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Leishmania amazonensis INFECTION INDUCES CHANGES IN POTASSIUM PERMEABILITY OF MACROPHAGE-LIKE CELLS
![Camacho Marcela 1,2 , Forero María Elisa 1 , Marín Mariela 1 , Corrales Alexandra 1 , Llano Isabel 3 , Alison Agnew 4 , Moreno Herman 1 3 AG Zellulare Neurobiologie Max-Planck Institut für biophysikalische Chemie Göttingen, Germany Leishmania amazonensis INFECTION INDUCES CHANGES IN POTASSIUM PERMEABILITY OF MACROPHAGE-LIKE CELLS 2 Departamento de Biología, Universidad Nacional de Colombia, Bogotá, Colombia, email: mcamacho@ciencias.ciencias.unal.edu.co 1 Laboratorio de Biofísica, Centro Internacional de Física Bogotá, Colombia, email: biofisic@latino.net.co INTRODUCTION Leishmania species are intracellular obligatory parasites of macrophages that infect 15 million people worldwide. After entry into its mammalian host, the parasite is phagocytosed by macrophages and confined to a lysosome-like compartment (reviewed by Russell, 1995), known as the parasitophorus vacuole (PV). Parasite survival depends from three concentric membranes: the parasite surface membrane, the membrane of the PV, and the surface membrane of the macrophage. In several parasite-host systems, the parasite changes the plasma membrane of its host cell, thus modifying ion homeostasis to secure fast uptake of nutrients and discharge of waste products (Gero & Upston 1992). In the case of Leishmania, little information is available on macrophage ion homeostasis alterations due to infection. There is evidence for changes in macrophage intracellular calcium (Eilam, et al 1985), but changes in the intracellular concentrations or transport properties of other ions have not been reported. Our results show that macrophages altered their membrane potential (Vm) and potassium permeability as a result of infection and these responses differ from those generated by phagocytosis. P M PV A ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Fig 1. Light microscopy photograph of infected J774.1 macrophages (EECACC No. 91051511) with a recording pipette. (M) macrophage. (A) amastigote. (PV) (parasitophorus vacuole). (P) recording pipette. J774.1 were maintained as a monolayer in 25 cm 2 flasks at 37 o C, 5 % CO 2 in RPMI, 10 % FBS and 100 U/ml penicillin/100 g/ml spreptomycin (GIBCO). A L. amazonensis isolate kindly donated by Dr. Nancy Saravia (CIDEIM, Cali, Colombia) was used. Promastigotes, were cultured at 24 o C in 25 cm 2 flasks in Schneider´s medium, 10 % FBS (GIBCO) up to their infective stage. 1 x 10 7 J774.1 cells were exposed to promastigotes or to 3 mm latex beads (Sigma) a ratio of 1:10 respectively, and incubated at 34 o C, 5 % CO 2 for 4 hours. Non adherent promastigotes or beads were washed out. DISCUSSION Infected macrophages shifted their Vm from an early depolarization to hyperpolarization as infection progressed. Similar shifts have been observed during differentiation (Brent et al. 1996), and activation (Gamalei et al. 1991) of macrophages and other leukocytes. Our evidence suggest that depolarization is specific of phagocytosis rather than infection since this was detected only in early infection (less than 6 hours) and in cells with latex beads. But why a macrophage depolarizes? There is evidence of this event after activation and phagocytosis. Interestingly oxygen radicals depolarize macrophage plasma membrane (Holevisnky & Nelson 1995) and have been implicated in intracellular pathogen killing by macrophages (Murray 1981). Thus, it is possible that L. amazonensis entry into the macrophage fully activates these cells and the depolarization detected is the result of oxygen radical production. However, it has been documented that as infection progresses Leishmania down regulates this response. Macrophages hyperpolarize after days of adherence onto glass (data not shown, Gallin & Sheehy 1985) and after phagocytosis of latex beads (Companioni et al. 1983). In this study, we found that time of adherence altered Vm, therefore this was kept constant (24 hours adherence). However, we did not observe hyperpolarization due to phagocytosis. It was only associated with infection. Moreover, the hyperpolarization found was accompanied by an increase in inward Ip density that was not detected in control or cells with latex beads. Whether hyperpolarization is important for parasite survival is a question that requires further investigation Vm (mV) Hours post-infection -60 -40 -20 0 0 24 48 72 C P I e I * * * * * Leishmania infection induces hyperpolarization of the macrophage plasma membrane, and this appears to be infection specific To document permeability changes induced by Leishmania infection in macrophages, membrane potential (Vm) and ion currents were measure in these cells. The average Vm in control cells was -39.4 ± 4 mV (n=11). After 24 hours post infection (pi) Vm changed by 38 % and by 48 pi the cells had a Vm 58% more hyperpolarized than controls (Fig 2, left pannel). All infected groups differed significantly from control cells. In contrast, cells that ingested latex beads (P) depolarized their plasma membrane by 30% (n=7). These results suggest that the hyperpolarization observed is specific of Leishmania infection. Fig 2. Membrane potential of control, infected and phagocytic cells. Control and treated cells in suspension were allowed to adhere onto sterile glass coverslips and kept in 35 mm petri dishes at 34 o C for 24 hours prior to electrophysiological studies. Coverslips were placed in a recording chamber kept at 20 o C on a Zeiss IM35 microscope stage. Cells were bathed in (mM): 145 NaCl, 5 KCl, 1 CaCl 2 , 2 MgCl 2 , 10 HEPES-Na, 5 glucose (pH 7.34, 300 mOsm). Membrane currents were recorded in whole cell configuration (Hamill et al . 