Eirunn Knudsen

Lysophosphatidic acid induces human natural killer cell chemotaxis and intracellular calcium mobilization

We have investigated the effects of the bioactive lipid lysophosphatidic acid (LPA) on several functions of activated human natural killer (NK) cells. Flow cytometric and immunoblot analyses show that these cells express LPA1, LPA2 and LPA3. LPA but not its precursor phosphatidic acid (PA) induces the chemotaxis of NK cells, an activity that is inhibited by prior treatment of the cells with pertussis toxin (PTX). In addition, LPA induces the mobilization of intracellular calcium, an effect that is markedly inhibited by PTX, but is not inhibited by the addition of EGTA. PA also induces calcium flux in NK cells, but with much lower efficacy than LPA. Cross‐desensitization experiments demonstrate that LPA and PA utilize different receptors. Moreover, LPA or PA but not sphingosine 1‐phosphate, enhances IFN‐γ secretion by activated NK cells. Our results may shed some light on the findings that activated NK cells are found at the sites of tumor growth.

Modulation of natural killer cell cytotoxicity and cytokine release by the drug glatiramer acetate.

Abstract.Glatiramer acetate (GA or Copaxone) is a drug used to treat experimental autoimmune encephalomyelitis in mice and multiple sclerosis in human. Here, we describe a new mechanism of action for this drug. GA enhanced the cytolysis of human NK cells against autologous and allogeneic immature and mature monocyte-derived dendritic cells (DCs). This drug reduced the percentages of mature DCs expressing CD80, CD83, HLA-DR or HLA-I. In contrast, it did not modulate the percentages of NK cells expressing NKG2D, NKp30, or NKp44. Nonetheless, anti-NKp30 or anti-CD86 inhibited GA-enhanced human NK cell lysis of immature DCs. Hence, CD86, and NKp30 are important for NK cell lysis of immature DCs, whereas CD80, CD83, HLA-DR and HLA-I are important for the lysis of mature DCs when GA is used as a stimulus. Further, GA inhibited the release of IFN-γ 24 h but increased the release of TNF-α 48 h after incubation with NK cells.

Macrophage‐dependent regulation of neutrophil mobilization and chemotaxis during development of sterile peritonitis in the rat

Abstract: Pro‐inflammatory cytokines attract leukocytes to inflamed tissues and activate them. Few attempts have been made to identify the sources of cytokines in vivo. We examined the importance of peritoneal macrophages in the mobilization and homing of neutrophils to a sterile peritonitis in the rat, with emphasis on their cytokine production. Macrophages, present in virtually all tissues, are known to be easily activated and to serve as an important source of cytokines. Flow cytometric analysis of cells stained intracellularly with tagged antibodies against various cytokines revealed that the peritoneal macrophages were stimulated to produce the following cytokines: interleukin (IL)‐1β, macrophage inflammatory protein‐2 (MIP‐2), and keratinocyte‐derived cytokine (KC). High numbers of neutrophils, activated on arrival into the peritoneal cavity, also produced IL‐1β, whereas lower numbers contained interleukin‐6, tumor necrosis factor‐α, MIP‐2, KC, and MIP‐1α. This marked activation of peritoneal neutrophils was also reflected by increased surface expression of CD11b. On the other hand, peritoneal macrophages expressed high basal levels of CD11b, which were reduced 24 h after the onset of inflammation. In rats selectively depleted of macrophages by i.p. injection of liposome‐containing clodronate, the massive influx of neutrophils to the peritoneal cavity was markedly reduced, as was the rapid mobilization of mature bone marrow neutrophils. Local macrophages are important both for the accumulation of neutrophils in the inflamed peritoneal cavity and for the early mobilization of neutrophils from the bone marrow. Macrophage‐derived IL‐1β, MIP‐2, and KC are possible mediators of neutrophil homing to inflamed tissues.

Gene therapy with hypoxia-inducible factor 1 alpha in skeletal muscle is cardioprotective in vivo

