Rapid Communication Interferon beta-1b inhibits reactive oxygen species production in peripheral blood monocytes of patients with relapsing-remitting multiple sclerosis

NEUROCHEMISTRY International Neurochem[ Int[ 22 "0887# 090Ð091

Rapid Communication

Interferon beta!0b inhibits reactive oxygen species production in peripheral blood monocytes of patients with relapsing!remitting multiple sclerosis Miguel Lucasa\\ Olga Sanchez!Solin½ob\ Francisca Solanoa and Guillermo Izquierdoc a

Molecular Biolo`y Service of the Vir`en Macarena University Hospital\ Seville\ Spain b Scherin` Espan½a S[A[\ Madrid\ Spain c Neurolo`y Service of the Vir`en Macarena University Hospital\ Seville\ Spain Received 5 February 0887^ accepted 8 February 0887

Abstract We studied the rate of reactive oxygen species "ROS# production by monocytes {ex vivo| in a cohort of healthy individuals\ in a group of MS patients undergoing treatment with interferon beta!0b and another group of MS patients who refused treatment with interferon beta!0b[ The rate of ROS production in healthy individuals was slightly lower than in non!treated MS patients[ The lower rate of ROS production was obtained in monocytes of MS patients treated with interferon beta!0b[ These results indicate that the treatment of relapsing!remitting MS patients with interferon beta!0b rendered the NADPH oxidase of the monocytes less sensitive to trigger reactive oxygen species "ROS#[ Þ 0887 Elsevier Science Ltd[ All rights reserved[

0[ Introduction Oxygen!derived free radicals are a highly reactive chemi! cal species involved in a variety of diseases\ including neurodegenerative disorders "Knight\ 0886#[ Superoxide anion "O− 1 #\ hydroxyl radical "OH = # and hydrogen per! oxide "H1O1#\ known as reactive oxygen species "ROS#\ are produced by the reduction of molecular oxygen to water in mitochondria\ by some amino oxidase!catalysed reactions\ and during the activation of phagocytic NADPH oxidase[ The nervous system has a high meta! bolic rate and is very rich in oxidizable substrates\ viz[ catecholamines and polyunsaturated lipids\ in addition to DNA "Halliwell and Gutteridge\ 0874#[ The ROS can damage and degrade myelin in vitro by lipid peroxidation "Konat and Wiggins\ 0874#\ and data have suggested a role of ROS in the pathogenesis of multiple sclerosis "MS# "LeVine\ 0881#[ It has been shown that interferons can regulate the production of superoxide anion in cultured rat microglia "Colton et al[ 0881#\ but similar activity in the central

 Corresponding author[ Departamento de Bioqu(mica\ Facultad de Medicina\ Avda[ Sanchez Pizjuan 3\ 30998 Sevilla\ Spain[ Fax] 234 344 63 70^ e!mail] LucasÝcica[es 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 0 3 Ð X

nervous system has not yet been described^ the inac! cessibility of the brain to biochemical monitoring and the extreme reactivity of free oxygen radicals hinder in vivo studies on the relation between ROS and in~ammation in the central nervous system[ Nonetheless\ at least one study has demonstrated the production of H1O1 in rat brain in vivo "Halliwell and Gutteridge\ 0874#[ The purpose of the present work was to study the activity of the NADPH!oxidase of monocytes of relaps! ing!remitting MS patients with and without treatment with interferon beta!0b[ This is a _rst approach to the hypothesis of the involvement of ROS production by brain!resident monocyte!derived cells in MS[

1[ Patients and methods Monocytes from peripheral blood were isolated by a sin! gle procedure\ with minimal handling of the cells[ Blood "09 ml# was drawn from] "a# healthy individuals "H\ n  03#^ "b# MS patients treated with interferon beta!0b "T\ n  7#^ and "c# non!treated\ control\ MS patients "C\ n  7#[ Mononuclear cells were separated by _coll gradi! ent\ washed\ and resuspended in phosphate bu}ered saline "PBS# supplemented with 0[1 mM CaCl1 and 0[1

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M[ Lucas et al[:Neurochem[ Int[ 22 "0887# 090Ð091

mM MgCl1[ Cells were incubated in plastic tubes that _t in the chemiluminometer chamber[ After 0 h at room temperature\ non!adhered cells were discarded by care! fully washing the wall of the tubes[ The cells were sup! plemented with 0 ml of PBS containing calcium and magnesium "see above#\ and the chemiluminescence response was measured following the activation of the cells with 099 nM PMA "Lucas and Solano\ 0881#[ We measured the production of ROS in the 0st and in the 1nd min after challenging the freshly isolated mon! ocytes with PMA\ which mimics the e}ect of diacyl! glycerol on protein kinase C[