1981) with an Axopatch-1C amplifier (Axon Instruments). Pipettes from non-heparinised hematocrit capillaries (2.5-5 M ) were filled with (mM): 140 K glutamate, 2 KCl, 5 EGTA-K, 0.5 CaCl 2 , 4 MgCl 2 , 10 HEPES-K, 3 ATP-Na 2 , 0.5 GTP-Na (pH 7.34, 300 mOsm). During whole cell recording series resistance was not greater than 10 M and it was left uncompensated. Currents were filtered at 1 kHz and digitized at 200 s/point with a DigiData 1200 Interface (Axon Instruments, Foster City, CA, US). Data acquisition and analysis were performed with the pclamp6 software (Axon Instruments). Statistical results are given as mean ± s.e.m. To compare data from control and treated cells, multiple group comparisons were performed using ANOVA; p values < than 0.05 were considered significant. When used, n refers to number of cells included in the analysis. After attaining the whole cell configuration, the amplifier was set to the current-clamp mode and potential at which the current was zero was determined. This value is referred to as resting membrane potential (Vm). Data represent the average Vm ± SE. (*) p < 0.05. Cell groups: (C) control, (P) 48 hours post-phagocytosis, (I e ) 6 hours and (I) 48 hours post-infection. Outward Ip density (pA/pF) 0 24 48 72 Hours post-infection 5 15 25 35 C P I Fig 3. Outward peak current density (pA/pF) of control, infected and phagocytic cells obtained as described in Fig 2, using the voltage-clamp mode. Data represent average current density elicited by a voltage step of 50 mV applied from a holding potential of – 60 mV ± SE. (*) p < 0.05. Cell groups: (C) control, (P) 48 hours post-phagocytosis and (I) 6 hours post-infection. Macrophage depolarization associates with phagocytosis Outward peak current (Ip) density, was not altered by Leishmania infection. None of the infected groups tested was significantly different from the control group where we detected an outward Ip density of 18.5 ± 2.9 pA/pF (Fig 3 left pannel). In phagocytic cells (P) we detected a 28 % decrease in outward Ip density (n=4), but this was not significantly different from controls (Fig 3 right pannel. However, depolarization was present in early infection where Vm was 37% lower than control cells (n=17, I e , Fig 2 right pannel) suggesting that this Vm decrease may be the result of phagocytosis. Inward Ip density (pA/pF) Hours post-infection C P I -40 -30 -20 -10 0 0 24 48 72 * * * * Fig 4. Inward peak current density (pA/pF) of control, infected and phagocytic cells obtained as described before. Data represent average current density elicited by a voltage step of -130 mV applied from a holding potential of – 60 mV ± SE. (*) p < 0.05. Cell groups: (C) control, (P) 48 hours post-phagocytosis and (I) 48 hours post-infection. Macrophage hyperpolarization during infection associates with an increase of inward current density In J774.1 cells, K + inward rectifier channels (KIR) appear to set the Vm, therefore changes in the properties and/or density of inward rectifying K + currents (I KIR ) are likely to account for the hyperpolarization observed after infection. I KIR was studied under conditions resembling physiological ones, i.e ., low K + outside and low Cl - inside. I KIR amplitude was larger in infected cells at all potentials above the holding potential. By 24 hours pi, I KIR density had increased 96%, from its control value of -14.9 ± 2.4 pA/pF (n= 12) to -29.2 ± 3.8 pA/pF (n=7). For the 48 and 72 hours pi groups, I KIR density remained significantly larger than that of control cells. As expected, phagocytic (P) cells did not increase I KIR density. Therefore, I KIR increased density in infected cells appears to be specific of this process. 4 School of Biology University of Leeds Leeds, Great Britain AGKNOWLEDGEMENTS This work was supported by Universidad Nacional de Colombia and financed by the Colombian Institute of Sciences, Colciencias, (grant NO. 2228-05-601-95) and by the grant from The Wellcome Trust to Dr. M. Camacho (grant NO. 04102/Z/96/CSD/JMacQ). We thank Dr. Nancy Saravia for providing parasites and for advice throughout the course of this work and Fundaciòn Universitaria Manuela Beltràn for supporting M.E. Forero.](https://image.slidesharecdn.com/posterneworleans-090409082857-phpapp02/85/leishmania-amazonensis-infection-induces-changes-in-potassium-permeability-of-macrophagelike-cells-1-320.jpg)
