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.     

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.     

Cell-matrix entanglement and mechanical anchorage of fibroblasts in three-dimensional collagen matrices

Fibroblast-3D collagen matrix culture provides a physiologically relevant model to study cell–matrix interactions. In tissues, fibroblasts are phagocytic cells, and in culture, they have been shown to ingest both fibronectin and collagen-coated latex particles. Compared with cells on collagen-coated coverslips, phagocytosis of fibronectin-coated beads by fibroblasts in collagen matrices was found to be reduced. This decrease could not be explained by integrin reorganization, tight binding of fibronectin beads to the collagen matrix, or differences in overall bead binding to the cells. Rather, entanglement of cellular dendritic extensions with collagen fibrils seemed to interfere with the ability of the extensions to interact with the beads. Moreover, once these extensions became entangled in the matrix, cells developed an integrin-independent component of adhesion. We suggest that cell–matrix entanglement represents a novel mechanism of cell anchorage that uniquely depends on the three-dimensional character of the matrix.     

Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibroblasts

Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopulations on the basis of functional attributes. We have studied the role of the membrane-anchored hydrolytic enzyme tissue-nonspecific alkaline phosphatase (TN-AP) and the placental alkaline phosphatase (PL-AP) in collagen phagocytosis and in the deletion of cells by apoptosis. Rat-2 cells, which do not constitutively express AP, were transfected with full-length rat TN-AP or PL-AP cDNAs to determine the impact of the TN-AP collagen-binding domain on cell function. Various levels of expression were driven by early (strong) or late (weak) SV40 promoters in the plasmid construct. Controls were transfected with plasmids that did not contain AP cDNA. AP expression in transfected cells was confirmed by Northern blotting, histochemical analysis, and SDS-PAGE analysis of membrane-anchored enzyme released by phosphatidyl inositol phospholipase C. Low levels of TN-AP expression increased cell spreading slightly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluorescence beads per cell. In cells transfected with PL-AP (i.e., no collagen-binding domain), collagen phagocytosis was not affected. Internalization of BSA beads was also not affected by either AP isozyme, indicating that AP was selective for integrin-mediated phagocytosis. In single cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocalization was not detected in cells transfected with PL-AP. Phagocytosis was inhibited by antibodies to the α2 integrin and to AP but not by levamisole, an inhibitor of AP phosphohydrolytic activity. High-level TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G₁ DNA content, nuclear condensation, and nuclear budding. In AP-positive cultures, there was a greatly increased number of floating cells; nick-labeling of DNA by terminal transferase and biotinylated dUTP showed a 15-fold increase of stained cells. These data indicate that low-level TN-AP expression enhances collagen phagocytosis, presumably through the TN-AP collagen-binding domain. High-level AP expression promotes cell deletion by apoptosis. We suggest that the expression of AP by fibroblasts indicates a novel role for this enzyme in collagen degradation by phagocytosis. J. Cell. Physiol. 172:323–333, 1997. © 1997 Wiley-Liss, Inc.     

Methylglyoxal inhibits the binding step of collagen phagocytosis

Bacterial infection-induced fibrosis affects a wide variety of tissues, including the periodontium, but the mechanisms that dysregulate matrix turnover and mediate fibrosis are not defined. Since collagen turnover by phagocytosis is an important pathway for matrix remodeling, we studied the effect of the bacterial and eukaryotic cell metabolite, methylglyoxal (MGO), on the binding step of phagocytosis by periodontal fibroblasts. Type 1 collagen was treated with various concentrations of methylglyoxal, an important glucose metabolite that modifies Arg and Lys residues. The extent of MGO-induced modifications was authenticated by amino acid analysis, solubility, and cross-linking. Cells were incubated with fluorescent beads coated with collagen, and the percentage of phagocytic cells was estimated by flow cytometry. MGO inhibited collagen binding (20% of control for 10 mm MGO) in a time- and concentration-dependent manner. MGO-induced inhibition of binding was prevented by aminoguanidine, which blocks the formation of collagen cross-links. MGO reduced collagen binding strength and blocked intracellular calcium signaling. MGO modified the Arg residue in the critical alpha2beta1 integrin-binding recognition sequence of triple helical collagen peptides, whereas MGO-induced cross-linking of Lys residues played only a small role in binding inhibition. Thus, MGO modifications of Arg residues in collagen could be a key factor in the impaired degradation of collagen that promotes fibrosis in chronic infections, such as periodontitis.     

