A new role for chondrocytes as non-professional phagocytes. An in vitro study

Chondrocytes are capable of engulfing latex particles, cell detritus, and necrotic and apoptotic remains in vitro. It is conceivable that chondrocytes might be involved in the clearance by phagocytosis of different materials within the cartilage. In fact, so far there is no evidence for the presence of "professional phagocytes" (macrophages and neutrophils) in this tissue. Chondrocyte suspensions obtained from rat knees and hips were cultured to assess phagocytosis of latex particles (1 microm), articular cartilage detritus, and necrotic and apoptotic chondrocyte remains (induced by VP-16 1 mM). We observed that chondrocytes phagocytosed latex particles as evaluated by confocal microscopy and flow cytometry. In addition, we observed that chondrocytes phagocytosed articular cartilage detritus and necrotic and apoptotic VP-16 induced-chondrocytes, as observed by bright field microscopy and transmission electron microscopy.     

Phagocytosis--the mighty weapon of the silent warriors

Professional phagocytes, comprising polymorphonuclear neutrophils and monocyte/macrophage cells, play an important role in the host defense. Any defect in their function exposes the organism to microbial intruders terminating in fatal diseases. The functional responses of the phagocytes to bacterial and fungal infections include chemotaxis, actin assembly, migration, adhesion, aggregation, phagocytosis, degranulation, and reactive oxygen species production. Superoxide generation by phagocytic NADPH oxidase is an imperative step toward bacterial killing. Phagocytes participate in inflammatory reactions and exert tumoricidal activity. They are supported by serum factors such as immunoglobulins, cytokines, complement, the acute phase reactant C-reactive protein, production of antibacterial proteins, and others. In addition to their principal task to eliminate bacteria, they are engaged in removing damaged, senescent, and apoptotic cells. Engulfed cell debris, large particles such as latex beads, fat, and oil droplets, are examples of phagocytic activity illustrated in the present review with transmission and scanning electron microscope micrographs. Numerous factors, such as diseases and stressful conditions, affect the engulfing activity of the professional phagocytes. Our experience regarding the impaired phagocytic capacity of cells in patients with diabetes and chronic renal failure is discussed. The results obtained in our laboratory from experiments detecting the effect of strenuous physical exercise, hypothermia, fasting, and abdominal photon irradiation on the phagocytic capacity of human polymorphonuclear neutrophils and rat peritoneal macrophages are hereby summarized and the reports on those subjects in the recent literature are reviewed. A variety of assays are applied for quantifying phagocytosis. Flow cytometry based on incubation of phagocytic cells with fluorescent conjugated particles and measuring the amount of fluorescence as an indicator of the engulfing capacity of the cells is a useful method. A direct visualization of the ingested particles using light or electron microscopy is a valuable tool for estimation of phagocytic function. In our hands, the use of semithin sections of embedded phagocytes following their incubation with latex particles provided satisfactory results for measuring the total number of phagocytic cells, as well as the internalizing capacity of each individual cell. Microbiological assays, the nitroblue tetrazolium test, quantitation of antibody- and antigen-mediated phagocytosis, as well as methods reviewed in detail in other reports are additional applications for determination of this intricate process.     

Phagocytosis of apoptotic cells by liver: a morphological study

The present review deals with the morphological features of the removal of apoptotic cells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form of phagocytosis, playing a major role in the general tissue homeostasis in physiological and pathological conditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences for neighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immune system. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecular mechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine) of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, often operating in cooperation. The data discussed in the present review are drawn from studies of phagocytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as well as in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, and endothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors (galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorganization that favors the engulfment of the apoptotic cells. The "flags" for the identification of apoptotic cells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydilserine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions between modified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways that drive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion).     

Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration

The literature concerning Schwann cells (SCs) and macrophages in myelin phagocytosis during Wallerian degeneration is reviewed. SCs carry out the first step in the removal of myelin by segmenting myelin and then incorporating the degraded myelin. The recruited macrophages then join in the myelin-phagocytosis event, appearing to make full use of their original phagocyte abilities until the end of myelin clearance. The molecular mechanisms of the two cells underlying myelin phagocytosis are thought to be different; myelin phagocytosis by SCs being lectin-mediated, i.e., opsonin-independent, whereas that of macrophages is mainly opsonin-dependent. It is important to note that SCs and macrophages cooperatively accomplish myelin phagocytosis.     

