Alon Krispin

Translocation of Active Heparanase to Cell Surface Regulates Degradation of Extracellular Matrix Heparan Sulfate upon Transmigration of Mature Monocyte-Derived Dendritic Cells

After Ag capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. To migrate from peripheral tissue such as the epidermis to regional lymph nodes, Ag-bearing epidermal Langerhans cells must move through an extracellular matrix (ECM) of various compositions. The nature of their capacity to transmigrate via ECM is not well understood, although MIP-3β and CCR7 play critical roles. We were interested in verifying whether heparanase, a heparan sulfate-degrading endo-β-d-glucuronidase that participates in ECM degradation and remodeling, is expressed and functional in monocyte-derived DCs. Using immunohistochemistry, confocal microscopy, RT-PCR, Western blot analysis, assays for heparanase activity, and Matrigel transmigration, we show that heparanase is expressed in both nuclei and cytoplasm of immature DCs, and that gene expression and synthesis take place mainly in monocytes and early immature DCs. We also found that both nuclear and cytoplasm fractions show heparanase activity, and upon LPS-induced maturation, heparanase translocates to the cell surface and degrades ECM heparan sulfate. Matrigel transmigration assays showed a MIP-3β-comparable role for heparanase. Because heparan sulfate glycosaminoglycans play a key role in the self-assembly, insolubility, and barrier properties of the ECM, the results of this study suggest that heparanase is a key enzyme in DC transmigration through the ECM.

‘Danger’ effect of low-density lipoprotein (LDL) and oxidized LDL on human immature dendritic cells

Dendritic cell (DC) maturation may accelerate autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, and may contribute to accelerated atherosclerosis seen in these patients. The immune system responds to both exogenous and endogenous ‘dangerous’ signals that can induce dendritic cell maturation. We have found that autologous plasma contains danger signals that induce up‐regulation of major histocompatibility complex (MHC) class II and co‐stimulatory molecules in immature DCs (iDCs). The objective of this study was to determine whether low‐density lipoprotein (LDL) and/or oxidized LDL (oxLDL) constitute danger signals, and to assess the effect of exposure to LDL and oxLDL following monocyte differentiation into iDCs in lipoprotein‐deficient serum (LPDS). IDCs were generated in the presence of autologous plasma or LPDS. Expression of maturation and migration molecules was evaluated using flow cytometry, and morphology was assessed by light microscopy. Pro‐ or anti‐apoptotic effect was determined using annexin V and propidium iodide binding. Phagocytosis of apoptotic cells was evaluated using autologous plasma or LPDS. LDL and oxLDL were clearly able to slightly up‐regulate levels of HLA‐DR and co‐stimulatory molecule CD86. High oxLDL concentrations (50–100u2003µg/ml) were associated with expression of additional maturation molecules. Moreover, iDCs that were prepared in LPDS showed partial maturation following exposure to LDL and oxLDL, and improved tolerogenic apoptotic cell uptake. This study suggests that oxLDL, and to some extent LDL, are at least partly responsible for the iDC ‘danger’ response induced by autologous plasma.

Constitutive Neutrophil Apoptosis: Regulation by Cell Concentration via S100 A8/9 and the MEK – ERK Pathway

Programmed cell death (PCD) is a fundamental mechanism in tissue and cell homeostasis. It was long suggested that apoptosis regulates the cell number in diverse cell populations; however no clear mechanism was shown. Neutrophils are the short-lived, first-line defense of innate immunity, with an estimated tu200a=u200a1/2 of 8 hours and a high turnover rate. Here we first show that spontaneous neutrophil constitutive PCD is regulated by cell concentrations. Using a proteomic approach, we identified the S100 A8/9 complex, which constitutes roughly 40% of cytosolic protein in neutrophils, as mediating this effect. We further demonstrate that it regulates cell survival via a signaling mechanism involving MEK-ERK via TLR4 and CD11B/CD18. This mechanism is suggested to have a fine-tuning role in regulating the neutrophil number in bone marrow, peripheral blood, and inflammatory sites.

