Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.     

Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes

Degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen-coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of ³H-Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead-labeled phagosomes in single, pre-mitotic cells demonstrated that > 90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis-inducing drugs nifedipine or dilantin. These cultures exhibited marked (15–75%), dose-dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis. © 1993 Wiley-Liss, Inc.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

Inhibition by theophylline of phagocytosis in Acanthamoeba castellanii.

Theophylline inhibited the phagocytosis of latex beads by Acanthamoeba castellanii (Neff). The cells recovered the ability to engulf beads after 1-2 hr of exposure to theophylline. Cells which have been exposed to 25 mM theophylline for a period of inhibition and recovery were not inhibited further by incubation with a fresh medium containing the same concentration of theophylline. However, the medium in which the cells recovered was as effective as a fresh medium in inhibiting phagocytosis in a fresh batch of cells, suggesting that the development of insensitivity to theophylline inhibition resides with the cells themselves. Dibutyryl cyclic AMP also inhibited bead uptake.     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

Measurement of phagocytosis using fluorescent latex beads

Fluorescent monodisperse latex beads and a computer-centered spectrofluorimeter were used to devise a sensitive new assay for phagocytosis. LM fibroblasts, a transformed cell line with a high endocytic rate, were exposed to fluoresbrite beads and the following parameters were investigated: incubation time, incubation temperature and bead/cell ratio. The bead uptake was linear for 60 min over a wide range of bead/cell ratios up to 130 beads/cell. Phagocytosis was inhibited at 4 degrees C, by incubation in the presence of colchicine, and by glucose deprivation. Scanning and transmission electron microscopy were used to confirm that at 37 degrees C both bead adsorption and internalization occurred while at 4 degrees C only bead adsorption but not endocytosis occurred. Large bead sizes (0.86 and 1.72 micrometer diameter) were most useful due to higher fluorescence and higher signal to noise ratios than smaller beads (0.25 and 0.57 micrometer diameter). Beads (0.86 micrometer diameter) were taken up at a rate of 4.4 beads/cell/h at 37 degrees C when a bead/cell ratio of 70 was used. The uptake was zero when assayed at zero time. These criteria establish that fluoresbrite beads provide a useful new fluorimetric assay for phagocytosis.     

Interaction of fibronectin-coated beads with attached and spread fibroblasts : Binding, phagocytosis, and cytoskeletal reorganization

After 15 min incubations, binding of 0.8-, 6-, and 16-microns fibronectin-coated latex beads occurred primarily at the margins of chick embryo fibroblasts that previously were attached and spread on fibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-micron beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and alpha-actinin patches adjacent to the sites where the beads were bound. The formation of alpha-actinin patches could be detected with 6- or 16-microns beads shortly after initial bead binding to the cells, but a similar reorganization of alpha-actinin in response to the binding of 0.8-micron beads was not detected. The patches of alpha-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy (SEM) to be sites of cell interaction with 6- but not 0.8-micron beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-microns beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interaction.     

Binding and phagocytosis of fibronectin-coated beads by BHK cells: receptor specificity and dynamics

Studies on the receptor specificity and dynamics involved in fibroblast phagocytosis of latex beads revealed the following: 1) Ligands other than fibronectin such as concanavalin A (ConA) and serum spreading factor, when coated on latex beads, were found to promote phagocytosis of the beads. This indicates that fibroblast phagocytosis, like spreading, is a ligand-receptor mediated phenomenon not specifically requiring fibronectin (pFN); 2) Anti-pFN antibodies were found to inhibit the ability of cells to ingest pFN-coated beads that previously were bound on the cell surfaces. Consequently, binding of beads to the cell surfaces per se is not a sufficient signal to promote ingestion of the beads; 3) Finally, divalent cations protected receptor function necessary for phagocytosis of pFN-coated beads from proteolysis by trypsin, as previously was found for receptors involved in cell attachment and spreading on pFN-coated culture dishes. Recovery experiments carried out with cells whose surface receptors had been destroyed indicated that there was an internal (or cryptic cell surface) pool of receptors that amounted to at least 50% of the receptors normally found on the cell surface. After complete destruction of the cell surface and cryptic pools of receptors, reappearance of receptors required for bead binding and phagocytosis required several hours and did not occur in the absence of new protein synthesis.     

Immunophenotyping using gold or silver nanoparticle-polystyrene bead conjugates with multiple light scatter

BACKGROUND: The type of antibody-conjugated polystyrene (PS) latex beads for use as light scatter shift agents for targeted lymphocyte populations in whole blood has been expanded to include gold and silver nanoparticle-aminodextran-PS latex bead conjugates with antibodies. The linkers between antibody and colloidal metal were an aminotrithiol ligand or aminodextran polymer molecules. METHODS: A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10-20 degrees ) and UMALS (20-65 degrees ) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead-fluorescent marker experiments. RESULTS: Two mutually exclusive cell populations, CD4+ and CD8+ lymphocytes, have been simultaneously enumerated in blood by using a mixture of CD4-PS, CD8-Au-PS or CD4-Au-PS, CD8-PS beads, and one laser line, 633 nm, excitation. Similar measurements were made with mixtures of CD4-PS, CD8-Ag-PS or CD4-Ag-PS, CD8-PS beads. Also, simultaneous use of bead and fluorescent markers mixed with whole blood was demonstrated with CD4-PS beads and with the CD4-RD1/CD8-FITC dual marker. CONCLUSIONS: Enumeration of CD4 and CD8 lymphocytes in whole blood by light scatter parameters only compared well with standard analyses with fluorescent markers. In simultaneous bead-fluorescent marker labeling of lymphocytes, the labeled bead had to be mixed first with cells in whole blood.     