Electron energy loss spectroscopy for analysis of inhaled ultrafine particles in rat lungs

Epidemiologic studies have associated cardiovascular morbidity and mortality with ambient particulate air pollution. Particles smaller than 100 nm in diameter (ultrafine particles) are present in the urban atmosphere in very high numbers yet at very low mass concentration. Organs beyond the lungs are considered as targets for inhaled ultrafine particles, whereby the route of particle translocation deeper into the lungs is unclear. Five rats were exposed to aerosols of ultrafine titanium dioxide particles of a count median diameter of 22 nm (geometric standard deviation, GSD 1.7) for 1 hour. The lungs were fixed by intravascular perfusion of fixatives immediately thereafter. TiO(2) particles in probes of the aerosol as well as in systematic tissue samples were analyzed with a LEO 912 transmission electron microscope equipped with an energy filter for elemental microanalysis. The characteristic energy loss spectra were obtained by fast spectrum acquisition. Aerosol particles as well as those in the lung tissue were unambiguously identified by electron energy loss spectroscopy. Particles were mainly found as small clusters with a rounded shape. Seven percent of the particles in the lung tissue had a needle-like shape. The size distribution of the cluster profiles in the tissue had a count median diameter of 29 nm (GSD 1.7), which indicates no severe clustering or reshaping of the originally inhaled particles. Electron energy loss spectroscopy and related analytical methods were found to be suitable to identify and localize ultrafine titanium dioxide particles within chemically fixed and resin-embedded lung tissue.     

Assessment of particle retention and clearance in the intrapulmonary conducting airways of hamster lungs with the fractionator1

A modified version of the fractionator was used to estimate the total number of polystyrene microspheres retained in the airways of hamster lungs at two different time points after inhalation. A systematic three-stage subsampling procedure with known sampling fractions was adopted. First, each lung was cut into slices, from which primary disectors were sampled systematically with a known sampling fraction. From each primary disector, smaller sub-disectors were subsampled, and the corresponding sampling fraction was estimated by point counting. Finally, a few particles were counted at the microscopic level in the sub-disectors, and the final estimate of total particle number (which is unbiased irrespective of any tissue deformations) was easily computed as a product of the counted number times the reciprocal of the successive sampling fractions. The error variance of each estimate was assessed from the data using a new estimator. An average of 6% of the deposited particles were retained on the epithelial surface of the intrapulmonary conducting airways shortly after the inhalation, from which at least one-third was already phagocytosed by macrophages. After 24 h, an average of 87% of the particles retained shortly after the inhalation had been cleared. The proportion of particles ingested by macrophages had increased to at least 87%, in three out of four animals studied.     

Staining of phagocytized Cryptococcus neoformans with DAPI

The DAPI (4',6-diamidino-2-phenylindole)-fluorochrome was used to demonstrate phagocytized Cryptococcus neoformans using the mouse peritoneal cavity technique. These yeast cells were chosen because they are large and their capsules exhibit a deep yellow fluorescence which contrasts very well with the blue fluorescent nuclei of the phagocytes (preferentially macrophages). In other words, DAPI stains both, acid mucopolysaccharides and nuclear DNA. The capacity of the phagocyte nuclei to surround or even enclose the yeast cells was the most remarkable result. Generally, the application of DAPI in these phagocytosis experiments provides valuable information rapidly, easily and specifically.     

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.     

A novel three-dimensional tissue equivalent model to study the combined effects of cyclic mechanical strain and wear particles on the osteolytic potential of primary human macrophages in vitro.

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.     

The formation of spherical wear particles

A number of recent papers report the formation of spherical wear particles during sliding. In this paper a model is developed in which wear particles formed by adhesive wear processes are trapped in cavities in the sliding surfaces and become smoothed by burnishing processes. It is shown that the reported spherical particle diameters and lengths of sliding are consistent with this model. According to the model, spherical particles are only to be anticipated in slow uniaxial sliding, in fretting and within cracks of a material being fatigued.     