Altered dendritic cells with tolerizing phenotype in patients with systemic lupus erythematosus

Earlier we showed the generation of tolerizing human monocyte‐derived DC following interaction with iC3b‐opsonized apoptotic cells. In this study we examine the generation of DC with our previously described tolerogenic phenotype in patients with the systemic autoimmune disease systemic lupus erythematosus (SLE). Monocyte‐derived DC were generated in 71 SLE patients, characterized, and then tested for clearance of iC3b‐opsonized 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanineperchlorate‐stained apoptotic cells using flow cytometry, and for autologous T‐cell activation using autologous mixed lymphocyte reaction (AMLR), at the same time as controls. Compared with healthy, age‐ and gender‐matched controls, SLE patients showed upregulation of MHC class II, with a mean expression of 130.5%±36.8% (p<0.007); CD86 in immature DC from SLE patients, generated in autologous human or control plasma, were also upregulated, with mean expression 106.6%±18.0% (p<0.03). A significant (>20%) reduction in iC3b‐apoptotic cell uptake, together with increased autologous mixed lymphocyte reaction, was seen in 75% of SLE patients. Mean 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanineperchlorate‐stained apoptotic cell acquisition was 70.0%±24% (p<0.0001) compared with healthy controls. Altered generation of a tolerizing DC phenotype was seen in at least one third of SLE patients following interaction with iC3b‐opsonized apoptotic cells. These results suggest that a substantial portion of SLE patients fail to generate DC with a tolerizing phenotype.

Calcium, leukocyte cell death and the use of annexin V: fatal encounters

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane’s outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results.

What do we mean when we write "senescence," "apoptosis," "necrosis," or "clearance of dying cells"?

The clearance of dying cells has become an important field of research. Apart from a significant increase in our understanding of the mechanisms for uptake, cell clearance is a basic mechanism in tissue homeostasis, cancer, resolution of inflammation, induction of tolerance, and autoimmunity. Phagocytosis of dying cells is a complex process, involving many interacting molecules on the dying cell and the phagocyte, and in the microenvironment. Although much is known on the subject, there are many questions and unknown variables that remain under investigation. Naturally, different terms were developed, among which some are misused, leading sometimes to pseudoconflicts of understanding. Several receptors were described as “phosphatidylserine receptor: are they all equal?” We will revise terms such as apoptosis, primary and secondary necrosis, lysed cells, senescent cells, clearance of apoptotic cells, efferocytosis, and more. We will try to point out misnomers, misunderstandings, and contradictions, and to define a consensual vocabulary.

Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells.

Identification and Characterization of Two Human Monocyte-Derived Dendritic Cell Subpopulations with Different Functions in Dying Cell Clearance and Different Patterns of Cell Death.

Human monocyte-derived dendritic cells (mdDCs) are versatile cells that are used widely for research and experimental therapies. Although different culture conditions can affect their characteristics, there are no known subpopulations. Since monocytes differentiate into dendritic cells (DCs) in a variety of tissues and contexts, we asked whether they can give rise to different subpopulations. In this work we set out to characterize two human mdDC subpopulations that we identified and termed small (DC-S) and large (DC-L). Morphologically, DC-L are larger, more granular and have a more complex cell membrane. Phenotypically, DC-L show higher expression of a wide panel of surface molecules and stronger responses to maturation stimuli. Transcriptomic analysis confirmed their separate identities and findings were consistent with the phenotypes observed. Although they show similar apoptotic cell uptake, DC-L have different capabilities for phagocytosis, demonstrate better antigen processing, and have significantly better necrotic cell uptake. These subpopulations also have different patterns of cell death, with DC-L presenting an inflammatory, “dangerous” phenotype while DC-S mostly downregulate their surface markers upon cell death. Apoptotic cells induce an immune-suppressed phenotype, which becomes more pronounced among DC-L, especially after the addition of lipopolysaccharide. We propose that these two subpopulations correspond to inflammatory (DC-L) and steady-state (DC-S) DC classes that have been previously described in mice and humans.

Role of class A human thrombospondins in the clearance of dying cells and tolerance induction

Mammalian thrombospondins (TSPs) are a group of large, secreted, calcium‐binding glycoproteins of complex spatial structure that mediate a wide range of intercellular activities and participate in cell–matrix interactions. This family includes five proteins, divided into two subfamilies, that possess different roles and tissue expression. TSPs have complex roles in mediating cellular processes. Apoptotic cell and phagocyte interactions show a dynamic structure with expanding complexity. However, a vast majority of the consequences of these interactions can be mediated by a single protein. One of these signaling molecules is TSP‐1, which binds to a wide variety of integrin and nonintegrin cell surface receptors and mediates both engulfment and immune modulation. This mechanism is not only important in homeostasis but may also be a major mechanism for inflammation downregulation and in avoiding autoimmunity.