extracellular superoxide dismutase ( sod3 ) is an enzyme known to catalyze dismutation of the highly reactive , superoxide anion into longer - lived and more stable hydrogen peroxide .
the consequences of sod3 action in the cells reach beyond the antioxidative functions , as it has been shown to downregulate inflammation , stimulate cell proliferation during tissue injury recovery , and to counteract apoptosis [ 4 , 5 ] by affecting cytokine production , cell signal transduction , and expression of survival - related genes .
the anti - inflammatory properties of sod3 have been studied in models of pulmonary disease and peritonitis [ 2 , 6 ] .
previous work with collagen - induced arthritis ( cia ) suggests that both genetic transfer of the sod3 gene   as well as a small molecular sod mimetic have the ability to ameliorate arthritis .
the arthritis ameliorating effect of sod3 was later confirmed using sod3 knock - out mice .
these results were explained on the basis that sod3 acts as an antioxidant and catalyses dismutation of superoxide into hydrogen peroxide , thus reducing inflammation - induced oxidative stress and restoring the oxidant balance in the arthritic joints [ 10 , 11 ] .
this explanation , however , is difficult to reconcile with the finding that animals naturally deficient in the induced oxidative burst in fact develop more severe arthritis [ 12 , 13 ] .
the most potent producer of superoxide , the substrate for sod3 , is the well - characterized phagocytic nox2 complex .
in inflamed tissues nox2 complex produces massive amounts of superoxide upon activation in a process called phagocyte oxidative burst .
in addition to nox2 , superoxide is produced from various other cellular sources , such as from the mitochondria during cellular respiration and by other members of the nox enzyme family .
however , it should be noted that during inflammation these superoxide producers are not nearly as efficient superoxide producers as the nox2 complex . in the current work we studied the role of sod3 in collagen - induced arthritis ( cia ) to understand whether the therapeutic effect of sod3 on arthritis operates through attenuating the biological effects of the induced oxidative burst produced by the nox2 complex . to avoid artifacts introduced by chemical inhibitors of nox2 complex [ 1416 ]
thus we used wild - type ( ncf1 ) and ncf1 mutated ( ncf1 ) mice on b10.q background .
these strains differ at only one snp in the ncf1 gene , which makes the ncf1 strain unable to produce oxidative burst .
the mutated mouse is more susceptible to induced arthritis due to hyperactivated t cells , and also increased susceptibility to thioglycollate peritonitis has been reported in the ncf1 knockout mouse .
our results confirm the previously documented anti - inflammatory role of sod3 and additionally , for the first time , we show that it can downregulate both cia and peritonitis even in the absence of functional nox2 complex and phagocyte oxidative burst .
the previously described ncf1 ( protein also called p47phox ) mouse , which carries a point mutation globally and completely abolishing nox2 complex derived ros production , has been backcrossed onto the b10.q background   and shown to contain only the causative mutation using a 10  k snp typing chip .
the mice were housed under specific pathogen - free conditions in climate - controlled environment and fed standard rodent chow and water ad libitum at turku university central animal facility . all experimental mice
were sex- and age - matched , treatment groups were blinded , and experimental groups were mixed in cages in all experiments .
the experiments were performed in accordance with the national and eu guidelines and the study was approved by the oulu section of the national animal experiment board ( elinkoelautakunta , ella ) with ethical approval numbers eslh-2008 - 02873 , eslh-2008 - 07941 , and esavi-0000497/041003/2011 . replication deficient adenoviral e1-partially - e3-deleted adbglii vectors ( developed from serotype ad5 ) expressing rabbit sod3 ( ade - sod3 ) or bacterial -galactosidase lacz ( ade - lacz )   were used in both in vitro and in vivo experiments .
collagen - induced arthritis ( cia ) was induced under isoflurane anesthesia by injecting 100  g rat type ii collagen ( purified from chondrosarcoma )   emulsified in complete freund 's adjuvant intradermally at the base of the tail .
arthritis was boosted day 19 with 50  g rat type ii collagen emulsified in incomplete freund 's adjuvant intradermally at the base of the tail .
disease development was evaluated macroscopically three times a week before the booster immunization and daily after the boost .
one point was given for each swollen toe or joint and five points for a swollen ankle , each paw having the maximum of fifteen points .
in the arthritis experiments ade - sod3 , ade - lacz ( vector control ) , and pbs ( injection control ) were injected locally in the left front paw in 25  l injection volume containing 2,5  10 pfu virus .
injections were performed right after the booster immunization during the same anesthesia at day 19 , before the onset of clinically apparent arthritis .
briefly , mice were pretreated i.p . with 0.5  10  pfu ade - sod3 , ade - lacz , or pbs three days before peritonitis induction with 5% proteose peptone ( bd difco , sparks , md , usa ) and 10  ng il-1 ( r&d systems , minneapolis , mn , usa ) in 1  ml pbs .