Ferrography: Its application to the study of human joint wear

Synovial fluid aspirates of 20 arthroplastic and 150 osteoarthritic joints were analyzed for evidence of wear particles. Ferrography, an industrial technique for the separation of particulate matter from samples of lubricating solutions, allows extraction of wear particles from synovial fluid. Bichromatic polarized microscopy and scanning electron microscopy permit identification and characterization of metallic, polyethylene and acrylic wear particles from arthroplastic joints as well as biological wear fragments of bone, cartilage, meniscus and synovium from osteoarthritic joints. With both techniques, the number and morphology of the wear particles within the synovial fluid specimens correlate with the rate and mechanism of wear as confirmed by examination of the joint implant or articular surfaces. Toxicity of the various types of wear particles was assessed by cytological examination of the fluid aspirate. Of significant interest is the identification of active phagocytosis of wear particles by synovial fluid white blood cells. This finding may implicate the particles as initiators of secondary inflammatory responses, as occurs in other arthritic diseases.Analysis of aspirated synovial fluid appears to be a useful method for studying the rates, mechanisms and biological responses to wear in both arthroplastic and degenerative joints. In surgical joint replacement, this technique holds much promise, not only as a test for wear and toxicity, but also as a means of assisting in the selection of materials and designs for superior articular implants. In osteoarthritic joints, this analysis provides a method for early diagnosis, serial assessment of therapy and prognostication concerning the future course of the disease. Possibly of most significance, in contrast to previous studies on intact articular surfaces, is the ability to study the principal site of degenerative changes, namely the wear particles. This ability may aid in the elucidation of the underlying cause of osteoarthritis.     

Endocytosis: The different energy requirements for the uptake of particles by small and large vesicles into peritoneal macrophages

The uptake of various, different-sized particles by macrophages was studied using the electron microscope. In addition to observing normal cells, cells were examined which had been inhibited by exposing them to low temperature (4°C), and to a number of metabolic poisons. It was found that large particles (> 01 μm) enter the cells and are contained in large vesicles (0·1–5 μm). Small particles (< 50 nm) may also enter the cells by this process. They enter most frequently, however, by passing into small (～ 70 nm) vesicles. These may later coalesce and their contents adhere to give a second kind of large vesicle. The various inhibitors prevented the ingestion of the large particles (and of the small particles en masse) into large vesicles, but did not prevent their initial adsorption onto the plasma membranes. They did not prevent nearly normal numbers of small particles from entering the pre-existing small vesicles, nor their subsequent fusion into the second class of large vesicles.     

The surface structure of mouse peritoneal cells--a study with the scanning electron microscope

Normal and stimulated peritoneal cells have been examined with the Stereo-scan scanning electron microscope. Normal lymphocytes were identified by their small size; some had numerous small stubby or pointed processes on their surfaces. Macrophages were larger and had ridge-like processes. Many of the stimulated cells showed no significant deviation from normal. Others, all macrophages, showed a marked deviation from the normal spherical shape. Ridge-like processes were more prominent on their surfaces, and large flange-like processes and a few finger-like processes were seen. This suggests that on stimulation peritoneal cells become more deformable and therefore irregular in shape, and that their surfaces become rougher.     

Low-temperature scanning electron microscopy of particle-exposed mouse lung

Inflated frozen mouse lungs were examined using low-temperature scanning electron microscopy (LTSEM) following bulk fracture under vacuum. Various aspects of pulmonary architecture were identified and correlated with structures revealed by SEM following conventional fixation and preparation techniques. Surface etching of selected samples was performed by radiant heating, revealing characteristic cytoplasmic, nuclear and extracellular lattice patterns resulting from ice crystal formation during freezing. These patterns aided in distinguishing between intra- and extracellular spaces. Pulmonary fluids such as mucus and surfactant were identified. Iron oxide particles were introduced into the lungs of some animals by intratracheal instillation and were subsequently identified in frozen-hydrated lung tissue using characteristic X-ray identification and mapping techniques. Particles were observed both intra-and extracellularly and were commonly found in large deposits. These observations confirm the utility of LTSEM techniques for examination of particles within pulmonary tissue. Particle exposure by intratracheal instillation was found to result in a non-uniform distributional pattern.     