Role of the cellular attachment domain of fibronectin in the phagocytosis of beads by human gingival fibroblasts in vitro

Summary To study the role of phagocytosis in periodontal tissues, internalization of fibronectin-coated latex beads by Gin-1 fibroblast populations was investigated. Demonstration of phagocytosis by internalization of beads was confirmed by immunofluorescence microscopy, electron microscopy, and flow-cytometry. The percent of cells phagocytosing beads measured by flow-cytometry was negligible at 4° and 23°C, but increased to approximately 17% at 37°C. As measured by automated image analysis, the percentage of phagocytosing cells increased linearly from 8 to 22 with increasing fibronectin concentration of the incubation solution from 30 ng to 300 g/ml. Similar linear increases in the percentage of phagocytosing cells were observed when beads were incubated with cells for periods ranging from 2 h to 2 days. To examine the role of the Arg-Gly-Asp receptor in mediating phagocytosis, fibronectin-coated beads were first coated with either Gly-Arg-Gly-Asp-Ser-Pro or Gly-Arg-Gly-Glu-Ser-Pro peptides at concentrations of 0.125, 0.5, and 1 mg/ml, or with control vehicle, and then incubated with cells. Phagocytosis was completely blocked at 1 mg/ml of the Gly-Arg-Gly-Asp-Ser-Pro peptide, but the Gly-Arg-Gly-Glu-Ser-Pro peptide showed no significant inhibition compared to control values. Blocking antibodies to the cell attachment domain of the fibronectin molecule also reduced the percentage of phagocytosing cells significantly. The data show that these phagocytic assays are sensitive enough to detect the influence of incubation temperature and time, cellular heterogeneity, ligand type, and ligand concentration on the percentage of phagocytosing cells. Further, the mechanisms which determine internalization of fibronectin-coated beads rely in part on the initial binding of ligand to the Arg-Gly-Asp receptor present on fibroblasts.     

Cyclosporin inhibition of collagen remodeling is mediated by gelsolin

Cyclosporin A (CsA) inhibits collagen remodeling by interfering with the collagen-binding step of phagocytosis. In rapidly remodeling connective tissues such as human periodontium this interference manifests as marked tissue overgrowth and loss of function. Previous data have shown that CsA inhibits integrin-induced release of Ca²⁺ from internal stores, which is required for the binding step of collagen phagocytosis. Because gelsolin is a Ca²⁺-dependent actin-severing protein that mediates collagen phagocytosis, we determined whether gelsolin is a CsA target. Compared with vehicle controls, CsA treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. Collagen degradation by phagocytosis in cultured gelsolin wild-type fibroblasts was blocked by CsA, comparable to levels of vehicle-treated gelsolin-null fibroblasts. In wild-type cells treated with CsA, collagen binding was similar to that of gelsolin-null fibroblasts transfected with a gelsolin-severing mutant and treated with vehicle. CsA blocked collagen-induced Ca²⁺ fluxes subjacent to bound collagen beads, gelsolin recruitment, and actin assembly at bead sites. CsA reduced gelsolin-dependent severing of actin in wild-type cells to levels similar to those in gelsolin-null fibroblasts. We conclude that CsA-induced accumulation of collagen in the extracellular matrix involves disruption of the actin-severing properties of gelsolin, thereby inhibiting the binding step of collagen phagocytosis. adhesion molecules; fibroblasts; knockout mice; actin     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Serum-induced inhibition of the phagocytic activity of cultured macrophages IC-21

Fetal bovine serum has been shown to considerably inhibit phagocytosis of non-opsonized 2-μm fluorescent latex beads by cultured macrophages IC-21. Phagocytic activity was assessed using fluorescent microscopy and specially devised ImageJ plugins. Phagocytosis percent, PP (percentage of the bead-containing cells in the cell population under study), and phagocytosis index, PI (mean number of beads per cell in the bead-containing population), were about 2 times lower in the cells incubated in the presence of 10% serum as compared to the respective parameters for the cells incubated in serum-free medium (55 ± 5% vs. 92 ± 1% and 2.0 ± 0.2 vs. 4.3 ± 0.2 beads/cell). The effect of serum was dose-dependent. Albumin (10 mg/ml) did not mimic the effect of serum, suggesting that fatty acid extraction was not the cause of the serum-induced inhibition. Serum is a source of exogenous cholesterol, therefore we checked if cholesterol removal could stimulate phagocytosis. Cholesterol-sequestering agent methyl-β-cyclodextrin (mβCD) in concentrations of 5–7 mM indeed caused an increase of the phagocytic activity but at 10 mM exerted an inhibitory effect in serum-free medium. Connexin channel blocker carbenoxolone (CBX, 250–500μM) in most cases inhibited phagocytosis; the presence of serum or mβCD modulated the CBX effects. The data indicate an important role of serum in regulation of the macrophage phagocytic activity. Stimulating effect produced by serum removal may partly be accounted for by a decrease of cholesterol concentration, which in turn may alter the functioning of integral proteins involved in the mechanisms of phagocytosis.     

