A Role for Potassium Permeability in the Recognition,Clearance, and Anti-inflammatory Effects of Apoptotic Cells

The benefits of programmed cell death by apoptosis are the safe and efficient clearance of damaged, infected, or surplus cells, primarily mediated by tissue-resident macrophages or tissue-infiltrating blood monocytes that differentiate into macrophages. Microglial cells are macrophages of the brain parenchyma, important immune surveillance cells that respond to various injuries and diseases of the brain. It is often stated that how a macrophage interacts with an apoptotic cell defines subsequent inflammatory responses, i.e., will engulfment be beneficial or detrimental for tissue repair, regeneration, and immunity. Our focus has been to better understand how macrophages discriminate between living and dying cells. Following our initial findings with platelet endothelial cell adhesion molecule (PECAM)-1, our studies have revealed a key role for potassium ion permeability in regulating integrin-dependent binding of apoptotic cells by macrophages and their subsequent response to proinflammatory stimuli. Specifically, apoptotic cells represent a depolarizing stimulus for macrophages where PECAM-1-mediated cell–cell interactions delay subsequent membrane repolarization. It is salient that potassium leak represents an early feature of cells destined to die by apoptosis that could trigger depolarization of macrophages that lie in close apposition. We speculate that how a tissue-resident macrophage responds to strong depolarizing stimuli has wider implications for inflammation and autoimmunity.     

Preadipocyte conversion to macrophage. Evidence of plasticity

Preadipocytes are present throughout adult life in adipose tissues and can proliferate and differentiate into mature adipocytes according to the energy balance. An increasing number of reports demonstrate that cells from adipose lineages (preadipocytes and adipocytes) and macrophages share numerous functional or antigenic properties. No large scale comparison reflecting the phenotype complexity has been performed between these different cell types until now. We used profiling analysis to define the common features shared by preadipocyte, adipocyte, and macrophage populations. Our analysis showed that the preadipocyte profile is surprisingly closer to the macrophage than to the adipocyte profile. From these data, we hypothesized that in a macrophage environment preadipocytes could effectively be converted into macrophages. We injected labeled stroma-vascular cells isolated from mouse white adipose tissue or 3T3-L1 preadipocyte cell line into the peritoneal cavity of nude mice and investigated changes in their phenotype. Preadipocytes rapidly and massively acquired high phagocytic activity and index. 60-70% of preadipocytes also expressed five macrophage-specific antigens: F4/80, Mac-1, CD80, CD86, and CD45. These values were similar to those observed for peritoneal macrophages. In vitro experiments showed that cell-to-cell contact between preadipocytes and peritoneal macrophages partially induced this preadipocyte phenotype conversion. Overall, these results suggest that preadipocyte and macrophage phenotypes are very similar and that preadipocytes have the potential to be very efficiently and rapidly converted into macrophages. This work emphasizes the great cellular plasticity of adipose precursors and reinforces the link between adipose tissue and innate immunity processes.     

Morphological analysis of osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

Under the influence of RANKL, in the presence of M-CSF, monocyte/macrophage precursor cells entered the osteoclast lineage and expressed the osteoclast marker tartrate-resistant acid phosphatase (TRAP). These cells were motile and began to differentiate by contacting and fusing together, initially forming cells with several nuclei. All sizes of cells continued to fuse, forming larger cells with more than 6 and as many as 50 nuclei. The degree of osteoclastogenesis was related to the concentration of RANKL. High cell density changed osteoclast morphology from a more rounded form with cytoplasm extended all round the cell to a form with cytoplasm concentrated around the nuclei and more restricted multiple cytoplasmic extensions. At optimal cell density and RANKL concentrations the large numbers of rounded cells fused into large cytoplasmic masses. On reaching a critical size, osteoclasts assumed a spread morphology with a peripheral ring structure. Most of the nuclei were associated with the peripheral ring. When cytoplasmic masses were present, rings also formed within the mass, often with no contact with the cell periphery. All forms of RANKL-induced osteoclastogenesis were blocked by the endogenous decoy receptor osteoprotegerin and were also strongly reduced by calcitonin, with the later arriving morphological categories being the first to disappear.     