Fine structure and function of kupffer cells

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.     

Staining of acid mucopolysaccharides appearing on and in various cell types by DAPI.

It could be shown that DAPI, a newer fluorochrome (4',6-diamidino-2-phenylindole), is able to stain various types of acid mucopolysaccharides, e.g. heparin and agar mucopolysaccharides. Due to their content of heparin, mast cells and basophil cells could be stained rapidly. Other cells e.g. mononuclear phagocytes and malignant epithelial cells, could be loaded with these acid mucopolysaccharides and subsequently stained by DAPI.     

An alkali digestion method to expose connective tissue fibers: A scanning electron microscopy study of rat lung

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 × 5 × 5 mm were immersed in 2.5 M NaOH at 25°C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.     

Age-related modifications of macrophages influenced by “inflammageing” in graft <i>vs.</i> host disease

Most studies focus on the adaptive immune cells in the GVHD pathogenesis, while little is known about innate immune cells in GVHD occurrence and development, especially macrophages. Meanwhile, a higher incidence of graft versus host disease (GVHD) is also found in the elderly patients. Though advances have been made in the modification of macrophages influenced by the inflamm-ageing, there is still no review on the role of macrophages in GVHD and the association between GVHD and the altered macrophages by inflamm-ageing. In this review, we focus on the potential age-related modifications of macrophage in GVHD, which contributes to the change of morbidity and mortality of GVHD. Via literature review, we found that the infiltration of macrophages is associated with GVHD and macrophages are modified in inflamm-ageing state, including the proliferation, migration, phagocytosis, antigen presentation, interaction with other immune cells, and pro-fibrosis. We suppose that altered macrophage functions in inflamm-ageing state contribute to GVHD in elderly patients.     

Analysis of nanometre-sized pyrogenic particles in the scanning transmission electron microscope

The chemical reactions that take place at surfaces as well as internally in nanoscale particles are of great scientific interest. Such reactions control the catalytic properties of small metal particles and thus are technologically very important. In order to allow enhancement of the performance of such catalytic systems, an understanding of the processes taking place at the atomic scale is necessary. Z-contrast imaging and electron energy-loss spectroscopy (EELS) in the dedicated scanning transmission electron microscope can give atomic-scale information and thus provide a unique opportunity to study such nanoscale systems. Here, iron particles from known positions in a ferrocene-seeded flame and their effects on soot formation are studied. Using EELS, spatial variations in oxidation state of the metal atoms are detected across individual nanometre-sized particles collected from the post-flame region. The surfaces of these particles are found to be less oxidized than their centres. Additionally, the particles are imaged with atomic resolution allowing their structure to be identified. Using these results and those for particles collected from in-flame regions, direct evidence is provided for the catalytic role of Fe-containing particles in the removal of carbonaceous soot produced during combustion of hydrocarbon-based fuels.     

Measuring the sizes of nanospheres on a rough surface by using atomic force microscopy and a curvature-reconstruction method

One of the advantages of atomic force microscopy (AFM) is that it can accurately measure the heights of targets on flat substrates. It is difficult, however, to determine the shape of nanoparticles on rough surfaces. We therefore propose a curvature-reconstruction method that estimates the sizes of particles by fitting sphere curvatures acquired from raw AFM data. We evaluated this fitting estimation using 15-, 30-, and 50-nm gold nanoparticles on mica and confirmed that particle sizes could be estimated within 5% from 20% of their curvature measured using a carbon nanotube (CNT) tip. We also estimated the sizes of nanoparticles on the rough surface of dried cells and found we also can estimate the size of those particles within 5%, which is difficult when we only used the height information. The results indicate the size of nanoparticles even on rough surfaces can be measured by using our method and a CNT tip.