after 18 hours peritoneal infiltrating cells were collected with 10  ml ice - cold rpmi cell culture medium .
cells from the peritoneal lavage were counted and cytocentrifuged , slides were stained with reastain diff - quick ( reagena , toivala , finland ) , and differential counting was performed under a standard light microscope .
experiments were pooled and the total cell numbers are presented as percentual increase from the pbs injection control .
all peritonitis results were normalized to adjust the vector control group ( ade - lacz ) mean to 100 .
cos-7 cells stably expressing all the essential components of the nox2 complex , namely , cybb ( gp91phox ) , cyba ( p22phox ) , ncf2 ( p67phox ) , and ncf1 ( p47phox ) were provided by dr .
cells were cultured in dulbecco 's complete medium ( gibco ) , 10% fetal calf serum , and penicillin - streptomycin ( invitrogen , paisley uk ) .
extracellular superoxide production was quantified two days after transduction ( moi 4 ) with adenoviral constructs ( ade - sod3 and ade - lacz ) or medium control directly on the 96-well cell culture plate using an isoluminol - enhanced chemiluminescence method [ 22 , 23 ] .
briefly , the cells were washed with pbs , and 100  l of isoluminol buffer was added in each well .
isoluminol buffer contained isoluminol ( 10  g / ml , sigma - aldrich ) and horse radish peroxidase - type ii ( 4  u / ml , sigma - aldrich ) dissolved in pbs with pma ( 200  ng / ml , dissolved in dmso , sigma - aldrich ) and data collection was initiated immediately and followed at 37c as produced luminescence signal ( tecan infinite m200 , tecan , mnnedorf , switzerland ) for 30 minutes .
red blood cells were lysed from heparinized whole blood with hypotonic lysis buffer and leukocytes were surface stained with apc conjugated anti - gr-1 ( rb6 - 8c5 ) and efluor 450 conjugated anti - cd11b ( m1/70 ) antibodies ( ebioscience ) .
cells were suspended in high - glucose d - mem ( gibco ) with antibiotics without fcs and incubated for 10  min at 37c with 3  m dihydro - rhodamine 123 ( dhr-123 ; molecular probes and invitrogen life technologies ) followed by 20  min activation at 37c with 200  ng / ml pma ( sigma - aldrich ) .
the cells were washed into pbs and acquired on lsr ii flow cytometer equipped with facs diva software ( bd biosciences ) .
live cells were gated on the cell type , and geometric means of respective populations were analyzed with flowing software ( cell imaging core , university of turku ) .
isoflurane anesthetized animals with equal arthritis scores were injected intraperitoneally with 20  mg / kg l-012 probe ( wako chemicals , germany ) dissolved in pbs .
the luminescent signal was detected with ivis 50 bioluminescent system ( xenogen , usa ) that consists of an anesthesia unit built in a light tight chamber equipped with a ccd camera .
rna was isolated from the treated paws collected d25 according to the manufacturer 's instructions with tri reagent ( sigma - aldrich ) .
the isolated rna was dnase treated with deoxyribonuclease i ( fermentas ) in the presence of ribolock rnase inhibitor ( fermentas ) .
the rna was used for reverse transcription reaction performed with revert - aid m - mulv ( fermentas ) .
the acquired cdna was subjected to q - pcr with ade - sod3 ( fw : gtg tgc tcc tgc ctg ctc , rev : ctg ctc cac cgt gtc tga g ) and -actin specific primers ( fw : cta agg cca acc gtg aaa ag , rev : acc aga ggc ata cag gga ca ) , and the gene expression level was analyzed using sybr green pcr master mix ( applied biosystems ) , icycler iq multicolor real - time pcr detection system ( bio - rad ) , and the icycler ( version 3.1 ) software .
ade - sod signal was normalized against -actin expression levels and the results are reported as fold change from the ade - lacz group mean denoted as 1 .
statistical significance was analyzed by using two - tailed student 's t - test or if more than two groups were analyzed one - way anova with lsd post hoc analysis was run using ibm spss statistics 19 software ( spss inc . ) , p < 0.05 is considered as statistically significant .
adenoviral sod3 gene delivery reduced the superoxide predominant ros signal 37% compared to the ade - lacz control virus when investigated two days after transduction with ade - sod3 and ade - lacz viruses ( figure 1 ) .
mice started to develop mild , clinically apparent arthritis at day 20 and made a full response , mean arthritis scores reaching 7 ( out of the maximum of 15 ) in ade - laz treated and 3 points in ade - sod3-treated mice some days later .
the mean disease score in the treated paws was lower in the ade - sod3-treated group when compared to the ade - lacz - injected control group , and the difference reached statistical significance at d26 ( figure 2(a ) ) .
