Kristin L. Sand

DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis

Polyunsaturated fatty acids (PUFAs) are normal constituents of the diet, but have properties different from other fatty acids (e.g., through generation of signaling molecules). N-3 PUFAs reduce cancer cell growth, but no unified mechanism has been identified. We show that docosahexaenoic acid (DHA; 22:6 n-3) causes extensive changes in gene expression patterns at mRNA level in the colon cancer cell line SW620. Early changes include unfolded protein response (UPR) and increased levels of phosphorylated eIF2α as verified at protein level. The latter is considered a hallmark of endoplasmic reticulum (ER) stress and is abundantly present already after 3 h. It may coordinate many of the downstream changes observed, including signaling pathways for cell cycle arrest/apoptosis, calcium homeostasis, cholesterol metabolism, ubiquitination, and proteasomal degradation. Also, eicosapentaenoic acid (EPA), but not oleic acid (OA), induced key mediators of ER stress and UPR at protein level. Accumulation of esterified cholesterol was not compensated for by increased total levels of cholesterol, and mRNAs for cholesterol biosynthesis as well as de novo synthesis of cholesterol were reduced. These results suggest that cytotoxic effects of DHA are associated with signaling pathways involving lipid metabolism and ER stress.

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.

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

BackgroundPrevious studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca2+ and Ca2+-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F2α.ResultsPretreatment of the cells with the Ca2+ chelators BAPTA-AM or EGTA, as well as the Ca2+ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF2α.ConclusionThe present data indicate that Ca2+ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.

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.

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.

Pronectin Promotes Calcium Signaling by Interferon-γ Human Neutrophils via G-Protein and Sphingosine Kinase-Dependent Mechanisms

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-γ (IFN-γ) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-γ-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-γ responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-γ-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine I-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-γ partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-γ. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-γ induced calcium signals.

Interleukin-17 (IL-17) Expression Is Reduced during Acute Myocardial Infarction: Role on Chemokine Receptor Expression in Monocytes and Their in Vitro Chemotaxis towards Chemokines

The roles of immune cells and their soluble products during myocardial infarction (MI) are not completely understood. Here, we observed that the percentages of IL-17, but not IL-22, producing cells are reduced in mice splenocytes after developing MI. To correlate this finding with the functional activity of IL-17, we sought to determine its effect on monocytes. In particular, we presumed that this cytokine might affect the chemotaxis of monocytes important for cardiac inflammation and remodeling. We observed that IL-17 tends to reduce the expression of two major chemokine receptors involved in monocyte chemotaxis, namely CCR2 and CXCR4. Further analysis showed that monocytes pretreated with IL-17 have reduced in vitro chemotaxis towards the ligand for CCR2, i.e., MCP-1/CCL2, and the ligand for CXCR4, i.e., SDF-1α/CXCL12. Our results support the possibility that IL-17 may be beneficial in MI, and this could be due to its ability to inhibit the migration of monocytes.

C‐Reactive Protein Triggers Calcium Signalling in Human Neutrophilic Granulocytes via FcγRIIa in an Allele‐Specific Way

C‐reactive protein (CRP) binds to Fcγ‐receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single‐cell digital imaging system. Receptor expression and polymorphism were studied by real‐time RT‐PCR, flow cytometry and standard PCR. C‐reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN‐γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP‐induced calcium signals increased. IFN‐γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN‐γ, can augment calcium signalling by CRP in low‐responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.

Effects of exercise on leukocytosis and blood hemostasis in 800 healthy young females and males

AIM To investigate the effects of exercise on healthy individuals of both genders. METHODS This study lasted 6 years and involved about 800 healthy people. Individuals were divided into females and males and further sub-divided into two groups; in the first group individuals run (or skied in the winter time) and then rested for 3 h, whereas individuals in the second group intensely cycled for 5 min. The status of health was determined by measuring the sedimentation rate and the intensity of exercises by measuring the heart rate. Blood samples were collected before and after exercise. RESULTS We observed that in the first group a significant increase of the total white blood cells, segmented neutrophils, band neutrophils, eosinophils and to a lesser extent lymphocytes but not monocytes in the blood circulation. However, all cell types were increased in the circulation after 5 min intense exercise. No differences in the pattern of cell increase were observed among the genders. Activated partial thromboplastin time (APTT) and D-dimer were also measured in the blood of individuals who cycled intensely for 5 min to determine the coagulation and fibrinolytic activities in the blood. APTT is reduced and D-dimer values significantly increased after intense exercise. However, APTT was statistically lower in males than females, whereas no differences in the D-dimer values were observed among the genders. CONCLUSION Our results indicate that exercise whether leisure or strenuous affects leukocytosis and hemostasis in both genders. A major advantage of this study is the high numbers of individuals involved and the inclusion of both females and males values.

Intricacies of Redoxome Function Demonstrated with a Simple In vitro Chemiluminescence Method, with Special Reference to Vitamin B12 as Antioxidant

The homeostatic control of the redox system (the redoxome) in mammalian cells depends upon a large number of interacting molecules, which tend to buffer the electronegativity of cells against oxidants or reductants. Some of these components kill – at high concentration – microbes and by‐stander normal cells, elaborated by professional phagocytes. We examined whether a simple, in vitro chemiluminescence set‐up, utilizing redox components from human polymorphonuclear neutrophils (PMN) and red blood cells (RBC), could clarify some unexplained workings of the redoxome. PMN or purified myeloperoxidase (MPO) triggers formation of reactive oxygen species (ROS), quantified by light emission from oxidized luminol. Both PMN and RBC can generate abundant amounts of ROS, necessitating the presence of a high‐capacity redoxome to keep the cellular electronegativity within physiological limits. We obtained proof‐of‐principle evidence that our assay could assess redox effects, but also demonstrated the intricacies of redox reactions. Simple dose–responses were found, as for the PMN proteins S100A9 (A9) and S100A8 (A8), and the system also revealed the reducing capacity of vitamin B12 (Cbl) and lutein. However, increased concentrations of oxidants in the assay mixture could decrease the chemiluminescence. Even more remarkable, A9 and NaOCl together stimulated the MPO response, but alone they inhibited MPO chemiluminescence. Biphasic responses were also recorded for some dose–response set‐ups and are tentatively explained by a ‘balance hypothesis’ for the redoxome.