The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Translocation of chromatin proteins to nucleoli-The influence of protein dynamics on post-fixation localization.

It is expected that the subnuclear localization of a protein in a fixed cell, detected by microscopy, reflects its position in the living cell. We demonstrate, however, that some dynamic nuclear proteins can change their localization upon fixation by either crosslinking or non-crosslinking methods. We examined the subnuclear localization of the chromatin architectural protein HMGB1, linker histone H1, and core histone H2B in cells fixed by formaldehyde, glutaraldehyde, glyoxal, ethanol, or zinc salts. We demonstrate that some dynamic, weakly binding nuclear proteins, like HMGB1 and H1, may not only be unexpectedly lost from their original binding sites during the fixation process, but they can also diffuse through the nucleus and eventually bind in nucleoli. Such translocation to nucleoli does not occur in the case of core histone H2B, which is more stably bound to DNA and other histones. We suggest that the diminished binding of some dynamic proteins to DNA during fixation, and their subsequent translocation to nucleoli, is induced by changes of DNA structure, arising from interaction with a fixative. Detachment of dynamic proteins from chromatin can also be induced in cells already fixed by non-crosslinking methods when DNA structure is distorted by intercalating molecules. The proteins translocated during fixation from chromatin to nucleoli bind there to RNA-containing structures.

STRIDE-a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells.

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

The subcellular localization of bHLH transcription factor TCF4 is mediated by multiple nuclear localization and nuclear export signals.

Transcription factor 4 (TCF4) is a class I basic helix-loop-helix (bHLH) transcription factor which regulates the neurogenesis and specialization of cells. TCF4 also plays an important role in the development and functioning of the immune system. Additionally, TCF4 regulates the development of Sertoli cells and pontine nucleus neurons, myogenesis, melanogenesis and epithelial-mesenchymal transition. The ability of transcription factors to fulfil their function often depends on their intracellular trafficking between the nucleus and cytoplasm of the cell. The trafficking is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). We performed research on the TCF4 trafficking regulating sequences by mapping and detailed characterization of motifs potentially acting as the NLS or NES. We demonstrate that the bHLH domain of TCF4 contains an NLS that overlaps two NESs. The results of in silico analyses show high conservation of the sequences, especially in the area of the NLS and NESs. This high conservation is not only between mouse and human TCF4, but also between TCF4 and other mammalian E proteins, indicating the importance of these sequences for the functioning of bHLH class I transcription factors.

Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm-l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm-l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein-enriched region flanked by protein-depleted regions. It can be explained as a result of the Ostwald-like ripening process and intrinsic properties of Stm-l, and bears some analogy to the daily growth layers of the otolith.

Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain-containing protein 4 (NPAS4).

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic ß-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.

Molecular determinants of Drosophila immunophilin FKBP39 nuclear localization.

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro.

Entry of Human Coronavirus NL63 into the Cell.

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and ß) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 µM camptothecin, 10 µM etoposide or 0.2 µM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Mapping of the Sequences Directing Localization of the Drosophila Germ Cell-Expressed Protein (GCE).

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. We performed confocal microscopy analysis of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed us to characterize the details of the subcellular traffic of this protein. We demonstrate that GCE possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the C-terminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins.

Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod-induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain-like protein. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase-8. Fingolimod-induced neutrophil death is independent of sphingosine-1-phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase-8 [Z-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone], receptor-interacting protein kinase 1 (necrostatin-1), receptor-interacting protein kinase 3 (necrosulfonamide), and heat shock protein 90 [geldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin]. Furthermore, NADPH oxidase 1 inhibition with diphenyleneiodonium chloride protects neutrophils against fingolimod-mediated cell death. Overall, these observations suggest that fingolimod acts through a mechanism involving the necrosome signaling complex and the oxidative stress machinery.

The nucleocapsid protein of human coronavirus NL63.

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells.

UNLABELLED: Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE: ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

Photosensitized damage inflicted on plasma membranes of live cells by an extracellular generator of singlet oxygen--a linear dependence of a lethal dose on light intensity.

We describe a study of the influence of a dose rate, i.e. light intensity or photon flux, on the efficiency of induction of a loss of integrity of plasma membranes of live cells in culture. The influence of a photon flux on the size of the light dose, which was capable of causing lethal effects, was measured in an experimental system where singlet oxygen was generated exclusively outside of live cells by ruthenium(II) phenantroline complex. Instantaneous, sensitive detection of a loss of integrity of a plasma membrane was achieved by fluorescence confocal imaging of the entry of this complex into a cell interior. We demonstrate that the size of the lethal dose of light is directly proportional to the intensity of the exciting light. Thus, the probability of a photon of the exciting light inflicting photosensitized damage on plasma membranes diminishes with increasing density of the incident photons.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.

Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25-250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

Inducing local DNA damage by visible light to study chromatin repair.

Dynamics of DNA repair and recruitment of repair factors to damaged DNA can be studied by live cell microscopy. DNA damage is usually inflicted by a laser beam illuminating a DNA-interacting photosensitizer in a small area of the nucleus. We demonstrate that a focused beam of visible low intensity light alone can inflict local DNA damage and permit studies of DNA repair, thus avoiding potential artifacts caused by exogenous photosensitizers.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes. Recognition of apoptotic cells is predominantly connected with CD16(+) monocytes (CD14(high) CD16(+) and CD14(dim) CD16(+)) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl-l-serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide-activated subpopulations of monocytes while in contact with apoptotic cells produce more anti-inflammatory cytokine interleukin-10 whereas the production of pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta is reduced.