Prostaglandin induced shape changes in fibroblasts grown in cell culture

PGE2 induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE2 at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hours. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE2 resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF2 alpha did not however induce similar changes. This difference between PGE2 and PGF2 alpha is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE2 may have the ability to induce shape changes in cells.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

An Intra-Axonal Protozoon in the Spinal Cord of the Toad Bufo arenarum (Hensel)

SYNOPSIS. A protozoon was found in myelinated axons of the spinal cord and brain of the toad, Bufo arenarum. Examination with the light microscope using Giemsa, Feulgen, PAS and methylene blue technics revealed a primary cell as large as 30 μ in diameter and containing up to 80 nuclei. Electron micrographs showed that the protozoon ranged from 2 μ to 30 μ in diameter and that larger specimens contained numerous secondary cells (2 μ) in addition to multiple nuclei. A few specimens were found in which the secondary cells had long processes with microtubules. Multiple nuclei together with secondary cells suggest that it may be a schizont form of a sporozoon.
The protozoon was found most frequently in axons of the perimedullary plexus just beneath the pia. These axons are without degenerative changes, are up to 3 times the diameter of the largest normal myelinated fibers. The myelin appears normal altho there are fewer laminae than in myelin of other large nerve fibers. The protozoon apparently causes axonal swelling but does not block the fibers completely.
Light microscopic attempts to locate similar forms or other stages in the life cycle by examining blood, skin lesions, spleen, liver, small intestine, dorsal and ventral roots, or sensory ganglia were unsuccessful.
Examination of spinal cords which had been mechanically severed excluded the possibility of confusing the protozoa with multinucleated macrophages. Altho observations do not prove their mode of entrance to the nervous system, the preponderance of protozoa in the peripherally located perimedullary plexus suggests that the path may be by way of the cerebrospinal fluid or along the endoneurium.     

Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.     

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.     

Scanning and transmission electron microscopic study of the supraependymal macrophages in the lateral ventricles of the toad brain

Transmission and scanning electron microscopy of the lateral ventricles of the toad brain revealed the presence of supraependymal cells that have the features of macrophages. Based solely on their surface morphology three different cell forms could be identified. The most frequently observed cells are flat and multipolar, and have a smooth or ruffled surface. The second type is spherical with a ruffled surface and occurs either singly, in which case it lacks processes, or in clusters from which processes radiate. The third type has surface blebs and numerous thin, smooth processes. However, when specimens that had been examined in the scanning electron microscope are viewed in the transmission electron microscope, all cells appear to belong to a single cell type. All cells viewed closely resemble macrophages in that they contain nuclei with clumped chromaffin, single cisternae of rough endoplasmic reticulum, numerous dense bodies, and many Golgi complexes. In addition, when horseradish peroxidase (HRP) was perfused into the ventricles, reaction product was found a short time thereafter within cytoplasmic vacuoles, and after a longer period within dense bodies. Because of their ultrastructural resemblance to macrophages and their capacity to ingest HRP, we suggest that these cells function as phagocytes and, as such, act to remove foreign materials from the cerbrospinal fluid.     

Light- and Electronmicroscopic Observations on the Blood Cells of the Atlantic Hagfish,Myxine glutinosa (L.)

In the blood of Myxine glutinosa three cell lines may be distinguished: erythrocytes, granulocytes and agranulocytes. The erythrocytes are remarkably large, oval and nucleated. They are well defined in all their developmental stages by a characteristic micropinocytosis. They originate from blast cells which proliferate in the circulating blood. The blast cells probably form from agranulate stem cells of the intestinal myeloid tissue. The granulocytes constitute about half of the leucocytes. They are neutrophilic with a lobated nucleus. The granulocytopoiesis takes place in the intestinal myeloid tissue. The agranulocytes mainly include two cell types, termed spindle cells and lymphocyte-like cells. These cell types, however, transform into each other. Macrophages occur essentially in the peritoneal cavity rarely in the blood. Transition forms between macrophages and granulocytes may exist. The blood also contains cells which on morphological grounds have been termed thrombocytes. Whether these cells are identical with those necessary for clotting of the blood remains to be proved. With the exception of erythroblasts, the different lines of blast cells are difficult to identify and distinguish from each other. Possibly all lines of blood cells originate from agranulate lymphocyte-like stem cells, most of which are produced by the intestinal myeloid tissue.     