AIMS Gene therapy of a peripheral organ to protect the heart is clinically attractive. The transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) transactivates cardioprotective genes. We investigated if remote delivery of DNA encoding for HIF-1α is protective against myocardial ischemia-reperfusion injury in vivo. MAIN METHODS DNA encoding for human HIF-1α was delivered to quadriceps muscles of mice. One week later myocardial infarction was induced and four weeks later its size was measured. Echocardiography and in vivo pressure-volume analysis was performed. Coronary vascularization was evaluated through plastic casting. HL-1 cells, transfected with either HIF-1α or HMOX-1 or administered bilirubin or the carbon monoxide (CO) donor CORM-2, were subjected to lipopolysacharide (LPS)-induced cell death to compare the efficacy of treatments. KEY FINDINGS After four weeks of reperfusion post infarction, animals pretreated with HIF-1α showed reduced infarct size and left ventricular remodeling (p<0.05, respectively). Fractional shortening was preserved in mice pretreated with HIF-1α (p<0.05). Invasive hemodynamic parameters indicated preserved left ventricular function after HIF-1α (p<0.05), which also induced coronary vascularization (p<0.05). HIF-1α downstream target heme oxygenase 1 (HMOX-1) was upregulated in skeletal muscle, while serum bilirubin was increased. Transfection of HL-1 cells with HIF-1α or HMOX-1 and administration of bilirubin or CORM-2 comparably salvaged cells from lipopolysacharide (LPS)-induced cell death (all p<0.05). SIGNIFICANCE HIF-1α gene delivery to skeletal muscle preceding myocardial ischemia reduced infarct size and postischemic remodeling accompanied by an improved cardiac function and vascularization. Similar to HIF-1α, HMOX-1, bilirubin and CO were protective against LPS-induced injury. This observation may have clinical potential.

Splenic natural killer cell activity in two models of experimental neurodegenerative diseases

Natural killer (NK) cells are antitumour/anti‐viral effectors and play important roles in shaping the immune system, but their role in neurodegenerative diseases is not clear. Here, we investigated the fate of these cells in two neurodegenerative diseases. In the first model, the activity of NK cells was examined in mice with experimental autoimmune encephalomyelitis (EAE) treated with glatiramer acetate (GA or Copaxone), a drug used to treat EAE in animals and multiple sclerosis in human. The second disease model is twitcher (Galctwi/Galctwi) mice, which represents an authentic model of human Krabbe’s disease. Administration of GA ameliorated EAE in SJL mice corroborated with isolating NK cells that expressed higher killing than cells isolated from vehicle‐dosed animals against immature or mature dendritic cells (DCs). However, this drug showed no effect on the numbers of NK cells or the expression of CD69 molecule. On the other hand, NK cells either disappeared from the spleens or were present in low numbers in the white pulp areas of Galctwi/Galctwi mice, which have increased D‐galactosyl‐β1–1′‐sphingosine (GalSph) levels. Analysis by confocal microscopy shows that NK cells found in the spleens of Galctwi/Galctwi mice were apoptotic. Incubating NK cells in vitro with GalSph induced the apoptosis in these cells, confirming the results of twitcher mice. Our results provide the first evidence showing that amelioration of EAE in mice is corroborated with NK cell lysis of antigen‐presenting DCs, whereas NK cell distribution into the spleen is altered in a devastating lipid disorder corroborated with induction of their apoptosis.

Sequestration patterns of transfused rat neutrophilic granulocytes under normal and inflammatory conditions.

Abstract: The fate of polymorphonuclear neutrophilic granulocytes (PMN) after their mobilization from the bone marrow of healthy individuals is not clearly understood. It has been suggested that there is a continuous utilization of these cells in widespread, subclinical inflammatory foci, where they are ultimately degraded. The goal of the present experiments was to determine whether an alternative ecotaxis (“homing”) exists, namely sequestration and degradation of PMN by mononuclear phagocytes exposed to the bloodstream in the liver, spleen and bone marrow. Blood PMN were collected from donor rats, labelled with 51Cr, and injected i.v. into 2 syngeneic rats, one of them having an induced sterile peritonitis. After various time intervals up to 18 h, the rats were killed and exsanguinated. As expected, we found cell‐bound radioactivity in the inflamed peritoneal cavities, and also a high amount of radioactivity in liver, spleen, and bone marrow. The bone marrow uptake of PMN appeared to be much lower in the inflammation rats than in the normal controls. These findings were confirmed in PMN transfer experiments using PVG rats congenic for the RT7 alloantigenic system. Here, transfused blood leukocytes were traced with fluorescent, monoclonal HIS41 antibodies and flow cytometry. A possible corticosteroid effect on the bone marrow sequestration could not be substantiated. Uptake and degradation of PMN takes place in organs containing phagocytes exposed to the bloodstream. Sequestration of PMN in the bone marrow is apparently down‐regulated in inflammatory states, perhaps increasing the PMN availability to inflamed tissue.