Silent cleanup of very early apoptotic cells by macrophages

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-L-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.     

Role of integrins in regulation of collagen phagocytosis by human fibroblasts

Phagocytosis of collagen fibrils by fibroblasts is an important pathway for degradation of extracellular matrix in mature connective tissues. To study regulatory mechanisms in phagocytosis, 2-μm fluorescent beads coated with either collagen (COL) or bovine serum albumin (BSA) were incubated with human gingival fibroblasts in vitro. For these studies single cell suspensions were prepared by trypsinization, and bead internalization and collagen receptor expression were assessed by flow cytometry. After 3-h incubations, up to 8-fold more cells internalized COL beads than BSA-coated beads. Increased collagen coating concentration was associated with elevated proportions of cells that internalized COL beads, and was observed also in the presence of competing fibronectin-coated beads. The number of beads per cell and the percent of phagocytic cells increased proportionally with higher bead loadings. At > 4 beads per cell a maximum of ∼︁80% of cells were phagocytic. Cells reacted with mAbs against the α1, α2, and α3 integrin subunits were, respectively, 5%, 98% and 93% positively stained above background controls. All cells that internalized COL beads exhibited α2 staining but there were large proportions of phagocytic cells that were not stained for α1. In unfixed cells, bead internalization caused an immediate reduction of surface staining of membrane-bound α2 by ∼︁55% which returned to control levels within 3 h, indicating that cell-surface α2 was internalized by phagocytosis. Preincubation of cells with up to 8 COL beads per cell reduced the proportion of phagocytic cells and the number of internalized beads after a second COL bead incubation 4 h later. To assess the relationship between the percent of phagocytic cells and α2 integrin levels, serum starvation and cycloheximide experiments were conducted. Compared to controls, serum starvation for 24 h induced a 3.2-fold increase of cells internalizing COL beads but did not alter α2 staining levels. In contrast, 3 h cycloheximide treatment reduced α2 staining to 60% of control levels and this treatment also inhibited COL bead internalization. GRGDTP peptide as well as mAbs against the α1 and α2 subunits significantly reduced internalization of COL beads by 1.8 to 2.6-fold, whereas GRGESP peptide and α3 mAb exerted no effect. Internalization of BSA beads was not affected by any of these treatments. Collectively, these data indicate that the α2 integrin, along with other, as yet unidentified components, is likely involved in COL bead internalization. The α2 integrin subunit is rapidly recycled or synthesized following a phagocytic load. In contrast, the α1 integrin is not directly required for phagocytosis but may regulate the internalization step. © 1996 Wiley-Liss, Inc.     

Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6- micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand- coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.     

Collagen phagocytosis by fibroblasts is regulated by decorin

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.     

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Deregulation of Collagen Phagocytosis in Aging Human Fibroblasts: Effects of Integrin Expression and Cell Cycle

Intracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with increased lysomomal enzyme content in fibroblasts, we examined the regulation of collagen phagocytosis by integrin expression and the cell cycle in anin vitrofibroblast aging model. Two different fibroblast lines (CL1; CL2) at the fourth subculture were passaged up to replicative senescence to model aging processesin vitro.Cells were incubated with collagen-coated or BSA-coated green fluorescent beads for 3 h to assess α₂β₁-integrin-mediated or nonspecific phagocytosis, respectively. Single-cell suspensions were stained with DAPI and sulforhodamine 101 to separate cycling G₁and noncycling G₀cells. Staining for α₂-integrin, bead internalization, and bivariate analyses of DNA/protein content were measured by three-color flow cytometry. Serum deprivation was used to induce increases in the proportion of G₀cells. For G₁cells, the proportion of collagen phagocytic cells was >50% for all passages and collagen beads were internalized >5-fold more frequently than BSA beads. In contrast, G₀cells with diploid DNA content but low protein content exhibited greatly reduced phagocytic capacity (<10% of cells internalized collagen or BSA beads), the number of beads per cells was 4-fold less, and α₂integrin expression was very low compared to G₁cells. The proportion of collagen phagocytic cells and the proportion of α₂-integrin-positive cells increased with transit through the cell cycle. At higher passage numbers mean cell volume and cytoplasmic granularity were reduced 30% but at replicative senescence cells with large surface area and subdiploid DNA predominated. The proportion of collagen and BSA phagocytic G₁cells increased 1.5- and 5-fold, respectively, and the number of beads per cell increased <3-fold. However, surface α₂-integrin staining remained unchanged. These data indicate that the collagen and nonspecific internalization pathways were greatly upregulated, independent of cell cycle phase, and that cellular agingin vitrostrongly influences the specificity and rate of phagocytic processes in fibroblasts. We suggest that age-related loss of collagen in connective tissues undergoing turnover may be a manifestation of a deregulated increase of collagen phagocytosis in which the net loss of degraded collagen exceeds new synthesis.     

Body muscles of the lamprey. Some structural features of the T system and sarcolemma

Electron microscopic studies confirm and extend the conclusions derived previously from a quantitative biochemical study of the phagocytosis of polystyrene and polyvinyltoluene latex beads by Acanthamoeba (1). Latex beads 1.305, 1.90, and 2.68 µ in diameter are ingested individually, with each bead tightly surrounded by a membrane derived from the plasma membrane. Latex beads 0.557, 0.264, 0.126, and 0.088 µ in diameter are accumulated at the surface of the ameba and then phagocytosed, with many beads tightly packed within one membrane-bounded vesicle.     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.