Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).     

Growth regulation of transformed T cells by nonactivated macrophages: the role of Ia expression

We studied the influence of unactivated mouse peritoneal macrophages on the proliferative capacity of a spontaneously transformed MRL-lpr/lpr T cell clone. Macrophages, 25%, induced a reduction in proliferative rate from 20% to 95% measured by [3H]thymidine incorporation and microscopic cytometry. MHC-compatible (H-2k) macrophages caused growth inhibition reciprocal to the amount of Ia expression on the macrophage. Thus, with increasing preculture of the macrophages there was both decreasing Ia and increasing suppression. H-2-incompatible macrophages had maximal inhibitory capacity without preincubation. Macrophages derived from the peritoneum of MRL-lpr/lpr mice were less suppressive than macrophages from other H-2k mice. In contrast to the case of activated macrophages in other studies, in the present system there was no killing of T cells, only reduction in proliferation. The inhibitory effect of the macrophages correlated with the spontaneous formation of rosettes between the macrophages and the T cell clone. The number of rosettes forming a single layer of T cells around the macrophages, but not the number of rosettes with multiple layers of cells, was reciprocally related to the amount of Ia expression. The results suggest that macrophages bear a surface structure that influences and modulates the growth of T cells.     

Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine.

Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.     

Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of [3H]-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with [3H]-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the [3H]-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.     

Clonal distribution of osteoprogenitor cells in cultured chick periostea: Functional relationship to bone formation

Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Clusters were predominantly AP-positive and located close to bone. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation. These data indicate that a very small population of cycling osteoprogenitor cells is essential for bone formation in vitro and give rise to relatively small numbers of clonally distributed progenitors with limited proliferative capacity. The progeny of these clusters undergo restricted migration and differentiate into osteoblasts.     

Hemorrhage induces enhanced Kupffer cell cytotoxicity while decreasing peritoneal or splenic macrophage capacity. Involvement of cell-associated tumor necrosis factor and reactive nitrogen.

Studies indicate that simple hemorrhage produces a profound depression of cell-mediated immunity, thereby contributing to an enhanced susceptibility to septic challenge in the host. However, it remains unknown whether or not the macrophages' cytotoxic capacity is altered after hemorrhage. To study this, C3H/HeN mice were bled to and maintained at a blood pressure of 35 mm Hg for 60 min, and adequately resuscitated. Mice were then killed at 2 or 24 h after hemorrhage to obtain peritoneal macrophage, splenic macrophage, and Kupffer cells. Cytotoxicity was assessed by determining the capacity of these macrophages to lyse [3H]TdR labeled WEHI-164 clone 13 or P815 tumor target cells (WEHI-164, sensitive to both soluble and cell-associated TNF vs P815 cells, insensitive to soluble TNF). Peritoneal and splenic macrophages from hemorrhaged animals exhibited a significantly reduced cytotoxic capacity, whereas Kupffer cells' ability to kill the target cells was enhanced. Similarly, the Kupffer cells' capacity to release TNF and IL-1, as well as express cell-associated forms of this cytokine are significantly enhanced on macrophages isolated 2 h after hemorrhage, whereas peritoneal macrophages are not. Furthermore, antibodies directed at mouse TNF but not against murine IL-1 alpha or murine IL-6 were able to oblate the enhanced target cell lysis of unfixed, as well as paraformaldehyde fixed (metabolically inactive) Kupffer cells. Studies using inhibitors (GN-monomethyl-arginine, superoxide dismutase, catalase, and ibuprofen) of other TNF-inducible mechanisms of target cell killing indicated that only the inhibition of the release of reactive nitrogen consistently depressed the cytotoxic capacity of Kupffer cells from hemorrhaged mice. Thus, the increased Kupffer cell cytotoxicity from hemorrhaged mice is most likely mediated through the expression of cell-associated TNF and the release of reactive nitrogen.     

Changes of colon epithelium proliferation due to individual aging with cyclin proliferating cell nuclear antigen (PCNA/cyclin) immunostaining compared to [3H]-thymidine radioautography

We report a change in the proliferative activity of mouse colonic epithelium due to development and aging. In order to measure the proliferative activity, colonic epithelium was immunostained for cyclin proliferating cell nuclear antigen (PCNA/cyclin), which appears from the Gl to the S phase of the cell cycle, and compared with labeling obtained by [³H]-thymidine radioautography. Litter mice of six age groups from the fetal period (embryonic day 19), newborn period (postnatal day 1), suckling period (postnatal day 5), weaning period (postnatal dy 21), adult period (2 month old) to the senescent period (11 month old) were examined by immunohistochemistry. The descending colons were fixed in methacarn (method-Carnoy) and embedded in paraffin. Sections were stained for PCNA/cyclin activity using 19A2 monoclonal antibody and the avidin-biotin peroxidase complex (ABC) technique. For radioautography, litter mice of nine age groups using in vivo intraperitoneal administration of [³H]-thymidine. The labeling indices of colonic epithelial cells in the proliferative zone were then analyzed and compared between the two investigative methods. Our results show that the prliferative activity of mice colon was high in the fetal and newborn periods and almost constant from the suckling period to senescence, as demonstrated by both PCNA/cyclin immunohistochemistry and [³H]-thymidine radioautography. The labeling index seen by PCNA/cyclin immunohistochemistry was, however, higher than that seen by [³H]-thymidine radioautography.     