2[ Results and discussion The activation of NADPH oxidase in monocytes by PMA showed a very clear response after a time lag close to 04 s[ The rate of chemiluminescent oxidation of lum! inol reached maximal values in 2Ð3 min and thereafter declined back to basal values[ The rate of ROS pro! duction in healthy individuals was slightly lower "stat! istically not signi_cant# than in non!treated MS patients[ The most striking _nding was the lower rate of monocyte activation in the monocytes obtained from beta!inter! feron treated patients^ the di}erence between the treated group and the control "MS non!treated patients# and healthy groups was statistically signi_cant both 0 minute and 1 minutes after PMA stimulation "see Fig[ 0#[ These results indicate that the treatment of relapsing! remitting MS patients with interferon beta!0b rendered the NADPH oxidase of the monocytes less sensitive to the triggering agent of ROS[ The mechanism of this e}ect is not yet known\ but a down!regulation of protein kinase C\ essential in NADPH activation\ can be argued[ Although not statistically signi_cant\ the production of ROS was higher in MS controls "non!treated patients# than in the healthy group[ It is known that microglia and macrophages\ both monocyte!derived cells\ are involved in the pathogenesis of in~ammatory processes in MS[ Whether or not the present _ndings in peripheral blood monocytes can be extrapolated to brain!resident\ mon! ocyte!derived cells\ viz[ microglia and macrophages\ remains to be studied[

Acknowledgments Partially supported by Grant 85:9167 from the Fondo de Investigaciones Sanitarias and by the Fundacion Espa! n½ola de Esclerosis Multiple[

Fig[ 0[ ROS production in monocytes[ We studied the rate of ROS production by monocytes {ex vivo| in three groups of individuals] a# a cohort of healthy individuals "n  03#^ "b# a group of MS patients undergoing treatment with interferon beta!0b "n  7#\ and "c# another group of MS patients who refused treatment with interferon beta!0b "n  7#[ The samples of treated MS patients were taken at 9[14\ 9[4\ 0\ 2\ 5\ 8 and 01 months\ and at 9\ 0\ 2\ 5\ 8 and 01 months for non!treated MS patients[ The cohort of 03 healthy volunteers was analysed over 01 months[ For statistical comparison\ the data of each group were pooled[ Values refer to the rate of luminol oxidation "cpm:0999 cells# in the 0st "light grey boxes# and in the 1nd "dark grey boxes# minute following the activation of monocytes by PMA[ The results are shown as mean and SEM[ The statistical signi_cance of the di}erences of the means of healthy "H#\ control "C# and treated "T# groups was determined by the Student|s t!test[ "i# Rate of ROS production in the 0st min] H vs T\ P  9[92^ C vs T\ P  9[990^ H vs C\ P  9[4[ "ii# Rate of ROS pro! duction in the 1nd min] H vs[ T\ P  9[92^ C vs[ T\ P  9[990^ H vs[ C\ P  9[3[

References Colton\ C[A[\ Yao\ J[\ Keri\ J[E[\ Gilbert\ D[\ 0881[ Regulation of microglial function by interferons[ J Neuroimmunol 39\ 78Ð87[ Halliwell\ B[\ Gutteridge\ M[C[\ 0874[ Oxygen radicals and the nervous system[ Trends Neurosci 7\ 11Ð18[ Knight\ J[A[\ 0886[ Reactive oxygen species and the neurodegenerative disorders[ Ann Clin Lab Sci 16\ 00Ð14[ Konat\ G[W[\ Wiggins\ R[C[ 0874[ E}ect of reactive oxygen species on myelin membrane proteins[ J Neurochem 34\ 0002Ð07[ LeVine\ S[M[\ 0881[ The role of reactive oxygen species in the patho! genesis of multiple sclerosis Med Hypotheses 28\ 160Ð3[ Lucas\ M[\ Solano\ F[\ 0881[ Coelenterazine is a superoxide anion! sensitive chemiluminescent probe\ its usefulness in the assay of res! piratory burst in neutrophils[ Anal Biochem 195\ 162Ð166[