the treatment effect was only seen in the treated paw , while there were no differences between the treatment groups in sum scores of the three untreated paws ( see supplementary figure 1(a ) in supplementary material available online at doi : 10.1155/2012/730469 ) highlighting the local character of the used gene therapy vector expressing sod3 .
both virus vector treated groups showed elevated arthritis scores in the treated paws when compared to the pbs - injected control paws in the injection control group .
this difference is illustrated in figure 2(b ) , where ade - lacz - treated paws are shown to have larger increase in arthritis score than the ade - sod3-treated paws .
starting from d20 the ncf1 mice started to develop arthritis with significantly higher mean score than the wild - type mice .
interestingly , the mutated mice without functional nox2 complex derived superoxide production also responded to ade - sod3 treatment .
ade - sod3-treated paws of the ncf1 mice had significantly lower mean disease score than the ade - lacz - treated vector control paws at days 24 and 25 after arthritis induction .
similarly to the wild - type mice , the difference in the mean arthritis score in nox2 complex deficient mice was observed when sod3 was highly expressed from the adenoviral vector ( figure 3(a ) ) .
no differences were observed between the treatment groups in the sum scores of the untreated paws ( supplementary figure 1(b ) ) again supporting the local character of the immunomodulatory effect of ade - sod3 treatment .
similarly to the wild - type mice , ade - sod3 treatment in ncf1 paws induced significantly smaller difference between the virus - treated and pbs - injected paws than ade - lacz virus injection ( significant d24 and d25 after immunization ) , again confirming the arthritis limiting effect of sod3 ( figure 3(b ) ) .
the experiment was repeated and as the experiments were well reproduced , data from both experiments was combined for analysis .
sod3 expression significantly reduced the number of peritoneal infiltrating cells in proteose peptone and il-1 induced peritonitis in wild type mice ( figure 4(a ) ) .
the decrease in infiltrating leukocytes was mainly due to a lowered number of infiltrating macrophages ( figure 4(b ) ) , which well corresponds with the macrophage phase of peritonitis taking place three days after virus injection and 18 hours after peritonitis induction .
both virus - treated groups had more infiltrating cells than the pbs - injected control mice . similarly as in the wild - type mice ade - sod3 was shown to reduce the number of infiltrating cells in the peritoneal cavity of ncf1 mice when compared to ade - lacz - treated mice .
the difference was due to a significantly lowered number of infiltrating macrophages in the ade - sod3-treated mice ( figures 5(a ) and 5(b ) ) . in the wild - type mouse
both severely inflamed paws ( right hind leg and right front paw ) emitted strong ros - induced l-012 luminescence signal , while in spite of the severe inflammation , no luminescent signal was detected from the ncf1 mice ( severe arthritis with arthritis score of 15 in both front paws and milder symptoms in the left hind leg ) ( figure 6(a ) ) .
monocytes ( cd11b - pos , gr-1-lo , or gr-1-neg ) and granulocytes ( gr-1-hi , cd11b - pos ) from ncf1 mice were unable to generate efficient ros production upon pma stimulation , while phagocytes from the wild - type animals responded to pma stimulation and induced significant increase in dhr-123 derived fluorescence signal detected by flow cytometry ( figures 6(c ) and 6(d ) ) .
quantitative rt - pcr revealed a a 5.5-fold ( fc , fold change ) expression of adenovirally produced sod3 mrna in the ade - sod3-treated joints collected at d25 when compared to the ade - lacz - treated vector control paws ( figure 6(b ) ) .
sod3 is an enzyme that has been shown to give rise to therapeutic responses in damaged tissues such as reduced ischemia - reperfusion injury , arthritis , peritonitis , hind limb injury , and lung injury   models .
these tissue healing promoting and anti - inflammatory effects induced by sod3 are accompanied by reduced macrophage infiltration , inhibition of oxidative fragmentation of the extracellular matrix matrix , decreased apoptosis [ 4 , 5 ] , and enhanced cell proliferation .
the beneficial effects of sod3 are mostly explained by its antioxidant properties and reduction of oxidative stress in the injured tissues .
however , in this report we show that overexpression of sod3 downregulated inflammation even in the absence of phagocyte oxidative burst , thus highlighting the capacity of sod3 to affect cellular processes independently of nox2 complex ' superoxide production .
the adenoviral gene expression system used in this work reaches its maximal expression around three days after the injection , after which the vector is eliminated by the immune system and the expression of the transgene slowly decreases to undetectable levels 14 days after the initial injection .
we confirmed the presence of virally delivered sod3 mrna in the paws d25 and thus confirmed that the decrease in arthritis severity coincided with substantial adenovirus driven sod3 expression .
