major structural proteins of nematode cuticles and basement membranes (basal laminae). The collagen proteins that form these structures differ in their biochemical and physical properties and are encoded by distinct gene families. Nematode basement membrane collagens are large proteins that show strong homology to basement membrane collagens of vertebrates. There appear to be 2 nonidentical basement membrane collagen genes in nematodes. Cuticle collagens are about one-sixth the size of basement membrane collagens and are encoded by a large family of 20-150 nonidentical genes. Cuticle collagens can be subdivided into 4 families based upon certain structural features in the proteins. The mature, extracellular forms of both types of collagen proteins are extensively cross-linked by disulfide bonds and are largely insoluble in the absence of a thiol-reducing agent. Cuticle collagens also are cross-linked by nonreducible covalent bonds that involve tyrosine residues. The experimental studies that have led to our current understanding of the structures of basement membrane and cuticle collagens are reviewed. Some previous questions about the physical properties of these proteins are reexamined in light of the primary sequence information now available for the proteins.     

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.     

The epithelial basal lamina of the isolated colonic mucosa: scanning and transmission electron microscopy

The morphology of the rat colonic basement membrane was studied under control conditions and after induction of secretion by distension in the Ussing chamber. The basement membrane was visualized by removing the epithelium with the aid of Ca2+ chelation combined with vibration. Specimens with and without metallic coating were studied with SEM and TEM, respectively. The surface of the basement membrane in the surface region of the epithelium, i.e. between the mouths of the crypts, is rough. In contrast, the deeper parts of the crypts have a rather smooth basal lamina, which is finely pleated. Fenestrations of the basal lamina with a diameter of 0.5-1 microns were found frequently at the surface region, but less frequently in the crypts. Fenestrations with diameters larger than 2 microns were never observed. After the distension experiments and long-time incubation in vitro (5 h), the size of the fenestrations remained unchanged, however, their number increased compared to the controls.     

Larval development in the Homoscleromorpha (Porifera, Demospongiae)

Abstract. Embryonic development from coeloblastula to fully developed larva was investigated in 8 Mediterranean homoscleromorph species: Oscarella lobularis, O. tuberculata, O. microlobata, O. imperialis, Plakina trilopha, P. jani, Corticium candelabrum , and Pseudocorticium jarrei. Morphogenesis of the larva is similar in all these species; however, cell proliferation is more active in species of Oscarella than in Plakina and C. candelabrum. The result of cell division is a wrinkled, flagellated larva, called a cinctoblastula. It is composed of a columnar epithelium of polarized, monoflagellated cells among which are scattered a few non-flagellated ovoid cells. The central cavity always contains symbiotic bacteria. Maternal cells are also present in O. lobularis, O. imperialis , and P. jarrei. In the fully developed larva, cell shape and dimensions are constant for each species. The cells of the anterior pole have large vacuoles with heterogeneous material; those of the postero-lateral zone have an intranuclear paracrystalline inclusion; and the flagellated cells of the posterior pole have large osmiophilic inclusions. Intercellular junctions join the apical parts of the cells, beneath which are other specialized cell junctions. A basement membrane underlying the flagellated cells lines the larval cavity. This is the first observation of a basement membrane in a poriferan larva. The basal apparatus of flagellated cells is characterized by an accessory centriole located exactly beneath the basal body. The single basal rootlet is cross striated. The presence of a basement membrane and a true epithelium in the larva of Homoscleromorpha—unique among poriferan clades and shared with Eumetazoa—suggests that Demospongiae could be paraphyletic.     

Ultrastructural changes in salivary glands of Pseudolynchia canariensis (Dipteria: Hippoboscidae) infected with sporozoites of Haemoproteus columbae

Salivary gland cells of Pseudolynchia canariensis are ultrastructurally similar to those of many other diptera. Heavy infections with Haemoproteus columbae sporozoites cause disruption of the basement membrane, basal plasma membrane, and disorganization of the functional organelles of the cell. Cytoplasmic microtubules appear to increase in number.     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.     

Mouse mammary epithelial cells produce basement membrane and cell surface heparan sulfate proteoglycans containing distinct core proteins

Cultured mouse mammary (NMuMG) cells produce heparan sulfate-rich proteoglycans that are found at the cell surface, in the culture medium, and beneath the monolayer. The cell surface proteoglycan consists of a lipophilic membrane-associated domain and an extracellular domain, or ectodomain, that contains both heparan and chondroitin sulfate chains. During culture, the cells release into the medium a soluble proteoglycan that is indistinguishable from the ectodomain released from the cells by trypsin treatment. This medium ectodomain was isolated, purified, and used as an antigen to prepare an affinity-purified serum antibody from rabbits. The antibody recognizes polypeptide determinants on the core protein of the ectodomain of the cell surface proteoglycan. The reactivity of this antibody was compared with that of a serum antibody (BM-1) directed against the low density basement membrane proteoglycan of the Englebarth-Holm-Swarm tumor (Hassell, J. R., W. C. Leyshon, S. R. Ledbetter, B. Tyree, S. Suzuki, M. Kato, K. Kimata, and H. Kleinman. 1985. J. Biol. Chem. 250:8098-8105). The BM-1 antibody recognized a large, low density heparan sulfate-rich proteoglycan in the cells and in the basal extracellular materials beneath the monolayer where it accumulated in patchy deposits. The affinity-purified anti-ectodomain antibody recognized the cell surface proteoglycan on the cells, where it is seen on apical cell surfaces in subconfluent cultures and in fine filamentous arrays at the basal cell surface in confluent cultures, but detected no proteoglycan in the basal extracellular materials beneath the monolayer. The amino acid composition of the purified medium ectodomain was substantially different from that reported for the basement membrane proteoglycan. Thus, NMuMG cells produce at least two heparan sulfate-rich proteoglycans that contain distinct core proteins, a cell surface proteoglycan, and a basement membrane proteoglycan. In newborn mouse skin, these proteoglycans localize to distinct sites; the basement membrane proteoglycan is seen solely at the dermal-epidermal boundary and the cell surface proteoglycan is seen solely at the surfaces of keratinocytes in the basal, spinous, and granular cell layers. These results suggest that although heparan sulfate-rich proteoglycans may have similar glycosaminoglycan chains, they are sorted by the epithelial cells to different sites on the basis of differences in their core proteins.     

An acellular human amnionic membrane model for in vitro culture of type ii pneumocytes: The role of the basement membrane in cell morphology and function

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with ³H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8–10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48–60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7–8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of ³H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

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.