Mechanism of cyclosporin-induced inhibition of intracellular collagen degradation

The immunosuppressant cyclosporin A (CsA) markedly inhibits collagen degradation by an intracellular phagocytic pathway in fibroblasts, an effect that can lead to massive gingival overgrowth. We used a collagen bead model of collagen phagocytosis to determine whether CsA inhibits internalization by blocking efflux of calcium from endoplasmic reticulum (ER) and mitochondrial calcium stores. CsA caused dose-dependent inhibition of phagocytosis of collagen-coated (but not bovine serum albumin-coated) beads. Chelation of intracellular Ca(2+) with BAPTA/AM or inhibition of Ca(2+)-ATPase of ER stores with thapsigargin reduced collagen bead phagocytosis. Measurement of intracellular calcium by ratio fluorometry showed increases in response to collagen-coated beads. Preincubation with CsA or thapsigargin caused a >3-fold decrease in intracellular calcium elevations in response to stimulation with collagen beads. Direct measurements of Ca(2+) in mitochondrial and ER stores showed that CsA only slightly inhibited collagen bead-induced discharge of calcium from mitochondria, but almost completely blocked discharge from ER stores. We reduced the numbers of mitochondria with chronic ethidium bromide treatment to test for the importance of ER/mitochondrial interactions. In these cells, CsA delayed collagen bead-induced calcium discharge from mitochondria. Collectively, these data indicate that CsA inhibits collagen phagocytosis by blocking calcium release from ER stores and may perturb functional interactions between the ER and mitochondria that regulate calcium stores.     

A critical role for the membrane-type 1 matrix metalloproteinase in collagen phagocytosis

Degradation of collagen is important for the physiological remodeling of connective tissues during growth and development as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodeling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. However, although various steps in the phagocytic pathway have been characterized, the enzyme required to initially fragment collagen fibrils for subsequent phagocytosis has not been identified. We have used laser confocal microscopy, transmission electron microscopy, and biochemical assays to show that human fibroblasts initiate degradation of collagen through the collagenase activity of the membrane-bound metalloproteinase MT1-MMP. Degradation of natural and reconstituted collagen substrates correlated with the expression of MT1-MMP, which was localized at sites of collagen cleavage at the surface of the cells and also within the cells, whereas collagen degradation was abrogated when MT1-MMP expression was blocked by small interfering RNA treatment. In contrast to MT1-MMP, the gelatinolytic activity of MMP-2 was not required for collagen phagocytosis. These studies demonstrate a pivotal role of catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation.     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

Cathepsin B Is Up-Regulated and Mediates Extracellular Matrix Degradation in Trabecular Meshwork Cells Following Phagocytic Challenge

Cells in the trabecular meshwork (TM), a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment). Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB). Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.     

Phagosomal membrane lipids of LM fibroblasts

Summary Murine fibroblasts, LM cells, were cultured in suspension or monolayer in a chemically defined medium without serum and exposed to polystyrene beads. The LM cells endocytized the beads in direct proportion to the bead/cell ratio and the bead surface area. However, equal volumes of beads irrespective of size or surface area were internalized. The lipid composition of the phagosome membrane differed significantly from the parent primary membrane in having higher contents of phosphatidylcholine, phosphatidylserine, and sterol but lower contents of sphingomyelin and lysophosphatidylcholine. When phagosomes isolated from suspension-cultured LM fibroblasts were exposed to trinitrobenzenesulfonic acid at 4°C, 55±1.6% of the phagosomal membrane phosphatidylethanolamine was trinitrophenylated. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane was not affected by the bead/cell ratio, bead diameter, or exposure time of LM fibroblasts to the beads. When cells were reacted with trinitrobenzenesulfonic acid at 4°C prior to phagocytosis, the amount of trinitrophenylphosphatidylethanolamine was greater in the isolated phagosomes than in the parent primary plasma membrane. Culturing LM fibroblasts in suspension or monolayer had no effect on the asymmetric distribution of phosphatidylethanolamine across primary plasma membrane bilayers. The data are consistent with the observation that LM fibroblasts grown either in suspension or monolayer internalize polystyrene beads at selective sites in the surface membrane.     

Role of the small GTPase activating protein IQGAP1 in collagen phagocytosis

Many adult connective tissues undergo continuous remodeling to maintain matrix homeostasis. Physiological remodeling involves the degradation of collagen fibers by the intracellular cathepsin‐dependent phagocytic pathway. We considered that a multidomain, small GTPase activating protein, IQGAP1, which is involved in the generation of cell extensions, is required for collagen phagocytosis, possibly arising from its interactions with cdc42 and the actin‐binding protein Flightless I (FliI). We examined the role of IQGAP1 in collagen phagocytosis by human gingival fibroblasts (HGFs) and by IQGAP1+/+ and IQGAP1?/? mouse embryonic fibroblasts. IQGAP1 was strongly expressed by HGFs, localized to vinculin‐stained cell adhesions and sites where cell extensions are initiated, and colocalized with FliI. Immunoprecipitation showed that IQGAP1 associated with FliI. HGFs showed 10‐fold increases of collagen binding, 6‐fold higher internalization, and 3‐fold higher β1 integrin activation between 30 and 180 min after incubation with collagen. Compared with IQGAP1+/+ fibroblasts, deletion of IQGAP1 reduced collagen binding (1.4‐fold), collagen internalization (3‐fold), β1 integrin activation (2‐fold), and collagen degradation (1.8‐fold). We conclude that IQGAP1 affects collagen remodeling through its regulation of phagocytic degradation pathways, which may involve the interaction of IQGAP1 with FliI.     

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.     

Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.     

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.     

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.