Kinetics of Kupffer cells as shown by parabiosis and combined autoradiographic/immunohistochemical analysis

Leucocytes from syngeneic rats were labeled with tritiated thymidine and donor and recipient rats were connected by a bilateral arteriovenous shunt. Based on the time-dependent label index and labeling intensity, it was concluded that Kupffer cells, the resident macrophages of the liver, have a half-life of 12.4 days and originate from monocytes undergoing one mitosis within 8.4 days after immigration into the liver. The labeled cells were easily identified as Kupffer cells by their selective immunoreactivity with the monoclonal antibody Ki-M2R which is specific for phagocytosing macrophages in the rat. The applicability of combined autoradiography/immunohistochemistry for the identification of other poorly defined macrophage subpopulations is shown.     

Induction of splenic granulopoiesis in vitro.

Murine spleen cells were cultured in vitro to study the induction of committed granulopoietic stem cell (CFU-C) proliferation and maturation. Marbrook-type diffusion cultures were established with and without the addition of colony-stimulating activity (CSA) and harvested at intervals up to 14 days for viable and differential cell counts, [3H]TdR autoradiography, and quantitation of CFU-C by the agar plate method. Without CSA there was poor cell viability and little proliferative capacity. In CSA-stimulated cultures there was a prominent rise in viable cell counts and [3H]TdR labeling indices rose from a mean of 2% at 0 time to 47% after 5 days in vitro. CFU-C increased by 70-fold in these cultures. Peak numbers of CFU-C, immature cells, and [3H]TdR-labeled cells occurred at about 7 days. Thereafter, mature granulocytes and macrophages predominated in culture. Because the liquid spleen cell culture system begins in a resting state and undergoes a wave of proliferative activity in response to CSA, it can provide a useful model system for studying phenomena associated with stem cell activation and differentiation in vitro.     

Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by approximately 80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.     

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.     

Uridine catabolism in Kupffer cells, endothelial cells, and hepatocytes

Kupffer cells, endothelial cells, and hepatocytes were separated by centrifugal elutriation. The rate of uracil formation from [2-14C]uridine, the first step in uridine catabolism, was monitored in suspensions of the three different liver cell types. Kupffer cells demonstrated the highest rate of uridine phosphorolysis. 15 min after the addition of the nucleoside the label in uracil amounted to 51%, 13%, and 19% of total radioactivity in the medium of Kupffer cells, endothelial cells, and hepatocytes, respectively. If corrected for Kupffer cell contamination, hepatocyte suspensions demonstrated similar activities as endothelial cells. In contrast to non-parenchymal cells, hepatocytes continuously cleared uracil from the incubation medium. The lack of uracil consumption by Kupffer cells and endothelial cells points to uracil as the end-product of uridine catabolism in these cells. Kupffer cells and endothelial cells did not produce radioactive CO2 upon incubation in the presence of [2-14C]uridine. Hepatocytes, however, were able to degrade uridine into CO2, beta-alanine, and ammonia as demonstrated by active formation of volatile radioactivity from the labeled nucleoside. There was almost no detectable formation of thymine from thymidine or of cytosine, uracil, or uridine from cytidine by any of the different cell types tested. These results are in line with low thymidine phosphorolysis and cytidine deamination in rat liver. Our studies suggest a co-operation of Kupffer cells, endothelial cells, and hepatocytes in the breakdown of uridine from portal vein blood with uridine phosphorolysis predominantly occurring in Kupffer cells and with uracil catabolism restricted to parenchymal liver cells.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the proliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [3H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE2. The underlayer of tumor macrophages P388 D1 resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [3H] thymidine uptake by EL-4 cells. These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Cell Reactions Following Acute Brain Injury: A Review

The proliferative behavior of glia following a cerebral stab wound in adult rats is reviewed. Proliferation was determined by both PCNA and [³H]thymidine labeling. Microglia were the first cells to divide and constituted the bulk of dividing cells. Both ramified and ameboid microglia divided. A smaller number of astrocytes entered the cell cycle a day later and were shown to derive from differentiated reactive cells. No differentiated oligodendroglia were labeled by thymidine, although a small number of dividing immature oligodendroglia could be detected in cultures of cells labeled in vivo. Recent studies of the properties of oligodendroglial precursors in brain repair mechanisms are discussed. The results so far support our conclusion that differentiated oligodendrocytes do not divide.     