FTY720 and SEW2871 reverse the inhibitory effect of S1P on natural killer cell mediated lysis of K562 tumor cells and dendritic cells but not on cytokine release

The aims of this study are to examine the effect of sphingosine 1-phosphate (S1P) on IL-2-activated natural killer (NK) cell lysis of K562 tumor cells and immature dendritic cells (iDCs), and to investigate the mechanisms involved in S1P activity. Our results show that S1P protected K562 cells or iDCs from NK cell lysis, which was reversed by FTY720 and SEW2871, the antagonists of S1P1. S1P did not modulate the expression of NKG2D, NKp30, NKp44 or CD158 on the surface of NK cells, and neither affected the expression of CD80, CD83, or CD86 on the surface of DCs. In contrast, it increased the expression of HLA-I and HLA-E on DCs, an activity that was inhibited by FTY720 or SEW2871. Similarly, the inhibitory effect of S1P for NK cell lysis of K562 cells was directed toward S1P1 expressed on the tumor cells but not on NK cells. Further analysis indicates that NK cells secreted various cytokines and chemokines with various intensities: (1) low (IL-4, IL-6, IL-12, TNF-α and MCP-1); (2) intermediate (IL-1β, IL-10, TGF-β1, and IL-17A); (3) high (IFN-γ, and MIP-1α); and (4) very high (MIP-1β). S1P significantly reduced the release of IL-17A and IFN-γ from NK cells, but this inhibition was S1P1-independent. These results indicate that S1P is an anti-inflammatory molecule, and that S1P1 is important for the interaction among NK cells and tumor cells or DCs leading to up-regulation of HLA-I and HLA-E on the surface of DCs, but not in S1P inhibition of the release of inflammatory cytokines from NK cells. Further, the results suggest that FTY720 and SEW2871 may potentially be used as prophylactic and/or therapeutic drugs to treat cancer patients.

G‐CSF enhances the proliferation and mobilization, but not the maturation rate, of murine myeloid cells

Objectives:  Whether G‐CSF enhances the maturation of neutrophilic granulocytes or just accelerates the mobilization of mature and maturing granulocytes from bone marrow to blood, or both, is not clear. Using an in vivo culture system where such mobilization cannot take place, we previously showed that G‐CSF did not accelerate maturation. To further clarify the role of G‐CSF, we now have examined its effect on murine granulopoiesis in situ.

Macrophages in spleen and liver direct the migration pattern of rat neutrophils during inflammation

Abstract:  Objective: The exact fate of polymorphonuclear neutrophilic granulocytes (PMN; neutrophils) after their mobilization from the bone marrow is not known. It is believed that they, after a relatively short lifespan (1–3 d), become apoptotic and phagocytosed by macrophages. We have recently shown that transfused neutrophils sequestrate not only in lungs, liver and spleen, but also to a large extent in the bone marrow, possibly because of uptake by macrophages. Hence, we studied if inactivation of macrophages would alter the pattern of neutrophil migration. Methods: We used transfused congenic or syngeneic neutrophils in rats with or without sterile peritonitis, induced by a casein preparation (Bacto‐Tryptone). To perturb macrophage function, we either killed them with liposome‐encapsulated clodronate or overloaded them with inert phagocytosable particles. Transfused neutrophils were tracked with flow cytometric or radiometric methods. Results: Not more than a small portion of the neutrophils migrated to the inflamed peritoneal cavity under any circumstance. Their ecotaxis to liver and spleen was reduced in rats with liver and spleen macrophages either congested with polystyrene particles or depleted by clodronate. The bone marrow uptake and blood retention of transfused neutrophils were increased in macrophage‐depleted rats 18 h after transfusion. In rats depleted of liver macrophages only, the sequestration in the liver was reduced, without detectably changed uptake in bone marrow and spleen. Conclusion: Macrophages are instrumental to the neutrophil migration stream in the organism, and their function in this regard is robust and not easily decreased by inert phagocytosable particles or a killing agent.

Calprotectin (S100A8/S100A9) and myeloperoxidase: co-regulators of formation of reactive oxygen species.

Inflammatory mediators trigger polymorphonuclear neutrophils (PMN) to produce reactive oxygen species (ROS: O2-, H2O2, ∙OH). Mediated by myeloperoxidase in PMN, HOCl is formed, detectable in a chemiluminescence (CL) assay. We have shown that the abundant cytosolic PMN protein calprotectin (S100A8/A9) similarly elicits CL in response to H2O2 in a cell-free system. Myeloperoxidase and calprotectin worked synergistically. Calprotectin-induced CL increased, whereas myeloperoxidase-triggered CL decreased with pH > 7.5. Myeloperoxidase needed NaCl for CL, calprotectin did not. 4-hydroxybenzoic acid, binding ∙OH, almost abrogated calprotectin CL, but moderately increased myeloperoxidase activity. The combination of native calprotectin, or recombinant S100A8/A9 proteins, with NaOCl markedly enhanced CL. NaOCl may be the synergistic link between myeloperoxidase and calprotectin. Surprisingly- and unexplained- at higher concentration of S100A9 the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function. We propose that the ∙OH is predominant in ROS production by calprotectin, a function not described before.