similarly , pretreating the mice three days before induction of peritonitis allowed us to analyze the effect of sod3 during substantial sod3 expression in the peritoneal cavity .
the percentual treatment effect in both inflammation models was comparable with the effect previously obtained with transgenic sod3 overexpression in pulmonary emphysema .
similarly , the degree of sod3-induced macrophage infiltration inhibition in peritonitis was similar as reported previously . even though sod3 is an important local regulator of the acute inflammatory reaction , it is obvious that there are a number of other factors affecting inflammation severity in vivo .
when the nox2 complex is not functional , there are still a number of other enzymes and enzyme complexes producing superoxide in the inflamed tissue .
nox1 has been reported to worsen hyperoxia - induced acute lung injury in mice , and nox4 has been suggested to stimulate microglial il-6 expression   and to hamper neurodegeneration after poststroke ischemia reperfusion injury .
however , only nox2 is abundantly expressed on phagocytes and thus recruited to inflammatory foci .
other nox family members are expressed on other cell types than phagocytes and their ros production is not upregulated during inflammation .
in addition to the nox family enzymes , superoxide is also produced during mitochondrial respiration at levels corresponding to the general metabolic rate in the tissue .
mitochondrial superoxide has also been linked to tlr2/4 signaling   and is also suggested as a pathologic mechanism in tissue injury .
all these superoxide generating processes , however , can not compensate for the massive production of ros by the nox2 complex during inflammation as the arthritic paws of ncf1 mice emitted no detectable luminescence signal when probed with l-012 dye ( figure 6(a ) ) .
l-012 reacts with any radical to produce light and can not be used to distinguish between superoxide and hydrogen peroxide in vivo . as a tool to study the nox2 complex dependency of sod3 , we used ncf1 mutated mice , in which a splice site point mutation abolishes the production of ncf1 protein leading to complete loss of nox2 complex derived oxidative burst . in line with previous reports , arthritis severity was significantly higher in ncf1 animals when compared to b10.q wild - type mice
. mutated mice also developed arthritis quicker after the booster allowing us to work with a more homogenous and extremely well reproducible disease model .
better arthritis synchronization in the mutated mouse model together with larger treatment groups resulted in less variability and increased statistical power in data analysis . in proteose
the use of ncf1 mutant mice allowed us to avoid pit falls associated with the use of chemical nox2 complex inhibitors such as dpi and apocynin , which are not specific for the nox2 complex , do not provide full suppression of superoxide production and additionally profoundly affect various other cellular processes [ 1416 ] .   in both arthritis and peritonitis , adenoviral gene expression vectors locally enhanced inflammation .
the arthritis enhancing effect was restricted to the treated paw , as the virus - injected groups did not differ from the pbs - injected control group when their nontreated paws were analyzed .
this is in line with previous reports where intra - articular injections of adenoviral gene expression vectors have been shown to induce increased paw swelling and elevated levels of inflammation mediators [ 33 , 34 ] .
intravenous injection route has not given rise to enhanced arthritis [ 3537 ] , but has triggered liver inflammation ; liver being the primary target of systemically administered adenoviral vectors .
the immunogenicity of the adenoviral gene expression systems is well documented in the literature [ 3840 ] .
sod3 polymorphisms are associated with copd , coronary artery disease , myocardial infarction   as well as acute lung injury and related mortality .
we report that sod3 limits inflammation in cia and peritonitis both in the presence and in the absence of phagocyte oxidative burst .
the anti - inflammatory function of sod3 is not compromised by the lack of functionality of the nox2 complex as both ncf1 and ncf1 mice develop milder inflammation when treated with sod3 .
thus , we conclude that the anti - inflammatory effect of sod3 is not dependent on superoxide produced by the nox2 complex derived phagocyte oxidative burst and thus acts via other signaling pathways .
 extracellular superoxide dismutase ( sod3 ) , an enzyme mediating dismutation of superoxide into hydrogen peroxide , has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues . 
 in inflamed tissues , superoxide is produced by the phagocytic nox2 complex , which consists of the catalytic subunit nox2 and several regulatory subunits ( e.g. , ncf1 ) . to analyze whether sod3 can regulate inflammation in the absence of functional nox2 complex 
 , we injected an adenoviral vector overexpressing sod3 directly into the arthritic paws of ncf1/ mice with collagen - induced arthritis . 
 sod3 reduced arthritis severity in both oxidative burst - deficient ncf1/ mice and also in wild - type mice . 
 the nox2 complex independent anti - inflammatory effect of sod3 was further characterized in peritonitis , and sod3 was found to reduce macrophage infiltration independently of nox2 complex functionality . 
 we conclude that the sod3-mediated anti - inflammatory effect on arthritis and peritonitis operates independently of nox2 complex derived oxidative burst . 