Uptake and processing of immunoglobulin-coated liposomes by subpopulations of rat liver macrophages

In vivo uptake and processing by liver macrophages (Kupffer cells) of liposomes, covalently coated with rabbit immunoglobulin (Ig liposomes) was studied following intravenous injection in rats. Rabbit Ig liposomes were labeled with trace amounts of cholesteryl[14C]oleate and [3H]cholesteryl hexadecyl ether. 1 h after injection of the liposomes, the non-parenchymal cells were isolated and subjected to centrifugal elutriation with stepwise-increasing flow rates; thus, five sub-fractions of Kupffer cells were obtained ranging in size from 9 to 14 micron in diameter. The cells were assayed for peroxidase activity and protein content. Rabbit Ig liposomes were taken up preferentially by Kupffer cells with diameters larger than 11 micron, which constitute less than 25% of the total Kupffer cell population. The intralysosomal degradation of the ingested liposomes was monitored by measuring the 3H/14C ratio of the cells. Due to the rapid release from the cells of the [14C]oleate formed from the cholesteryl[14C]oleate and the virtually complete retention of the non-metabolizable [3H]cholesteryl hexadecyl ether the 3H/14C ratio of the cells increases with proceeding hydrolysis of the liposomes. Thus, we were able to show that, in vivo, the Kupffer cells of the larger size classes, are not only more active in liposome uptake, but are also substantially more active in liposome degradation than smaller cells. The maintenance of the observed heterogeneity of rat liver Kupffer cells, with respect to liposome uptake under in vitro culture conditions, was examined. Subfractions were maintained in monolayer culture for 2 days and incubated with rabbit Ig liposomes. Binding and uptake of liposomes by the cells was monitored by measuring cell-associated radioactivity at 4 degrees C and 37 degrees C, respectively. In contrast to our in vivo results, we observed maximal in vitro liposome binding and uptake in those subfractions containing small cells (10-11 micron diameter), while the fractions containing cells larger than 12 micron, which were more active in vivo, were substantially less active than the smaller cells. The maximum we observed was even more pronounced when the liposome concentration was increased. We conclude that liver macrophage subfractions that barely participate in liposome uptake from the bloodstream in vivo, possess the potential to develop the capacity in vitro to phagocytose rabbit Ig-coated liposomes to extents equal to or even higher than the cells belonging to those subfractions containing the phagocytically most active cells under in vivo conditions.     

Temporal differences in desmin expression between myoblasts from embryonic and adult chicken skeletal muscle

Desmin expression by myoblasts cultured from embryonic and adult chicken breast muscle was examined employing indirect immunofluorescence. The study was performed in conjunction with [3H]thymidine autoradiography and analysis of skeletal myosin expression in order to determine whether the desmin-expressing cells were terminally differentiated. Following 2 h of labeling with [3H]thymidine, 0.55%, 2.60%, and 15.10% of the cells in mass cultures from 10-day-old embryos, 18-day-old embryos and adults, respectively, incorporated [3H]thymidine and were desmin-positive but did not express skeletal-muscle-specific myosin. Using the same approach we determined that 0.07%, 1.25%, and 7.59% of the mononucleated cells in myogenic clones from 10-day-old embryos, 18-day-old embryos and adults, respectively, were desmin-positive, myosin-negative, [3H]thymidine-positive. We suggest that these desmin-positive, myosin-negative myoblasts are proliferating cells, and we conclude that the progeny of adult myoblasts exhibit more desmin-expressing cells of this type than embryonic myoblasts do.     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore

Clostridium difficile toxin A causes severe intestinal inflammation and fluid secretion in rabbit ileum and is chemotactic for neutrophils in vitro. The mechanism of intestinal injury produced by toxin A appears to involve direct epithelial cell damage as well as recruitment of an inflammatory cell response. The current study was undertaken to determine if toxin A can directly stimulate a proliferative response in lymphocytes. Highly purified toxin A, in the presence of the calcium ionophore, ionomycin, stimulated substantial [3H]thymidine incorporation by murine splenic lymphocytes, which was maximal at 10(-9) M toxin A and 800 ng/ml ionomycin. Removal of T cells with anti-Thy-1.2 antibody plus complement had no effect on the proliferative response induced by toxin A. However, [3H]thymidine incorporation in response to toxin A was significantly inhibited (P less than 0.001) by the removal of macrophages from splenocyte suspensions and was restored by the addition of peritoneal macrophages or cell-free supernatant from toxin A-treated macrophage cultures. Analysis of the toxin A-treated macrophage supernatants showed high levels of IL-1, but not IL-2 or IL-4. The combination of recombinant IL-1 plus ionomycin was found to stimulate [3H]thymidine incorporation by T cell-depleted splenic lymphocytes. These results suggest that toxin A stimulates the release of IL-1, and possibly other factors, from macrophages which can costimulate murine B lymphocytes.