Appearance and distribution of fetal brain macrophages in mice

Summary A study on the localization of fetal and neonatal brain macrophages of mice from embryonic day 10 (E10) to postnatal day 21 (P21) was carried out immunohistochemically using a monoclonal antibody against a macrophage differentiation antigen (Mac-1) and the labeled avidin-biotin technique. In the central nervous system, the macrophages recognized first were mainly located in the choroid plexuses of the fourth and lateral ventricles at E14. Their number increased at E17–P3 and gradually decreased thereafter. In the cerebral parenchyma, a few macrophages appeared at E14 in the matrix cell layer. They were also detected in the migrating zone at E15, E17 and in the cortical plate at E19. Mapping of positive cells at the stage of neuroblast formation (E15, E17, E19) disclosed the precise distribution of cerebral macrophages. The macrophages that appeared first in the choroid plexuses at E15 may be derived from the subarachnoid vessels, which extend into the stroma of the choroid plexuses when the matrix cell layer invaginates into the lateral ventricle to form the choroid plexuses. Almost all of the macrophages recognized in the cerebral parenchyma disappeared at P9 when the cytoarchitecture seemed to be completed. In the cerebellum, which develops later than the cerebrum, macrophages appeared after birth and were located mainly in the internal granular layer. The brain macrophages always appeared in the regions where cell proliferation and brain remodeling are most active at each stage. These findings suggest that fetal and neonatal brain macrophages may play an important role in scavenging degenerated cells and cell debris during histogenesis of the central nervous system.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Infection of Macrophages in Culture by Leptomonads of Leishmania donovani

SYNOPSIS. Peritoneal macrophages from hamsters were monolayered on coverslips in Leighton tubes. Twenty-four hours later these were transferred to a perfusion chamber. Leptomonads were added with fresh medium and the infection process observed with the aid of phase contrast. In the perfusion chamber free-swimming leptomonads attached to the macrophage by the tip of their flagella. Shortly after this initial attachment the macrophage extended a narrow pseudopodium around the flagellum which eventually reached and enveloped the body of the parasite. Upon complete envelopment the pseudopod containing the leptomonad was retracted into the central body of the macrophage. When first seen in the granular endoplasm of the macrophage, most of the leptomonads appeared to be surrounded by vacuoles. In most cases these vacuoles disappeared in a few minutes making it difficult to distinguish the parasite from the host cell cytoplasm.
Leptomonads also were added directly to Leighton tube cultures, and the coverslips with the adherent macrophages and parasites were removed, fixed and stained periodically during the infection process. In these preparations most of the parasites were in clumps in the vicinity of macrophages. Details of the ingestion of the clumps could not be seen, but occasionally a single organism was seen with its flagellum and part of its body enclosed by an extended pseudopod. Most of the intracellular leptomonads were in large vacuoles. Forms intermediate between elongate leptomonads and LD bodies were surrounded by smaller vacuole-like spaces. The halo-like vacuoles most frequently seen around LD bodies may have been fixation artifacts. Under favorable conditions the leptomonads transformed to LD bodies in 1–4 hours, but it was 48 hours before a population increase could be found.     

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.     

Brain macrophages in cerebellar cell cultures

Brain macrophages were studied in dispersed monolayer cultures of post-natal mouse cerebella. The phagocytotic activity, binding of immunoglobulins, presence and characteristics of nucleoside diphosphatases as well as argentophilia and lipid distribution were tested, with results similar to those for brain macrophages in vivo and for other macrophages in vitro. The macrophages in the cultures showed four different forms with continuous transition from one form to the following and subsequent cell death. No signs of proliferation of this cell type was found. Dynamic investigations showed repelling of neuronal growth cones by certain macrophages, resulting in the formation of areas devoid of neuronal and glial processes around the macrophages. An in vitro model for these cells, described by other authors as being important in post-natal reshaping of the brain, is presented and investigated in this study.     

Microvilli and blebs as sources of reserve surface membrane during cell spreading

The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

Role of telomerase in reactivation of macrophage nuclei in heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

Phenotypic Change of an SV40-Transformed Mouse Macrophage Line,BB-W-531–2, Induced by Different Cultural Methods

A simian virus 40 (SV40)-transformed macrophage clone which was established from BALB/cAnN mouse bone marrow cells was used to study the effect of different cultural conditions on the expression of macrophage properties. The macrophage clone, BB-W-531–2 line, expressed and maintained the macrophage properties, immune phagocytosis or Fc- and complement receptors, under the growth-inhibiting conditions of confluent density and of cultivation on bacteriologic dishes with reduced adhesiveness. However, the cells lost their ability to express the macrophage properties dependent upon cell density after repeated culture splits in the growing phase. These cells regained that ability when they were cocultured with cells having macrophage properties. These results suggest that there is a possible correlation between reduced multiplication and the expression of macrophage properties, and that macrophage properties which have been suppressed or blocked may be induced by diffusible factor(s) produced by macrophages.